Antibodies capable of binding oX40 in combination therapy

Abstract

This invention provides a combination therapy using an antibody capable of binding to OX40 in combination with a PD-1 inhibitor to reduce or prevent the progression of tumors or cancer or to treat tumors or cancer.

Description

Technical Field

[0001] This invention relates to a combination therapy using an antibody capable of binding to OX40 in combination with a PD-1 inhibitor to reduce or prevent tumor progression or to treat cancer. Background Technology

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

[0003] The only known ligand for OX40 is the type II transmembrane glycoprotein OX40L (TNFSF4; CD252). OX40L is not persistently expressed, but its expression can be induced on dedicated 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) and non-hematopoietic cells (such as endothelial cells and smooth muscle cells) (Croft et al. Immunol Rev. 2009 May; 229(1):173-91).

[0004] In addition to their membrane-bound forms, both OX40 and OX40L exist in soluble forms (sOX40 and sOX40L), which have previously been associated with autoimmune diseases. The biological activity of these soluble forms is supported by reports that sOX40 can compete with membrane-expressed OX40 for interaction with OX40. This can suppress inflammatory responses and thus mimic Treg function (Laustsen et al., Arthritis Res Ther. 2014 Oct 30; 16(5):474).

[0005] The OX40 protein contains three complete cysteine-rich domains (CRDs; CRD1, CRD2, and CRD4) and a truncated CRD (CRD3; Willoughby et al., Mol Immunol. 2017 Mar; 83:13-22). OX40L binding encompasses CRD1-3. OX40L is a trimer, and each trimer associates with three OX40 molecules on the T cell surface. The formation of the trimer receptor-ligand complex ensures the clustering of OX40 cytoplasmic domains, which contain QEE motifs characteristic of many TNFR family members, creating docking sites for TNFR-associated factor (TRAF) adaptor proteins. These interactions link receptor binding to activation of various signaling pathways, such as via activation of NF-κB and PI3K / AKT (Willoughby et al., Mol Immunol. 2017 Mar; 83:13-22).

[0006] In humans, the binding of OX40L to OX40 leads to downstream signaling, enhancing the expression of cyclin A, cyclin-dependent kinase, Bcl-2 anti-apoptotic molecules, and various cytokines (such as IL-2) and their receptors. These effects ultimately result in the proliferation and survival of effector T cells. In addition, OX40 signaling promotes the generation of memory T cells and inhibits the function of Tregs (Croft et al. Immunol Rev. 2009 May; 229(1):173-91). In mice, several in vivo studies have demonstrated that OX40 antagonism induces FoxP3 expression in naïve CD4+ T cells and inhibits IL-10 expression in induced Tregs. Moreover, after interaction with Tregs, agonistic OX40 antibodies help deplete tumor-infiltrating OX40-expressing Tregs via antibody-dependent cytotoxicity (ADCC) induced by bone marrow and NK cells (Choi et al. J ImmunotherCancer. 2020 Oct; 8(2):e000966). However, under certain conditions, such as when IFN-γ and IL-4 are absent, OX40 signaling has been reported to induce Treg proliferation (Ruby et al. J Immunol. 2009 Oct 15; 183(8):4853-7).

[0007] Experiments have been conducted to verify whether targeting the OX40-OX40L interaction by blocking OX40 or OX40L has therapeutic benefits for autoimmune diseases, including EAE, SLE, RA, colitis, AEU, type 1 diabetes, MS, graft-versus-host disease (GVHD), and inflammatory bowel disease (IBD) (Fu et al., Acta Pharm Sin B. 2020 Mar; 10(3):414-433).

[0008] A small retrospective study using melanoma cell lines and patient samples demonstrated that lower levels of OX40 in the tumor microenvironment (TME) were associated with worse prognosis after anti-PD-1 therapy, particularly in patients with fewer tumor-infiltrating lymphocytes (TILs). Activating OX40 signaling through combination therapy with an anti-OX40 agonist monoclonal antibody (mAb) and adoptive T-cell therapy helped restore or enhance T-cell-mediated antitumor responses, resulting in survival benefits in mice carrying prostate tumors. The antitumor activity of OX40 mAbs was associated with T-cell infiltration into the tumor and intratumoral proliferation of effector T cells (He et al., Int Immunopharmacol. 2020 Dec; 89(Pt B):107097).

[0009] In addition, several agonistic IgG1 antibodies against human OX40 have been identified for the treatment of cancer.

[0010] WO2009 / 079335A1 revealed the human OX40-binding antibody 11D4, which exhibited agonistic activity in various murine tumor models. The antibody clone 11D4 induced T cell activation after incubation with primary T cells pre-incubated with anti-human CD3 or healthy donor PBMC samples. 11D4 blocked the binding of the natural ligand OX40L and also showed binding to cynomolgus monkey T cells. Gutierrez et al. reported that the 11D4-based clinical candidate antibody BMS-986178, when administered as monotherapy in a phase 1 / 2a clinical trial, did not induce dose-limiting toxicities or objective responses in patients with advanced cancer (Gutierrez et al., Clin Cancer Res. 2021 Jan 15; 27(2):460-472).

[0011] Other OX40 antibodies described as inducing T cell activation by linking human OX40 include clones 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).

[0012] Optimizing effector function by modifying the Fc region of an antibody can improve the effectiveness of therapeutic antibodies for treating cancer or other diseases, such as enhancing the antibody's ability to induce an immune response against antigen-expressing cells. These effects are described, for example, in WO2013 / 004842 A2; WO2014 / 108198 A1; WO2018 / 146317; WO2018 / 083126; and WO2018 / 031258 A1.

[0013] US2014 / 0377284A1 reveals the human OX40-binding antibody clone 12H3 (also known as SF2), which does not block the binding of OX40L to OX40. The SF2 antibody induces T cell activation and shows an inhibitory effect on Tregs in vitro. Zhang et al. demonstrated that the agonist and effector functions of the antibody clone SF2 can be enhanced by different engineering methods, including the introduction of the hexamerization-enhancing mutant E345R (Zhang et al. J Biol Chem. 2016 Dec 30; 291(53):27134-27146).

[0014] WO2016 / 164480A1 reveals human OX40-binding antibody clones (including clone h3C8) that have been molecularly engineered to confer enhanced agonistic activity. Engineering strategies include introducing enhanced hexamerization mutations E345R, E430G, S440Y, or different combinations thereof. Additionally, inert Fc mutations such as L234A-L235A-P329G are introduced.

[0015] US20190276549A1 describes a method for engineering antibodies to have increased Fc-Fc interactions, while further containing a mutation that reduces the binding of complement factor C1q to the Fcγ receptor, to produce antibodies that target non-depleted immune cells and have agonistic properties independent of Fcγ receptor binding.

[0016] Other engineered methods for developing human OX40 agonist constructs have been described, including creating a hexavalent construct by covalently linking the Fc domains of six single-domain antibodies (WO2017 / 019805A1) and a fusion protein consisting of the extracellular domain (ECD) of PD-1, which is linked to the extracellular domain (ECD) of the OX40L trimer via an IgG4 Fc-linked protein.

[0017] PD-1 (also known as CD279) is an immunomodulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. The protein PD-1 has two naturally occurring ligands, called PD-L1 (also known as CD274) and PD-L2 (also known as CD273). PD-L1 is expressed in a wide range of cancers, including melanoma, lung cancer, kidney cancer, bladder cancer, esophageal cancer, gastric cancer, and others. Therefore, in cancer, the PD-L1 / PD-L1 system, through interaction with PD-1, can suppress T lymphocyte proliferation, cytokine release, and cytotoxicity, thereby providing cancer cells with an opportunity to avoid T cell-mediated immune responses.

[0018] Monoclonal antibodies are known to be suitable for modulating the activity of the PD-1 / PD-L1 axis. PD-1 / PD-L1 interaction can be inhibited by the following antibodies: antibodies targeting PD-1, such as pembrolizumab (also known as MK-3475, lambolizumab, or Keytruda) and nivolumab (also known as ONO-4538, BMS-936558, or Opdivo), or monoclonal antibodies developed to bind to PD-L1, such as atezolizumab (also known as MPDL3280A, RG7446, or Tecentriq).

[0019] Among them, Garber et al. discussed the opportunity for combination therapy, which consists of agonist antibodies targeting co-stimulatory receptors on T cells (such as 4-1BB (CD137), OX40, glucocorticoid-induced tumor necrosis factor receptor family-associated receptor (GITR) and independent co-stimulatory receptors (ICOS)) and monoclonal antibodies blocking the PD-1 / PD-L1 axis (Garber et al. Nat Rev Drug Discov. 2020 Jan;19(1):3-5).

[0020] WO2016200835A1 provides a method for treating or delaying cancer progression in an individual, which involves administering an anti-human OX40 agonist antibody and an anti-PDL1 antibody to the individual.

[0021] The effects of combining anti-OX40 antibody therapy with anti-PD-1 therapy were studied in mice with pancreatic tumors (Ma et al., Combination of PD-1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer. Gastroenterology. 2020 Jul; 159(1):306-319.e12). Furthermore, the combination of anti-PD-1 and anti-OX40 therapy enhanced the antitumor effect in a mouse model of lung cancer (Lao et al., OX40 enhances T cell immune response to PD-1 blockadetherapy in non-small cell lung cancer Int Immunopharmacol. 2022 Jul:108:108813).

[0022] However, despite these and other efforts in this art, there remains a need for therapeutically potent OX40 antibodies, provided as combination therapies with antibodies that block immune checkpoints, possessing enhanced agonism and / or increased potency and / or effectiveness even when the number of FcγR-expressing cells is limited. Therefore, an object of the present invention is to provide a potent and highly agonistic anti-OX40 antibody that induces greater T cell proliferation and activation, independent of the number of FcγR-expressing cells providing secondary cross-linking to OX40 clusters on the cell membrane. Therefore, another object of the present invention is to provide an anti-OX40 antibody that does not require cross-linking of FcyR-expressing cells to activate the co-stimulation of OX40-mediated immune responses. Another object of the present invention is to provide an anti-OX40 antibody that binds to both human OX40 and cynomolgus monkey OX40. Another object of the present invention is to provide an OX40 agonist antibody that induces OX40 agonism through enhanced IgG1 hexamer formation, independent of secondary cross-linking in a C1q or FcyR-independent manner. In the context of cancer, these antibodies may enhance anti-tumor immunity. Another object of the present invention is to provide a method for enhancing the potency of OX40 agonist antibodies by combining these agonists with PD-1 inhibitors. There remains a need for anti-OX40 antibodies that exhibit highly potent agonistic activity to enhance anti-tumor immune responses. Summary of the Invention

[0023] This invention relates to antibodies capable of binding OX40 in combination therapy. The inventors were surprised to discover a synergistic effect between anti-OX40 antibodies (i.e., IgG1-CD134-003-HC6LC2-RR) and anti-PD-(L)1 antibodies.

[0024] In one aspect, this disclosure provides a method for treating a subject's disease, preferably a method for reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject i) an antibody capable of binding OX40; and ii) a PD-1 inhibitor.

[0025] In one aspect, this disclosure provides a kit comprising i) an antibody capable of binding OX40, ii) a PD-1 inhibitor, and optionally (iii) one or more additional therapeutic agents.

[0026] In one aspect, this disclosure provides a kit for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in a subject or treating a subject's cancer, the kit comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor.

[0027] In one aspect, this disclosure provides pharmaceutical compositions comprising i) an antibody capable of binding OX40; ii) a PD-1 inhibitor; and iii) optionally a pharmaceutically acceptable carrier.

[0028] In one aspect, this disclosure provides a pharmaceutical composition for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in a subject or treating a subject's cancer, the pharmaceutical composition comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor.

[0029] In one aspect, this disclosure provides an antibody capable of binding OX40 for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject i) an antibody; and ii) a PD-1 inhibitor.

[0030] In one aspect, this disclosure provides a PD-1 inhibitor for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject i) an antibody capable of binding OX40; and ii) a PD-1 inhibitor.

[0031] In one aspect, this disclosure provides the use of an antibody capable of binding OX40 in the preparation of a medicament, preferably in combination with a PD-1 inhibitor for reducing or preventing tumor progression in a subject or for treating cancer in a subject.

[0032] In one aspect, this disclosure provides the use of a PD-1 inhibitor in the preparation of a medicament, preferably in combination with an antibody capable of binding OX40 for reducing or preventing tumor progression in a subject or for treating cancer in a subject.

[0033] In one aspect, this disclosure provides (i) the use of an antibody capable of binding OX40 and (ii) a PD-1 inhibitor in the preparation of a medicament, preferably for reducing or preventing tumor progression in a subject or for treating cancer in a subject.

[0034] In one aspect, this disclosure provides a medical preparation comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor. Attached Figure Description

[0035] Figure 1 The binding of the anti-human OX40 antibody to (A) human and (B) cynomolgus monkey OX40 expressed on OX40-transfected HEK293F cells is shown, as determined by flow cytometry. The unbinding antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment performed in one of the two experiments.

[0036] Figure 2 The half-maximum effective concentration (EC50) of the anti-human OX40 antibody for binding to human and cynomolgus monkey OX40 expressed on OX40-transfected HEK293F cells is shown, as determined by flow cytometry. Data shown are mean EC50 + SD from the two experiments performed. The black dashed line represents the mean EC50 for binding to IgG1-CD134-003-HC6LC2-RR containing human OX40.

[0037] Figure 3 The sequence alignment of the OX40 shuffle constructs with wild-type human and mouse OX40 (TNR4) is shown. Amino acids in the shuffle constructs that differ from 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.

[0038] Figure 4 This shows the sequence alignment of human and mouse OX40 (TNR4). Amino acids in mouse OX40 that differ from the human OX40 sequence are highlighted in black.

[0039] Figure 5The 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 the individual CRDs of human OX40 replaced with mouse analogs (C to F), as determined by flow cytometry, is shown. The non-binding antibody IgG1-b12-RR is included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment from two experiments performed.

[0040] Figure 6 The binding of the 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 the individual CRDs of human OX40 replaced with mouse analogs (C to F), as determined by flow cytometry, is shown. The non-binding antibody IgG1-b12-RR is included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment from two experiments performed.

[0041] Figure 7 The binding of the 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 the individual CRDs of human OX40 replaced with mouse analogs (C through F), as determined by flow cytometry, is shown. The non-binding antibody IgG1-b12-RR is included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment from two experiments performed.

[0042] Figure 8The binding of the anti-human OX40 antibodies IgG1-CD134-GBR830, IgG1-CD134-Hu106, and IgG1-CD134-IBI101 to ExpiCHO-S cells transiently transfected to express mouse OX40 (A), human OX40 (B), or one of the individual CRDs of human OX40 replaced with mouse analogs (C to F), as determined by flow cytometry, is shown. The non-binding antibody IgG1-b12-RR is included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment from two experiments performed.

[0043] Figure 9 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 the individual CRDs of human OX40 replaced with mouse analogs (C to F), as determined by flow cytometry, is shown. The non-binding antibody IgG1-b12-RR is included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from a representative experiment from one of the two experiments performed.

[0044] Figure 10 The relative binding of the anti-human OX40 antibody 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) is shown, as measured by flow cytometry. The data shown are mean fluorescence intensities (MFI) measured at an antibody concentration of 5 μg / mL, normalized to the MFI of antibody IgG1-CD134-A4453. The black dashed line represents individual measurements, and the horizontal bars represent the mean and SD of two experiments performed.

[0045] Figure 11The figures show reporter cells expressing OX40 activated with IgG1-CD134-003-HC6LC2-RR and variants of antibodies IgG1-CD134-h3C8(A), IgG1-CD134-RG7888(B), IgG2s-CD134-SF2-E345R(C), IgG1-CD134-007-RR, IgG1-CD134-012-RR(D), and IgG1-CD134-11D4(E). The non-binding antibody IgG1-b12-RR was included as a negative control. The data shown are the mean RLU from a single measurement, two replicates (IgG1-CD134-003-HC6LC2-RR in Figures A-D), or four replicates (IgG1-CD134-003-HC6LC2-RR in Figure E) performed in one experiment.

[0046] Figure 12 This demonstrates the half-maximal effective concentration (CMP) of the anti-human OX40 antibody for activating reporter cells overexpressing human OX40. 50 (This shows individual ECs derived from two to five independent experiments.) 50 Values, the horizontal line represents the average EC50 of all experimental combinations. 50 The black dashed line represents the average EC50 of IgG1-CD134-003-HC6LC2-RR. 50 .

[0047] Figure 13 The effects of IgG1-CD134-003-HC6LC2-RR and variants of antibodies IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2s-CD134-SF2 on the proliferation of activated primary human CD4+(A) and CD8+(B) T cells, as analyzed by flow cytometry, are shown. The unconjugated antibody IgG1-b12-RR was included as a negative control. Data shown are mean expansion indices ± SD of repeated measurements of (A) CD4+ and (B) CD8+ T cells from one representative donor from four to eight tested donors. The black dashed line represents the expansion index of CD4+ or CD8+ T cells cultured in the absence of anti-human OX40 antibody. (C) In polyclonal activated healthy human donor CD8+ - CD4 within PBMC + The T cell expansion index, and (D) in polyclonal activated healthy human donor CD4 - CD8 within PBMC + The T-cell expansion index, as analyzed on day four. Data were derived from a representative donor from one of the four donors tested in the three experiments conducted.

[0048] Figure 14 The effects of variants of IgG1-CD134-003-HC6LC2-RR and antibodies IgG1-CD134-h3C8 and IgG1-CD134-RG7888 on the percentage of CD4+ central memory T cells within 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 ± SD of repeated measurements of CCR7+CD45RA- cells in (A) CD4+ T cells and (B) CD8+ T cells from a representative donor from one of the six donors tested. The black dashed line represents the percentage of CCR7+CD45RA- cells in CD4+ T cells cultured in the absence of anti-human OX40 antibody.

[0049] Figure 15 The binding of IgG1-CD134-003-HC6LC2-RR, its variant without the Fc indolent 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 is shown, 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 in a single experiment.

[0050] Figure 16 The binding of IgG1-CD134-003-HC6LC2-RR, its variant without the Fc indolent 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 is shown, 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 in a single experiment.

[0051] Figure 17The binding of the anti-human OX40 antibody to ExpiCHO-S cells transiently transfected to express FcγRIa is shown. Binding was demonstrated by flow cytometry analysis of IgG1-CD134-003-HC6LC2-RR, its variant without Fc mutation (i.e., IgG1-CD134-003-HC6LC2) and its parental chimeric antibody (i.e., IgG1-CD134-003) (A), and antibodies IgG2-CD134-SF2 (a variant) (B), IgG1-CD134-11D4, IgG1-CD134-INCAGN1949, and IgG1-CD134-IBI101 (C), IgG1-CD134-h3C8 (D), and IgG1-CD134-RG7888 (E). The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean of fluorescence intensity (gMFI) from a representative experiment from one of the two experiments conducted.

[0052] Figure 18 The results showed that IgG1-CD134-003-HC6LC2-RR was activated in primary human CD4 cells after anti-CD3 / CD28 bead activation. + and CD8 + The binding capacity on T cells was analyzed by quantitative flow cytometry using saturated concentrations of IgG1-CD134-003-HC6LC2-RR 1, 2, or 3 days after T cell activation. The data presented are the mean number of antibody binding sites from three donors ± SD, with the symbol indicating the number from an individual donor.

[0053] Figure 19 The results showed that IgG1-CD134-003-HC6LC2-RR was associated with activated human CD4+. + and CD8 + Dose-dependent binding of T cells was assessed. Human PBMCs were cultured for 2 days in the presence of anti-CD3 / CD28 antibody and subsequently 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 using anti-human Fcγ antibody. Data shown are mean ± SD of replicate wells from a representative donor from one of the three donors tested.

[0054] Figure 20 The results showed IgG1-CD134-003-HC6LC2-RR or soluble OX40L (sOX40L) and activated human CD4+.+ and CD8 + T-cell binding. Human CD4 cells activated for two days with anti-CD3 / CD28 beads. + and CD8 + T cells were incubated with IgG1-CD134-003-HC6LC2-RR or control antibody IgG1-b12-RR in the presence and absence of sOX40L at a saturated concentration (2 μg / mL). Analysis was performed by flow cytometry. (A) with CD4 + and CD8 + Antibodies that bind to T cells. (B) and CD4. + and CD8 + T-cell binding to sOX40L. The data shown are geometric mean fluorescence intensity (mean gMFI) ± SD from duplicate wells of a representative donor from one of the three donors.

[0055] Figure 21 The image shows reporter cells expressing OX40 activated with anti-human OX40 antibody. (A) Mean bioluminescence ± SD as alternative OX40 agonist activity, 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 the presence and absence of FcγRIIb-CHO-K1 cells. + RLU ± SD in Jurkat reporter T cells. Data shown are from one experiment. (B) OX40 cells incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-11D4, IgG1-CD134-RG7888, IgG1-CD134-IBI101 or the unbound control antibody IgG1-b12-RR in the presence and absence of IgG1-CD134-003-HC6LC2-RR. + Mean bioluminescence ± SD in Jurkat reporter T cells. Dashed lines represent the mean RLU values ​​of untreated wells. Data shown are from a representative experiment of two studies.

[0056] Figure 22 This study demonstrates the expression of cell surface markers associated with human T cell activation after two or five days of incubation with either IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR in polyclonal activated healthy donor PBMC samples. The expression of CD4+, 4-1BB, CD25, HLA-DR, or PD-1 is also observed. +(A, C, E, G) and CD8 + The percentage ± SD of T cells (B, D, F, H) was determined by flow cytometry. Data shown were selected from day 2 or 5, depending on the date on which the maximum effect of IgG1-CD134-003-HC6LC2-RR on each marker was observed. Dashed lines indicate the percentage of marker-expressing T cells incubated only with CD3 antibodies. All data shown were derived from a representative donor from one of the seven donors tested in three experiments. CD4+ cells expressing 4-1BB, CD25, or HLA-DR were expressed upon stimulation with 5 µg / mL of IgG1-CD134-003-HC6LC2-RR, IgG1-b12-RR, or OX40 agonist reference antibody analogs IgG1-CD134-RG7888, IgG1-CD134-11D4, and IgG1-CD134-IBI101. + (IK) and CD8 + Percentage of T cells (LM) ± SD, as analyzed by flow cytometry two and five days after stimulation. The data shown are mean normalized data ± SD of pooled data from four to seven donors evaluated in two to three independent experiments.

[0057] Figure 23 The kinetics of cytokine concentrations in the supernatant of polyclonal activated healthy human donor PBMC samples incubated with IgG1-CD134-003-HC6LC2-RR or the non-binding control antibody IgG1-b12-RR for 1, 2, 3, 4, or 6 days are shown. Mean calculated concentrations ± SD of (A) TNFα, (B) IL-2, (C) IFNγ, and (D) IL-13, as determined by multi-task ECLIA. Data shown were derived from a representative donor from one of the three donors tested.

[0058] Figure 24 This displays the cytokine concentrations in the supernatant of polyclonal activated healthy human donor PBMC samples incubated for four days with either IgG1-CD134-003-HC6LC2-RR or the non-binding control antibody IgG1-b12-RR. The PBMC samples were either not depleted or depleted of CD4+ prior to incubation. + or CD8 + T cells. Mean calculated concentrations ± SD of (A) TNFα, (B) IL-2, (C) IFNγ and (D) IL-13, as determined by multi-task ECLIA. Data shown were derived from a representative donor from one of the three donors tested.

[0059] Figure 25The table shows the concentrations of cytokines in the supernatant of polyclonal activated healthy human donor PBMC samples incubated for four 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. The mean calculated concentrations ± SD of (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα, as determined by multi-task ECLIA, are shown. Data presented were derived from one of the two donors tested.

[0060] Figure 26 The table shows the concentrations of cytokines in the supernatant of polyclonal activated healthy human donor PBMC samples incubated for four 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. The mean calculated concentrations ± SD of (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα, as determined by multi-task ECLIA, are shown. Data presented were derived from one of the two donors tested.

[0061] Figure 27 The concentrations of cytokines in the supernatant of polyclonal activated healthy human donor PBMC samples incubated with IgG1-CD134-003-HC6LC2-RR, IgG2sCD134-SF2-E345R, or the unbound control antibody IgG1-b12-RR are shown. Mean calculated concentrations ± SD of (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα, as determined by multi-task ECLIA. Data shown were derived from one of the two donors tested.

[0062] Figure 28 The CD8+ level was observed on day 4 after treatment with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR, as measured by antigen-specific human T-cell proliferation assay. + T cell proliferation, as assessed by flow cytometry. (A) CLDN6-specific CD8+ expressing OX40, incubated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR from one of the three donors tested in two experiments. +Mean expansion index ± SD of T cell replicate wells. The black dashed line represents the baseline value, such as iDC:CD8 without antibody incubation (culture medium only). + As determined by T cell co-culture. (B) and expression of autologous CLDN6 (CLDN6) + ) or simulated transfection (CLDN6) - The expression of OX40 CLDN6-specific CD8 by iDC incubation in the presence of 2 µg / mL IgG1-CD134-003-HC6LC2-RR was enhanced. + Mean expansion index ± SD of T cell replicate wells. Data for all three donors tested are shown.

[0063] Figure 29 The diagram shows a lack of binding between IgG1-CD134-003-HC6LC2-RR and FcγRIa-expressing human monocyte-derived M2c-like macrophages, as analyzed by flow cytometry. 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 was observed after incubation for 15 min (A) and 24 h (B). Binding is shown relative to the background control (binding only to secondary antibodies, indicated by black dashed lines). Dots represent three individual donors measured in two independent experiments, and bar graphs and error bars represent the mean fold change and SD of the three donors, respectively.

[0064] Figure 30 The binding of IgG1-CD134-003-HC6LC2-RR to the human neonatal receptor FcRn is shown, as analyzed by SPR. The data presented are sensor plots of the interaction between FcRn and IgG1-CD134-003-HC6LC2-RR at pH 6.0 (A to E) and pH 7.4 (F), with IgG1-CD134-003-HC6LC2-RR tested at different concentrations, as indicated in each subgroup. The data shown are from a representative experiment from three experiments at pH 6.0 and from a representative experiment from two experiments at pH 7.4.

[0065] Figure 31 The results showed that IgG1-CD134-003-HC6LC2-RR and IgG1-CD52-E345R were associated with activated CD4+. + and CD8 +T cell binding and its binding to C1q. (A to C) Primary human T cells were stimulated with anti-CD3 / CD28 beads and subsequently incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E345R, or IgG1-b12-RR. Flow cytometry was used to assess antibody binding to cells. The binding of antibodies to CD4+ is shown. + (A) and CD8 + (B and C) T cell binding. Figure C shows the same IgG1-b12-RR data as Figure B, but with a different Y-axis range. Data are expressed as mean gMFI ± SD from duplicate wells of a representative donor from one of the three donors tested in three experiments. C1q with OX40-bound IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E345R, or IgG1-b12-RR on activated CD4 + (D) and CD8 + (E) Binding to the cell membrane of T cells, as analyzed by flow cytometry. All data shown are mean gMFI ± SD from replicate wells of a representative donor from one of the three donors tested in three experiments.

[0066] Figure 32 Demonstrates the binding of monovalent and bivalent anti-human OX40 antibodies to activated human T cells. Human CD4 cells activated for three days using anti-CD3 / CD28 beads were then... + (A) and CD8 + (B) T cells were incubated with IgG1-CD134-003-HC6LC2-RR, monovalent OX40 antibody BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, IgG1-CD134-003-RR-K409R, IgG1-CD134-003, IgG1-CD134-003-HC6LC2, or the unbound control antibody IgG1-b12-RR and their binding was assessed by flow cytometry. The data shown are the mean gMFI ± SD from duplicate wells of a representative donor from one of the four donors tested.

[0067] Figure 33 The results showed that BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, and IgG1-CD134-003-RR-K409R were present at OX40. +Functional activity in T cell reporter assays and polyclonal T cell proliferation assays. (A) Bioluminescence as an alternative to OX40 agonist activity is OX40 incubated with BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-RR-K409R or IgG1-b12-RR. + RLU in T cells with Jurkat reporter. Data shown are from a representative experiment out of a total of four experiments. (B) Enhanced CD4+ in polyclonal T cell proliferation assays after incubation with BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, or IgG1-b12-RR at concentration ranges. + T cell proliferation, as analyzed by flow cytometry, is presented as mean ± SD of replicate wells. The data shown are from one of three donors tested in one experiment, representing CD4+. + T-cell expansion index.

[0068] Figure 34 The expression of membrane OX40 and the level of soluble OX40 (sOX40) in polyclonal activated healthy human donor PBMC cultures were shown after treatment with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR. (A) OX40 in CD44 repeat wells. + and CD8 + (A) Mean ± SD of sOX40 expression on the cell surface of T cells, as assessed by flow cytometry. (B) Mean ± SD of sOX40 concentration in the supernatant of repeat wells, as measured by ECLIA. The data shown are from a representative donor from one of three donors tested in a similar experimental setup.

[0069] Figure 35 The results showed that reporter cells expressing OX40 were activated with IgG1-CD134-003-HC6LC2-RR in the presence of soluble OX40 (sOX40). OX40 + Jurkat reporter cells were cultured for 5 h in the presence of different concentrations of IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR and sOX40. Bioluminescence activity as an alternative OX40 agonist was measured as the mean RLU ± SD of replicate wells. The data shown are derived from one of the two experiments.

[0070] Figure 36The results showed that, in a polyclonal T-cell proliferation assay, after treatment with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR in the presence or absence of sOX40, polyclonal activated CD4+ cells... + and CD8 + T cell proliferation. The data shown are from repeat wells (A) CD4. + T cell expansion index, and (B) CD8 + The mean ± SD of the T cell expansion index, as determined by flow cytometry on day 4. Data were derived from a representative donor from one of four donors tested in one experiment.

[0071] Figure 37 The total huIgG level in the plasma of SCID mice following intravenous administration of anti-human OX40 antibody was shown, as determined by ECLIA in serially obtained plasma samples. Data are expressed as mean huIgG concentration ± SD of three mice in each treatment group, except for mice receiving 0.125 mg / kg or 1.25 mg / kg (N=2) of IgG1-CD134-003-RR-K409R, 0.125 mg / kg of IgG1-CD134-003-FEAL (N=1), or at 12.5 mg / kg of IgG1-CD134-003-HC6LC2-RR (N=2). No values ​​below the lower limit of quantitation (LLOQ) were shown (N=3 in mice after day 8 of receiving 0.125 mg / kg IgG1-CD134-003-RR-K409R, and N=1 in mice after day 14 of receiving 0.125 mg / kg IgG1-CD134-003-FEAL). The dashed line represents the predicted WT huIgG plasma concentration based on the two-compartment model. The horizontal dashed line represents the LLOQ and upper limit of quantitation (ULOQ) determined by ECLIA. Data shown are from one experiment.

[0072] Figure 38 The pharmacokinetic parameters of the antibody drugs 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, and the total huIgG level was measured by ECLIA in serially obtained plasma samples to calculate CL(A), t½(B), and C. max (C). Individual mouse values ​​are plotted as horizontal bars representing the mean ± SD for each treatment group.

[0073] Figure 39Tumor volume measured in human OX40 knock-in (hOX40 KI) mice carrying MC38 tumors. Mice were treated intraperitoneally on day 0 with different concentrations of IgG1-CD134-003-HC6LC2-RR or 20 mg / kg of IgG1-b12-FEAL. (A) Tumor volume measured on day 10 after the start of treatment. Individual mouse values ​​are plotted as horizontal bars representing the mean ± SD for each treatment group. Statistically significant differences between treatment groups were assessed using Mann-Whitney analysis. (P<0.01). (B) Mean tumor volume (±SEM) measured over time in treated hOX40 KI mice carrying MC38 tumors, as shown in the figure. (C) Proportion of progression-free survival in treated mice carrying MC38 tumors (e.g., tumor volume <500 mm). 3 The Kaplan-Meier curve (as defined) is shown in the figure. Significance was calculated using Mantel-Cox analysis (compared to the IgG1-b12-FEAL control group). P<0.05 and P<0.001).

[0074] Figure 40 The percentage and absolute number of T cells in peripheral blood samples from hOX40 KI mice carrying MC38 tumors treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL are shown, as analyzed by flow cytometry. (The text also mentions CD3 cells, but the context is unclear.) + Percentage of T cell population (A, C) and CD4 + (A, B) and CD8 + The absolute number of T cells (C, D) (B, D). Points in the graph represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using Man Whitney analysis. P<0.05; P<0.01).

[0075] Figure 41 CD4+ expressing the proliferation markers Ki67(A), CD25(B), IA / IE(C), PD-1(D), and 4-1BB(E) +The percentage of T cells, as analyzed by flow cytometry, was observed in peripheral blood samples collected on day 5 (after two treatments) from hOX40 KI mice carrying MC38 tumors treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Dots in the figure represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using a Man Whitney analysis. P<0.05; P<0.01).

[0076] Figure 42 Demonstrates proliferation and tumor-specific (ADPGK tetramer-specific) CD8 + The percentage and absolute number of T cells. The data shown is (A) CD8 expression of the proliferation marker Ki67. + Percentage of T cells, (B) CD8 + Ki67 + Absolute cell number, (C) tumor-specific CD8 + The percentage of T cells and (D) tumor-specific CD8 + The absolute number of T cells was analyzed by flow cytometry in peripheral blood samples collected on day 5 (after two treatments) from hOX40 KI mice carrying MC38 tumors treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Dots in the figure represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using a Man Whitney analysis. P<0.01).

[0077] Figure 43 CD8+ expressing the proliferation markers CD25(A), IA / IE(B), PD-1(C), and 4-1BB(D) + The percentage of T cells, as analyzed by flow cytometry, was observed in peripheral blood samples collected on day 5 (after two treatments) from hOX40 KI mice carrying MC38 tumors treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Dots in the figure represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using a Man Whitney analysis. P<0.05; P<0.01).

[0078] Figure 44 Plasma cytokine concentrations are shown in hOX40 KI mice carrying MC38 tumors treated with IgG1-CD134-003-HC6LC2-RR. Plasma samples were collected on day 0 (before treatment) and on days 2 and 5 after one or two treatments with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Cytokine analysis in plasma samples was performed using ECLIA. Data shown are concentrations in four individual mice and mean concentrations of (A) IFNγ, (B) IP-10, (C) IL-2, (D) IL-4, (E) MCP-1, (F) IL-10, (G) IL-27p28, and (H) TNFα. Data shown are derived from one experiment.

[0079] Figure 45 The image shows the number of hOX40 KI mice carrying MC38 collected from mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL per mm. 2 Number of intratumoral immune cells in tumor tissue. Per mm 2 (A) CD3 in tumor tissue + Cells, (B) CD3 + Ki67 + Cells, (C) CD4 + Cells, (D) CD8 + Cells, (E) Granzyme B + Cells and (F) human OX40 + Cell count, as determined by quantitative immunohistochemical analysis of tumor tissue. In the graph, dots represent individual mice, and bars and error bars represent the mean ± SD. As determined by the Man Whitney test. P<0.05.

[0080] Figure 46 Tumor volume measured in hOX40 KI mice carrying MC38 tumors. Mice were treated intraperitoneally on day 0 with different concentrations of IgG1-CD134-003-HC6LC2-RR or 20 mg / kg of IgG1-b12-FEAL. (A) Tumor volume measured on day 15 after the start of treatment. Individual mouse values ​​are plotted as horizontal bars representing the mean ± SEM of each treatment group included in an experiment. Statistically significant differences between treatment groups were assessed using Man Whitney analysis. P<0.05; (P<0.01). (B) Mean tumor volume (±SEM) measured over time in treated hOX40 KI mice carrying MC38 tumors, as shown in the figure. (C) Proportion of progression-free survival in treated mice carrying MC38 tumors (e.g., tumor volume <500 mm). 3 The Kaplan-Mail curve (as defined) is shown in the figure. Significance was calculated using Mantel-Cox analysis (compared to the IgG1-b12-FEAL control group). P<0.05).

[0081] Figure 47 The concentrations of cytokines and other soluble factors in the supernatant of mixed lymphocyte response (MLR) assays stimulated alone or in combination with IgG1-CD134-003-HC6LC2-RR or pembrolizumab are shown, as evaluated by Luminex. Antibody IgG1-b12-RR and IgG4 isotype controls are included as non-binding isotype controls for IgG1-CD134-003-HC6LC2-RR and pembrolizumab, respectively. The mean ± SD concentrations of IL-13 (A), sOX40 (B), IL-2 (C), GM-CSF (D), TNFα (E), and granulysin (F) are shown from duplicate wells of a representative donor pair from one of the four donor pairs tested in one experiment.

[0082] Figure 48 The chart shows the mean fold change (FC) ± SD of the concentrations of specified cytokines and soluble factors in the supernatant of an MLR assay performed with combined stimulation of 1 µg / mL IgG1-CD134-003-HC6LC2-RR and 1 µg / mL IgG1-PD1 from four donors compared to untreated (without Tx), as analyzed using Luminex. Light gray bars indicate soluble factors with FC > 1.5 observed in three of the four tested donors, and dark gray bars indicate soluble factors with FC > 1.5 observed in all four of the four donors.

[0083] Figure 49 The mean median score is shown using a ZIP synergy analysis, based on the concentration of a specified soluble factor measured in the supernatant of an MLR assay performed with combined stimulation of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1, as analyzed by Luminex. Dashed lines represent the mean median scores of -10 and 10. A ZIP score ≥10 indicates synergy.

[0084] Figure 50The effect of MLR stimulation on IL-2 secretion in the supernatant of MLR assays is shown. (A) Dose-response curves of mean IL-2 concentration ± SD measured in the supernatant of MLR assays stimulated by IgG1-CD134-003-HC6LC2-RR, IgG1-PD1, or IgG1-CD134-003-HC6LC2-RR with IgG1-PD1, as analyzed by Luminex. Dashed lines represent cytokine levels measured in untreated control wells. (B) Matrix plots show the fold change (FC) of IL-2 secretion in response to treatment with IgG1-CD134-003-HC6LC2-RR alone, IgG1-PD1 alone, and IgG1-PD1 with IgG1-PD1 at concentrations within a given range, compared to untreated. (C) A matrix plot of ZIP synergistic scores calculated from IL-2 secretion in response to treatment with a combination of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 at a specified concentration. A ZIP score ≥10 indicates synergistic effect. The mean synergistic score of IL-2 secretion and the 25th and 75th percentiles are shown. The data shown are from a representative donor pair from four donor pairs tested in two experiments.

[0085] Figure 51The effect of MLR stimulation on GM-CSF secretion in the supernatant of MLR assays is shown. (A) Dose-response curves of mean GM-CSF concentration ± SD measured in the supernatant of MLR assays stimulated by IgG1-CD134-003-HC6LC2-RR, IgG1-PD1, or IgG1-CD134-003-HC6LC2-RR and IgG1-PD1, as analyzed by Luminex. Dashed lines represent cytokine levels measured in untreated control wells. (B) Matrix plot showing the fold change (FC) in GM-CSF secretion induced by individual IgG1-CD134-003-HC6LC2-RR, individual IgG1-PD1, and combinations of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 at specific concentration ranges compared to untreated GM-CSF secretion. (C) Matrix plot of ZIP synergy scores calculated in response to GM-CSF secretion from treatment with combinations of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 at specific concentration ranges. A ZIP score ≥10 indicates synergy. The mean synergy score and 25% and 75% quantiles of GM-CSF secretion are shown. The data shown are from a representative donor pair from one of the four donor pairs tested in the two experiments.

[0086] Figure 52The effect of IL-12p40 secretion in the supernatant of MLR assays stimulated by a combination of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 or either compound alone is shown. (A) Dose-response curves of mean IL-12p40 concentration ± SD measured in the supernatant of MLR assays stimulated by IgG1-CD134-003-HC6LC2-RR, IgG1-PD1, or a combination of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1, as analyzed by Luminex. Dashed lines represent cytokine levels measured in untreated control wells. (B) Matrix plot showing the fold change (FC) in IL-12p40 secretion induced by individual IgG1-CD134-003-HC6LC2-RR, individual IgG1-PD1, and combinations of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 at specific concentration ranges compared to untreated. (C) Matrix plot of ZIP synergy scores calculated in response to treatment with combinations of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 at specific concentrations. A ZIP score ≥10 indicates synergy. The mean synergy score and 25% and 75% quantiles of IL-12p40 secretion are shown. The data shown are from a representative donor pair from four donor pairs tested in two experiments.

[0087] Figure 53 The effect of the combination of IgG1-CD134-003-HC6LC2-RR and pembrolizumab on the proliferation of CD3 antibody-stimulated T cells is shown, as assessed by flow cytometry analysis of CellTrace Violet dilutions. A representative donor's CD4 count is visualized as a line graph. + (A) and CD8 + Mean expansion index of T cells (B). (C) CD4 + T cells (C) and CD8 + The dose-response matrix for T cells (D) represents the average relative expansion index, expressed as a percentage ± SD of the maximum expansion index achieved by each of the ten reactive donors (C) tested in five experiments or by each of the six reactive donors (D) tested in four experiments.

[0088] Figure 54 This demonstrates the effect of the combination of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 on the proliferation of CD3 antibody-stimulated T cells, as assessed by flow cytometry analysis of CellTrace Violet dilutions. It also shows the effect of CD4+.+ T cells (A) and CD8 + The dose-response matrix for T cells (B) represents the average relative expansion index, expressed as a percentage ± SD of the maximum expansion index achieved by each of the three donors tested in an experiment.

[0089] Figure 55 This study demonstrates the synergistic effect of IgG1-CD134-003-HC6LC2-RR combined with pembrolizumab or IgG1-PD1 on enhancing polyclonal T cell proliferation. (A, B) The synergistic effect of the combination of IgG1-CD134-003-HC6LC2-RR and pembrolizumab on enhancing polyclonal T cell proliferation was studied using the ZIP synergistic scoring model, with a ZIP synergistic score ≥10 indicating a synergistic effect. The study also shows the CD4 counts of all ten reactive donors tested in five experiments (A) or all six reactive donors tested in four experiments (B). + T cell proliferation (A) and CD8 + Mean co-scores and 25th and 75th percentiles for T cell proliferation (B). (C, D) The co-score effect of the combination of IgG1-CD134-003-HC6LC2-RR and IgG1-PD1 on enhancing polyclonal T cell proliferation was studied using a ZIP co-score model. The results show the CD4+ of three donors tested in one experiment. + T cell proliferation (C) and CD8 + Mean co-scores and 25% and 75% quantiles of T cell proliferation (D).

[0090] Figure 56 This displays cytokine levels in the supernatant of polyclonal T cell proliferation assays stimulated with IgG1-CD134-003-HC6LC2-RR, pembrolizumab, or a combination thereof. Cytokine levels in cell culture supernatants of polyclonal T cell proliferation assays after treatment with IgG1-CD134-003-HC6LC2-RR alone, pembrolizumab alone, a combination thereof, or IgG1-b12-RR were measured using ECLIA. Single measurements of TNFα (A), IL-6 (B), IFNγ (C), and IL-10 (D) from a representative donor from one of the three donors tested in the three experiments are shown.

[0091] Figure 57This displays cytokine levels in the supernatant of polyclonal T cell proliferation assays after stimulation with IgG1-CD134-003-HC6LC2-RR, IgG1-PD1, or a combination thereof. Cytokine levels in the cell culture supernatant of polyclonal T cell proliferation assays after treatment with IgG1-CD134-003-HC6LC2-RR alone, IgG1-PD1 alone, a combination thereof, or IgG1-b12-RR were measured using ECLIA. Single-measurement levels of TNFα (A), IL-6 (B), CXCL10 (C), IFNγ (D), and CXCL9 (E) from a representative donor from one of the three donors tested in one experiment are also displayed.

[0092] Figure 58 The CD8+ level was observed in antigen-specific T cell proliferation assays after treatment with IgG1-CD134-003-HC6LC2-RR, either alone or in combination with pembrolizumab. + T cell proliferation. The combination of IgG1-CD134-003-HC6LC2-RR (10 μg / mL) and pembrolizumab (0.0044, 0.0133, or 0.8 μg / mL) showed efficacy against CD8+ in antigen-specific T cell proliferation assays. + The effect on T cell proliferation was assessed by flow cytometry and compared with the combination of unbound antibody IgG1-b12-RR (10 µg / mL) and pembrolizumab. Unbound antibody IgG1-b12-RR (10 µg / mL) was included as a negative control, both as a monotherapy and as a co-culture with non-electroplated iDCs (mock) in the absence of antibody treatment. (A) CD8+ from duplicate wells of a representative donor from one of the four donors tested. + Mean expansion index of T cells ± SD. The black dashed line represents the baseline value, such as iDCs (CD8) without antibody incubation (culture medium only). + Assays were performed on T-cell co-cultures. (B) For all tested donors, the fold increase in the amplification index of cells treated with IgG1-CD134-003-HC6LC2-RR in combination with pembrolizumab, relative to cells treated alone or in combination with pembrolizumab and IgG1-b12-RR. Data derived from individual donors are connected by lines, with a symbol representing the mean of replicate wells.

[0093] Figure 59 The CD8+ level was observed in antigen-specific T cell proliferation assays after treatment with IgG1-CD134-003-HC6LC2-RR, either alone or in combination with IgG1-PD1. +T cell proliferation. The combination of IgG1-CD134-003-HC6LC2-RR (10 μg / mL) and IgG1-PD1 (0.0044, 0.0133, or 0.8 μg / mL) in antigen-specific T cell proliferation assays showed an effect on CD8+. + The effect on T cell proliferation was assessed by flow cytometry. Unbound antibody IgG1-b12-RR (10 µg / mL) was included as a negative control, both as a monotherapy and as a co-culture of non-electroplated iDCs (mock) in the absence of antibody treatment. (A) CD8+ from duplicate wells of a representative donor from one of the four donors tested. + Mean expansion index of T cells ± SD. The black dashed line represents the baseline value, such as iDCs (CD8) without antibody incubation (culture medium only). + Assays were performed on T-cell co-cultures. (B) For all tested donors, the fold increase in the expansion index of cells treated with IgG1-CD134-003-HC6LC2-RR in combination with IgG1-PD1, relative to cells treated alone or in combination with IgG1-PD1 and IgG1-b12-RR. Data derived from individual donors are connected by lines, with the symbol representing the mean of replicate wells.

[0094] Figure 60 Tumor volume measured in human OX40 and PD-1 double knock-in (hOX40 / hPD-1 dKI) mice carrying MC38 tumors. Mice were treated intraperitoneally on day 0 with different concentrations of 5 mg / kg IgG1-CD134-003-HC6LC2-RR, 10 mg / kg pembrolizumab, 10 mg / kg IgG1-PD1, or a combination of IgG1-CD134-003-HC6LC2-RR and pembrolizumab or IgG1-PD1, or 10 mg / kg IgG1-b12-FEAL. (A) Mean tumor volume (± SEM) measured over time in treated hOX40 / hPD-1 dKI mice carrying MC38 tumors, as shown in the figure. (B) Tumor volume measured on day 11 after the start of treatment. Statistically significant differences between treatment groups were assessed using a Man Whitney analysis. P<0.05; P<0.01; P<0.001; P<0.0001). (C) Progression-free survival in hOX40 / hPD-1 dKI mice, such as those with tumor volume less than 500 mm. 3 The percentage of mice is defined as shown in the Kaplan-Mail curve. The cross (“treatment”) indicates the date of administration (IgG1-CD134-003-HC6LC2-RR: days 0, 3, 7, 10, 14, 17; pembrolizumab: days 0, 3, 7, 10).

[0095] Figure 61 The figure shows the percentage and absolute number of T cells in peripheral blood samples collected from treated hOX40 / hPD-1 dKI mice carrying MC38 tumors, as analyzed by flow cytometry. (A) CD3 + The absolute number of T cells is represented by the number of cells per μL of peripheral blood collected from mice on day 4 after two treatments. CD3 + T cells (B), CD4 + T cells (C), CD8 + T cells (D) and CD25 + FOXP3 + The absolute number of cells (E) is presented as the number of cells per μL of peripheral blood collected on day 8 after mice received three treatments. CD45 in peripheral blood samples collected on day 8 after the start of treatment. + T cells (F) and CD4 cells within the cell population + T cells (G) and CD8 + T cell (H) percentage. Points in the graph represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using a Man Whitney analysis. P<0.05; P<0.01).

[0096] Figure 62 The study showed that on day 8 after three treatments, CD4+ cells with effector memory (EM), central memory (CM), or naive T-cell phenotypes were present in peripheral blood samples collected from hOX40 / hPD-1 dKI mice carrying MC38 tumors. + and CD8 + The percentage of T cells, as analyzed using flow cytometry. (A) In CD4 + (A) and CD8 +(B) Percentage of EM, CM, and naïve T cells within the T cell population. Points in the figure represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using Man Whitney analysis. P<0.05; P<0.01).

[0097] Figure 63 The percentage of proliferative T cells in peripheral blood samples collected from hOX40 / hPD-1 dKI mice carrying MC38 tumors on day 8 after three treatments, as analyzed by flow cytometry. (Ki67) + CD4 + T cells (A), CD8 + T cells (B) and CD25 + FOXP3 + Percentage of cells. Points in the graph represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistical significance between treatment groups was assessed using Man Whitney analysis. P<0.05; P<0.01).

[0098] Figure 64 Tumor-specific CD8 was observed in peripheral blood samples collected from hOX40 / hPD-1 dKI mice carrying MC38 tumors on day 8 after three treatments. + The percentage of T cells, as analyzed using flow cytometry. Tumor-specific CD8. + T cells were characterized by positive staining for ADPGK-tetramer. Statistically significant differences between treatment groups were calculated using Man Whitney analysis. P<0.05; P<0.01). In the figure, the dots represent individual mice, and the bars and error bars represent the mean ± SD of all animals included in an experiment.

[0099] Figure 65 The hOX40 / hPD-1 dKI mice carrying MC38 tumors expressed CD44, a marker of T cell activation, after treatment as indicated. + and CD8 + The percentage of T cells. This indicates the expression of CD25 in peripheral blood samples collected from treated mice on day 8 after three treatments. (A) IA / IE (B) 4-1BB (C) and Human OX40 (hOX40) (D) CD4 + and CD8 + The percentage of T cells and CD25 expression ( CD8 of E), IA / IE (F), 4-1BB (G) and hOX40 (H) + The percentage of T cells, as analyzed using flow cytometry. Points in the graph represent individual mice, and bars and error bars represent the mean ± SD of all animals included in a single experiment. Statistically significant differences between treatment groups were assessed using Man Whitney analysis. P<0.05; P<0.01).

[0100] Figure 66 Plasma cytokine concentrations in hOX40 / hPD-1 dKI mice carrying MC38 tumors are shown in the figure. Plasma samples were collected on day 8 after mice received three treatments with the indicated compounds. Cytokine analysis in plasma samples was performed using ECLIA. Data shown are concentrations of (A) IL-2, (B) IL-5, (C) IL-10, (D) TNFα, (E) IFNγ, (F) IL-27p28, (G) MCP-1, (H) MIP-1α, (I) IL-4, and (J) CXCL-10 in five individual mice, and mean concentrations ± SD. Data were derived from one experiment. Statistically significant differences between treatment groups were assessed using Man Whitney analysis. P<0.05; P<0.01).

[0101] Figure 67 The images show the results of data collected per mm from hOX40 / hPD-1 dKI mice carrying MC38, treated with IgG1-CD134-003-HC6LC2-RR, pembrolizumab, IgG1-PD1, or a combination of IgG1-CD134-003-HC6LC2-RR and pembrolizumab or IgG1-PD1, or the unbound control antibody IgG1-b12-FEAL. 2 Tumor tissue density and immune cell density. (A) Proliferative (CD3) - Ki67 + (B) CD3 cells + Cells, (C) CD4 + Cells, (D) CD8 + Cells, (E) human OX40 (hOX40) +Cells, (F) Human PD-1 (hPD-1) + Cells, (G) proliferating T cells (CD3) + Ki67 + (H) Granulase B (GZMB) + Cell density (per mm) 2 (cells), and (I)PD-L1 + The percentage of tissue surface area, as determined by quantitative immunohistochemical analysis on excised tumor tissue. Points in the figure represent individual mice. Data shown are cell numbers and mean concentrations ± SD for 5 individual mice. p<0.05, p<0.01, as determined by the Man Whitney Laboratory.

[0102] Figure 68 The images show that PBMC samples from four cancer patients (patients #1 to #4) previously treated with anti-PD-1 antibodies expressed (A) PD-1 and (B) OX40 CD4+. + and CD8 + The percentage of T cells, as determined by flow cytometry prior to the start of the MLR assay (baseline characteristics). (C) IL-2 concentrations in the supernatant of a 96-h MLR assay performed in the presence of IgG1-CD134-003-HC6LC2-RR, pembrolizumab, or a combination of IgG1-CD134-003-HC6LC2-RR and pembrolizumab, as assessed by Luminex. IgG1-b12-RR is included as a non-binding isotype control for IgG1-CD134-003-HC6LC2-RR and pembrolizumab, respectively. Mean concentration ± SD or single concentration of the control is shown in the replicate wells. Dashed lines indicate IL-2 concentrations in the MLR assay without antibody treatment.

[0103] Figure 69 This chart shows the IL-2 concentration measured in the supernatant of a 96-h MLR assay performed on healthy donors using three donors, either alone or in combination with 1 μg / mL of pembrolizumab, nivolumab, cimipril, or atezolizumab, within a certain concentration range. The mean concentration ± SD is shown in the replicate wells. Dashed lines indicate IL-2 concentrations induced by 1 μg / mL of pembrolizumab (Pembro), nivolumab (Nivo), cimipril (Cemi), or atezolizumab (Atezo), or by antibody-free treatment (No ab).

[0104] Figure 70A heatmap showing the average concentration of IL-2 measured in the supernatant of a 96-h MLR assay performed on healthy donors PBMCs of 1(A), 2(B), and 3(C) in the presence of IgG1-CD134-003-HC6LC2-RR or atezolizumab (Atezo) or a combination thereof within a given concentration range. The average concentration is shown in duplicate wells.

[0105] Figure 71 A matrix plot showing the ZIP synergistic score calculated from IL-2 secretion measured in the supernatant of a 96-h MLR assay performed using healthy donor PBMCs in the presence of IgG1-CD134-003-HC6LC2-RR or atezolizumab or a combination thereof at specific concentration ranges. A ZIP score ≥ 10 indicates synergistic effect. The matrix plot shows the synergistic score of IL-2 secretion. Data for the three donor pairs (A to C) are shown. Figure 70 The donors described herein are the same.

[0106] Figure 72 The results showed that single-dose IgG1-CD134-003-HC6LC2-RR (0.006 μg / mL or 10 μg / mL), single-dose unbound control antibody IgG1-b12-RR (10 μg / mL), single-dose pembrolizumab, nivolumab, cimipril, or atezolizumab, or IgG1-CD134-003-HC6LC2-RR, or IgG1-b12-RR in combination with any of the following antibodies, were co-cultured with autologous immature DCs expressing CLDN6, and electroporated with hOX40, hPD-1, and CLDN6-specific TCRs in the presence of previously determined concentrations to induce low (low), moderate (medium), or strong (high) effects; PD-1-b12-RR (10 μg / mL), single-dose pembrolizumab, nivolumab, cimipril, or atezolizumab, or IgG1-CD134-003-HC6LC2-RR, or PD-(L)1 antibody. + A heatmap of T-cell amplification index. In the heatmap below: amplification index after treatment with a single dose of EC90 concentration of pembrolizumab, nivolumab, cimipril, or atezolizumab. The data shown are the average amplification index from five replicate wells of healthy human donors. Detailed Implementation

[0107] definition

[0108] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen. The antibodies of this invention comprise the Fc domain of an immunoglobulin and an antigen-binding region. Antibodies typically contain two CH2-CH3 regions and a linker region, such as a hinge region, or at least one Fc domain. Therefore, the antibodies of this invention may comprise an Fc region and an antigen-binding region. Variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant region or "Fc" region of an antibody mediates 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 of the classical pathway of complement activation. As used herein, unless contradicted by context, the Fc region of an immunoglobulin typically contains at least one CH2 and CH3 domain of the immunoglobulin CH and may include a linker region, such as a hinge region. The Fc region typically dimers via, for example, disulfide bonds connecting two hinge regions and / or non-covalent interactions between two CH3 regions. The dimer can be a homodimer (where the two Fc region monomers have the same amino acid sequence) or a heterodimer (where the two Fc region monomers have one or more different amino acids). The Fc region fragment of a full-length antibody can be generated, for example, by digesting a full-length antibody with papain, as is well known in the art. As defined herein, an antibody contains, in addition to the Fc region and antigen-binding region, one or both of the immunoglobulin CH1 and CL regions. Antibodies can also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term "bispecific antibody" refers to an antibody that is specific to at least two different and typically non-overlapping epitopes. These epitopes may be on the same or different targets. If the epitopes are on different targets, these targets may be on the same cells or different cells or cell types. As indicated above, unless otherwise stated or explicitly contradicted by the context, the term "antibody" as used herein includes antibody fragments that contain at least a portion of the Fc region and retain the ability to bind specifically to an antigen. Such fragments can be provided by any known technique, such as enzyme cleavage, peptide synthesis, and recombinant expression. The antigen-binding function of antibodies has been shown to be performed by fragments of full-length antibodies. Examples of binding fragments covered in the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described in Genmab WO2007059782); heavy-chain antibodies consisting of only two heavy chains and naturally occurring in animals such as camels (e.g., Hamers-Casterman (1993) Nature 363:446; ThioMabs, Roche, WO2011069104); and chain-exchange engineered domains (SEED or Seed-body), which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205).Triomab (Pharma / Fresenius Biotech, Lindhofer et al., 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792); Enzyme scaffold (Zymeworks / Merck, WO2012 / 058768); mAb-Fv (Xencor, WO2011 / 028952); Xmab (Xencor); Bivariate domain immunoglobulin (Abbott, DVD-Ig, US Patent No. 7,612,181); Bivariate domain biheaded antibody (Unilever; Sanofi Aventis, WO20100226923); Bifunctional antibody (ImClone / Eli Lilly); Knob-in-well antibody form (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); Electrostatic steering antibody (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545); CrossMAbs (Roche, WO2011117329); LUZ-Y (Genentech); Biclonic (Merus, WO2013157953); Dual-targeting domain antibody (GSK / Domantis); dual-target antibodies or dual-action Fabs (Genentech, NovImmune, Adimab); cross-linked Mabs (Karmanos CancerCenter); covalently fused mAbs (AIMM); CovX-body (CovX / Pfizer); FynomAbs (Covagen / Janssenilag); 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, DIG, and PIG bodies (Pharmabcine); dual-affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538); BEAT (Glenmark); Zybodies (Zyngenia); fusion proteins containing a polypeptide sequence fused to an antibody fragment containing an Fc region, such as scFv fusion proteins, such as BsAb from ZymoGenetics / BMS, and HERCULES from Biogen Idec. (US007951918); SCORPIONS (Emergent BioSolutions / Trubion and Zymogenetics / BMS); Ts2Ab (MedImmune / AZ (Dimasi, N. et al. JMol Biol, 2009. 393(3): p. 672-92); scFv fusion protein (Genentech / Roche); scFv fusion protein (Novartis); scFv fusion protein (Immunomedics); scFv fusion protein (Changzhou Adam Biotech Inc., CN 102250246); TvAb (Roche, WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008 / 003116); and dual scFv fusion proteins. Unless otherwise specifically specified, the term antibody should be understood to include monoclonal antibodies (such as human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (such as bivalent monospecific antibodies), bispecific antibodies, any allotype and / or allotype antibodies; antibody mixtures (recombinant polyclonal antibodies), such as those produced by techniques explored by Symptomogen and Merus (Oligoclonics), such as polymeric Fc proteins described in WO2015 / 158867 and fusion proteins described in WO2014 / 031646. Although these different antibody fragments and forms are generally included within the meaning of antibody, they are common and independent features of this invention, exhibiting different biological properties and effects.

[0109] "Agonistic antibodies" of natural receptors are compounds that bind to the receptor to form receptor-antibody complexes and activate the receptor, thereby triggering pathway signaling and further biological processes.

[0110] The terms “agonistic” and “agonistic” are used interchangeably in this document and refer to or describe antibodies that can directly or indirectly substantially induce, promote or enhance the biological activity or activation of OX40. "Activating OX40 antibody" may optionally be an antibody that can activate the OX40 receptor through a mechanism similar to that of OX40 ligands (referred to as OX40L) (CD134L, OX-40L, TNLG2B, OX4OL, GP34, CD252 antigen, CD134 ligand, TAX-activated glycoprotein 1, CD252, OX40 ligand, OX40L, TNFSF4, tumor necrosis factor ligand superfamily member 4, glycoprotein Gp34, TNF superfamily member 4, TXGP1, Tax-activated glycoprotein 1 (34kD), tumor necrosis factor (ligand) superfamily member 4, tumor necrosis factor (ligand) superfamily member 4, tumor necrosis factor superfamily member 4), which leads to the activation of one or more intracellular signaling pathways, including the activation of the NF-κB and MAPK8 / JNK pathways.

[0111] As described in this article, “OX40 antibody” or “anti-OX40 antibody” is an antibody that specifically binds to the protein OX40, especially human OX40.

[0112] As used herein, a “variant” refers to a protein or polypeptide sequence that differs from a parent or reference sequence by one or more amino acid residues. A variant may, for example, have at least 80%, such as 90%, 95%, 97%, 98%, or 99%, sequence identity with the parent or reference sequence. Alternatively, a variant may differ from the parent or reference sequence by 12 or fewer mutations (such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1), such as substitution, insertion, or deletion of amino acid residues. Therefore, the terms “variant antibody” or “antibody variant” used interchangeably herein refer to an antibody that differs from a parent or reference antibody, for example, from one or more amino acid residues in the antigen-binding region, Fc region, or both. Similarly, a “variant Fc region” or “Fc region variant” refers to an Fc region that differs from the parental or reference Fc region by one or more amino acid residues, optionally differing from the parental or reference Fc region by 12 or fewer mutations (such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1), such as substitution, insertion, or deletion of amino acid residues. The parental or reference Fc region is typically the Fc region of a human wild-type antibody, which may be a specific isotype depending on the context. The dimerized form of the variant Fc region can be a homodimer or a heterodimer, for example, in which one amino acid sequence of the dimerized Fc region contains a mutation, while the other amino acid sequence is identical to the parental or reference wild-type amino acid sequence. Examples of amino acid sequences of wild-type (typically parental or reference sequence) IgG CH and variant IgG constant regions containing the Fc region amino acid sequence are listed in Table 3.

[0113] As used herein, the terms “immunoglobulin heavy chain” or “heavy chain of immunoglobulins” refer to one of the heavy chains of immunoglobulins. A heavy chain typically consists of a variable region (abbreviated VH) defining the isotype of the immunoglobulin and a constant region (abbreviated CH). The constant region typically consists of three domains: CH1, CH2, and CH3. As used herein, the term “immunoglobulin” refers to a structurally related class of glycoproteins composed of two pairs of polypeptide chains (a pair of low-molecular-weight light (L) chains and a pair of heavy (H) chains), all four chains potentially linked together by disulfide bonds. The structure of immunoglobulins has been fully characterized (see Fundamental Immunology Ch. 7 Paul, W., 2nd ed. RavenPress, NY 1989). Within the structure of an immunoglobulin, two heavy chains are linked together via disulfide bonds in a region called the “hinge region.” Similar to the heavy chain, each light chain typically consists of several regions: variable regions (VL) and constant regions. The constant region typically consists of a single domain (CL). Furthermore, the VH and VL regions can be further subdivided into highly variable regions (or highly variable regions in sequence and / or structure-defined ring forms), also known as complement-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences used in this paper are based on the IMGT definition (see Lefranc MP et al., Nucleic Acids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).

[0114] When used herein, the terms “half-molecule,” “Fab arm,” and “arm” refer to a pair of heavy-chain-light chains. When a bispecific antibody is described as comprising a half-molecule antibody “derived from” a first antibody and a half-molecule antibody “derived from” a second antibody, the term “derived from” means that the bispecific antibody is produced by recombinating the half-molecules from each of the first and second antibodies using any known method. In this context, “recombination” is not intended to be limited to any particular recombination method and therefore includes all methods described below for producing bispecific antibodies, including, for example, recombination using “half-molecule exchange” (also described herein as “Fab-arm exchange”) and the DuoBody® method, as well as recombination at the nucleic acid level and / or co-expression of two half-molecules in the same cell.

[0115] As used herein, the terms "antigen-binding region" or "binding site" or "antigen-binding domain" refer to an antibody region capable of binding to an antigen. This binding region is typically defined by the antibody's VH and VL domains, which can be further subdivided into highly variable regions (or highly variable areas, whose sequence and / or structurally defined loop form can be highly variable), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Antigens can be any molecule, such as polypeptides, and may be present on cells, bacteria, or viruses. Unless contradicted by the context, the terms "antigen-binding region," "antigen-binding site," and "antigen-binding domain" are used interchangeably within the context of this invention.

[0116] Unless contradicted by the context, the terms “antigen” and “target” are used interchangeably in the context of this invention.

[0117] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target. When measured by biolayer interferometry using an antibody as a ligand and an antigen as an analyte, this binding typically corresponds to 1E. 6 M or smaller, such as 5E 7 M or smaller, 1E 7 M or smaller, such as 5E 8 M or smaller, such as 1E 8 M or smaller, such as 5E 9 M or smaller, or such as 1E 9 M or smaller K D The binding affinity, and the antibody corresponds to K D The affinity of the antigen to bind to the predetermined antigen is at least ten times lower than the affinity to bind to non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. For example, at least 100 times lower, at least 1,000 times lower, at least 10,000 times lower, or at least 100,000 times lower.

[0118] As used in this article, the term "K" D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, and is determined by k..." d Divide by k a To obtain.

[0119] As used in this article, the term "k" d (sec) -1 This refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as k. off Value or off-rate.

[0120] As used in this article, the term "k" a (M) -1 x sec -1 This refers to the association rate constant of a specific antibody-antigen interaction. This value is also known as k. on Value or association rate (on-rate).

[0121] As used herein, the term "OX40" refers to the human protein designated 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 to 28 are the signal peptide, and amino acid residues 29 to 277 are the mature polypeptide.

[0122] In the cynomolgus macaque (Macaca fascicularis), the OX40 protein has the amino acid sequence shown in SEQ ID NO: 51.

[0123] The term "antibody-binding region" refers to the antigenic region, which contains the epitope that binds to the antibody. Antibody-binding regions can be determined by methods such as epitope binding using biolayer interferometry, alanine scanning, or shuffling assays (using an antigen construct in which the antigenic region is exchanged with an antigenic region of another species, and determining whether the antibody still binds to the antigen). The amino acids within the antibody-binding region that interact with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and crystallography of the antibody bound to its antigen.

[0124] The term "epitaph" refers to an antigenic determinant that an antibody specifically binds to. Epitopes typically consist of surface groups of a molecule, such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that antibodies do not bind to the former in the presence of denaturing solvents, but they do bind to the latter. Epitopes may contain amino acid residues that directly participate in binding, as well as other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked or covered by the antibody when binding to the antigen (in other words, the amino acid residues are within or adjacent to the footprint of the specific antibody).

[0125] As used herein, the terms “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” “mAb,” or similar terms refer to antibody molecules consisting of a single molecular weight. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting single binding specificity, possessing variable and constant regions derived from human immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas fused with immortalized cells, including B cells derived from transgenic or inverted chromosome non-human animals (such as transgenic mice or rats) with genomes containing human heavy-chain and light-chain transgenes. Monoclonal antibodies can also be produced from recombinantly modified host cells or from cell extracts obtained through in vitro transcription and / or translation using nucleic acid sequences supporting the encoding antibody.

[0126] As used herein, the term "isotype" refers to a class of immunoglobulins (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene, or any of its allotypes, such as IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype can combine with a kappa(κ) or lambda(λ) light chain.

[0127] When the term "full-length antibody" is used herein, it means that the antibody is not a fragment, but a specific isotype containing all the domains of the isotype normally found in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of IgG1 antibody. Compared to full-length parental or wild-type antibodies, the heavy and light chain constant and variable domains in full-length variant antibodies may contain amino acid substitutions that improve the functional properties of the antibody. The full-length antibody according to the invention can be produced by a method comprising the steps of: (i) cloning a CDR sequence into a suitable vector containing the complete heavy chain sequence and the complete light chain sequence, and (ii) expressing the complete heavy chain sequence and the light chain sequence in a suitable expression system. The full-length antibody is produced with the knowledge of a person skilled in the art, starting from the CDR sequence or the full-length variable region sequence. Therefore, a person skilled in the art will know how to produce a full-length antibody according to the invention.

[0128] As used herein, the term "human antibody" is intended to include antibodies comprising variable and frame regions derived from human immunoglobulin sequences and constant structural domains of human immunoglobulins. Human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another non-human species (such as a mouse) has been grafted onto a human frame sequence.

[0129] As used herein, “humanized antibody” refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that co-form the antigen-binding site into a homologous human receptor frame region (FR) (see, for example, WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to substitute frame residues from the parent antibody (i.e., the nonhuman antibody) into the human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, humanized antibodies may contain a nonhuman CDR sequence, primarily a human frame region (which optionally contains one or more amino acid reversion mutations to a nonhuman amino acid sequence), and a full-length human constant region. Optionally, additional amino acid modifications, which are not necessarily reversion mutations, may be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0130] As used herein, the terms "Fc region" or "Fc domain" are used interchangeably and refer to the region of the heavy chain constant region, which includes at least the hinge region, CH2 region, and CH3 region extending from the N-terminus to the C-terminus of the antibody. 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.

[0131] It should be understood that the terms "parental polypeptide" or "parental antibody" are the same as the polypeptide or antibody according to the present invention, but wherein the parental polypeptide or parental antibody is not mutated, unless otherwise stated or clearly contradicted by the context. For example, antibody IgG1-CD134-003 is a parental antibody of IgG1-CD134-003-P329R-E345R.

[0132] As used herein, the term "hinge region" refers to the hinge region of the immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the Eu number (Eu index), as described in Kabat, EA, et al., *Sequences of proteins of immunological interest. 5th Edition - USDepartment of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689* (1991). However, the hinge region can also be any of the other subtypes described herein.

[0133] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of the immunoglobulin heavy chain. Therefore, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118 to 215 according to Eu numbering proposed by Kabat (ibid.). However, the CH1 region can also be any of the other subtypes described herein.

[0134] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of the immunoglobulin heavy chain. Therefore, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231 to 340 according to Eu numbering proposed by Kabat (ibid.). However, the CH2 region can also be any of the other subtypes described herein.

[0135] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of the immunoglobulin heavy chain. Therefore, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341 to 447 according to Eu numbering proposed by Kabat (ibid.). However, the CH3 region can also be any of the other subtypes described herein.

[0136] As used herein, the terms “Fc-mediated effector function” or “Fc effector function” are used interchangeably and are intended to refer to the result of the binding of a peptide or antibody to its target or antigen on the cell membrane, wherein the Fc-mediated effector function is attributable to the Fc region of the peptide 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-γ receptor (FcγR) binding, (vi) antibody-dependent FcγR-mediated antigen crosslinking, (vii) antibody-dependent phagocytosis (ADCP), (viii) complement-enhanced cytotoxicity, (ix) binding to the complement receptor of an antibody-mediated opsonized antibody, (x) opsonization, and (xi) combinations of any of (i) to (x).

[0137] The terms “reduced Fc effector function” or “reduced Fc-mediated effector function” used herein are used interchangeably and are intended to refer to a reduction in the Fc effector function of an antibody when compared directly to the Fc effector function of the parent peptide or antibody in the same assay.

[0138] As used herein, the terms "inert," "dormant," or "inactive" mean at least the inability to bind one or more FcγRs, to induce Fc-mediated FcγR crosslinking, to induce FcγR-mediated target antigen crosslinking via two Fc regions of an individual antibody, or to bind the Fc region of C1q. Therefore, in certain embodiments of the invention, the Fc region is inert. Consequently, in certain embodiments, some or all of the Fc-mediated effector functions are reduced or completely absent.

[0139] As used herein, the term “oligomerization” is intended to refer to the process of converting monomers into a limited degree of polymerization. Antibodies according to the invention can form oligomers, such as hexamers, via non-covalent association of Fc regions after binding to a target on, for example, cell surfaces. Oligomerization of anti-OX40 antibodies via Fc:Fc interactions after binding to the cell surface can increase OX40 clustering, leading to activation of intracellular OX40 signaling. The ability of antibodies containing E345R or E430G mutations to form oligomers (such as hexamers) after binding to the cell surface can be evaluated, as described in de Jong RN et al., PLoS Biol. 2016 Jan 6; 14(1):e1002344. Fc-Fc mediated antibody oligomerization occurs via intermolecular association of Fc regions between adjacent antibodies after binding to a target on the (cell) surface, and is increased by the introduction of E345R or E430G mutations (according to Eu index numbers).

[0140] As used in this article, “clustering” refers to the oligomerization of antibodies through non-covalent interactions.

[0141] As used herein, the term "Fc-Fc enhancement" refers to increasing the binding strength between the Fc regions of two antibodies containing Fc regions or stabilizing their interaction, resulting in the formation of oligomers, such as hexamers, on the cell surface. This enhancement can be achieved by specific amino acid mutations (such as E345R or E430G) in the Fc region of the antibody. In the context of this invention, the term "monovalent antibody" refers to an antibody molecule that interacts with a specific epitope on an antigen using only one antigen-binding domain (e.g., a Fab arm). In the context of bispecific antibodies, "monovalent antibody binding" refers to a bispecific antibody binding to only one specific epitope on an antigen using only one antigen-binding domain (e.g., a Fab arm).

[0142] In the context of this invention, the term "monospecific antibody" refers to an antibody that has binding specificity to only one epitope. An antibody can be a monospecific monovalent antibody (i.e., carrying only one antigen-binding region) or a monospecific bivalent antibody (i.e., an antibody having two identical antigen-binding regions).

[0143] The term "bispecific antibody" refers to an antibody containing two distinct antigen-binding domains, such as two different Fab arms or two Fab arms with different CDR regions. In the context of this invention, bispecific antibodies are specific for at least two distinct epitopes. These epitopes may be on the same or different antigens or targets. If the epitopes are on different antigens, these antigens may be on the same or different cells, cell types, or structures, such as the extracellular matrix or vesicles and soluble proteins. Bispecific antibodies can therefore be cross-linked with multiple antigens, such as two different cell types. Specific bispecific antibodies of this invention are capable of binding to OX40 and a second target.

[0144] The term "bivalent antibody" refers to an antibody with two antigen-binding regions, which binds to one or two epitopes on one or two targets or antigens, or to one or two epitopes on the same antigen. Therefore, bivalent antibodies can be monospecific bivalent antibodies or bispecific bivalent antibodies.

[0145] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. An amino acid is an organic compound containing an amine functional group (-NH₂) and a carboxyl functional group (-COOH), along with the characteristic side chain (R group) of each amino acid. In the context of this invention, amino acids can be classified based on their structure and chemical characteristics. Therefore, amino acid categories may be reflected in one or both of the following tables:

[0146] Table 1. Major classifications based on the structure and general chemical characteristics of the R group.

[0147]

[0148] Table 2. Physical and functional categories of amino acid residue substitution

[0149]

[0150] The substitution of one amino acid with another amino acid can be classified as conservative substitution or non-conservative substitution. In the context of this invention, "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structure and / or chemical characteristics. This means that one amino acid residue is substituted with another amino acid residue of the same category as defined in either of the two tables above: for example, leucine can be substituted with isoleucine, since both are aliphatic branched hydrophobic compounds. Similarly, aspartic acid can be substituted with glutamic acid, since both are small, negatively charged residues.

[0151] In the context of this invention, substitutions in antibodies are represented as follows:

[0152] Original amino acid - position - substituted amino acid;

[0153] The accepted amino acid nomenclature mentioned uses three-letter codes or one-letter codes (including the codes "Xaa" or "X") to represent any amino acid residue. Therefore, Xaa and X can generally represent any of the 20 naturally occurring amino acids. As used herein, "naturally occurring" refers to any 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 symbol "K409R" or "Lys409Arg" indicates that the antibody contains lysine replaced by arginine at amino acid position 409.

[0154] Replacing an amino acid at a given position with any other amino acid is called:

[0155] Original amino acid - position; or for example, "K409".

[0156] The modification of the original amino acid and / or substituted amino acids may include one or more, but not all, amino acids, and these modifications may be separated by commas or slashes. For example, lysine replaced by arginine, alanine, or phenylalanine at position 409 is:

[0157] "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409 replaced with R, A or F".

[0158] In the context of this invention, this naming is interchangeable but has the same meaning and purpose.

[0159] Furthermore, the term "substitution" includes substitution with any one or the other nineteen natural amino acids, or substitution with other amino acids, such as non-natural amino acids. For example, substitution of amino acid K at position 409 includes each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. Incidentally, this is equivalent to the designation 409X, where X indicates any amino acid other than the original amino acid. Such substitutions may also be designated as K409A, K409C, etc., or K409A,C, etc., or K409A / C / etc. The same applies in a similar manner to each and every position mentioned herein, specifically including any of such substitutions herein.

[0160] The antibodies according to the present invention may also contain deletions of amino acid residues. Such deletions may be represented as "del", and include, for example, written as K409del. Thus, in these embodiments, the lysine at position 409 has been deleted from the amino acid sequence.

[0161] As used herein, the term "host cell" is intended to refer to the cell in which the expression vector has been introduced. It should be understood that these terms are intended not only to the specific subject cell but also to the cell's progeny. Because specific modifications can occur in progeny due to mutations or environmental influences, these progeny cells may actually differ from the parent cells but 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, NSO cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli, and other eukaryotic hosts such as plant cells and fungi.

[0162] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express antibodies or target antigens, such as CHO cells, PER.C6 cells, NS0 cells, HEK293 cells, Expi293F cells, plant cells, or fungi, including yeast cells.

[0163] 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) executed 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) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "Longest Consistency" (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0164] (Consistent residues × 100) / (Alignment length - total number of gaps in alignment).

[0165] The retention of similar residues can also be measured, or alternatively, by a similarity score, such as by using a BLAST procedure (e.g., BLAST 2.2.8 available from NCBI, using standard settings BLOSUM62, open gap=11, and extended gap=1). Suitable variants typically exhibit at least about 45% similarity to the parental sequence, such as 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%).

[0166] As used herein, the term "effective cell" refers to an immune cell involved in the effector phase of an immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effective cells express Fc receptors (FcγR) or complement receptors and perform specific immune functions. In some embodiments, effective cells (such as, for example, natural killer cells) can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kuffour cells expressing FcγR are involved in specifically killing target cells and / or presenting antigens to other components of the immune system, or 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. The C3 cleavage product is a ligand for a complement receptor (CR) (such as CR3) expressed on bone marrow cells. Recognition of the complement fragment by CRs on effector cells promotes enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation yields the C3 fragment on target cells. These C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells may phagocytose target antigens, target particles, or target cells dependent on antibody binding, mediated by FcγR expressed on effector cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, it has been found that FcγRI expression is upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-carrying cells against the target. Effector cells may phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation yields a C3 fragment on target cells. These C3 cleavage products can directly promote phagocytosis via effector cells or indirectly promote phagocytosis by enhancing antibody-mediated phagocytosis. In this particular embodiment, where the antibody has an inert Fc region, the antibody does not induce Fc-mediated effector function.

[0167] As used herein, “effective T cells” or “Teffs” or “Teff” refers to T lymphocytes that perform immune responses (such as killing tumor cells and / or activating anti-tumor immune responses that can lead to the elimination of tumor cells from the body). Examples of Teff phenotypes include CD3. + CD4 + and CD3 + CD8 +Teff may secrete, contain, or express markers such as IFNγ, granzyme B, and ICOS. It should be recognized that Teff may not be entirely limited to these phenotypes.

[0168] As used herein, “memory T cells” refers to T lymphocytes that remain in the body long after infection has been removed. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It should be recognized that memory T cells may not be entirely limited to these phenotypes.

[0169] As used herein, "regulatory T cells," "Tregs," or "Treg" refer to T lymphocytes that typically regulate the activity of other T cells and / or other immune cells by inhibiting their activity. An example of the Treg phenotype is the CD3+ T lymphocyte. + CD4 + CD25 + CD127dim. Tregs can further express Foxp3. It should be recognized that Tregs may not be entirely limited to this phenotype.

[0170] As used herein, “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 the surface. C1 is a complex composed of six recognition proteins, C1q, and the heterotetramer C1r2C1s2 of a serine protease. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions starting with C4 cleavage into C4a and C4b and C2 cleavage into C2a and C2b. C4b is deposited and, together with C2a, forms an enzyme called C3 convertase, which cleaves complement component C3 into C3b and C3a, with C3a forming C5 convertase. This C5 convertase cleaves C5 into C5a and C5b and deposits the last component onto the membrane, thus triggering the late events of complement activation, in which terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). In the specific embodiments described herein, in which the antibody has an inert Fc region, the antibody does not induce complement activation.

[0171] Complement activation can be assessed using C1q binding efficacy and CDC kinetics assays (as described in WO2013 / 004842 and WO2014 / 108198) or by the C3b and C4b cell deposition assays described in Beurskens et al., J Immunol April 1, 2012 vol. 188 no.7, 3532-3541.

[0172] As used herein, the term "C1q binding" is intended to refer to the binding of C1q to an antibody that has already bound to its antigen. Antibody-antigen binding should be understood to occur both in vivo and in vitro in the context described herein. C1q binding can be assessed, for example, by using antibodies immobilized on artificial surfaces or by using antibodies that bind to predetermined antigens on the surface of cells or viral particles, as described in Example 8 herein. Binding of C1q to antibody oligomers should be understood herein as a multivalent interaction resulting in high avidity binding. For example, reduced C1q binding due to the introduction of mutations into the antibodies of the present invention can be measured by comparing the C1q binding of the mutated antibody to the C1q binding of its parent antibody (the antibody of the present invention without the mutation in the same analysis).

[0173] The term "treatment" refers to the application of an effective amount of the therapeutically active antibody of this invention for the purpose of relieving, improving, suppressing or eradicating (curing) symptoms or disease states.

[0174] The term "effective dose" or "therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dosage and duration. The therapeutically effective dose of an antibody can vary depending on factors such as an individual's disease state, age, sex, weight, and the antibody's ability to induce the desired response in the individual. Therapeutic effective dose is also the amount in which the therapeutic benefit outweighs any toxic or harmful effects of the antibody variant.

[0175] The "programmed death-1 (PD-1)" receptor is an immunosuppressive receptor belonging to the CD28 family.

[0176] As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, allotypes, and species homologs of hPD-L1, such as macaque (cynomolgus monkey), African elephant, wild boar, and mouse PD-L1 (see, for example, NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), and analogs that share at least one common epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO: 97 (mature sequence) and SEQ ID NO: 100, where amino acids 1 to 18 are expected to be signal peptides. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, allotypes, and species homologs of hPD-L2, and analogs that share at least one common epitope with hPD-L2. PD-1 ligands (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells (such as dendritic cells or macrophages) and other immune cells. Binding of PD-1 to either PD-L1 or PD-L2 leads to downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can shut down PD-1-expressing T cells, resulting in suppression of the anticancer immune response. The interaction between PD-1 and its ligands leads to a reduction in tumor-infiltrating lymphocytes, reduced T-cell receptor-mediated proliferation, and immune evasion by cancer cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and has an additive effect when the interaction between PD-1 and PD-L2 is blocked.

[0177] The term "PD-1" is associated with programmed cell death-1 and includes any variants, conformations, allotypes, and species homologs of PD-1 expressed naturally in cells or in cells transfected with the PD-1 gene. Preferably, "PD-1" is associated with human PD-1, and particularly with proteins having the amino acid sequence listed in SEQ ID NO: 98 (NCBI reference sequence: NP_005009.2). Alternative names for "PD-1" include CD279 and SLEB2.

[0178] The term “PD-1” includes post-translational variants, allotypes and species homologs of human PD-1 that are naturally expressed in cells or expressed in cells / cells transfected with the PD-1 gene.

[0179] The term “PD-1 variant” shall include (i) PD-1 splice variants, (ii) PD-1 post-translational modified variants, particularly variants with different N-glycosylation states, and (iii) PD-1 conformational variants. Such variants may include soluble forms of PD-1.

[0180] PD-1 is a type I membrane protein belonging to the immunoglobulin superfamily (The EMBO Journal (1992), vol. 11, issue 11, pp. 3887-3895). The human PD-1 protein comprises an extracellular domain consisting of amino acids 24 to 170 of the sequence listed in SEQ ID NO: 98, a transmembrane domain consisting of amino acids 171 to 191 of the sequence listed in SEQ ID NO: 98, and a cytoplasmic domain consisting of amino acids 192 to 288 of the sequence shown in SEQ ID NO: 98. The term “PD-1 fragment” as used herein shall encompass any fragment of the PD-1 protein, preferably an immune fragment. The term also includes, for example, the aforementioned domains of the full-length protein or any fragments of such domains, particularly immune fragments. The preferred amino acid sequence of the extracellular domain of the human PD-1 protein is listed in SEQ ID NO: 99 of the sequence listing.

[0181] The Fc region may have a lysine residue at its C-terminus. This lysine residue is derived from a naturally occurring sequence found in humans from which the Fc region originates. During the production of recombinant antibody cell cultures, this terminal lysine residue can be cleaved by proteolysis with an endogenous carboxypeptidase to obtain a constant region with the same sequence but lacking the C-terminal lysine residue. For antibody manufacturing purposes, the DNA encoding this terminal lysine residue can be omitted from the sequence, resulting in the production of antibodies without the lysine residue. Antibodies produced with or without a nucleic acid sequence encoding a terminal lysine residue are substantially identical in sequence and function because, for example, when antibodies are produced using a CHO-based production system, the terminal lysine residue is typically highly processed (Dick, LW et al., Biotechnol. Bioeng. 2008; 100: 1132-1143). Therefore, it should be understood that proteins (such as antibodies) according to the present invention can be produced with or without encoding a terminal lysine residue. According to the present invention, it should also be understood that a sequence having a terminal lysine (such as a constant region sequence having a terminal lysine) can be understood as a sequence without a corresponding terminal lysine, and a sequence without a terminal lysine can also be understood as a sequence with a corresponding terminal lysine.

[0182] Specific implementation scheme of the present invention

[0183] In one aspect, this disclosure provides a method for treating a subject's disease, preferably a method for reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject i) an antibody capable of binding to human OX40; and ii) a PD-1 inhibitor.

[0184] In one embodiment of the invention, the antibody capable of binding OX40 includes an antigen-binding region comprising a heavy chain variable (VH) region, wherein CDR1, CDR2, and CDR3 comprise sequences as listed in SEQ ID NO: 16, 17, and 18, respectively, and a light chain variable (VL) region, wherein CDR1, CDR2, and CDR3 comprise sequences as listed in SEQ ID NO: 20, DAS, and 21, respectively. This provides an anti-OX40 antibody capable of binding to humans. In one embodiment of the invention, the antibody binds to OX40 on T cells and is agonistic upon binding to its target. This provides an antibody that stimulates T cell activation and proliferation. The antibody can further stimulate T cell memory formation and survival. Such an antibody can be used, for example, to treat cancer. The antibody can further bind to cynomolgus monkey OX40, which can be used for toxicological studies of the antibody. Therefore, in one embodiment of the antibody according to the invention, the antibody is capable of binding to cynomolgus monkey OX40 having the sequence of SEQ ID NO: 51.

[0185] In another embodiment of the invention, the antibody is capable of binding to human OX40 having the sequence SEQ ID NO: 52. Human OX40 comprises four extracellular domains named CRD1, CRD2, CRD3, and CRD4, respectively. Without being bound by theory, it is envisioned that the binding of the antibody to the CRD1 domain enhances the ability to activate T cells. In one embodiment of the invention, the antibody binds to the CRD1 domain of human OX40.

[0186] It is well known in this art that binding affinity is important for antibody function. Therefore, the affinity of an antibody for its homoantigen may be too high or too low for the antibody to activate the desired intracellular pathway. In one embodiment, the antibody has a binding affinity of 1 x 10⁻⁶ for human OX40. -9 M to 6 x 10 -9 M's binding affinity K D In one implementation, the antibody has a 2 x 10⁻⁶ phosphate group against human OX40. -9 M to 5 x 10 -9 M's binding affinity K D In one implementation, the antibody has a 3 x 10⁻⁶ phosphate group against human OX40. -9 M to 4 x 10 -9 M's binding affinity K D In another embodiment, the antibody has a 3.4 x 10⁻⁶ phosphate group against human OX40. -9 M's binding affinity K D .

[0187] In another embodiment of the present invention, the antibody is a humanized or chimeric antibody.

[0188] The immunogenicity of antibodies can be reduced through humanization, thereby decreasing the level of anti-drug antibody (ADA) responses. Therefore, in one embodiment of the present invention, the antibody is a humanized antibody.

[0189] In one embodiment of the invention, the antibody comprises a VH region having the sequence listed in SEQ ID NO: 15 and a VL region having the sequence listed in SEQ ID NO: 19. This provides an antibody comprising a humanized VH region as listed in SEQ ID NO: 15 and a humanized VL region as listed in SEQ ID NO: 19. It is well known in the art that mutations can be introduced into proteins (such as antibodies) having intact sequences and three-dimensional structures without loss of function. Therefore, in some embodiments of the invention, variants with mutations in the frame regions of the VH and / or VL sequences are also contemplated, such as specific variants of the VH and / or VL regions listed in SEQ ID NO: 15 and SEQ ID NO: 19, respectively. Compared to the frame regions (i.e., FR1, FR2, FR3, and FR4 of the parental VH and / or VL sequences), variants may differ in one or more amino acids, for example, in one or more frame regions, but still allow the antigen-binding region to retain at least a considerable proportion (at least about 90%, 95%, or more) or even all the affinity and / or specificity of the parental antibody. Typically, such functional variants retain sequence identity equivalent to the parental sequence. Exemplary variants include variants differing from the respective parental VH or VL regions by 5 or fewer (e.g., 5, 4, 3, 2, or 1) amino acid residues (such as substitutions). Exemplary variants include variants that differ primarily from the parental VH and / or VL regions by conserved amino acid substitutions; for example, 5 (e.g., 5, 4, 3, 2, or 1) amino acid substitutions in the variant may be conserved. In a further embodiment of the invention, the antibody may contain up to 1, 2, or 3 mutations in the VH frame region and / or VL frame region, respectively. These mutations may be substitutions. Preferably, such substitutions do not significantly alter the binding affinity and / or binding specificity of the anti-OX40 antibody of the present invention. Therefore, the present invention comprises variants of the anti-OX40 antibody of the present invention having the same functional characteristics as antibodies comprising the VH region CDR sequences as listed in SEQ ID NO: 16, 17 and 18 and the VL region CDR sequences as listed in SEQ ID NO: 20, DAS and 21.

[0190] In another embodiment of the invention, the antibody comprises a VH region containing at least 80% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 85% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 90% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 95% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 96% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 97% of the sequence identical to that of the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 98% of the sequence identical to the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing at least 99% of the sequence identical to the VH region listed in SEQ ID NO: 15. In another embodiment of the invention, the antibody comprises a VH region containing the sequence listed in SEQ ID NO: 15.

[0191] In another embodiment of the invention, the antibody comprises a VL region containing at least 80% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 85% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 90% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 95% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 96% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 97% of the sequence identical to that of the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 98% of the sequence identical to the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing at least 99% of the sequence identical to the VL region listed in SEQ ID NO: 19. In another embodiment of the invention, the antibody comprises a VL region containing the sequence listed in SEQ ID NO: 19.

[0192] In another embodiment of the invention, the antibody comprises VH and VL regions, which respectively comprise the sequences listed in SEQ ID NO: 15 and SEQ ID NO: 19.

[0193] In one embodiment, the antibody of the present invention is an isolated antibody.

[0194] 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). CH preferably comprises a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0195] The antibody according to the present invention may contain a light chain constant region, which is a human κ light chain. In another embodiment, it may contain a human λ light chain constant region.

[0196] The antibody according to the invention preferably further comprises an Fc region, which is a human IgG isotype. In some embodiments, the IgG is a modified human IgG containing one or more amino acid substitutions. Human IgG is present in various isotypes, such as IgG1, IgG2, IgG3, or IgG4. The antibody according to the invention preferably further comprises a heavy chain constant region, which is a human IgG1 isotype. Thus, in one embodiment, the antibody comprises a human IgG1 Fc region.

[0197] The antibody according to the present invention preferably contains the human IgG1 Fc region, which includes the P329R mutation and the E345R mutation, wherein the amino acid positions are numbered according to the Eu number.

[0198] Human IgG1 isotypes exist in various allotypes, such as IgG1m(f), IgG1m(a), IgG1m(x) and IgG1m(z), and allotypes can also be written as IgG1mf, IgG1ma, IgG1mx and IgG1mz, respectively.

[0199] In one embodiment of the present invention, the antibody comprises an Fc region selected from human IgG1mf, human IgG1ma, human IgG1mx, or human IgG1mz allotypes. In one embodiment of the present invention, the Fc region is a human IgG1mf allotype. In one embodiment of the present invention, the antibody comprises an Fc region having the sequence listed in SEQ ID NO: 1.

[0200] The antibody according to the invention preferably comprises a modified human IgG1 constant region. This human IgG1 includes an Fc region comprising CH2 and CH3 regions. By modifying the IgG1 constant region in the Fc region, it is possible, for example, to modulate the Fc effector function of the antibody or increase Fc-Fc interactions, thereby making the antibody prone to forming clusters, such as hexamers. In one embodiment of the invention, the human IgG1 or modified human IgG1 is selected from IgG1mf, IgG1ma, IgG1mx, or IgG1mz. In one embodiment, it is a modified IgG1 having at least two mutations. In another embodiment, it is IgG1mf having at least two mutations. In yet another embodiment, it is IgG1ma having at least two mutations. In a further embodiment, it is IgG1mx having at least two mutations. In yet another embodiment, it is IgG1mz having at least two mutations. In a particular embodiment, the IgG is a modified human IgG comprising two or more amino acid substitutions in the Fc region. In one embodiment, it may be human IgG1 comprising two or more amino acid substitutions in the Fc region. In a further embodiment of the invention, IgG1mf comprises two or more amino acid substitutions in the Fc region. In a further embodiment of the invention, IgG1mf comprises three amino acid substitutions in the Fc region. In a further embodiment of the invention, IgG1mf comprises four amino acid substitutions in the Fc region. In a further embodiment of the invention, IgG1mf comprises five amino acid substitutions in the Fc region. In one embodiment, the IgG1 Fc region has a maximum of three amino acid substitutions. In one embodiment, the IgG1mf Fc region has a maximum of three amino acid substitutions. In one embodiment, the IgG1mf Fc region has a maximum of four amino acid substitutions. In one embodiment, the IgG1mf Fc region has a maximum of five amino acid substitutions.

[0201] In a further embodiment of the invention, the modified human IgG1 heavy chain constant region contains up to five amino acid substitutions in the Fc region. In another embodiment, it contains up to four amino acid substitutions. In another embodiment, it contains up to three amino acid substitutions. In yet another embodiment, it contains up to two amino acid substitutions.

[0202] Mutations in the amino acid residues at the E345 position in the human IgG1 heavy chain (wherein the amino acid residues are numbered according to Eu numbers) can improve the ability of antibodies to induce CDC and other effector functions. Without being bound by theory, it is believed that introducing E345R substitution can stimulate antibody oligomerization, thereby modulating Fc-mediated effector functions to, for example, enhance C1q binding, complement initiation, CDC, ADCP, internalization, or other related functions that may provide in vivo therapeutic effects. In one embodiment, the antibody according to the invention comprises an Fc region contained in the sequence listed in SEQ ID NO: 2.

[0203] In one embodiment, 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 OX40.

[0204] This provides antibodies with enhanced Fc-Fc interactions, which can lead to antibody-dependent aggregation of OX40 on the cell surface after antibody binding, thereby increasing the excitability of the antibodies of the present invention.

[0205] In one embodiment of the invention, the antibody comprises a mutated human IgG1 Fc region or a mutated human IgG1 CH region comprising both E345R and P329R mutations. Hereinafter, it is mentioned that mutations in the Fc region can equally apply to mutations in the human IgG1 CH region, and vice versa.

[0206] As described herein, when numbered according to Eu designations, the position of the amino acid to be mutated in the Fc region can be given relative to (i.e., “corresponding to”) its position in the naturally occurring (wild-type) human IgG1 heavy chain. Therefore, if the parental Fc region already contains one or more mutations and / or if the parental Fc region is, for example, the IgG1, IgG1mf, IgG1ma, IgG1mz, or IgG1mz Fc region, the amino acid position (such as, for example, E345) corresponding to the amino acid residue in the human IgG1 heavy chain numbered according to Eu designations can be determined by alignment. Specifically, the parental Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue in the human IgG1 heavy chain sequence corresponding to the position E345. Any wild-type human IgG1 constant region amino acid sequence can be used for this purpose, including any of the different human IgG1 allotypes listed in Table 3.

[0207] In one embodiment of the invention, modifications in the Fc region of IgG1 induce increased OX40 activating activity compared to the same antibody but containing the same isotype or allotype of IgG Fc region (e.g., IgG1). This can be achieved, for example, by introducing an R amino acid at the amino acid position corresponding to position E345, thus introducing the E345R mutation into the human IgG1 heavy chain according to Eu numbering.

[0208] In a preferred embodiment, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to the Eu number is R. Therefore, the antibody of the present invention may contain E345R substitution in the Fc region.

[0209] This provides antibodies with enhanced Fc-Fc interactions, which can lead to antibody-dependent clustering of OX40 on the cell surface after antibody binding, thereby increasing the excitability of the antibodies of the present invention.

[0210] In another embodiment of the antibody of the present invention, an amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu number is replaced by an R amino acid, thereby introducing the P329R mutation into the human IgG1 heavy chain according to Eu number.

[0211] In a further embodiment of the invention, the antibody has an amino acid residue R at position P329 in the human IgG1 heavy chain according to Eu numbering. Therefore, the antibody of the present invention contains a P329R substitution in the Fc region. Without being bound by theory, it is believed that the antibody of the present invention containing the E345R mutation in the Fc region has an increased serum clearance rate. The inventors have found that further introducing mutations (such as P329R) at position 329 restores the clearance rate of the antibody of the present invention to the level of antibodies containing the wild-type sequence of the human IgG1 Fc region (i.e., without any mutations in the human IgG1 Fc region). Furthermore, the P329R mutation reduces the antibody's ability to bind to FcγR receptors (such as FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa).

[0212] In a preferred embodiment of the invention, the Fc region contains P329R and E345R mutations at positions corresponding to P329 and E345 in the human IgG1 heavy chain according to Eu numbering. This provides antibodies with increased OX40 receptor agonism and comparable pharmacokinetic properties (such as, for example, serum clearance) compared to antibodies containing the same VH and VL regions and containing the same IgG1 heavy chain constant regions except for the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345.

[0213] Therefore, embodiments of the present invention provide OX40-binding antibodies that, when compared with the pharmacokinetic properties of antibodies containing the same VH and VL regions but containing the constant region of the wild-type IgG1 heavy chain (such as those listed in SEQ ID NO: 1), exhibit increased receptor agonism upon binding to OX40 and further possess comparable pharmacokinetic properties (such as similar or even identical pharmacokinetic properties). In other words, the present invention provides OX40-binding antibodies whose pharmacokinetic properties are not significantly different from those of OX40-binding antibodies containing the same OX40 heavy chain constant region except for the wild-type IgG1 heavy chain constant region.

[0214] In one implementation, the parental Fc region and / or the human IgG1 CH region are wild-type human IgG1 isotypes.

[0215] In one embodiment of the present invention, the parental human IgG1 Fc region and / or human IgG1 CH region is a wild-type human IgG1mf allotype. In one embodiment of the present invention, the parental human IgG1 Fc region and / or human IgG1 CH region is a wild-type human IgG1ma allotype. In one embodiment of the present invention, the parental human IgG1 Fc region and / or human IgG1 CH region is a wild-type human IgG1mx allotype. In one embodiment of the present invention, the parental human IgG1 Fc region and / or human IgG1 CH region is a wild-type human IgG1mz allotype.

[0216] Therefore, apart from the listed mutations E345R and P329R, the mutated Fc region could be the human IgG1 Fc region.

[0217] In another embodiment, the present invention provides an antibody comprising a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 59, 60, and 61. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 58. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 59. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 60. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 61.

[0218] In one embodiment, the antibody according to the present invention comprises:

[0219] The VH region contains the amino acid sequence listed in SEQ ID NO: 15.

[0220] The VL region contains the amino acid sequence listed in SEQ ID NO: 19, and

[0221] The Fc region contains the amino acid sequence listed in SEQ ID NO:3.

[0222] In another embodiment, the antibody according to the invention comprises:

[0223] The VH region contains the amino acid sequence listed in SEQ ID NO: 15.

[0224] The VL region contains the amino acid sequence listed in SEQ ID NO: 19, and

[0225] The CH region contains the amino acid sequence listed in SEQ ID NO: 58.

[0226] In another embodiment, the antibody according to the invention comprises: a heavy chain comprising the amino acid sequence listed in SEQ ID NO: 13, and a light chain comprising the amino acid sequence listed in SEQ ID NO: 14.

[0227] In yet another embodiment, the present invention provides an antibody comprising a modified heavy chain constant region, such that the antibody induces Fc-mediated effector function to a lesser extent compared to the same antibody other than the modification. An example is the OX40 binding antibody of the present invention comprising P329R and E345R substitutions. Compared to antibodies comprising the same sequence except for the absence of P329R substitution, and also compared to antibodies comprising the same sequence (such as wild-type IgG1 heavy chain) except for the absence of P329R and E345R substitutions, these antibodies induce one or more Fc-mediated effector functions to a lesser extent. In one embodiment, the Fc-mediated effector function is reduced by at least 20%. In another embodiment, the Fc-mediated effector function is reduced by at least 30%. In another embodiment, the Fc-mediated effector function is reduced by at least 40%. In another embodiment, the Fc-mediated effector function is reduced by at least 50%. In another embodiment, the Fc-mediated effector function is reduced by at least 60%. In another embodiment, the Fc-mediated effector function is reduced by at least 70%. In another embodiment, Fc-mediated effector function is reduced by at least 80%. In another embodiment, Fc-mediated effector function is reduced by at least 90%. In another embodiment, the antibody does not induce one or more Fc-mediated effector functions. The reduced or completely uninduced one or more Fc effector functions may be selected from the group consisting of complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), Clq binding, and FcγR binding. Therefore, in one embodiment, relative to the same antibody but having the wild-type IgG1 HC constant region, the antibody of the present invention induces CDC by at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or by at least 90%. In another embodiment, the antibody of the present invention does not induce CDC.

[0228] In another embodiment, the antibody of the present invention induces CDCC by at least 20% compared to the same antibody, but having the wild-type IgG1 HC constant region, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or by at least 90%. In another embodiment, the antibody of the present invention does not induce CDCC.

[0229] In another embodiment, the antibody of the present invention induces ADCC at least 20% less than an antibody that is identical but has a wild-type IgG1 HC constant region, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. In another embodiment, the antibody of the present invention does not induce ADCC.

[0230] In another embodiment, the antibody of the present invention induces ADCP at least 20% less than that of the same antibody, but having the same wild-type IgG1 HC constant region, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. In another embodiment, the antibody of the present invention does not induce ADCP.

[0231] In another embodiment, compared to the same antibody but having the wild-type IgG1 HC constant region, the antibody of the present invention induces C1q binding at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. In another embodiment, the antibody of the present invention does not induce C1q binding.

[0232] In another embodiment, compared to the same antibody but having the wild-type IgG1 HC constant region, the antibody of the present invention induces FcγR binding at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%. In another embodiment, the antibody of the present invention does not induce FcγR binding. Preferably, FcγR binding is determined as in Example 8.

[0233] In one embodiment, compared with antibodies containing the same amino acid sequence but without the P329R substitution, the antibody of the present invention has reduced C1q binding and reduced FcγR binding.

[0234] In one implementation scheme, except for the mutations listed herein, the antibody according to any aspect or implementation scheme herein is a human antibody.

[0235] In one embodiment of the present invention, the antibody is a monovalent antibody.

[0236] In another embodiment, the antibody is a bivalent antibody.

[0237] Furthermore, the antibody of this invention can be a monospecific antibody.

[0238] In one embodiment, the antibody according to any aspect or embodiment of this document 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.

[0239] In a preferred embodiment, in addition to the optional listed mutations in the Fc region, the antibody according to any aspect or embodiment herein is an IgG1 antibody, such as a full-length IgG1 antibody, such as a human full-length IgG1 antibody, optionally a human monoclonal full-length bivalent IgG1,κ antibody, such as a human monoclonal full-length bivalent IgG1m(f),κ antibody.

[0240] The antibody according to the invention is preferably 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 contemplated. Therefore, unless contradicted by the context, the antibody according to any aspect or embodiment herein may be a monospecific antibody or a bispecific antibody.

[0241] Therefore, 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 comprising different epitopes capable of binding to 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 comprising different targets. These targets may be on cells different from or the same as OX40.

[0242] In one embodiment of the invention, the antibody is capable of binding to human OX40 having the sequence listed in SEQ ID NO: 52. In another embodiment, the antibody of the present invention is capable of further binding to cynomolgus monkey OX40, such as that listed in SEQ ID NO: 51.

[0243] In a further embodiment of the present invention, the antibody is capable of binding to human T cells expressing OX40.

[0244] In another embodiment of the present invention, the antibody is able to bind to OX40-expressing cynomolgus monkey T cells.

[0245] In another embodiment of the invention, the antibody has increased agonistic activity compared to wild-type parental antibodies that do not have P329R and E345R mutations.

[0246] In another embodiment of the invention, the antibody induces increased T cell proliferation.

[0247] In another embodiment of the invention, the antibody induces increased T cell proliferation compared to parental antibodies that do not have P329R and E345R mutations.

[0248] In one embodiment of the present invention, the full-length IgG1 antibody has a C-terminal lysine residue of a cleaved HC. Such antibodies are also considered "full-length antibodies".

[0249] In another embodiment of the invention, the antibody can induce the proliferation of human T cells, such as CD4+. + and CD8 + T cells, such as helper T cells and cytotoxic T cells, as described in Example 7.

[0250] In another embodiment of the present invention, the antibody can induce activation of T cells expressing human OX40.

[0251] In another embodiment of the present invention, the antibody can induce activation of T cells expressing human OX40 in the absence of Fcγ receptor IIb crosslinking.

[0252] In another embodiment of the invention, the antibody is capable of inducing CD4+ and CD8+ cells with a central memory T cell phenotype. + T cell proliferation.

[0253] In one embodiment, the PD-1 inhibitor prevents PD-1-related inhibitory signaling. In one embodiment, the PD-1 inhibitor is an antibody or fragment thereof that disrupts or inhibits PD-1-related inhibitory signaling. In one embodiment, the PD-1 inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the PD-1 inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the PD-1 inhibitor is an inhibitory nucleic acid that disrupts or inhibits inhibitory signaling.

[0254] Inhibition or blockage of PD-1 signaling as described herein results in the prevention or reversal of immunosuppression of T-cell immunity and the establishment or enhancement of immunity against cancer cells. In one embodiment, inhibition of PD-1 signaling as described herein reduces or suppresses dysfunction of the immune system. In one embodiment, inhibition of PD-1 signaling as described herein reduces the degree of dysfunction of dysfunctional immune cells. In one embodiment, inhibition of PD-1 signaling as described herein reduces the degree of dysfunction of dysfunctional T cells.

[0255] In one embodiment, PD-1 is human PD-1. Preferably, PD-1 has or comprises the amino acid sequence as listed in SEQ ID NO: 98 or SEQ ID NO: 99, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence listed in SEQ ID NO: 98 or SEQ ID NO: 99, or is an immune fragment thereof.

[0256] In one implementation, PD-L1 is human PD-L1, particularly human PD-L1 contained in the sequence listed in SEQ ID NO: 97.

[0257] In one implementation, the PD-1 inhibitor prevents the interaction between PD-1 and PD-L1.

[0258] PD-1 inhibitors may be antibodies, their antigen-binding fragments, or constructs thereof comprising an antibody portion having the desired specificity of an antigen-binding fragment. Antibodies or their antigen-binding fragments are as described herein. Antibodies or antigen-binding fragments (which are PD-1 inhibitors) particularly include antibodies or their antigen-binding fragments that bind to PD-1 and antibodies or their antigen-binding fragments that bind to PD-L1. Antibodies or antigen-binding fragments may also be conjugated to other portions as described herein. Antibodies or their antigen-binding fragments are particularly chimeric antibodies, humanized antibodies, or human antibodies.

[0259] In one implementation, the antibody (which is a PD-1 inhibitor) is an isolated antibody.

[0260] In one embodiment, the PD-1 inhibitor is an antibody, fragment thereof, or construct that prevents the interaction between PD-1 and PD-L1.

[0261] PD-1 inhibitors can be repressive nucleic acid molecules, such as oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules and aptamers (e.g., DNA or RNA aptamers), particularly antisense oligonucleotides. In one embodiment, the PD-1 inhibitor as siRNA interferes with mRNA, thereby blocking translation, such as the translation of PD-1 protein.

[0262] In one embodiment, the PD-1 inhibitor is an antibody, its antigen-binding portion, or a construct thereof that disrupts or inhibits the interaction between the PD-1 receptor and one or more of its ligands (PD-L1 and / or PD-L2). Antibodies that bind to PD-1 or PD-L1 and disrupt or inhibit the interaction between PD-1 and one or more of its ligands are known in the art. In a particular embodiment, the antibody, its antigen-binding portion, or a construct thereof specifically binds to PD-1. In a particular embodiment, the antibody, its antigen-binding portion, or a construct thereof specifically binds to PD-L1. In a particular embodiment, the antibody, its antigen-binding portion, or a construct thereof specifically binds to PD-L2.

[0263] In a particular preferred embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, such as a PD-1 blocking antibody. In a particular preferred embodiment, the PD-1 inhibitor is an antibody that binds to PD-L1, such as a PD-L1 blocking antibody.

[0264] Exemplary PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), rambrizumab (e.g., disclosed in WO2008 / 156712 as hPD-109A and its humanized derivatives h409A1, h409A16 and h409A17), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrixe Bioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see US Patent No. 8,008,449; WO 2013 / 173223; WO 2006 / 121168), pembrolizumab (Keljuda; MK-3475; Merck; see WO 2008 / 156712), pidilizumab (CT-011; CureTech; see Hardy et al. 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; see WO2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO 2012 / 145493), TSR-042 (see WO2014 / 179664), cimiprimab (REGN-2810; Regeneron; H4H7798N; see US) 2015 / 0203579 and WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al. 2007, J. Hematol. Oncol.70: 136), AMP-224 (GSK-2661380; see Li et al. 2016, Int J Mol Sci17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), tislelizumab (BGB-A317; BeiGene; see WO 2015 / 35606, US Patent Nos. 9,834,606 and US2015 / 0079109), BI 754091, SHR-1210 (see WO2015 / 085847), antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 as described in WO 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al. 2017, J. Hematol. Oncol. 70: 136), STI-1110 (SorrentoTherapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al. J. Clin. Oncol. 36, no. 5_suppl(2018) 58), genolimzumab (CBT-501; see Patel et al. J. ImmunoTher. Cancer, 2017, 5(Suppl2):P242), sasanlimab (PF-06801591; see Youssef et al. Proc. Am. Assoc. Cancer Res. Ann.Meeting 2017; Cancer Res 2017; 77(13 Suppl): Abstract), toripalimab (JS-001; see US 2016 / 0272708), camrelizumab (SHR-1210; INCSHR-1210; see US 2016 / 376367; Huang et al. Clin. Cancer Res. 2018; 24(6):1296-1304), spartalizumab (PDR001; see WO 2017 / 106656; Naing et al. J. Clin. Oncol. 34, no. 15_suppl(2016) 3060-3060), BCD-100 (JSC BIOCAD, Russia; see WO 2018 / 103017), balstilimab (AGEN2034; see WO 2017 / 040790), sintilimab (IBI-308; see WO 2017 / 024465 and WO 2017 / 133540), ezabenlimab (BI-754091; see US 2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.5_suppl (2018) 212-212), zimberelimab (GLS-010; see WO 2017 / 025051), LZM-009 (see US 2017 / 210806), AK-103 (see WO 2017 / 071625, WO2017 / 166804 and WO 2018 / 036472), retifanlimab (MGA-012; see WO 2017 / 019846), Sym-021 (see WO 2017 / 055547), CS1003 (see CN107840887), anti-PD-1 antibodies (such as US 7,488,802, US 8,008,449, US 7,488,802, US 8,008,449, US 8,008,449, US 8,008,802 ... 8,168,757, WO 03 / 042402, WO 2010 / 089411 (further revealing anti-PD-L1 antibody), WO 2010 / 036959, WO 2011 / 159877 (further revealing antibody against TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO2012 / 145493 (further revealing anti-PD-L1 antibody), WO 2015 / 035606, WO 2014 / 055648 (further revealing anti-KIR antibody), US 2018 / 0185482 (further revealing anti-PD-L1 and anti-TIGIT antibody), US 8,008,449, US 8,779,105, US 6,808,710, US 8,168,757, US 2016 / 0272708 and US 8,354,509), small molecule antagonists of the PD-1 signaling pathway (such as those disclosed in Shaabani et al. 2018, Expert Op TherPat., 28(9):665-678 and Sasikumar and Ramachandra 2018, BioDrugs, 32(5):481-497), siRNAs targeting PD-1 (such as those disclosed in WO 2019 / 000146 and WO 2018 / 103501), soluble PD-1 proteins (such as those disclosed in WO 2018 / 222711), and oncolytic viruses containing soluble forms of PD-1 (such as those disclosed in WO 2018 / 222711). (As described in 2018 / 022831).

[0265] In a particular implementation, the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody or its antigen-binding fragment, which includes a complementarity-determining region (CDR) of one of the anti-PD-1 or anti-PD-L1 antibodies or antigen-binding fragments as described herein, such as the CDR of an anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment selected from the group consisting of: nivolumab, Amp-514, tislelizumab, cimipril, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0266] In a specific embodiment, the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region of one of the anti-PD-1 or anti-PD-L1 antibodies or antigen-binding fragments as described herein, such as the heavy chain variable region and light chain variable region of an anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment selected from the group consisting of: nivolumab, Amp-514, tislelizumab. Lizumab, Cimiprimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, Camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0267] In a specific implementation, the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody or its antigen-binding fragment selected from the group consisting of: nivolumab, Amp-514, tislelizumab, cimipril, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0268] In certain embodiments, the PD-1 inhibitor is an antibody that binds to PD-1 or PD-L1. In some preferred embodiments, the PD-1 inhibitor is an antibody that is an antagonist of the PD-1 / PD-L1 interaction. In some preferred embodiments, the PD-1 inhibitor is a PD-1 blocking antibody or a PD-L1 blocking antibody.

[0269] In certain embodiments, the PD-1 inhibitor is an antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, such as an antibody of the IgG1 isotype. In one embodiment, the PD-1 inhibitor is an antibody of the IgG1 isotype. In one embodiment, the PD-1 inhibitor is an antibody of the IgG2 isotype. In one embodiment, the PD-1 inhibitor is an antibody of the IgG3 isotype. In one embodiment, the PD-1 inhibitor is an antibody of the IgG4 isotype.

[0270] In certain implementations, the PD-1 inhibitor is a full-length antibody or antibody fragment, such as a full-length IgG1 antibody.

[0271] In a specific implementation, the PD-1 inhibitor is a monospecific antibody.

[0272] In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences listed in SEQ ID NO: 79, 80, and 81, respectively; and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences listed in SEQ ID NO: 82, LAS, and SEQ ID NO: 83, respectively. In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a VH region containing the amino acid sequence of SEQ ID NO: 84, and a VL region containing the amino acid sequence of SEQ ID NO: 85. In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain containing the amino acid sequence of SEQ ID NO: 86, and a light chain containing the amino acid sequence of SEQ ID NO: 87. In a preferred embodiment, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof.

[0273] In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1. The PD-1-binding antibody may comprise a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences; and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively contain or have the sequences listed in SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, and the LCDR1, LCDR2, and LCDR3 sequences respectively contain or have the sequences listed in SEQ ID NO: 93, QAS, and SEQ ID NO: 94. In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a VH region containing the amino acid sequence of SEQ ID NO: 88, and a VL region containing the amino acid sequence of SEQ ID NO: 92. In one embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain containing the amino acid sequence of SEQ ID NO: 95, and a light chain containing the amino acid sequence of SEQ ID NO: 96. Specific, but non-limiting, examples of such antibodies are IgG1-PD1 in Examples 29 to 32 of this application.

[0274] In some embodiments, the PD-1 inhibitor is a PD-1 blocking antibody. In some embodiments, the PD-1 inhibitor is a PD-L1 inhibitor, such as a PD-L1 blocking antibody.

[0275] In a specific implementation scheme, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680), acrixolimab, or their respective biosimilars. In specific implementation schemes, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiprimab, dostalimumab, refulimab, toripalimab, vopalimumab, spartazumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-514 (MEDI0680), axololimumab, or biosimilars thereof. In specific implementation schemes, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiprimab, dostalimumab, refulimab, toripalimab, or biosimilars thereof.

[0276] In a specific implementation, the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170, BMS-986189, or a biosimilar thereof. In a specific implementation, the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab, KN035, cosibelimab, or a biosimilar thereof. In a specific implementation, the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab, or a biosimilar thereof.

[0277] Subjects who wish to be treated according to this disclosure are preferably human subjects.

[0278] In one implementation, the tumor or cancer is a hematologic cancer or a solid tumor, preferably a solid tumor such as colorectal cancer.

[0279] In one implementation, the tumor or cancer is metastatic.

[0280] In one implementation, the treatment method comprises administering one or more additional therapeutic agents to the subject, such as one or more chemotherapeutic agents, particularly chemotherapeutic agents commonly used to treat tumors or cancers as described herein. For example, one or more chemotherapeutic agents include platinum-based compounds (such as cisplatin, carboplatin), taxane-based compounds (such as paclitaxel, nab-paclitaxel), nucleoside analogs (such as gemcitabine), antifolate agents (such as pemetrexed), and any combination thereof.

[0281] In one embodiment, the method includes systemic administration of an antibody and / or a PD-1 inhibitor to a subject, particularly by injection or infusion, such as intravenous injection or infusion.

[0282] In one aspect, this disclosure provides a kit comprising i) an antibody capable of binding OX40, ii) a PD-1 inhibitor, and optionally (iii) one or more additional therapeutic agents, such as one or more chemotherapeutic agents.

[0283] In one embodiment of the kit according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0284] In one embodiment of the kit according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0285] In one embodiment of the kit according to this aspect, the binder, PD-1 inhibitor, and one or more additional therapeutic agents, if present, are used for systemic administration, particularly for injection or infusion, such as intravenous injection or infusion.

[0286] In one aspect, this disclosure provides a kit for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating the subject's cancer, the kit comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor.

[0287] In one embodiment of the kit used according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0288] In one embodiment of the kit used according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0289] In one embodiment of the kit used according to this aspect, the method is as defined in any aspect or embodiment of this disclosure.

[0290] In one embodiment of the kit used according to this aspect, the tumor or cancer is as defined in any aspect or embodiment of this disclosure.

[0291] In one aspect, this disclosure provides pharmaceutical compositions comprising i) an antibody capable of binding OX40; ii) a PD-1 inhibitor; and iii) optionally a pharmaceutically acceptable carrier.

[0292] In one embodiment of the pharmaceutical composition according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0293] In one embodiment of the pharmaceutical composition according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0294] In one aspect, this disclosure provides a pharmaceutical composition for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating the subject's cancer, the pharmaceutical composition comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor.

[0295] In one embodiment of the pharmaceutical composition used according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0296] In one embodiment of the pharmaceutical composition used according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0297] In one embodiment of the pharmaceutical composition used according to this aspect, the method is as defined in any aspect or embodiment of this disclosure.

[0298] In one embodiment of the pharmaceutical composition used according to this aspect, the tumor or cancer is as defined in any aspect or embodiment of this disclosure.

[0299] In one aspect, this disclosure provides an antibody capable of binding to OX40 for use in a method of treating a subject’s disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating the subject’s cancer, the method comprising administering to the subject i) an antibody; and ii) a PD-1 inhibitor.

[0300] In one embodiment of the antibody used according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0301] In one embodiment of the antibody used according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0302] In one embodiment of the antibody used according to this aspect, the method is as defined in any aspect or embodiment of this disclosure.

[0303] In one embodiment of the antibody used according to this aspect, the tumor or cancer is as defined in any aspect or embodiment of this disclosure.

[0304] In one aspect, this disclosure provides a PD-1 inhibitor for use in a method of treating a subject’s disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating the subject’s cancer, the method comprising administering to the subject i) an antibody capable of binding OX40; and ii) a PD-1 inhibitor.

[0305] In one embodiment of the PD-1 inhibitor used according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0306] In one embodiment of the PD-1 inhibitor used according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0307] In one embodiment of the PD-1 inhibitor used according to this aspect, the method is as defined in any aspect or embodiment of this disclosure.

[0308] In one embodiment of the PD-1 inhibitor used according to this aspect, the tumor or cancer is as defined in any aspect or embodiment of this disclosure.

[0309] In one aspect, this disclosure provides the use of an antibody capable of binding OX40 in the preparation of a medicament, preferably in combination with a PD-1 inhibitor for reducing or preventing tumor progression in a subject or for treating cancer in a subject.

[0310] In one embodiment of the use of this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0311] In one embodiment of the use of this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0312] In one embodiment of the use of this aspect, a tumor or cancer is defined as in any aspect or embodiment of this disclosure.

[0313] In one aspect, this disclosure provides the use of a PD-1 inhibitor in the preparation of a medicament, preferably in combination with an antibody capable of binding OX40 for reducing or preventing tumor progression in a subject or treating cancer in a subject.

[0314] In one embodiment of the use of this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0315] In one embodiment of the use of this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0316] In one embodiment of the use of this aspect, a tumor or cancer is defined as in any aspect or embodiment of this disclosure.

[0317] In one aspect, this disclosure provides (i) the use of an antibody capable of binding to OX40 and (ii) a PD-1 inhibitor in the preparation of a medicament, preferably for reducing or preventing tumor progression in a subject or treating cancer in a subject.

[0318] In one embodiment of the use of this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0319] In one embodiment of the use of this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0320] In one embodiment of the use of this aspect, a tumor or cancer is defined as in any aspect or embodiment of this disclosure.

[0321] In one aspect, this disclosure provides a medical preparation comprising i) an antibody capable of binding OX40 and ii) a PD-1 inhibitor.

[0322] In one embodiment of the medical preparation according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure.

[0323] In one embodiment of a medical preparation according to this aspect, a PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0324] In one aspect, this disclosure provides an antibody capable of binding to human OX40 for treating a disease in a subject, the use comprising administering (i) an antibody; and (ii) a PD-1 inhibitor to the subject. In one embodiment of the use according to this aspect, the antibody is as defined in any aspect or embodiment of this disclosure. In one embodiment of the use according to this aspect, the PD-1 inhibitor is as defined in any aspect or embodiment of this disclosure.

[0325] Table 3: Sequence List

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] Other aspects of this disclosure are revealed herein.

[0339] Example

[0340] Example 1: Generation of anti-human OX40 antibody and its Fc variant

[0341] As described in WO2016 / 110584A1, nine rabbits used to generate anti-human OX40 antibodies were immunized with recombinant human OX40 (CD134) fused with the Fc portion of human IgG1 (Adipogen, catalog number AG-40B-0014; MAB Discovery GmbH). Following a series of immunizations, blood samples were collected at four different time points and B cells were enriched. Individual B cells were sorted by flow cytometry, and monoclonal cells were cultured and expanded at 37°C for seven days. After seven days, the culture supernatant was collected to assess the production of human OX40-specific antibodies, and the B cells were frozen and stored at -80°C until further use.

[0342] Will be combined with OX40's V H and V LThe identified unique sequence of the combination was transiently co-expressed in HEK293 cells to generate rabbit-human chimeric antibodies. Based on screening assays evaluating the antibodies' ability to bind to human OX40 and their agonistic activity, 30 chimeric antibodies were screened for further testing. For this purpose, the V... H and V L The region was genetically synthesized and cloned upstream of the human IgG1 constant region. Subsequently, antibodies were produced and purified in HEK293 cells. Flow cytometry was used to test the binding of the antibodies to human and cynomolgus monkey OX40. These antibodies were generated from the IgG1-P329R-E345R-K409R (IgG1-RR-K409R) backbone, and their agonistic activity was assessed using the OX40 bioluminescent reporter assay (Promega) and PBMC proliferation assay. Based on the results of the 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. Two of these clones (IgG1-CD134-003-RR-K409R and IgG1-CD134-012-RR-K409R) were subsequently humanized. For humanization, a combination of CDR transplantation and amino acid point mutations (Abzena) was used, and five humanized V antibodies were designed for each clone. L Chains (LC1 to LC5) and 6 humanized V H Chain (HC1 to HC6). Encode V H and V L All combinations of the strands (30 pairs per clone) and DNA plastids encoding the IgG1 constant region with an RR mutation in the heavy strand were transfected into Expi293F cells for antibody production. The supernatant from Expi293F cell cultures containing individual humanized OX40 antibody variants was used to assess the binding affinity of the antibodies to human and cynomolgus monkey OX40 by biolayer interferometry, and the binding affinity of the antibodies to activated primary human T cells (derived from five healthy donors) was assessed by flow cytometry.

[0343] Because the binding characteristics of all humanized antibody variants derived from IgG1-CD134-003-RR and IgG1-CD134-012-RR are very similar to those of the parental antibodies, 24 antibodies were further selected based on the highest sequence similarity to the closest human homologs and purified to compare their ability to bind to activated primary human T cells to induce OX40 signaling and enhance T cell proliferation. One humanized OX40-binding antibody (IgG1-CD134-003-HC6LC2-RR) was selected and further characterized in the experiments described in the following examples, along with chimeric antibodies IgG1-CD134-003, IgG1-CD134-007, and IgG1-CD134-012 with different mutations in the Fc domain.

[0344] The sequences of the anti-human OX40 antibodies used in this paper are 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, tavorimab), IgG1-CD134-ABBV368 (INN 11242, redominis), IgG1-CD134-IBI101 (INN 11200, kudarolimab), IgG1-CD134-GBR830 (INN11273, telazorlimab), IgG1-CD134-INCAGN1949 (US10259882B2 ...INCAGN1949 (US10259882B2, SEQ ID NO: 5 and 11), SEQ ID NOs: 61 and 20), IgG1-CD134-SF2 and IgG2-CD134-SF2 (US2014 / 0377284A1, VL1VH2, SEQ ID NOs: 78 and 80), IgG1-CD134-h3C8 (WO2016 / 164480A1, SEQ ID NOs: 118 and 119), IgG1-CD134-RG7888 (INN 10272, vonlerolizumab), and IgG1-CD134-49B4 (WO2019 / 086497A2, SEQ ID NOs: 40 and 41). V containing b12 H / V L Human IgG1 antibody (HIV1 gp120 specific antibody) was used as a negative control (Barbas et al. J Mol Biol. 1993 Apr 5; 230(3):812-2).

[0345] Example 2: Binding affinity of anti-human OX40 antibody to recombinant human and cynomolgus monkey OX40

[0346] 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 other anti-human OX40 IgG1 antibodies to recombinant human and cynomolgus monkey OX40 proteins was determined using label-free biolayer interferometry on an Octet HTX instrument (Sartorius).

[0347] The experiment was conducted with shaking at 1,000 RPM at 30°C. To determine the affinity of the OX40 antibody for human and cynomolgus monkey OX40, an anti-human IgG Fc capture (AHC) biosensor (Sartorius, catalog number 18-5060) was pre-conditioned for 5 seconds in 10 mM glycine (Sigma-Aldrich, catalog number 15527) buffer at pH 1.7, followed by neutralization in sample diluent (Sartorius, catalog number 18-1104) for 5 seconds; these two steps were repeated five times. Next, the AHC sensor was loaded with antibody (1 μg / mL of sample diluent) for 600 seconds. After baseline measurements (100 seconds) in the sample diluent, association (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 determined using a concentration range of 0.78 to 800 nM and a 2-fold dilution procedure.

[0348] The theoretical molecular weight of the antigen, based on its amino acid sequence, was used for calculation. A reference sensor was used for each antibody, which was incubated with a sample diluent substituted for the antigen. The AHC sensor was generated by exposure to 10 mM glycine buffer at pH 1.7 for 5 seconds, followed by neutralization in the sample diluent for 5 seconds; these two steps were repeated twice. The sensor was then reloaded with antibody for the next kinetic measurement cycle.

[0349] Data were acquired using data acquisition software v12.0 (Sartorius) and analyzed using data analysis software v12.0 (Sartorius). Data traces for each antibody were corrected by subtracting a reference sensor. The Y-axis was aligned with the baseline for the last 10 seconds, and inter-step correction and dissociation alignment were performed. Savitzky-Golay filtering was applied. Data traces with a reaction velocity <0.05 nm were excluded from the analysis. For K... D Antibodies with concentrations less than 50 nM were excluded from the analysis, while data traces with concentrations higher than 100 nM were excluded. Data were fitted using a 1:1 model with an association time set at 200 seconds and dissociation times set at 50 seconds, 200 seconds, and 1,000 seconds. Dissociation times were based on R... 2 The selection is based on visual inspection of values ​​and curves, and at least 5% signal attenuation during the dissociation step.

[0350] Affinity to human OX40 can be accurately determined by fourteen OX40 antibodies (Table 4; all tested antibodies except IgG1-CD134-SF2 and IgG1-CD134-49B4-G236R-E345R-K439E[RRE], due to unsatisfactory fitting curves), K D The values ​​are mainly in the nanomolar range.

[0351] 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) can be accurately measured in cynomolgus monkeys. The K value measured by these antibodies... D The values ​​are mainly in the nanomolar range (Table 4).

[0352] In summary, although antibody IgG1-CD134-RG7888 showed the highest affinity for human 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. A reliable explanation for the binding characteristics of antibody IgG1-CD134-SF2 to human and cynomolgus monkey OX40 cannot be determined.

[0353] Table 4. Binding affinity of anti-human OX40 antibody to human and cynomolgus monkey OX40.

[0354]

[0355] A binding was observed, but due to an imperfect curve fit, K... D k on and k dis The value is unreliable, resulting in no reliable interpretation when using a 1:1 model. ND: Undetectable.

[0356] Example 3: Evaluation of cross-blocking of anti-human OX40 IgG1 antibodies using biolayer interferometry

[0357] Antibody cross-blocking 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 on an Octet HTX instrument (Sartorius). The experiments were conducted at 30°C with simultaneous shaking at 1,000 RPM.

[0358] The amine-reactive second-generation (AR2G) biosensor (Sartorius, catalog number 18-5092) was activated for 300 seconds in 20 mM EDC solution (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; Sartorius, catalog number 18-1033) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt; Sartorius, catalog number 18-1067). The activated AR2G sensor was loaded with 20 µg / mL of primary antibody diluted in 10 mM acetate at pH 5.0 (Sartorius, catalog number 18-1069) or pH 6.0 (Sartorius, catalog number 18-1070) for 600 seconds and quenched for 300 seconds in 1 M ethanolamine at pH 8.5 (ETA; Sartorius, catalog number 18-1071). After a baseline measurement of 50 seconds in sample diluent (Sartorius, catalog number 18-1104), the AR2G biosensor containing the immobilized antibody was loaded with human OX40 fused to His-tag (100 nM diluted in sample diluent; Acro Biosystems, catalog number OX0-H5224) for 300 seconds. Association of the secondary antibody (10 µg / mL in sample diluent) was measured (300 seconds). The sensor was regenerated by three cycles of exposure to 10 mM glycine (Sigma-Aldrich, catalog number 15527) buffer at pH 2.5 for 5 seconds, followed by neutralization in sample diluent for 5 seconds.

[0359] Data were captured using data acquisition software v12.0 (Sartorius) and analyzed using data analysis software v12.0 (Sartorius). Data traces were corrected by subtracting a reference curve (using sample diluent instead of the second antibody) to correct for antigen dissociation from the immobilized first antibody. The Y-axis was aligned with the start of the association step and Savitzky-Golay filtering was applied. The corrected association reactions of the second antibody were plotted in matrix format (Table 5). Reactions ≥0.1 nm were considered non-blocking antibody pairs, while reactions <0.1 nm were considered blocking antibody pairs. Compared to the buffer control, some antibody pairs showed a reduced signal for the second antibody. This was considered antibody displacement, i.e., the second antibody replaced the interaction between the first antibody and the antigen (Abdiche et al., PLoS ONE 2017 Jan 6; 12(1): e0169535).

[0360] 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, but does not block 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. A 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.

[0361] 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 with each other. Similarly, antibodies IgG1-CD134-MEDI0562, IgG1-CD134-SF2, IgG1-CD134-RG7888, IgG1-CD134-ABBV368, and IgG1-CD134-INCAGN1949 all cross-blocked with each other. Antibody IgG1-CD134-A4453 did not cross-block any of the other antibodies tested.

[0362] Table 5. Antibody cross-blocking was determined using biolayer interferometry. The immobilized primary antibody is displayed vertically, and the secondary antibody horizontally. The corrected association reaction of the secondary antibody is shown. A reaction <0.1 nm is considered a blocking antibody pair (shown in gray boxes), and a reaction ≥0.1 nm is considered a non-blocking antibody pair (unlabeled). Compared to the buffer control, some antibody pairs showed a faintly reduced signal in the secondary antibody, indicating antibody displacement (shown in bold numbers). In some cases, the interaction between antibodies was manually adjusted to non-blocking after visual examination of the sensor map (shown in underlined numbers).

[0363]

[0364] Example 4: Binding of anti-human OX40 antibody to human and cynomolgus monkey OX40 expressed on cell surface

[0365] 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 (eleven IgG1 antibodies and one IgG2 antibody) to human and cynomolgus monkey OX40 expressed on the cell surface was analyzed by flow cytometry using transiently transfected FreeStyle 293-F suspension (HEK293F) cells. The non-binding antibody IgG1-b12-RR was used as a negative control.

[0366] HEK293F cells (ThermoFisher, catalog number R79007) were transiently transfected with 293fectin transfection reagent (ThermoFisher, catalog number 12347019) supplemented with 50 units of penicillin and 50 units of streptomycin (pen / strep; Lonza, catalog number 17-603E) and Opti-MEM® I reduced serum medium (ThermoFisher, catalog number 51985034) with Glutamax, according to the manufacturer's instructions, using mammalian expression vector pSB (SEQ ID NO: 52 and SEQ ID NO: 51, respectively) encoding full-length human or cynomolgus macaque OX40.

[0367] Transfected cells were seeded into 96-well plates (30,000 cells / well; ThermoFisher, catalog number 163320) and incubated sequentially, with a washing step in between using fluorescence-activated cell sorting (FACS) buffer 1 (containing 0.1% bovine serum albumin [BSA; Roche, catalog number 10735086001] and 0.02% sodium azide [NaN3; Bio-World, catalog number 41920044-3] supplemented with 2 mM EDTA (Sigma-Aldrich, catalog number 03690) and phosphate-buffered saline [PBS; Lonza, catalog number BE17-517Q] supplemented with 0.1% bovine serum albumin [BSA; Roche, catalog number 10735086001] and 0.02% sodium azide [NaN3; Bio-World, catalog number 41920044-3]) to be washed with fluorescently activated cell sorting (FACS) buffer 1 (PBS; Lonza, catalog number BE17-517Q) to be supplemented with 2 mM EDTA (Sigma-Aldrich, catalog number 03690). Cells were sequentially incubated at 4°C for 30 min with 50 µL of serially diluted anti-human OX40 antibody (10-fold dilution in EDTA-replenished FACS buffer 1, from 0.005 to 50 µg / mL). Then, they were incubated with 50 µL of R-phycoerythrin (R-PE)-conjugated goat anti-human IgGF(ab')2 (Jackson ImmunoResearch, catalog number 109-116-098, diluted 1:200) in EDTA-replenished FACS buffer 1 for 30 min. Cells were then washed with EDTA-replenished FACS buffer 1 and resuspended in 30 µL of the viability marker ToPro-3 (Invitrogen, catalog number T3605, diluted 1:10,000) in EDTA-replenished FACS buffer 1. The cells were subsequently analyzed on an iQue® 3 flow cytometer (Satorius). The data was analyzed using FlowJo software and visualized using GraphPad Prism.

[0368] All tested antibodies showed dose-dependent binding to OX40 in humans and cynomolgus monkeys. Figure 1A, B). The half-maximal effective concentrations (EC50) of IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R for OX40 in humans and cynomolgus monkeys are comparable to most other tested antibodies. Figure 2 Compared with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R, antibodies IgG2s-CD134-SF2-E345R, IgG1-CD134-GBR830, IgG1-CD134-Hu106, and IgG1-CD134-MEDI0562 showed higher EC50 binding to human OX40. 50 The binding of IgG2s-CD134-SF2-E345R, IgG1-CD134-GBR830, and IgG1-CD134-h3C8-E345R to OX40 in cynomolgus monkeys exhibits high EC50. 50 Only antibodies IgG1-CD134-49B4-E345R and IgG1-CD134-ABBV368 showed slightly lower EC50 binding to OX40 in humans and cynomolgus monkeys compared to IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R. 50 .

[0369] Most antibodies bind to human OX40 EC. 50 EC combined with crab-eating macaque OX40 50 There was no significant difference, except for antibodies IgG1-CD134-h3C8-E345R and IgG1-CD134-GBR830, which showed similar effects on EC40 in cynomolgus monkeys. 50 The value is higher than that of human OX40.

[0370] Example 5: Determination of key domains for binding between anti-human OX40 antibody and human OX40 using domain shuffling of OX40 molecules

[0371] The extracellular domain (ECD) of OX40 consists of four cysteine-rich domains (CRD1 to CRD4). To determine which CRDs are important for binding to human OX40 antibodies, DNA shuffling was performed between human and mouse OX40. Four shuffling constructs were prepared by replacing individual CRDs with mouse CRD analogs to encode human OX40 DNA. Figure 3The following constructs were generated in this manner: 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). Homology between human and mouse OX40 was limited. Figure 4 Therefore, if the binding of the CRD in human OX40 to an anti-OX40 antibody is important, then this binding is lost after the mouse analogue replaces this domain. Conversely, the binding of retained OX40 antibodies to OX40 molecules with domain remodeling demonstrates that the remodeled human OX40 domain is not important for binding.

[0372] Four revamped constructs, along with wild-type human 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 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 11 additional OX40 antibodies (ten IgG1 antibodies and one IgG2 antibody) to shuffled constructs expressed on cell surfaces and to wild-type human and mouse OX40, transfected cells (30,000 cells / well) were incubated in 96-well plates (ThermoFisher, catalog number 163320) with 50 µL serially diluted individual OX40 antibodies (in the final concentration range of 0.005 to 50 µg / mL for 10-fold dilution steps) in FACS buffer 1 at 4°C for 30 min. Cells were washed twice in FACS buffer 1 and then incubated in FACS buffer 1 at 4°C for 30 min in the dark with 50 µL of a secondary antibody R-PE conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog number 109-116-098; diluted 1:200). Next, cells were washed twice with FACS buffer 1 and resuspended in 30 µL of the viability marker ToPro-3 (Invitrogen, catalog number T3605; diluted 1:10,000) in FACS buffer 1 supplemented with 2 mM EDTA. Data were analyzed on iQue Screener Plus (Intelligent Corporation, USA). Data were analyzed using FlowJo software and visualized using GraphPadPrism. Antibody IgG1-b12-RR, which did not bind to the target, was included as a negative control.

[0373] All tests showed that the anti-human OX40 antibody bound to wild-type human OX40. Figures 5 to 9 ), and no antibodies tested showed binding to wild-type mouse OX40, except for the highest concentration of antibody IgG1-CD134-RG7888 ( Figure 6 A).

[0374] Antibody IgG1-CD134-A4453 is the only antibody that shows loss of binding to human OX40 containing mouse CRD4. Figure 7B, F), therefore used to normalize the binding data of other antibodies in order to combine the results of two separate experiments ( Figure 10 Combined normalized data showed that IgG1-CD134-003-HC6LC2-RR lost binding to human OX40 containing mouse CRD1. Figure 10 B), similar to IgG1-CD134-012-RR-K409R and antibody IgG1-CD134-11D4, but retains binding with all other shuffled constructs ( Figure 10 C to E). IgG1-CD134-007-RR and 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 10 C). 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 10 D).

[0375] These data together illustrate that CRD1 is important for the binding of IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-012-RR-K409R to human OX40, while for all tested antibodies except IgG1-CD134-11D4, other CRDs are important for binding to human OX40 (Table 6). CRD2 appears to be important for the binding of IgG1-CD134-007-RR and IgG1-CD134-h3C8-E345R to human OX40, while CRD3 appears to be important for IgG2s-CD134-SF2-E345R and IgG1-CD134-RG7888.

[0376] Table 6. Binding regions crucial for the binding of anti-human OX40 antibodies to human OX40.

[0377]

[0378] Example 6: Agonistaltic activity of anti-human OX40 antibody in cell-based OX40 reporter assay

[0379] The OX40 agonist activity of different anti-human OX40 antibodies with a hexamerization-enhancing mutation (E345R) and further composed of an active or inactive Fc backbone 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 assembly and constitutively express human OX40, resulting in luciferase production in response to OX40 agonism. OX40 + Jurkat cells were thawed and incubated overnight in assay buffer (RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum [FBS; catalog number J121A]) at 37°C / 5% CO2 in opaque white 96-well flat-bottomed culture plates (30,000 cells / well; PerkinElmer, catalog number 6005680) at 37°C / 5% CO2. The following day, a series of antibody dilutions (from 0.000026 to 10 µg / mL in assay buffer) were prepared. Figure 11 [AD] or a final concentration range of 0.00064 to 50 µg / mL [ Figure 11 E) was added to the wells and incubated at 37°C / 5% CO2 for 5 h. 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 reagent, prepared by mixing Bio-Glo luciferase assay buffer (Promega, catalog number G719A) and Bio-Glo luciferase assay matrix (Promega, catalog number G720A) according to the manufacturer's instructions, was added at a 1:1 ratio to a solution containing OX40. +Jurkat cells and a series of antibody dilutions were incubated in each well at RT in the dark for 5 to 10 min. Luminescence was measured using an EnVision multi-label reader (PerkinElmer) and presented as relative luminescence units (RLU) in bar graphs generated using GraphPadPrism software. EC 50 The value is calculated using GraphPad Prism.

[0380] IgG1-CD134-003-HC6LC2-RR variants of IgG1-h3C8 antibody exhibited hexamerization enhancement (E345R) of either an inert Fc backbone (LALAPG) or a Fc backbone showing no C1q binding and reduced FcγR binding, inducing stronger reporter cell activation (K322A) expressing human OX40. Figure 11 A). This is exemplified by the higher maximum level of cell activation induced by IgG1-CD134-003-HC6LC2-RR compared to the IgG1-h3C8 variant, as evidenced by the higher maximum RLU signal observed with the former antibody ( Figure 11 A) and slightly lower EC was observed with IgG1-CD134-003-HC6LC2-RR. 50 value( Figure 12 This is reflected in the fact that IgG1-CD134-003-HC6LC2-RR is superior to two hexamerization-enhancing variants of the IgG1-RG7888 antibody (IgG1-CD134-RG7888-E345R-LALAPG and IgG1-CD134-RG7888-K322A-E345R) in inducing reporter cell activation. Figure 11 B, 12). IgG1-CD134-003-HC6LC2-RR also induced a higher maximum activation level than the IgG1-RG7888 variant. Figure 12 Furthermore, IgG1-CD134-003-HC6LC2-RR induced stronger reporter cell activation than the Fc indolent variant of IgG2-CD134-SF2 carrying a hexamerization-enhancing mutation, with higher maximal RLU signal and lower ECG. 50 The value reflects (IgG2s-CD134-SF2-E345R); Figure 11 C, 12). Both IgG1-CD134-007-RR and IgG1-CD134-012-RR induced reporter cell activation, with IgG1-CD134-012-RR inducing higher maximal activation, comparable to IgG1-CD134-003-HC6LC2-RR. Figure 11 D, 12). No reporter cell activation was observed with antibody IgG1-CD134-11D4. Figure 11 E).

[0381] In summary, IgG1-CD134-003-HC6LC2-RR showed greater potency in inducing reporter cell activation overexpressing human OX40 than antibodies IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2-CD134-SF2, which are composed of E345R hexamerization-enhanced mutants and have an inert Fc backbone or an Fc backbone showing no C1q binding and reduced FcγR binding. Furthermore, IgG1-CD134-007-RR and IgG1-CD134-012-RR induced reporter cell activation, while activity was undetectable in this assay with antibody IgG1-CD134-11D4. Non-humanized variants of 007 and 012, as well as the humanized 003 clone, were tested. In preliminary PBMC proliferation assays, the functional activity of the 007 clone was lower than that of 003 and 012. After humanization, the 012 clone lost its functional activity in T cell proliferation assays.

[0382] Example 7: The ability of anti-human OX40 antibody to enhance T cell proliferation

[0383] To investigate the ability of IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR) to induce T cell proliferation and compare this ability with antibodies that exhibit hexamerization enhancement (E345R) and are further composed of an active Fc backbone, an inactive Fc backbone (L234A-L235A-P329G; LALAPG), or an Fc backbone showing no C1q binding and reduced FcγR binding (K322A), polyclonal T cell proliferation assays were performed using human peripheral blood mononuclear cells (PBMCs) from healthy donors.

[0384] PBMCs were obtained from the erythrocyte sedimentation rate (ESR) amber layer of healthy donors (Transfusionszentrale University Hospital, Mainz, Germany) using Ficoll-Paque density gradient separation (GE Healthcare, catalog number 17-1440-03). Where applicable, CD4 or CD8 microbeads (Miltenyi Biotec GmbH, catalog numbers 130-097-48 and 130-045-201) were used to separate CD4-depleted CD4-rich PBMCs. + or CD8 + T cells. The isolation procedure is usually performed according to the manufacturer's instructions, with slight modifications (reducing the size of the microbeads), and in principle, it is similar to CD4. - and CD8- The separation of PBMCs is the same. In short, after determining the cell number, the cell suspension is centrifuged and the supernatant is discarded. Cells are then added at a rate of 1 × 10⁻⁶ cells per 80 µL of buffer. 7 Each live cell was resuspended in MACS buffer (DPBS [ThermoFisher Scientific, catalog number 14190250], 5 mM EDTA [Sigma-Aldrich, catalog number 03690], 1% human albumin [CSL Behring, catalog number PZN-00504775]). Every 10 cells... 7 Add 12 µL of CD4 or CD8 microbeads to each cell. Thoroughly mix the cells and beads before incubation at 2–8 °C for 15 min and twice during incubation to ensure uniform labeling. The cell suspension is then washed with MACS buffer (8 min, 300 × g, RT) and filtered through a 30 µm cell filter (BD Biosciences, catalog number 340626). An LS column (Miltenyi Biotec GmbH, catalog number 130-042-401) is placed in a QuadroMACS separator on a MACS MultiStand (MiltenyiBiotec GmbH) and equilibrated with MACS buffer. Load the bead-labeled cells onto the column, allowing the suspension to flow by gravity to retain the bead-labeled cells in the column. The column is then washed three times with MACS buffer. The cells containing bead-labeled CD4 microbeads are then added to the column. + and CD8 + Cells were discarded. Unlabeled CD4+ cells in the effluent were removed. - and CD8 - Centrifuge the PBMC (8 min, 300×g, RT), then resuspend it in DPBS and count the cells.

[0385] PBMC samples are marked with CellTrace™ Violet (Thermo Fisher Scientific, catalog number C34557) according to the manufacturer's instructions for use.

[0386] CellTrace™ Violet-labeled PBMCs were added to 0.3 µg / mL of soluble anti-human CD3 antibody (STEMCELL) in a 96-well round-bottom plate. Technologies, catalog number 60011) and serially diluted solutions of 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 unbound negative control antibody IgG1-b12-RR (in the range of final antibody concentrations from 0.00098 to 10 µg / mL in 2- or 10-fold dilution steps) in the presence of 5% pooled human serum (OneLambda) Cells were incubated in Iscove's Modified Durbecco's GlutaMAX medium (Thermo Fisher Scientific, catalog number 31980030) at the incubation site of Inc. (catalog number A25761). After four days of culture, cells were stained with Viability dye (Thermo Fisher Scientific, catalog number 65-0865-14, 1:1,500 dilution) and 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 achieved using the BD FACSCelesta™ flow cytometer (BD Biosciences) with CD4+. + and CD8 + The cell count of CellTrace™ Violet dilution in T cells was assessed by flow cytometry. The amplification index was calculated using an integral formula from the proliferation modeling tool in FlowJo software. Additionally, CD4 counts were evaluated. + and CD8 + Central memory cells (CCR7) in T cells + CD45RA - )percentage.

[0387] IgG1-CD134-003-HC6LC2-RR enhances CD4 in a dose-dependent manner. + and CD8 + T cell proliferation has high efficiency ( Figure 13 IgG1-CD134-h3C8-E345R-LALAPG also enhances CD4. + and CD8 + T cell proliferation, but with reduced potency compared to IgG1-CD134-003-HC6LC2-RR. Figure 13 (A, 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 do not enhance CD4+. + or CD8 + T cell proliferation ( Figure 13 A, B). When testing IgG1-CD134-003-HC6LC2-RR enhanced CD4 in a large population of healthy donor PBMCs. + and CD8 + When assessing the T cell proliferation capacity, a consistent dose-dependent enhancement of CD4 was observed. + and CD8 + T cell proliferation can be used to calculate EC. 50 (n=17; Table 7).

[0388] Table 7: CD4+ enhanced by IgG1-CD134-003-HC6LC2-RR in polyclonal T cell proliferation assay + and CD8 + ECG T cell proliferation 50 The value is based on the results of 17 healthy human donors.

[0389]

[0390] To evaluate the increased CD4 count associated with IgG1-CD134-003-HC6LC2-RR + Does T cell proliferation depend on CD8? + The presence of T cells and vice versa, using depleted CD8 + or CD4 + Polyclonal T cell proliferation was measured using PBMC samples of T cells. Increased T cell proliferation induced by IgG1-CD134-003-HC6LC2-RR was observed on CD8 cells. + T cells are absent, and CD4 is present.+ T cells are retained, but CD4 cells are not. + T cells are absent and not CD8. + T cell retention Figure 13 C, D). This indicates CD4. + The presence of T cells is evidenced by the CD8+ induced by IgG1-CD134-003-HC6LC2-RR in polyclonal T cell proliferation assays. + T cell proliferation is essential.

[0391] In addition, IgG1-CD134-003-HC6LC2-RR treatment increases CCR7 in a dose-dependent manner. + CD45RA - Central Memory CD4 + and CD8 + Percentage of T cells ( Figure 14 IgG1-CD134-h3C8-E345R-LALAPG also increases central memory CD4 in a dose-dependent manner. + The percentage of T cells was increased, but efficacy was reduced compared to IgG1-CD134-003-HC6LC2-RR. In contrast, no significant difference in central memory CD4 was observed after treatment with IgG1-CD134-h3C8-K322A-E345R, IgG1-CD134-RG7888-K322A-E345R, or IgG1-CD134-RG7888-E345R-LALAPG. + percentage of T cells ( Figure 14 ).

[0392] Example 8: Binding of anti-human OX40 antibody to human and cynomolgus monkey Fcγ receptors

[0393] The binding of different anti-human OX40 antibodies to human Fcγ receptors (FcγRs) was analyzed using two methods: surface plasma resonance (SPR) and flow cytometry measurements, which were performed on anti-human OX40 antibodies bound to FcγRIa transfected ExpiCHO-S cells. These antibodies were composed of a mutation with enhanced hexamerization (E345R) and further composed of an active Fc backbone, an inactive Fc backbone (L234A-L235A-P329G; LALAPG or P329R), or an Fc backbone showing no C1q binding and reduced FcγR binding (K322A).

[0394] Regarding the former method, 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 other screened OX40-specific antibodies (see [link to relevant documentation]). Figures 15-16 The binding of HIV to the human FcγR variant was analyzed using the Biacore SPR system and compared with an anti-HIV gp120 antibody (IgG1-b12) with a wild-type Fc domain as a reference sample. The Biacore S-sensor chip CM5 (Cytiva, catalog number 29104988) was covalently coated with anti-His antibody using an amine conjugation 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 onto the surface of the anti-His chip in HBS-EP+ buffer (Cytiva, catalog number BR100669) until a capture reaction of 400 RU was achieved. After three starter cycles in HBS-EP+ buffer, antibody samples were injected, with 12 cycles per sample. Binding profiles were generated using antibody concentrations ranging from 0 to 3,000 nM for FcγRIa and from 0 to 10,000 nM for other FcγRs. Samples analyzed on FcR-coated surfaces (active surfaces) were also analyzed on FcγR-free parallel flow cells (reference surfaces) for background correction. Dissociation from anti-His-coated surfaces was achieved using surface regeneration with 10 mM glycine-HCl pH 1.5 (Cytiva, catalog number BR100354). Sensing maps were generated using Biacore Insight evaluation software (Cytiva) and four-parameter logic (4PL) fitting was applied to calculate the binding of individual human OX40-specific antibodies relative to the reference sample (IgG1-b12).

[0395] Regarding the latter method, ExpiCHO-S cells (ThermoFisher, catalog number A29127) were transiently transfected with human FcγRIa as follows. ExpiCHO-S cells (approximately 3.0 × 10⁶ cells) 6 Cells / mL, Thermo Fisher Scientific, Catalogue A29133) were cultured in ExpiCHO™ expression medium (Thermo Fisher Scientific, Catalogue A2910001). 10 µg of DNA encoding FcyR1a for each transfection was added to 0.39 mL of cold OptiPro™ serum-free medium (OptiPro...). TM SFM (ThermoFisher Scientific, catalog number A29131) was mixed, and 0.03 mL of ExpiFectamine CHO was added simultaneously. TM Add the reagent (ThermoFisher Scientific, catalog number A29131) to 0.37 mL of cold OptiPro. TM In SFM. Next, 0.4 mL of the mixture was added to the DNA / OptiPro mixture and incubated at RT for 1 to 5 min. Then, this mixture was added dropwise to cultured ExpiCHO-S cells. After incubation for 18 to 22 h, 0.06 mL of ExpiCHO was added to each culture flask. TM Enhancer (ThermoFisher Scientific, catalog number A29131) and 2.4 mL of ExpiCHO Feed (ThermoFisher Scientific, catalog number A29131). After transfection, cells were incubated at 37°C, 70% humidity, and 8% CO2 with shaking for 24 h, and then frozen in ExpiCHO™ expression medium supplemented with 10% DMSO (Sigma-Aldrich, catalog number D2438).

[0396] Transfected cells were plated in 96-well U-bottom plates (20,000 cells / well; ThermoFisher, catalog 163320) in RPMI 1640 supplemented with L-glutamine and 25 mM HEPES, containing 50 units of penicillin and 50 units of streptomycin [pen / strep; Lonza, catalog 17-603E] and 10% donor bovine serum with iron [DBSI; Gibco, catalog 20371-030]. Next, the cells were inoculated with 25 µL of serially diluted OX40-specific antibodies IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-HC6LC2, IgG1-CD134-003, or other OX40-specific antibodies (variants) (see [link to OX40-specific antibody 'variant')). Figure 17 Cells were incubated at 4°C for 30 min in FACS buffer 1 at a final concentration range of 0.00051 to 10 µg / mL (3-fold dilution). After incubation, cells were washed twice with FACS buffer 1 and then incubated at 4°C for 30 min in FACS buffer 1 with 50 μL of a solution of R-PE-labeled F(ab')2 fragment goat anti-human IgG, specifically the F(ab')2 fragment (Jackson ImmunoResearch, catalog number 109-116-097, diluted 1:200). Next, cells were washed twice with FACS buffer 1 and incubated at 20 min in FACS buffer 1 at 4°C with 30 μL of a solution of ToPro-3 (Invitrogen, catalog number T3605, diluted 1:10,000) at RT in the dark. All samples were measured on an iQue® 3 flow cytometer (Satorius). The data was analyzed using FlowJo software and visualized using GraphPad Prism.

[0397] In SPR measurements, IgG1-CD134-003-HC6LC2-RR showed low residual binding to the high-affinity receptor FcyRIa, while no binding of this antibody to FcyRIIa (H and R variants), FcyRII2b, or FcyRIIIa (F and V variants) was observed. Figures 15-16 The binding of IgG1-CD134-h3C8-K322A-E345R and IgG1-CD134-RG7888-K322A-E345R to FcyR in all tests was shown, although slightly less than that to the positive control IgG1-b12. Figures 15-16In contrast, variants of the antibody anchored to the E345R-LALAPG Fc backbone did not show binding to any of the tested FcyRs. Low residual binding of IgG1-CD134-003-HC6LC2-RR to cynomolgus monkey FcγRIa was observed at high antibody concentrations (greater than 100 nM [14.9 µg / mL]) using a similar method.

[0398] Analysis using FcyRIa-transfected ExpiCHO-S cells and flow cytometry showed that IgG1-CD134-003-HC6LC2-RR did not bind to FcyRIa. Figure 17 A). Antibody variants carrying the wild-type Fc domain were observed to bind to FcyRIa, 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, IgG1-CD134-RG7888-E345R, and variants of IgG1-CD134-h3C8 and IgG1-CD134-RG7888 anchored to the E345R-K322A mutation. Figure 17 A, C to E). Variants of IgG2 antibodies IgG2-SF2 and IgG2s-SF2-E345R, as well as variants of IgG1-h3C8 and IgG1-RG7888 anchored to the E345R-LALAPG mutation, were observed not to bind to FcyRIa. Figure 17 (B, D to E).

[0399] In summary, IgG1-CD134-003-HC6LC2-RR showed minimal binding (FcγRIa) or no binding (FcγRIIa, FcγRIIb and FcγRIIIa) to the human IgG Fcγ receptor.

[0400] Example 9: IgG1-CD134-003-HC6LC2-RR in activated primary CD4 + and CD8 + Antibody binding ability on T cells

[0401] In activated proto-human CD4 + and CD8 +The number of binding sites on the cell surface of T cells was quantitatively determined 24 h, 48 h, and 72 h after activation. First, total human T cells were enriched directly from the erythrocyte sedimentation rate (ESR) amber layer using RosetteSep® Human T Cell Enrichment Mixture (StemCell Technologies, catalog number 15021), followed by density centrifugation using a Ficoll gradient (PromoCell, catalog number C-44010 or Corning, catalog number 25-072-CI), both according to the manufacturer's instructions. Enriched T cells were washed once in PBS (GE Healthcare, catalog number SH3A3830.03 or Capricorn Scientific, catalog number SP-2121-500 mL), centrifuged at 300×g for 3 min, and resuspended in RPMI-1640 medium (Lonza, catalog number BE12-115F / Capricorn, catalog number RPMI-HA) supplemented with 10% heat-activated FBS [ATCC, catalog number 30-2020], 1% penicillin / streptomycin, and 1% L-glutamine [Gibco, catalog number RPMI-HA 25030-081]). The enriched T cells were then counted using a Cellometer Auto 2000 cell viability counter (Nexcelom Biosciences) with Cellometer ViaStain® AOPI solution in PBS (Nexcelom Biosciences, catalog number CS2-0106) to distinguish between live and dead cells. The cells are precipitated and then resuspended in the activation culture medium.

[0402] To induce T cell activation, anti-CD3 / CD28 beads (Dynabeads™ Human T-Activator CD3 / CD28; ThermoFisher Scientific, catalog number 11131D) were washed with PBS, and T cells and beads were resuspended in activation medium at a bead-to-cell ratio of 1:2. Next, 300,000 T cells (150 μL) with beads were seeded per well in 96-well round-bottom plates (ThermoFisher Scientific, catalog number 163320) and incubated at 37°C and 5% CO2 for one, two, and three days. The beads were then removed using a Dynal® bead separator (Invitrogen, catalog number 3019669), followed by washing of the T cells in activation medium. This bead removal step was repeated twice. Cells were counted using AOPI solution to distinguish between live and dead cells.

[0403] The number of IgG1-CD134-003-HC6LC2-RR binding sites was analyzed using a Human IgG Correction Kit (Biocytex, catalog CP010) and flow cytometry according to the manufacturer's instructions. Bead-activated T cells were plated in 96-well round-bottom plates (50,000 cells / well; ThermoFisher Scientific, catalog 163320). 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 (i.e., saturated antibody concentration) at 4°C for 30 min. Next, correction beads (included in the kit) containing a defined number of human IgG monoclonal antibodies were separately plated from the cells in FACS buffer 1 (15 μL beads per well). After washing in FACS buffer 1, cells and calibration beads were incubated with the antibody group described in Table 8 in FACS buffer 1 at 4°C for 30 min in the dark. The cell and bead suspensions were washed twice and resuspended in 80 μL of ToPro-3 activity marker (1:10,000; Invitrogen, catalog number T3605) diluted in FACS buffer 1, and measured on a BD FACSCelesta cell analyzer. Data were analyzed using FlowJo software. Compensation was performed using UltraComp eBEADS (Thermo Fisher Scientific, catalog number 01-2222-42). The antibody binding capacity (sABC) of IgG1-CD134-003-HC6LC2-RR, representing the number of IgG1-CD134-003-HC6LC2-RR binding sites per cell, was determined by interpolation from the standard curve using GraphPad Prism software.

[0404] Table 8: Analysis of activated CD4 + and CD8 + Antibody group at the IgG1-CD134-003-HC6LC2-RR binding site in T cell subsets

[0405]

[0406] With two CD4 stimulations per day + and CD8 + Compared to T cells, the number of IgG1-CD134-003-HC6LC2-RR binding sites was higher two and three days after stimulation. Figure 18 In addition, in CD4 +The number of IgG1-CD134-003-HC6LC2-RR binding sites (i.e., sABCs) on T cells was higher across all time points than on CD8. + T cells showed an increase of approximately 4.5 to 8.5 times (mean sABCs of CD4 after one day and three days of stimulation: 11,285 and 21,682, respectively; mean sABCs of CD8: 1,322 and 4,816, respectively).

[0407] Example 10: Binding of anti-human OX40 antibody to activated T cells expressing OX40

[0408] As described in Example 9, OX40 expression on T cells can be induced upon T cell activation, with maximum expression observed two or three days after activation. Here, the binding of anti-human OX40 antibody to T cells activated with anti-CD3 / CD28 antibody was evaluated using flow cytometry.

[0409] Healthy human donor PBMCs were purified substantially as described in Example 7. The cell concentration was adjusted to 2 × 10⁻⁶ in assay medium. 6 Cells / mL (final concentration) were seeded in 6-well plates (Greiner, catalog number 657160). PBMCs were stimulated with 0.3 µg / mL anti-CD3 (STEMCELL Technologies, catalog number 60011) and 0.5 µg / mL anti-CD28 antibody (BioLegend, catalog number 302934) and cultured at 37°C and 5% CO2 for two days. Next, PBMCs were harvested, counted, and cultured in FACS buffer 1 (DPBS containing 2% heat-inactivated FBS and 2 mM EDTA) to reach 2 × 10⁻⁶ cells / mL (final concentration) and plated in 6-well plates (Greiner, catalog number 657160). 6 Cells / mL concentration. OX40 expression on stimulated T cells was confirmed by staining with a commercially available anti-OX40 antibody (data not shown). Activated PBMCs were injected at 1 × 10⁶ cells per well. 5Cells were transferred to 96-well round-bottom plates and incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-IBI101, IgG1-CD134-11D4, IgG1-CD134-RG7888, or the unbound control antibody IgG1-b12-RR (4-fold dilution to final concentration: 0.00061 to 10 μg / mL). After 60 min at RT, cells were prepared for flow cytometry analysis. For this purpose, cells were washed twice with 150 µL of FACS buffer 1 and then incubated with 30 µL / well of a cell surface antibody mixture (Table 9) containing an antibody against human Fcγ (Table 9) and the immobilizable viable dye eFluor 780 (1:1,500). The staining procedure was performed at 4°C in the dark for 15 to 20 min. The stained cells were washed twice with 120 to 150 µL of FACS buffer 1 (5 min, 460 × g, RT) and then resuspended in 60 to 100 µL of FACS buffer 1 for direct flow cytometry analysis. Flow cytometry data were captured on a FACSymphony A3 and analyzed using FlowJo software.

[0410] Table 9: Antibody group used to evaluate the binding of anti-human OX40 antibodies to activated human T cells

[0411]

[0412] All tested anti-human OX40 antibodies bound, in a dose-dependent manner, to stimulated CD44 from all tested donors. + and CD8 + T cells (representative donors shown in) Figure 19 (in Chinese). ECGs of IgG1-CD134-003-HC6LC2-RR that bind to stimulated T cells. 50 The values ​​are in the high picomolar range, with an average EC50 of 0.140 nM / 0.021 µg / mL. 50 Value (CD4) + T cells; Table 10) and mean EC50 of 0.159 nM / 0.024 µg / mL 50 Value (CD8) + T cells; Table 10). Other anti-human OX40 antibodies and their ECGs. 50 The values ​​were in the same range (IgG1-CD134-11D4), slightly higher (IgG1-CD134-RG7888), or significantly higher (IgG1-CD134-IBI101), while IgG1-CD134-RG7888 reached a higher maximum binding plateau.

[0413] Table 10: Anti-human OX40 antibody and activated human CD4 + and CD8 + T cells bind to EC 50 Value. SD: Standard deviation.

[0414]

[0415] These results indicate that IgG1-CD134-003-HC6LC2-RR, which binds to activated human T cells expressing OX40, has similar or higher epigenetic affinity to the baseline OX40 agonist antibody (IgG1-CD134-11D4) or (IgG1-CD134-RG7888, IgG1-CD134-IBI101).

[0416] Example 11: Binding of IgG1-CD134-003-HC6LC2-RR with human OX40 in the presence of soluble OX40L

[0417] Most clinically developed OX40 agonist antibodies bind to the same region as OX40's natural ligand, OX40L, and antibodies binding to this region are 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.

[0418] To determine the relationship with activated human CD4 + and CD8 +The saturation concentration of sOX40L for T cell binding and the assessment of IgG1-CD134-003-HC6LC2-RR binding at this saturation concentration were determined by stimulating human PBMCs with anti-CD3 / CD28 beads for two days (essentially as described in Example 9, except that a 6-well plate [Greiner, catalog number 657160] and IMDM medium containing 5% pooled human serum [PHS, One Lambda Inc., catalog number A25761] were used). The stimulated PBMCs were precipitated, resuspended in FACS buffer 2 (DMPBS containing 2% heat-inactivated FBS [Sigma-Aldrich, catalog number F7524] and 2 mM EDTA [Sigma-Aldrich, catalog number 03690]) and seeded (100,000 cells / well) in 96-well round-bottom plates (VWR International GmbH, catalog number 734-197). Next, cells were incubated at RT with mouse Fc-tagged human sOX40L (Sino Biological, catalog number 13127-H04H) at a final concentration range of 0.00017 to 30 μg / mL (a three-fold dilution in FACS buffer 2 was used for stimulated cells, or 30 μg / mL for unstimulated cells) for 30 min to determine the saturation binding concentration of sOX40L. Alternatively, binding competition was determined by incubating IgG1-CD134-003-HC6LC2-RR and IgG1-b12-RR antibodies at a final concentration range of 0.00046 to 10 μg / mL (a 10-fold dilution in the first step, followed by a three-fold dilution in FACS buffer 2) for 30 min in the presence or absence of 2 μg / mL. Next, the cells were washed twice with 150 µL of FACS buffer 2 and incubated for 20 min at 4°C in the dark with 30 µL of APC-conjugated goat anti-mouse IgG F(ab')2 secondary antibody (diluted 1:500 in FACS buffer 2; Jackson Immuno Research, catalog number 109-546-098), alone or with AF488-conjugated goat anti-human IgG F(ab')2 secondary antibody (diluted 1:1,000 in FACS buffer 2; Jackson Immuno Research, catalog number 115-135-164). Next, the cells were washed twice with FACS buffer and incubated for 20 min at 4°C in the dark with 30 µL of the antibody group described in Table 11, diluted 1:1,500 in FACS buffer 2 containing the fixative viability dye eFluor 780.Cells were then washed twice with 120 to 150 µL of FACS buffer 2 and measured on a BD FACSCelesta flow cytometer using a BD high-throughput sampler. Subsequent analyses were performed using FlowJo software.

[0419] Table 11: Used for staining CD4 + and CD8 + T-cell antibody group

[0420]

[0421] The saturation concentration of sOX40L was determined to be 2 μg / mL. The maximum binding of IgG1-CD134-003-HC6LC2-RR to OX40 was not hindered in the presence of 2 µg / mL sOX40L. Figure 20 A), despite being related to CD4 + and CD8 + T cells bind to EC 50 The values ​​were approximately three times higher in the presence of sOX40L (Table 12). Conversely, sOX40L binding was lost in a dose-dependent manner in the presence of IgG1-CD134-003-HC6LC2-RR. Figure 20 B) indicates that IgG1-CD134-003-HC6LC2-RR may block the binding of sOX40L. This illustrates that OX40L and IgG1-CD134-003-HC6LC2-RR overlap and bind to the domain on OX40, but the presence of OX40L has a very small effect on the binding of IgG1-CD134-003-HC6LC2-RR (and vice versa).

[0422] Table 12: IgG1-CD134-003-HC6LC2-RR binds to activated human T cells in the presence of soluble OX40L, and vice versa.

[0423]

[0424] Example 12: Agonist activity of anti-human OX40 antibody in the presence or absence of cells expressing Fcγ receptor

[0425] Example 6 describes an assay performed using Jurkat cells transfected with human OX40 to study the potency of different anti-human OX40 antibodies. Here, the same assay was used to investigate the efficacy of inducing OX40 agonist activity in the presence or absence of FcγR-expressing cells. This study targeted IgG1-CD134-003-HC6LC2-RR and the following variants: having an Fc active backbone (IgG1-CD134-003; SEQ ID No: 9 and 10), or having an Fc backbone with the anchoring hexamerization-enhancing mutant E345R (as illustrated in the constant region of SEQ ID NO: 2), or having an Fc backbone with Fc-inert mutants L234F, L235E, and D265A other than the F405L mutant that promotes heterodimerization of the half-molecule and the other half-molecule anchoring the K409R mutant (Labrijn et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. PNAS 2013; 110(13):5145-50), as illustrated in the constant region of SEQ ID NO: 62. In individual experimental groups, the potency 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 non-binding control antibody IgG1-b12-RR was used as a negative control.

[0426] The assays were performed substantially as described in Example 6, with a few exceptions: Jurkat cells expressing OX40 were stimulated with anti-human OX40 antibody in or without CHO-K1 cells (Promega, catalog JA2255; FcγRIIb-CHOK1 cells) transfected to express human Fcy receptor 2B. Vials of FcγRIIb-CHO K1 cells were thawed and the cells were resuspended in 14.5 to 29 mL of RPMI 1640 medium (Promega, catalog JA2191) supplemented with 5% fetal bovine serum (FBS, provided by Promega kit). Cells were seeded at a density of 100 μL per well in 96-well white flat-bottomed assay plates (Fisher Scientific, catalog 10072151). After incubation at 37°C, 5% CO2 for 5–6 h, the culture medium in each well was discarded, and 60 μL of Jurkat effector cells expressing OX40 (with or without FcγRIIb-CHO K1 cells) was added to each well, followed by incubation for 16–20 h (37°C, 5% CO2). Additionally, luminescence was measured using a CLARIOstar® Plus microplate reader (BMG Labtech). Data were fitted to an sigmoid, 4PL (X is the logarithm of concentration) equation and presented as RLU in a line graph generated using GraphPad Prism software. EC 50 Values ​​are derived from the fitted curve.

[0427] IgG1-CD134-003-HC6LC2-RR induces potent dose-dependent OX40 agonist activity, independent of Fc involvement via cells expressing FcγRIIb. Figure 21A). In the absence of cells expressing FcγRIIb, strong OX40 agonist activity was observed only in 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 cells expressing FcγRIIb significantly enhanced the OX40 signaling activity of FcγR-binding anti-human OX40 antibodies (IgG1-CD134-003 and IgG1-CD134-003-E345R), while the addition of cells expressing FcγRIIb did not increase or only minimally increased the activity of anti-human OX40 antibodies with an inert Fc backbone (IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-003-FEAL).

[0428] Compared to IgG1-CD134-003-HC6LC2-RR, the anti-human OX40 antibodies IgG1-CD134-11D4, IgG1-RG7888, and IgG1-CD134-IBI101 induced OX40 agonist activity in the assays only in the presence of FcγR-expressing cells, not in their absence. Figure 21 B).

[0429] In summary, compared with other anti-human OX40 antibodies lacking hexamerization-enhancing mutations, IgG1-CD134-003-HC6LC2-RR induces potent dose-dependent OX40 agonist activity regardless of the cells expressing FcγR.

[0430] Example 13: Expression of T cell activation markers after incubation with IgG1-CD134-003-HC6LC2-RR

[0431] Using the T-cell proliferation assay described in Example 7, the expression of cell surface markers associated with T-cell activation was investigated after incubation of healthy human donor PBMCs with IgG1-CD134-003-HC6LC2-RR. Furthermore, the potency of the OX40 agonist reference antibody analogs IgG1-CD134-BMS986178, IgG1-CD134-RG7888, and IgG1-CD134-IBI101 in enhancing the expression of cell surface markers associated with T-cell activation was evaluated. The effects of 4-1BB, CD25, HLA-DR, and PD-1 on CD4+ were also measured. + and CD8 +T cell expression levels were determined by washing cells and staining them in 30 µL of FACS buffer in 96-well round-bottom plates (VWR International, catalog number 734-1797) with titrated amounts of antibody (Table 13), viability dye eFluor 780 (1:1,500; ThermoFisher Scientific, catalog number 65-0865-14), and brilliant stain buffer plus (1:10; BD Biosciences, catalog number 566385) to detect CD4, CD8, 4-1BB, CD25, HLA-DR, PD-1, and viability. The staining procedure was performed at 4°C in the dark for 15 min. 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 were acquired using a BD FACSymphony A3 flow cytometer with a BD high-throughput sampler. CD4 expression levels were measured. + and CD8 + The percentage of T cell populations expressing activation markers 4-1BB, CD25, HLA-DR, and PD-1 was determined using FlowJo and presented as a linear curve using GraphPad Prism.

[0432] Table 13: Antibody groups used to assess the expression levels of biomarkers associated with T cell activation

[0433]

[0434] Expression of activation markers was analyzed after two and five days of incubation with IgG1-CD134-003-HC6LC2-RR. In all donors, IgG1-CD134-003-HC6LC2-RR increased CD4 in a dose-dependent manner. + and CD8 + T cell proliferation, as assessed on day 5 (data not shown). IgG1-CD134-003-HC6LC2-RR was induced on day 2 or 5, or both days, with a dose-dependent increase in CD4+ expression of 4-1BB, CD25, and HLA-DR. + and CD8 + The percentage of T cells, but PD-1 was not present in most of the donors analyzed. Figure 22 (A to H).

[0435] Compared with IgG1-CD134-003-HC6LC2-RR, the tested OX40 agonist reference antibody analogs IgG1-CD134-RG7888, IgG1-CD134-BMS986178, and IgG1-CD134-IBI101 did not increase the expression of 4-1BB, CD25, or HLA-DR CD4. + and CD8 + Percentage of T cells ( Figure 22 IP).

[0436] Example 14: IgG1-CD134-003-HC6LC2-RR enhances cytokine secretion in polyclonal activated T cell proliferation assays using healthy human donor PBMCs.

[0437] After culturing for 1, 2, 3, 4 and / or 6 days, the cytokine concentrations in the supernatant collected from polyclonal T cell proliferation assays (using the universal assay settings described in Example 7; staining cells with CellTrace Violet [using CellTrace™ Violet Kit, Thermo Fisher Scientific, catalog number C34557] or CFSE [using Vybrant CFDA SE Cell Tracking Kit, Life Technologies, catalog number V12883]) were compared using the V-Plex Proinflammatory Panel 1 Human Kit (10-plex: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-1β). For multi-task ECLIA assays of IFN-γ, IL-1β, IL-6, and TNF-α (MSD, catalog number K15049D-2) or V-PLEX Human Pro-inflammatory Group 1 Kit (4-plex: IFN-γ, IL-1β, IL-6, and TNF-α, MSD, catalog number K15052D-2), follow the manufacturer's instructions. In short, after washing the MSD plate with 150 µL of PBST (dPBS with 0.05% Tween® 20 [Sigma-Aldrich, catalog number P7949]), add standards or samples (diluted 1:2 to 1:200) to the wells (50 µL / well) and incubate the plate at RT with continuous shaking for 2 h. Wash the plate three times with PBST and add the detection antibody (25 µL / well). After incubating at RT with continuous shaking for 2 h, wash the plate three times with PBST. Reading buffer (150 µL / well) was then added and the plate was immediately analyzed on a MESO QuickPlex SQ 120 reader. Additionally, the cytokine concentration in the supernatant collected after four days of self-culture was determined using the polyclonal T cell proliferation assay described in Example 7, exhausting the CD4+ or CD8+ T cells. Furthermore, the ability of IgG1-CD134-003-HC6LC2-RR to induce cytokine secretion after four days of culture was compared with 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. The non-binding antibody IgG1-b12-RR was used as a negative control. Results were presented as line graphs using GraphPad Prism.

[0438] In all donors, IgG1-CD134-003-HC6LC2-RR increased CD4 in a dose-dependent manner. + and CD8 + T cell proliferation (data not shown). In kinetic assays, IgG1-CD134-003-HC6LC2-RR increased cytokine secretion in a dose-dependent manner at the highest IgG1-CD134-003-HC6LC2-RR assay concentrations (0.2 and 2 μg / mL), targeting TNF after three to four days ( Figure 23 A) IL-2 levels decreased relatively rapidly from two days later, followed by days three through six. Figure 23 B), and targeting IFNγ ( Figure 23 C) and IL-13 Figure 23 D) The test was conducted two days later.

[0439] IgG1-CD134-003-HC6LC2-RR was detected on day four in depleted CD8+ cells. + T cells induce increased secretion of TNFα, IFNγ, and IL-13 in PBMCs, while depleted CD4+ cells... + T cells in PBMCs secrete little or no of these cytokines. Figure 24 ).

[0440] Variants of IgG1-CD134-h3C8 and IgG1-CD134-RG7888 (anchored to K322A-E345 or E345R-LALAPG mutations) and IgG2s-CD134-SF2-E345R ( Figure 25 , 26 Compared to 27), IgG1-CD134-003-HC6LC2-RR induced higher levels of TNFα, IL-2 and IL-13 cytokines, and a similar trend was observed with IFNγ, although there were relatively large differences between repeated measurements in the latter case.

[0441] In summary, the results presented in this paper indicate that IgG1-CD134-003-HC6LC2-RR enhances the release of pro-inflammatory cytokines in polyclonal T cell proliferation assays using healthy human donor PBMCs, and this release is CD4-dependent. + The presence of T cells. Moreover, IgG1-CD134-003-HC6LC2-RR enhanced cytokine secretion more effectively in this assay than other hexamer-enhanced anti-human OX40 antibodies tested (i.e. variants of IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2s-SF2).

[0442] Example 15: The ability of IgG1-CD134-003-HC6LC2-RR to enhance CD8+ T cell proliferation in antigen-specific T cell proliferation assay

[0443] IgG1-CD134-003-HC6LC2-RR enhances CD8 + The proliferative capacity of T cells was studied by flow cytometry of T cells stimulated with their homologous antigens. Human CD8+ cells were electroporated using TCRs with OX40 and a CLDN6-derived peptide that recognizes type 1 major histocompatibility (MHC-I) molecules. + T cells were co-cultured with autologous immature moDCs (iDCs) electroporated from full-length human CLDN6.

[0444] PBMCs were isolated from the erythrocyte sedimentation rate (ESR) layer of healthy human donors using a Ficoll-Paque density gradient, essentially as described in Example 7, and used for cell separation. Prior to cell separation, PBMCs were confirmed to be HLA-A by flow cytometry. 02 positive. MACS columns or AutoMACS Pro separators (both from Miltenyi Biotec GmbH) are used for cell isolation depending on availability. CD14 microbeads (Miltenyi Biotec GmbH, catalog number 130-050-201) are used for positive screening of CD14 from freshly isolated PBMCs. + Mononuclear cells and negative screening CD14 - Peripheral blood lymphocytes (PBL). CD8 microbeads are used to isolate CD8 from previously frozen PBL. + T cells. The isolation procedure is usually performed according to the manufacturer's instructions, with slight modifications (reducing the microbead volume; at a rate of 10...). 7 (12 μL beads per cell), and in principle, CD14 + / CD14 - Cell isolation and CD8 + The individual T cells are identical.

[0445] CD14 after isolation + Monocytes differentiate into iDCs. For this purpose, a maximum of 40 × 10⁴ cells were cultured in each T175 suspension culture flask (Greiner Bio-One GmbH, catalog number 661195). 6 CD14 +Monocytes were cultured for five days in a DC medium (RPMI 1640, 5% PHS, 1× minimum essential 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 a constant temperature incubator (37°C, 5% CO2). After three days of culture, half of the medium in each culture flask was replaced. Because the culture medium removed from the culture flask contained non-adherent monocytes, it was centrifuged (8 min, 300×g, RT), the supernatant was discarded, and the cell pellet was resuspended in fresh DC medium and then returned to the original flask along with 200 ng / mL GM-CSF and 200 ng / mL IL-4 (final concentration). Five days later, adherent cells were detached from the cell culture flask by incubation at 37°C with 10 mL DPBS containing 2 mM EDTA for 10 min, and harvested along with the adherent cells before further use. iDCs were washed with DPBS (8 min, 300×g, RT), counted, and frozen in FBS (Sigma-Aldrich, F7524) containing 10% DMSO (AppliChem GmbH, catalog number A3672, 0100) or resuspended in X-VIVO 15 medium for direct electroporation.

[0446] CD8 will be separated from PBL. + T cells and iDCs were electroporated using the ECM 830 electroporation system. CD8+ were then... + T cells were electroporated with 10 µg each of the α and β chains encoding the CLDN6-specific TCR (TCR#12α and TCR#12β) and 10 µg of RNA encoding OX40. iDCs were electroporated with 2 µg of CLDN6-encoding RNA or without RNA as a mimic control (Table 14).

[0447] Table 14: RNA used for electroporation

[0448]

[0449] Electroporation of iDC is typically performed in 250 µL of X-VIVO 15 medium at a concentration of 5 × 10⁻⁶. 6The cells were selected for treatment. T cells were cultured in 250 µL of X-VIVO 15 medium at a concentration of 15 × 10⁶ cells / cells. 6 Electroporation was performed at high cell density. Cells were pipetted into cuvettes (4.0 mm gap size; VWR International, catalog number 732-0023) under RT. RNA was added to the cells by pipetting and mixed. Immediately after mixing, T cells were electroporated at 500 V, 3 ms, and 1 pulse, and iDCs were electroporated at 300 V, 12 ms, and 1 pulse. Immediately after electroporation, 750 μL of pre-warmed assay medium (IMDM, 5% PHS) was added to the cells. To assess electroporation efficiency, 2.5 × 10⁶ cells were used per well. 5 Each iDC and 2 to 3 × 10 per hole 5 CD8 + T cells (non-electroplated cells and CFSE-stained electroporated cells, see below) were cultured in 150 μL of assay medium in 96-well round-bottom plates. The remaining iDCs were transferred to 6-well plates (Greiner, catalog number 657160) and cultured in 3 mL of assay medium per 6 well for both O / N and electroporation (37°C, 5% CO2). Electroporated T cells were transferred to 15 mL tubes and incubated for at least 2 hours (37°C, 5% CO2) before CFSE labeling.

[0450] CD8 electroporation + T cells were labeled with CFSE using the Vybrant CFDA SE Cell Tracking Kit (LifeTechnologies 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 precipitate was then washed with 20×10⁻⁶ ppm. 6 Cells were resuspended in DPBS at a concentration of 1 cells / mL. An equal volume of 1.6 µM CFSE solution (diluted from stock solution in DPBS) was added. Cells were incubated in an incubator (37°C, 5% CO2) for 10 min. The labeling reaction was terminated by adding twice the excess volume of FBS (Sigma-Aldrich, catalog number F7524), followed by resting at RT for 2 min. To wash the cells, the cell suspension was filled to the test medium and centrifuged (8 min, 300×g, RT). After CFSE labeling, cells were resuspended in 3 mL of test medium per well, transferred to 6-well plates, and incubated at O / N (37°C, 5% CO2).

[0451] Electroporated iDC and CFSE-marked CD8+ T cells were harvested after O / N incubation and counted using a C-Chip cell counting chamber and red fluorescent B solution. The concentration was adjusted to 1.5 × 10⁻⁶ in the assay medium. 6 T cells / mL and 1.5 × 10 5 iDC / mL. CD8 + T cells and iDCs were used at a ratio of 10:1 (7.5 × 10⁶ cells per well). 4 7.5 × 10 T cells and 7.5 × 10 3 Cells were seeded with iDCs in 96-well round-bottom plates. Serial dilutions of IgG1-CD134-003-HC6LC2-RR (0.0003 to 10 µg / mL, final concentration) were prepared for dose-response analysis of IgG1-CD134-003-HC6LC2-RR. IgG1-b12-RR at the same concentration as IgG1-CD134-003-HC6LC2-RR was used as a non-binding control antibody. The diluted antibody was added to the seeded cells. Assay medium was added as needed to reach a total volume of 150 µL per well. The plates were gently shaken on a vibrating platform (150 RPM, 1 min) and incubated for four days (37°C, 5% CO2). Cell proliferation was analyzed by flow cytometry as described below.

[0452] Cultured cells for proliferation analysis were stained in 96-well round-bottom plates with 30 µL of FACS buffer using CD8 antibody (BD Biosciences, catalog number 564116; diluted 1:400) and fixed-activity dye eFluor780 (ThermoFisher Scientific, catalog number 65-0865-14; 1:1,500). Staining and washing procedures were performed as described in Example 10. Flow cytometry data were acquired on a BD FACSCelesta flow cytometer using the BD™ high-throughput sampler. Flow cytometry data were analyzed using FlowJo software.

[0453] The amplification index values ​​were plotted using GraphPad Prism software, compared with their respective antibody concentrations, and the data were fitted with an S-shaped, 4PL (X is the logarithm (concentration)) equation. EC 50 Exporting self-fitted curves.

[0454] IgG1-CD134-003-HC6LC2-RR induces dose-dependent increase in purified CFSE-labeled OX40 and CDLN6-TCR-electroplated CD8+. + T cell proliferation was observed, and these cells were co-cultured with autologous CLDN6 electroporated iDCs for four days. Figure 28A) IgG1-CD134-003-HC6LC2-RR increases CD8 only in the presence of antigen stimulation. + T cell proliferation was confirmed, and TCR activation was identified as a prerequisite for OX40 co-stimulation, and IgG1-CD134-003-HC6LC2-RR did not induce resting T cell proliferation. Figure 28 B).

[0455] Example 16: Binding of IgG1-CD134-003-HC6LC2-RR to M2c-like macrophages derived from human monocytes expressing FcγRIa

[0456] To assess whether the minimal binding of IgG1-CD134-003-HC6LC2-RR to FcγRIa (as detected by SPR) as described in Example 8 may be biologically relevant, flow cytometry was used to evaluate the binding of IgG1-CD134-003-HC6LC2-RR to FcγRIa physiologically expressed in human monocyte-derived M2c-like macrophages.

[0457] For this purpose, human peripheral blood mononuclear cells (PBMCs) were purified from the erythrocyte sedimentation rate (ESR) amber layer of a healthy human donor (Sanquin Blood Supply Foundation, Netherlands) by density gradient centrifugation (at low braking at 800×g for 20 min) in LeucoSep™ tubes (Greiner, catalog number 227290) on lymphocyte separation medium (Promocell, catalog number C-44010) according to the manufacturer's instructions. The PBMC layer was carefully transferred to 50 mL tubes and washed with an excess volume of PBS (HyClone, catalog number SH3A3830.03). The purified PBMCs were precipitated by centrifugation (300×g for 10 min), washed, and resuspended in PBS. The cells were then counted on a Cellometer Auto 2000 cell viability counter (Nexcelom Bioscience) using Cellometer ViaStain™ AOPI staining solution in PBS (Nexcelom Bioscience, catalog number CS2-0106) to distinguish between live and dead cells.

[0458] Positive screening of human monocytes using CD14 microbeads (Miltenyi Biotec, catalog number 130-050-201) purified from PBMCs according to the manufacturer's instructions for use. CD14 count. + Cells and they were 1.0 × 10 6The cells were resuspended at a density of 10 cells / mL in CellGenix® GMP DC medium (CellGenix, catalog number 20801-0500) supplemented with 50 ng / mL macrophage colony-stimulating factor (M-CSF; Gibco, catalog number PHC9501).

[0459] To polarize monocytes into M2c-like macrophages, purified monocytes were plated on 100 mm thick plates with an UpCell™ surface. 2 Nunc™ Petri Dishes (8×10) 6 Cells / culture dish, at 1.0 × 10⁻⁶ 6 Cells were cultured at a density of cells / mL in CellGenix GMP DC medium supplemented with M-CSF (CellGenix, catalog number 20801-0500) as described in Thermo Fisher Scientific, catalog number 174902. Cells were cultured in this medium for 7 days (37°C / 5% CO2), followed by 3 days 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 allowed to detach from the culture dish surface by incubation at RT for 40–60 min. The detached macrophages were centrifuged (300 × g for 5 min), counted, and cultured at 1.5 × 10⁻⁶ cells / mL. 6 The cells were resuspended at a density of 10 cells / mL in CellGenix GMP DC medium.

[0460] The M2c-like phenotype of monocyte-derived macrophages and its associated FcγRIa expression were confirmed by flow cytometry. Cells were plated in 96-well round-bottom plates (75,000 cells / well; Thermo Fisher Scientific, catalog 163320), centrifuged, and washed twice in FACS buffer. Next, cells were incubated in FACS buffer at 4°C for 30 min in the dark with either 50 µL of FITC-labeled anti-human CD64 (FcγRIa) antibody (BioLegend, catalog 305006, diluted 1:25) or a mixture of 50 µL of antibodies used for characterizing human M2c macrophages (Table 15), followed by washing twice in FACS buffer.

[0461] Table 15: Antibody group used to confirm M2c phenotype

[0462]

[0463] Next, cells incubated with antibodies used for human M2c characterization were resuspended in 100 µL of FACS buffer supplemented with the activity marker 7-AAD (7-aminoactinomycin D; BD Pharmingen, catalog number 68981E; diluted 1:240) and measured on a BD FACSymphony™ cell analyzer (BD Biosciences). Cells incubated with FcγRIa antibodies were resuspended in 100 µL of FACS buffer supplemented with the activity marker DAPI (4′,6-diamidinyl-2-phenylindole; BD Pharmingen, catalog number 564907; diluted 1:5,000) and measured on a BD LSRFortessa™ cell analyzer.

[0464] 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 50 µL of IgG1-CD134-003-HC6LC2-RR, IgG1-b12, or IgG1-b12-RR in CellGenix GMP DC medium (final antibody concentration of 10 µg / mL) at 37°C / 5% CO2 for 15 min or 24 h. Next, the cells were washed twice with FACS buffer and incubated with 50 μL of R-PE conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog number 109116 097; diluted 1:200) in FACS buffer at 4°C for 30 min. 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 number 564907; diluted 1:5,000) and then measured on a BD LSRFortessa cell analyzer.

[0465] The study included IgG1-b12, containing the wild-type Fc domain, as a positive control for binding to M2c-like macrophages, and Fc-inert IgG1-b12-RR as a negative control. Although IgG1-b12 showed effective binding to FcγRIa-expressing M2c-like macrophages after 15 min and 24 h of incubation, binding to IgG1-CD134-003-HC6LC2-RR and IgG1-b12-RR was not observed at any time point in any of the three donors tested. Figure 29Furthermore, no binding was observed between IgG1-CD134-003-HC6LC2-RR and cynomolgus monkey FcγR1a expressed in cynomolgus monkey mononuclear cells (no data shown). Therefore, IgG1-CD134-003-HC6LC2-RR is considered to be unable to bind FcγR1a in a physiological environment.

[0466] Example 17: Binding of IgG1-CD134-003-HC6LC2-RR to neonatal Fc receptors

[0467] The binding of IgG1-CD134-003-HC6LC2-RR to immobilized neonatal Fc receptor (FcRn) was evaluated in vitro by SPR at pH 6.0 and pH 7.4. Aliquots of recombinant His-tagged FcRn protein (SinoBiological, catalogue number CT009-H08H-B) were diluted in PBS-P+ buffer at pH 7.4 (Cytiva, catalogue number 28995084) or in PBS-P+ buffer adjusted to pH 6.0 (with added hydrochloric acid [Sigma-Aldrich, catalogue number 30721-M]) and used at a flow rate of 10 μL / min and a contact time of 60 s to capture the FcRn protein onto the surface of a sensor chip coated with an anti-His antibody. This resulted in capture levels ranging from 35 to 60 RU.

[0468] After three start-up cycles in PBS-P+ buffer at pH 6.0 or pH 7.4, a series of antibody concentrations (6.25 to 100 nM diluted twice in PBS-P+ buffer at pH 6.0 or pH 7.4) were injected to generate binding profiles. Samples analyzed on surfaces with captured FcRn (active surfaces) were also analyzed on parallel flow cells without captured FcRn (reference surfaces) for background correction. The signal from the third start-up cycle containing HBS-EP+ as the (simulated) analyte was subtracted from the other sensor profiles to generate dual-reference data. At the end of each cycle, the surfaces were regenerated using 10 mM glycine HCl pH 1.5 (Cytiva, catalog number BR100354). Data were analyzed using the predefined "Multi-cycle kinetics with capture" evaluation method in Biacore Insight evaluation software (Cytiva).

[0469] At pH 6.0, dose-dependent binding of IgG1-CD134-003-HC6LC2-RR to FcRn was observed. Figure 30 (A to E), while no FcRn binding was observed at pH 7.4 ( Figure 30F). These results show that IgG1-CD134-003-HC6LC2-RR binds FcRn at pH 6.0, but binds at pH 7.4, as expected for IgG1 molecules.

[0470] Example 18: Evaluation of binding to C1q of transmembrane-bound IgG1-CD134-003-HC6LC2-RR

[0471] To confirm the absence of complement binding to the Fc domain of IgG1-CD134-003-HC6LC2-RR, flow cytometry was used to measure the binding of C1q to IgG1-CD134-003-HC6LC2-RR expressed on the membrane of activated human T cells. IgG1-CD52-E345R (VH SEQ ID NO: 64, VL SEQ ID NO: 68; constant region SEQ ID NO: 2) was included as a positive control, carrying the same hexamerization-enhancing mutation as IgG1-CD134-003-HC6LC2-RR but lacking the inert Fc mutation. The inert Fc non-binding control antibody IgG1-b12-RR was included as a negative control.

[0472] Human T cells were negatively screened using RosetteSep™ Human T Cell Enrichment Mixture (Example 9), and then purified from the erythrocyte sedimentation rate (ESR) of healthy volunteers (Sanquin Blood Supply Foundation, Netherlands) by density centrifugation (at low speed at 800×g for 20 min) on lymphocyte separation medium (Corning, catalog number 25-072-CI), all according to the manufacturer's instructions.

[0473] Purified T cells were washed with PBS (HyClone), precipitated, and resuspended in activation medium (RPMI 1640 [Lonza, BE12-115F] supplemented with 10% FBS [Sanquin, catalog number K1146], 50 units of penicillin, 50 µg / mL of streptomycin [Lonza, catalog number DE17-603E], and 1% L-glutamine [Lonza, catalog number BE17-605E] containing 25 mM HEPES and L-glutamine). Next, T cells were counted using a Cellometer Auto 2000 cell viability counter (Nexcelom Bioscience) with Cellometer ViaStain™ AOPI staining solution (Nexcelom Bioscience, catalog number CS2-0106) to distinguish between live and dead cells. Cells were then plated in 96-well round-bottom plates (Thermo Fisher Scientific, catalog number 170189; 3×10⁶ cells per 150 µL). 5 (cells / well). To induce OX40 expression, T cells were activated by incubating anti-CD3 / CD28 beads (Dynabeads™ Human T-Activator CD3 / CD28; Thermo Fisher Scientific, catalog number 11132D) at a 1:2 bead-to-cell ratio at 37°C for 72 h. After incubation, the beads were removed using a magnet, and the cells were pooled, washed once in PBS, and counted.

[0474] To evaluate the binding of C1q to cell-bound IgG1-CD134-003-HC6LC2-RR, CD3 / CD28-activated human T cells were plated in 96-well round-bottom plates (50,000 cells / well) and incubated with serially diluted IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E435R, or IgG1-b12-RR (at a final concentration of 0.00051 to 30 μg / mL from the 3-fold dilution step) in 80 µL of activation medium at 37°C for 15 min to allow antibody binding to the cells. Next, 20 μL of normal human serum (NHS; Sanquin; 20% final concentration) as the C1q source was added, and the mixture was incubated on ice for 45 min. Cells were washed twice with cold FACS buffer and then incubated with a mixture of 50 μL of FITC-conjugated rabbit anti-human C1q antibody (DAKO, catalog number F0254; 1:100 dilution; final concentration 20 μg / mL) and antibodies for human T cell characterization (Table 16) in FACS buffer at 4°C in the dark for 30 min.

[0475] Table 16: Antibody sets used for T cell characteristics

[0476]

[0477] Next, the cells were washed twice with cold FACS buffer, precipitated, and resuspended in 80 μL of FACS buffer supplemented with the viability marker TO-PRO™-3 iodide (Invitrogen, catalog number T3605; diluted 1:10,000). C1q binding was analyzed by flow cytometry on a BD FACSCelesta™ cell analyzer (BD Biosciences). Binding curves were analyzed using nonlinear regression analysis (S-shaped dose-response with variable slope) and GraphPad Prism software. Binding of the OX40 antibody to activated T cells was assessed after incubation with serially diluted antibody. Cells were washed twice with cold FACS buffer and incubated for 30 min at 4°C in the dark with a mixture of 50 μL of R-phycoerythroxene (R-PE)-conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog number 109-116-098; 1:200 dilution) and antibodies for human T cell characterization (Table 16). Next, the cells were washed twice with cold FACS buffer, precipitated, 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 on a BD FACSCelesta cell analyzer. Binding curves were analyzed using nonlinear regression analysis (S-shaped dose-response with variable slope) and GraphPad Prism software.

[0478] IgG1-CD134-003-HC6LC2-RR and IgG1-CD52-E345R with activated CD4 + and CD8 + T-cell binding was confirmed by flow cytometry. Figure 31 (A to C). Although C1q effectively binds to membrane-bound IgG1-CD52-E345R, no binding of C1q to membrane-bound IgG1-CD134-003-HC6LC2-RR was observed at any of the antibody concentrations tested. Figure 31 (D, E) No binding of C1q to cells incubated with IgG1-b12-RR was observed. These results indicate that C1q does not bind to membrane-bound IgG1-CD134-003-HC6LC2-RR.

[0479] To confirm that IgG1-CD134-003-HC6LC2-RR does not induce target-dependent fluid-phase complement activation, IgG1-CD134-003-HC6LC2-RR was incubated in normal human serum (NHS) and produced as a measure of complement activation at C4d, then measured by ELISA. The results demonstrated that IgG1-CD134-003-HC6LC2-RR does not induce fluid-phase complement activation in vitro (data not shown).

[0480] Example 19: Evaluation of monovalent versus bivalent binding of IgG1-CD134-003-HC6LC2-RR and its variants

[0481] To determine whether IgG1-CD134-003-HC6LC2-RR binds to OX40 monovalently or bivalently, the binding of IgG1-CD134-003-HC6LC2-RR to activated T cells was compared with the binding of functional monovalent OX40-specific antibodies (BsIgG1-b12-RR-F405LxCD134-003-RR-K409R; generated by controlled Fab arm exchange of parental antibodies IgG1-b12-RR-F405L [SEQ ID No 71, 75, and 78] and IgG1-CD134-003-RR-K409R [SEQ ID No 15, 19, and 63]). Healthy donor T cells were stimulated in vitro with anti-CD3 / CD28 beads for three days to induce OX40 expression. Subsequently, IgG1-CD134-003-HC6LC2-RR, the monovalent antibody BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, the chimeric control antibody IgG1-CD134-003, or the negative control antibody IgG1-b12-RR were combined with activated CD4+. + and CD8 + The binding of T cells was analyzed by flow cytometry.

[0482] Total human T cells were directly enriched from the negative selection of the erythrocyte sedimentation rate (ESR) amber layer, 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 on a Cellometer Auto 2000 cell viability counter (Nexcelom Biosciences) using Cellometer ViaStain® AOPI solution in PBS (Nexcelom Biosciences, catalog number CS2-0106) to distinguish between live and dead cells. The T cells were then pelleted and resuspended in activation medium.

[0483] T cells were activated by stimulating CD3 and CD28 with anti-CD3 / CD28 beads (Dynabeads™ Human T-Activator CD3 / CD28) for 72 h, as described in Example 9. Activated T cells were used directly or cryopreserved in a 1:1 mixture of IMDM medium with L-glutamine and HEPES (Lonza, catalog number 12-115F) and cryoprotective cryoprotective medium (Lonza; catalog number 12-132A) for further use.

[0484] Activated T cells were seeded (50,000 cells / well) in 96-well round-bottom plates (Thermo Scientific, catalog number 163320), washed once with PBS (GE Healthcare, catalog number SH3A3830.03) and once with FACS buffer. Cells were then resuspended in 50 μL of first-order antibody (IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405LxCD134-003-RR-K409R or control antibody IgG1-CD134003-RR-K409R, IgG1-b12-RR and IgG1-CD134-003) diluted in FACS buffer (concentration range of 0.0005 to 10 μg / mL for the three-fold dilution step) and incubated at 4°C for 30 min. Flow cytometry was performed on a BD FACSymphony A1 cell analyzer (BD Biosciences) and analyzed using FlowJo software.

[0485] The monovalent OX40-specific antibody BsIgG1-b12-RR-F405LxCD134-003-RR-K409R showed similarity to activated CD4+. + and CD8 + The dose-dependent binding of T cells to IgG1-CD134-003-HC6LC2-RR was compared with that to CD4. + and CD8 + T cells exhibit a high maximum binding rate to both, as defined by the gMFI Y-space ( Figure 32 Chimeric control antibody IgG1-CD134-003 showed similarity to OX40. + CD4 + and CD8 + The binding of T cells was similar to that of IgG1-CD134-003-HC6LC2-RR, indicating that the higher maximum binding was driven by the monovalent binding of BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, rather than by CDR chimeric traits or functionally irrelevant mutations in different Fc backbones. These data suggest that IgG1-CD134-003-HC6LC2-RR binds not only monovalently but also bivalently.

[0486] Example 20: Functional comparison of IgG1-CD134-003-HC6LC2-RR and its monovalent binding variants

[0487] As described in Example 19, IgG1-CD134-003-HC6LC2-RR binds not only monovalently but also bivalently. Here, the functional activity of IgG1-CD134-003-HC6LC2-RR and the monovalently bound variant BsIgG1-b12-RR-F405LxCD134-003-RR-K409R was compared by two separate assays, namely, based on the OX40 reporter assay and polyclonal T cell proliferation assay using healthy human donor PBMCs.

[0488] To measure OX40 agonist activity in the cell reporter assay, the OX40 Bioassay Kit (Thaw and Use Kit; Promega, catalog number JA2191) was used, essentially as described in Example 6. Briefly, after thawing, OX40+Jurkat cells were seeded at 60 µL per well in fresh 96-well white flat-bottomed assay plates (PerkinElmer, catalog number 6005680) and incubated for 16 to 20 h (37°C, 5% CO2). The following day, dilutions of IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405LxCD134-003-RR-K409R, and control antibodies IgG1-CD134-003-RR-K409R, IgG1-CD134-003, IgG1-CD134-003-FEAL, and IgG1-b12-RR were prepared in RPMI 1640 medium with 5% FBS (to a final concentration of 0.000457 to 1 µg / mL from the three-fold dilution step). 20 µL of the prepared antibody dilution series or medium alone was added to the wells, and the assay plate was incubated for 5 h (37°C, 5% CO2). After incubation, the assay plate was equilibrated to RT. Subsequently, 80 µL of Bio-Glo™ luciferase assay matrix was added to each well, and the plate was incubated for 10 min with constant shaking and in the dark. Luminescence was measured using an EnVision Multi plate reader (PerkinElmer). Data are presented in relative luminescence units (RLU) in line graphs generated using GraphPad Prism software. Data are fitted to an sigmoid, 4PL (X is the logarithm of concentration) equation.

[0489] To evaluate the ability of IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405LxCD134-003-RR-K409R and negative control antibody IgG1-b12-RR (concentration range between 0.0015 μg / mL and 30 μg / mL) in a polyclonal T cell proliferation assay, the procedure described in Example 7 was followed.

[0490] In a cell-based OX40 reporter assay, BsIgG1-b12-RR-F405LxCD134-003-RR-K409R induced OX40 agonist activity with approximately five times less potency than IgG1-CD134-003-HC6LC2-RR, indicating that the optimal agonist activity of IgG1-CD134-003-HC6LC2-RR is achieved via its ability to bind to both Fab arms. Figure 33A). In a polyclonal T cell proliferation assay, BsIgG1-b12-RR-F405LxCD134-003-RR-K409R enhanced CD4+ T cell proliferation ( Figure 33 B), however, requires a higher concentration to achieve the maximum effect compared to IgG1-CD134-003-HC6LC2-RR.

[0491] These results together indicate that the optimal bioactivity of IgG1-CD134-003-HC6LC2-RR is achieved through its ability to bind to OX40 with two Fab arms.

[0492] Example 21: Response to OX40 expression and shedding in cells incubated with IgG1-CD134-003-HC6LC2-RR in vitro

[0493] In response to IgG1-CD134-003-HC6LC2-RR therapy, CD4 + and CD8 +Changes in OX40 expression levels on T cells were analyzed in a polyclonal T cell proliferation assay using PBMCs obtained from ten healthy human donors (using the universal assay setup described in Example 7; PBMCs were stained with CellTrace™ Violet kit with CellTrace Purple [Thermo Fisher Scientific, catalog number C34557; staining description see Example 7] or Vybrant CFDA SE CellTrace Kit with CFSE [Life Technologies, catalog number V12883; staining description see Example 15]). After incubation with IgG1-CD134-003-HC6LC2-RR for two and five days as described in Example 13, transmembrane expression of OX40 in seven of the tested donors was determined by flow cytometry (antibody staining: CD4 antibody labeled with APC [ThermoFisher Scientific, catalog number 17-0048-42; diluted 1:100], CD8α antibody labeled with BV605 [BD Biosciences, catalog number 564116; diluted 1:400], and OX40 antibody labeled with PE [BDBiosciences, catalog number 340420; diluted 1:80]). The expression in the remaining three donors was evaluated after incubation for one to four days and six days (antibody staining: CD4 antibody labeled with PerCP-eFluor710 [ThermoFisher]). Scientific, catalog number 46-0047-42; diluted 1:100], PE-Cy7-labeled CD8α antibody [TONBO, catalog number 60-0088-T100; diluted 1:100], BV421-labeled OX40 antibody (BD Biosciences, catalog number 744881; diluted 1:80). In CD4 + and CD8 + OX40 expression on T cells was evaluated in a one-dimensional histogram analyzing geometric mean fluorescence intensity (gFMI) values.

[0494] Soluble OX40 (sOX40) has been detected in the serum of 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 believed to act as a decoy receptor by blocking the binding of OX40 ligand (OX40L) to transmembrane-bound OX40. OX40 expression on the day of detachment in response to in vitro treatment with IgG1-CD134-003-HC6LC2-RR was assessed by ECLIA. The concentration of sOX40 in the supernatant collected from polyclonal T cell proliferation assays was determined according to the manufacturer's protocol using a multi-task ECLIA with the U-PLEX Human OX40 / TNFRSF4 assay (MSD, catalog number K151T7K-2). In short, add 25 µL of biotinylated antibody diluted 100 μL to each well of an MSD Gold small-dot streptavidin plate and incubate at RT for 1 h. Wash the plate three times with PBST and add standards or samples (diluted 1:8 in diluent 58) to the wells (total 50 µL / well), and incubate the plate at RT with continuous shaking. Wash the plate three times with PBST and add the detection antibody diluted in diluent 3 (50 µL / well). Incubate the plate at RT with continuous shaking for 1 h and wash the plate three times with PBST. Then add Gold Read buffer B (150 µL / well) and analyze the plate immediately on a MESO QuickPlex SQ 120 imager (MSD).

[0495] IgG1-CD134-003-HC6LC2-RR (at a concentration of 0.01 µg / mL or higher) enhanced CD4 in three out of ten donors after two days of incubation. + OX40 expression was induced by CD3, and enhanced in six out of ten donors after three to six days of incubation or five days. + and CD8 + CD3-induced OX40 expression on T cells, and expression on CD8 cells after two and three days of incubation. + CD3-induced OX40 expression on T cells (e.g.) Figure 34A illustrates one of the three donors in total; donor variability was observed in other tested donors. Meanwhile, for all three donors testing sOX40 release, IgG1-CD134-003-HC6LC2-RR treatment increased the sOX40 concentration in the supernatant in a dose-dependent manner over time, indicating that IgG1-CD134-003-HC6LC2-RR induced sustained sOX40 release, which began on day 3 and continued to increase until day 6. Figure 34 B). Although partial interference with IgG1-CD134-003-HC6LC2-RR was observed by the ECLIA kit for sOX40 detection, increased sOX40 levels were clearly detected after treatment with IgG1-CD134-003-HC6LC2-RR at 0.2 and 2 μg / mL.

[0496] These data suggest that IgG1-CD134-003-HC6LC2-RR can enhance CD4+ + and CD8 + OX40 expression on T cell membranes, despite high donor variability, also enhances OX40 shedding from the plasma membrane.

[0497] Example 22: Evaluation of the interference of soluble OX40 on the activity of IgG1-CD134-003-HC6LC2-RR agonist

[0498] Example 21 describes how IgG1-CD134-003-HC6LC2-RR can enhance CD4+. + and CD8 + sOX40 membrane expression on T cells was enhanced, and OX40 shedding from the plasma membrane was also increased. This study investigated the effects of sOX40 on the ability of IgG1-CD134-003-HC6LC2-RR to induce OX40 agonism as observed in a cell-based OX40 reporter assay (see Example 6) and on its ability to increase T cell proliferation as observed in a polyclonal T cell proliferation assay (see Example 7).

[0499] To investigate whether the presence of sOX40 interferes with IgG1-CD134-003-HC6LC2-RR-mediated OX40 agonist activity, assays as described in Example 13 were performed on FcγRIIb-CHO K1 cells. IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR, alone or together with sOX40 (human OX40, AcroBiosystems, catalog number OX0-H5224), at final concentrations ranging from 0.0024 to 40 µg / mL (serial dilutions of 1:4), were added in a final concentration range ranging from 1 to 1,000 ng / mL (serial dilutions of 1:10), and luminescence was measured using a CLARIOstar Plus microplate reader. To investigate whether the presence of sOX40 interferes with T cell proliferation increased by IgG1-CD134-003-HC6LC2-RR, 0.002 to 40 μg / mL of IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR (serial dilutions of 1:4) were added alone or together with sOX40 (0.1 ng / mL to 1,000 ng / mL; serial dilutions of 1:10), further following the method described in Example 7, omitting CD45RA and CCR7 staining.

[0500] Although dose-dependent OX40 agonist activity induced by IgG1-CD134-003-HC6LC2-RR was retained in the presence of low concentrations of sOX40, reduced activity was observed at 1,000 ng / mL of sOX40, and a lower degree was observed at 100 ng / mL of sOX40. Figure 35 Similarly, in the presence of sOX40 concentrations up to 10 ng / mL, the dose-dependent increase in CD4+ induced by IgG1-CD134-003-HC6LC2-RR was retained. + and CD8 + T cell proliferation was observed, while at 100 ng / mL and 1,000 ng / mL sOX40, a decrease in CD4+ concentration was observed at up to 0.625 µg / mL IgG1-CD134-003-HC6LC2-RR. + and CD8 + Increased T cell proliferation ( Figure 36 No interference from sOX40 was observed in PBMC samples incubated with a higher concentration of IgG1-CD134-003-HC6LC2-RR (≥2.5 µg / mL).

[0501] In summary, interference with IgG1-CD134-003-HC6LC2-RR-induced OX40 agonism in reporter cells and polyclonal activated T cells was observed only at concentrations well above the clinically relevant levels.

[0502] Example 23: Preclinical Immunogenicity Assessment of IgG1-CD134-003-HC6LC2-RR

[0503] Immunogenicity assessment of the heavy chain (HC) and light chain (LC) of IgG1-CD134-003-HC6LC2-RR was performed using the amplification intelligence (AI) platform iTope-AI and computer simulations of TCED (Abzena). The immunogenic potential of IgG1-CD134-003-HC6LC2-RR was further analyzed in vitro using the EpiScreen DC:T cell proliferation assay (Abzena), which measured the proliferation of human CD4 cells co-cultured with MoDCs previously incubated with IgG1-CD134-003-HC6LC2-RR. + T cell proliferation.

[0504] Nine-mer peptides spanning eight overlapping amino acids across the protein sequences of IgG1-CD134-003-HC6LC2-RR HC and LC were generated using computer simulations. Using iTope-AI technology, favorable interactions were predicted between the amino acid side chains of each nine-mer and the 46 most common human leukocyte antigen (HLA) alleles (HLA-DR, DP, and -DQ allotypes) found globally. For peptides showing favorable interactions, the P1 anchor position indicates the first amino acid of its sequence. The binding fractions of individual peptides from each of the 46 allotypes were given from 0 (no binding) to 3 (strong binding), and these fractions for all allotypes were summed to provide the overall risk score for each nine-mer, called the position risk score. When the position risk scores of hybrid peptides (position risk score > 0) reached 1 to 2, 3 to 5, or 6+, respectively, these peptides were considered weak, moderate, or strong MHC class II binders. The total score for all protein sequences tested was calculated by summing the positional risk scores obtained for each individual peptide. 9-mer peptides that are completely homologous to human proteome sequences were excluded from the analysis because germline sequences are unlikely to have immunogenic potential due to T-cell tolerance. The total score for IgG1-CD134-003-HC6LC2-RR was then compared with the total score observed using benchmark mAbs (e.g., murine, chimeric, humanized, and human mAbs).

[0505] To assess the immunogenic risk of the IgG1-CD134-003-HC6LC2-RR HC and LC sequences, identified hybrid peptides were searched against peptide sequences in the TCED using a basic local alignment search tool (BLAST) to identify any high sequence homology with >10,000 peptides from unrelated proteins and antibodies that elicited stimulated T cell responses in previous in vitro EpiScreen studies (i.e., T cell epitopes) at Abzena.

[0506] CD4 was measured using the EpiScreen DC:T cell assay. + T-cell response (a key driver of memory-based immunogenicity) was used to assess the potential immunogenicity of IgG1-CD134-003-HC6LC2-RR in vitro. First, PBMCs were isolated from healthy donor leukocyte cones (within 24 hours of blood collection) obtained with the consent of the UK National Health Service Transfusion Service. Donors were characterized for HLA-DR haplotype using the Sequence-Specific Oligonucleotide (SSO) HLA typing method (VHBio). Using a cohort of 50 healthy PBMC donors, representatives of all known HLA alleles were selected, except for HLA-DP, which was excluded due to its low prevalence and potentially low expression levels.

[0507] Prior to use, IgG1-CD134-003-HC6LC2-RR was diluted to 0.75 μM (112.5 µg / mL) in MoDC medium (RPMI 1640 [ThermoFisher, The ... The T-cell response to limpethemocyanin (KLH) was used as a positive control. KLH (Pierce Life Technologies, catalog number 77600) was stored at -20°C as a stock solution of 10 mg / mL in distilled water. Aliquots of KLH were immediately thawed and then diluted to 1 mg / mL in MoDC medium. Herceptin® (Roche, catalog number HERC / 150 / 1 / BG) was used as a baseline antibody, known for its low clinical immunogenicity, and was stored at -80°C as a stock solution of 20 mg / mL. It was immediately thawed in MoDC medium and diluted to 125 μg / mL before use.

[0508] To prepare MoDC, CD14 + Monocytes were purified from donor PBMCs using a negative human monocyte purification kit (StemCell Technologies, catalog number 19058RF) and an automated cell purification system (RoboSep™ StemCell Technologies) according to the manufacturer's instructions. Monocytes were resuspended in MoDC medium at a density of 1.5 × 10⁶ cells per well. 6Cells were seeded in 2 mL of low-binding 24-well plates (final volume) and incubated at 37°C. On the second day, cells were fed by replacing 1 mL of MoDC medium with fresh medium. On the fourth day, cells were fed again with 1 mL of MoDC medium containing the following reagents (final volume): 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 the wells containing reagents, except for the addition of reagents. Cells were incubated for 1 hour (37°C / 5% CO2). After incubation, 0.01 µg / mL lipopolysaccharide (LPS; Sigma, catalog number L4391) was added to induce MoDC maturation. On day five, mature MoDCs were harvested, counted, and viability was assessed using trypan blue (Sigma, catalog number T8154) staining. Viability was expressed as the percentage of cells not stained with trypan blue out of the total cell count. Cell viability assessment confirmed that IgG1-CD134-003-HC6LC2-RR, KLH, and Herceptin controls did not affect the viability of MoDCs used in the EpiScreen analysis. Mature MoDCs were then irradiated with γ (40 Gy) before use in proliferation assays. Finally, autologous CD4+ was... + T cells use human CD4 + The T-cell enrichment kit (StemCell Technologies, catalog number 19052) and automated cell purification instruments are used to purify PBMCs from the same donor through negative screening.

[0509] After counting and assessing cell viability, 1×10 6 CD4 + T cells and 1×10 5 γ-irradiated MoDCs were co-cultured for 12 days in 24-well plates in medium (RPMI 1640 supplemented with human serum, 2-mercaptoethanol, and L-glutamine) at a T:DC ratio of 10:1. On days 9, 10, 11, and 12, the cultures were carefully resuspended, and 3 × 100 μL aliquots were transferred to 96-well round-bottom plates for pulse labeling. Cells were then cultured with a solution containing 1.0 µCi […]. 3Cells were pulsed with 100 μL of 1H-thymidine (PerkinElmer) culture medium and incubated at 37°C for 6 h, then harvested on filter pads using a TomTec Mach III cell harvester. Cell counts per minute (CPM) for each sample were determined using a MicroBeta Microplate β counter with MeltiLex™ (Perkin Elmer) scintillation counting in ParaLux™ low background counting mode. Immunogenicity was expressed as the stimulation index (SI), defined for each sample as the average CPM of wells containing compound-treated T cells / the average CPM of baseline (untreated control wells).

[0510] Stimulation Index (SI) = (Average CPM (treated wells)) / (Average CPM (untreated control wells))

[0511] Based on previous analyses by Abzena, an empirical threshold of SI ≥ 1.90 was established as the minimum signal-to-noise ratio threshold, allowing for maximum sensitivity without detecting a large number of false positives or omitting cryptic immunogenic events. Samples inducing a response greater than this threshold were assessed as positive for inducing T-cell immune responses in the test donor. For each sample at the given time points, CD4+ incubated with reagents loaded with mature DC samples was used. + Triple copies of T cells (CPM) and CD4 cells incubated solely with mature MoDCs cultured in culture medium. + Comparing triplicate CPMs of T cells, a positive proliferative response was considered statistically significant (P<0.05), using an unpaired two-sample Student's t-test. Donors who were positive at at least one time point during the time-course assay (SI≥1.90, P<0.05) were rated as positive donors, and the mean amplitude SI was calculated from the average of the positive donor responses.

[0512] iTope-AI analysis of the IgG1-CD134-003-HC6LC2-RR HC sequence anchored to P329R and E345R Fc mutations predicted a total of 12 non-species mixed-binding peptides in the HC constant region, classifying these peptides as strong (2), moderate (2), or weak (8) MHC class II binders (Table 17). The P329R mutation was located in one of the identified weak binders (P1 anchor sites C317 and K318; Table 17), but this peptide did not match 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-species mixed-binding peptides were predicted to be in the HC variable domain (VH), of which 2 were predicted to be moderate binders and 2 were predicted to be strong binders (Table 17). Peptides identified in frame (Fw) 3 (position risk score: 1) were found to be homologous to peptides in TCED. Overall, the total iTope score of IgG1-CD134-003-HC6LC2-RR HC was 45.

[0513] Table 17: Non-species hybrid CD134-003-HC6LC2-RR HC peptides identified by iTope-AI and TCED analysis. Each 9-mer peptide is indicated by its P1 anchoring position. Variable regions are numbered according to Kabat numbering, followed by linear numbering of constant regions. The positional risk scores of hybrid-bound peptides are categorized into weak (1 to 2), moderate (3 to 5), and strong (6+) affinity binders. Homologous peptides identified by TCED are listed.

[0514]

[0515] a The first amino acid of 9-mer peptide.

[0516] b The P1 position of the variable region is determined by the Kabat number; the linear numbering is used for the constant region.

[0517] c The underlined residues indicate the positions defined in IgG1-CD134-003-HC6LC2-RR VH CDR according to the IMGT definition.

[0518] d R = P329R mutation in the Fc region of IgG1-CD134-003-HC6LC2-RR; based on the location of the Eu number.

[0519] e The total score of the test sequence is calculated by summing the positional risk scores obtained by all individual peptides.

[0520] fSummary of HC TCED inquiry for non-species-binding peptides. List the homologous peptides identified from TCED and their matching P1>P9>P7 anchoring sites. The contribution to MHC class II binding at specific "anchoring" sites exhibits a hierarchical pattern, with the largest effect observed at P1>P9>P7≥P6≥P4.

[0521] iTope-AI analysis predicted the presence of 3 strong-binding, 2 moderate-binding, and 10 weak-binding non-species mixed 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). Three of the predicted peptides (two weakly-binding and one moderately-binding peptide) were identified as homologous peptides in TCED. The total iTope score of the IgG1-CD134-003-HC6LC2-RR LC sequence was 74. The total score of IgG1-CD134-003-HC6LC2-RR (the sum of the HC and LC scores) was 119, consistent with the baseline humanized mAb.

[0522] Table 18: Non-species hybrid IgG1-CD134-003-HC6LC2-RR LC peptides identified by iTope-AI and TCED analysis. Each 9-mer peptide is indicated by its P1 anchoring position. Variable regions are numbered according to Kabat numbering, followed by linear numbering of constant regions. The positional risk scores of hybrid-binding peptides are categorized into weak (1 to 2), intermediate (3 to 5), and strong (6+) affinity binders. Homologous peptides identified by TCED are listed.

[0523]

[0524] a The first amino acid of 9-mer peptide.

[0525] b The P1 position of the variable region is determined by the Kabat number; the linear numbering is used for the constant region.

[0526] c The underlined residues indicate the positions defined in IgG1-CD134-003-HC6LC2-RR VL CDR according to the IMGT definition.

[0527] d The total score of the test sequence is calculated by summing the positional risk scores obtained by all individual peptides.

[0528] e Summary of LC TCED inquiry for non-species-binding peptides. List the homologous peptides identified from TCED and the matching P1>P9>P7≥P6≥P4 anchoring sites.

[0529] Mature MoDCs loaded with IgG1-CD134-003-HC6LC2-RR induced a low frequency of positive T cell proliferation (SI ≥ 1.90), i.e., in 8% (4 / 50) of the donor population (Table 19). This is similar to the range of Herceptin (inducing proliferation in 8% [4 / 50] of the donor population), which is known to show low clinical immunogenicity and is below the 10% threshold set by Abzena based on historical EpiScreen data of proteins considered to have an increased risk of immunogenicity in clinical settings. The neoantigen KLH, including its use as a positive control for immunogenicity, showed a positive response rate of 88%. The mean amplitude SI (mean SI of positive donor response) of IgG1-CD134-003-HC6LC2-RR and Herceptin was comparable (SI = 2.66 ± 0.6 and SI = 2.64 ± 0.7, respectively), but lower than that of KLH (SI = 4.6 ± 2.54).

[0530] Table 19: CD4 induced by mature MoDCs loaded with IgG1-CD134-003-HC6LC2-RR, Herceptin, or KLH + Summary of T cell proliferation levels

[0531]

[0532] 1 The percentage of donors with a T-cell proliferative response at least one of the time points (SI ≥ 1.90, P < 0.05, where the mean amplitude SI is calculated from the mean of positive donor responses).

[0533] These data together indicate a low clinical immunogenicity risk for IgG1-CD134-003-HC6LC2-RR, which is within the range of known therapeutic antibodies that have shown low immunogenicity in clinical practice.

[0534] Example 24: Pharmacokinetic analysis of IgG1-CD134-003-HC6LC2-RR in mice in the absence of target binding.

[0535] 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 containing either the indolent mutations L234F-L235E-D265A and the DuoBody mutation F405L, or the same RR Fc mutation as IgG1-CD134-003-HC6LC2-RR except for the DuoBody mutation K409R (IgG1-CD134-003-FEAL and IgG1-CD134-003-RR-K409R, respectively). IgG1-CD134-003-HC6LC2-RR does not bind to mouse OX40, as shown in Example 5 and... Figure 5 As shown in A, this experiment was therefore designed to test the pharmacokinetic behavior of the described anti-human OX40 antibody in vivo in the absence of target binding. IgG1-CD134-003-FEAL and IgG1-CD134-003-RR-K409R also do not bind to mouse OX40 (data not shown). Severely combined immunodeficient female mice (SCID mice, Envigo; CB-17 / IcrHan®Hsd Prkdcscid) were housed in sterile, individually ventilated cages (IVCs) at the Central Laboratory Animal Research Facility (Gemeenschappelijk Dierenlaboratorium, GDL) of Utrecht University, five mice per cage, with sterile food and water provided randomly. Mice received tail tattoos for identification purposes upon arrival at GDL. Animal experiments were conducted in accordance with the Dutch Animal Protection Act (WoD), a translation of the Directive on "Animal Experimentation for Cancer Research" (2010 / 63 / EU) and the regulations (Inspection V&W, Zutphen, Netherlands, 1999), and were approved by the Utrecht Ethics Committee. Animals were housed and handled in animal facilities (GDL) certified by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and ISO 9001:2018, in accordance with the Good Animal Practices as defined by the Federation of European Associations for Laboratory Animal Science (FELASA).

[0536] At 10 weeks of age, female SCID mice (n=3 per group) were intravenously injected via the tail vein with 60 μL of a solution containing the aforementioned test antibody and control antibody, equivalent to approximately 0.125, 1.25, and 12.5 mg of antibody per kilogram of body weight, respectively. 40 µL 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 the total human IgG concentration was determined by ELISA. To obtain plasma, blood was collected in Microvette® CB 300 K2 EDTA vials (Sarstedt, catalog number 16.444.100) and centrifuged at 14,000 × g for 10 min without heat inactivation. 96-well MULTI-ARRAY standard plates (MSD, catalog number L15XA-3) were coated with 2 µg / mL mouse anti-huIgG capture antibody (IgG2amm-1015-6A05; Genmab, batch number 3093-008-EP) in PBS at 2–8°C for 16–24 hours. After washing the plates with PBS-T (PBS-Lonza, catalog number BE17-156Q, supplemented with 0.05% Tween 20-Sigma, catalog number P1379) to remove unbound antibodies, the unoccupied surfaces were blocked with 3% (w / v) BSA at RT for 60 ± 5 min using MSD blocking agent (Blocker) A in PBS-T, followed by washing with PBS-T. Calibration samples used to generate the reference curve are prepared by blending 2% pooled mouse plasma K2EDTA (BIOIVT, catalog number MSE00PLK2PNN) in the assay buffer with serially diluted 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). Quality control samples used to confirm the validity of individual assays are prepared by blending undiluted pooled mouse plasma K2EDTA with three antibody concentrations covering the following ranges of the reference curve: 0.5 µg / mL (for low-concentration quality control; LQC), 1.75 µg / mL (for medium-concentration quality control; MQC), and 15 µg / mL (for high-concentration quality control; HQC).

[0537] Next, the coated plates were incubated at RT for 90 ± 5 min with 50 μL of mouse plasma samples, calibration samples, and quality control samples diluted in assay buffer (supplemented with 1% [w / v] MSD blocking agent A [Meso Scale Discovery-MSD-, catalog number R93AA-1] in PBS-T [PBS-Lonza, catalog number BE17-156Q- supplemented with 0.05% Tween 20-Sigma, catalog number P1379]. After washing with PBS-T, the plate was incubated at RT for 90 ± 5 min with SULFO-TAG conjugated anti-huIgG detection antibody (IgG2amm-1015-4A01-ST; Genmab, batch number 210329_PSM_0053#001; IgG2amm-1015-4A01 antibody labeled with MSDGOLD SULFO-TAG NHS ester [MSD, catalog number R91AO-1]). After washing with PBS-T, an electrochemiluminescence (ECL) signal was generated by adding a read buffer containing tripropylamine (TPA) (MSD GOLD read buffer, catalog number R92TG-2) to the electrode surface of the plate, which was electrochemically stimulated on the SULFO-TAG immobilized antibody. Electrochemiluminescence (ECL) signals were measured at 620 nm on a MESO Sector S600 plate reader (MSD) and processed using SoftMax® Pro GxP software (MolecularDevices).

[0538] The predicted IgG concentration-time curves for WT huIgG in mice without target binding were based on a two-compartment PK model definition for WT huIgG antibodies with linear clearance, as described in the literature (Bleeker WK et al., 2001. Accelerated autoantibody clearance by intravenous immunoglobulintherapy: studies in experimental models to determine the magnitude and timecourse of the effect. Blood 98: 3136-3142). This model appropriately describes the PK distribution observed in previous internal studies in mice with other (unbound) huIgG molecules.

[0539] PK parameters were derived using Phoenix WinNonlin software (Certara) with a non-atrioventricular method of IV (or related extravascular) drug administration. The following parameters were calculated:

[0540] · C max - The maximum observed antibody concentration (μg / mL) is defined as the highest antibody concentration observed in a blood sample after antibody injection.

[0541] · t max -Observed C max The sampling time point (h).

[0542] · t ½ - The terminal elimination half-life (h) is determined by linear regression of at least three data points from the terminal elimination half-life of a log(concentration) versus time plot (d).

[0543] · AUC inf - The area under the plasma concentration-time curve from time point zero (t=0) to infinity (h μg / mL). At t=0 and the last measurement time point (t... last The area between 21 d) is calculated using the linear up-log-linear down trapezoidal method. From t last The area up to infinity was calculated using the elimination rate (λz) estimated between 3 and 21 days after antibody injection.

[0544] • CL - Systemic plasma clearance (mL / h / kg), expressed as dose / AUC inf Calculation. For mice with extravascular PK distribution, the calculated CL is the CL implicitly normalized to epigenetic bioavailability (F).

[0545] PK parameters are summarized as mean ± standard deviation (SD) for each treatment group, but t-values ​​are used instead. max Except for the median and range, the results are summarized. The results are visualized using GraphPad Prism.

[0546] IgG1-CD134-003-HC6LC2-RR exhibited typical PK properties consistent with intravenously administered non-target-binding human IgG antibodies in mice, showing maximum exposure at the earliest tested post-injection time point, followed by a two-phase decline. Figure 37 IgG1-CD134-003-HC6LC2-RR showed slightly lower total CL and slightly longer t compared to two control antibodies targeting chimeric OX40. ½Within the tested dose range, the C values ​​of all three antibodies were... max resemblance( Figure 38 (Table 20).

[0547] Table 20: Antibody pK parameters for each treatment group. This shows the antibody CL and t values ​​of three mice in each treatment group. ½ AUC inf and C max The mean ± SD, and antibody t max The median along with the range, unless otherwise specified.

[0548]

[0549] a The mean and SD are based on samples from two mice.

[0550] These data together indicate that the PK properties of IgG1-CD134-003-HC6LC2-RR are comparable to those of WT huIgG in the absence of target binding.

[0551] Example 25: In vivo antitumor efficacy of IgG1-CD134-003-HC6LC2-RR

[0552] Because of the lack of binding to mouse OX40 (mOX40) (as demonstrated in Example 5), the antitumor activity of IgG1-CD134-003-HC6LC2-RR was evaluated in C57BL / 6 mice genetically engineered to express the extracellular domain of human OX40 (hOX40 knock-in [KI] mice) possessing 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. Furthermore, pharmacodynamic changes in response to treatment with IgG1-CD134-003-HC6LC2-RR were studied in the peripheral blood of hOX40 KI mice carrying MC38.

[0553] hOX40 KI mice based on the C57BL / 6 background (strain C57BL / 6-Tnfrsf4) tm1(TNFRSF4)The HOX40 KI mouse (Catalogue No. 110014) was obtained from Beijing Biocytogen Co., Ltd. and is characterized by its humanized drug target (OX40) in immune-active mice. Mice were housed and transferred from Biocytogen Co., Ltd. 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 disposed of according to good animal practices as defined by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All experimental data management and reporting procedures were strictly in accordance with applicable Crown Bioscience, Inc. guidelines and standard operating procedures. 8- to 9-week-old female HOX40 KI mice were subcutaneously injected (SC) with 1 × 10⁻⁶ oz. 6 One MC38 cell (FuDan IBS cell center, catalog number 1101MOU-PUMC000523, 100 µL in PBS) was injected into the lower abdominal region. Tumor growth was assessed three times weekly using calipers. Tumor volume (mm) 3 The caliper measurement is calculated as ([length] × [width] × [width]) / 2, where the length is the longest tumor size and the width is the longest tumor size perpendicular to the length.

[0554] The study consisted of antitumor activity studies and pharmacodynamic (PD) studies. Mice were randomly assigned based on tumor volume using the Matched Distribution method (StudyDirector™ software). Of the 84 animals seeded with MC38 cells, those with tumor volume of 58.2 mm were initially selected. 3 Forty mice with an average tumor volume of 63.6 mm were selected and divided into four groups of 10 mice each to evaluate antitumor activity (Table 21). The remaining 44 animals were randomly assigned, with those having a tumor volume of 63.6 mm being selected. 3 Sixteen mice with the average tumor volume were selected and divided into four groups of four mice per group for PD analysis (referred to as PD Part 1) (Table 22). Because some blood clots formed in tubes during blood collection from these 16 animals (PD Part 1), it was decided to include another 16 mice for Part 2 of the study (PD Part 2). For this purpose, the remaining 28 mice were randomly assigned a third time seven days later, resulting in four groups of four mice each with a tumor volume of 146.81 mm. 3 The average tumor volume.

[0555] Table 21: Treatment groups and dosage levels (antitumor activity fraction) in hOX40 KI mice

[0556]

[0557] Table 22: Treatment groups and dosage levels in hOX40 KI mice (PD Parts 1 and 2)

[0558]

[0559] Treatment began on the day of randomization (day 0). Mice were administered IgG1-CD134-003-HC6LC2-RR (1, 5, or 20 mg / kg) or IgG1-b12-FEAL control antibody (20 mg / kg) via intraperitoneal injection (IP). The volume of administration per mouse was adjusted for body weight (10 μL / g in PBS). Tumor growth was assessed three times weekly after the start of administration. To assess antitumor activity over time, animals were administered twice weekly for three weeks (2QW × 3). To assess the pharmacodynamics of PD Parts 1 and 2, animals were administered twice daily on days 0 and 3.

[0560] Tumor growth inhibition (TGI) was measured based on the difference between the mean tumor volume on day 0 (at the start of administration) and the last day of administration for all groups, and was calculated for each treatment group using the following formula:

[0561]

[0562] Where Tt = mean tumor volume in the treatment group on the last day of completion across all groups, Ct = mean tumor volume in the control group on the last day of completion across all groups, T0 = mean tumor volume in the treatment group on day 0, and C0 = mean tumor volume in the control group on day 0. The TGI response of this study is classified in detail in Table 23.

[0563] Table 23: Classification of Tumor Growth Inhibition

[0564]

[0565] When the tumor volume exceeds 1,500 mm 3 Or, when an animal reaches another humane endpoint (e.g., weight loss > 20% relative to day 1 of administration [i.e., day 0], has tumor ulcers with a diameter greater than 5 mm, or observes severe clinical signs), the experiment on an individual mouse is terminated. Any animal exhibiting ulcers or necrotic tumors is immediately separated and housed individually.

[0566] For progression-free survival analysis, 500 mm was applied. 3 Tumor volume cutoff value. Tumor progression >500 mm 3The time points were calculated using GraphPad Prism with nonlinear regression curve fitting of individual tumor growth curves, assuming an exponential growth equation, and plotted as Kaplan-Maill curves. Progression-free survival between the treatment and control groups was analyzed using a nonparametric log-rank Mantel-Cox analysis, followed by paired comparisons using SPSS software.

[0567] To evaluate the pharmacodynamic effects of IgG1-CD134-003-HC6LC2-RR treatment, mice were euthanized after two doses, and changes in peripheral blood immune cell populations, including CD4+, were analyzed on day 5. + and CD8 + T cell proliferation and activation. On day 5 of the experiment, approximately 50 to 100 μL of whole blood was collected via cardiac puncture under isoflurane anesthesia and transferred to K2-EDTA tubes (BD, catalog number 36597499). Whole blood samples (both PD Part 1 [9 animals] and PD Part 2 [14 animals]; whole blood samples containing blood clots were excluded from the analysis; 50 to 100 μL) for cell surface staining were added to mouse BD FcBlock at 1 μg / mL. TM (Catalogue No. 553141; diluted in PBS + 0.09% sodium azide) Incubate at 4°C in the dark for 10 minutes. Then stain the cells with either the antibody group used for general immunophenotypes (Table 24) or the antibody group used for T cell characteristics (Table 25).

[0568] For general immunophenotypes, add the antibody mixture (except for the anti-FOXP3 antibody) detailed in Table 24, diluted in Fc blocking buffer, to each sample and stain at 4°C for 30 min. Add 2 mL of room temperature erythrocyte lysis buffer (1×; BioGems, catalog number 64010-00100) to each tube, incubate at RT in the dark for 10 min, and gently vortex to lyse the cells. Wash the samples twice with PBS, centrifuge at 300×g for 5 min, and discard the supernatant. After resuspending the cell pellet, add 200 μL of fixation / permeabilization buffer (eBioscience, catalog number 00-5523), vortex to mix, and incubate at room temperature in the dark for 30 min. Wash the cells twice with 1X permeabilization buffer (eBioscience, catalog number 00-5523; diluted in distilled H2O), then centrifuge and decant the supernatant. Add 100 µL of FOXP3 antibody in permeation buffer to each sample and incubate at RT in the dark for 30 min. After washing the cells twice with 2 mL of PBS, resuspend the cells in 250 μL of PBS and analyze them on a BD LSR Fortessa™ X-20 cell analyzer (BDBiosciences). Data were analyzed using Kaluza software. Absolute counts were calculated using 123 count eBeads (ThermoFisher Scientific, catalog number 01-1234-42). Add 100 µL of beads to each sample to be counted. Calculate the absolute count using the following formula:

[0569]

[0570] For T cell characterization, after blocking the cells, ADPGK tetramer was added to each sample, gently vortexed, and incubated at 4°C in the dark for 30 min. Then, the antibody mixture (except Ki67), diluted in Fc blocking buffer and detailed in Table 25, was added to each sample and stained at 4°C for 30 min. Cells were lysed and washed as described above. After resuspending the cell pellet, cells were stained with Ki67 as described above. After washing the cells twice with 2 mL of PBS, the cells were resuspended in 250 µL of PBS and analyzed on a BD LSR Fortessa X-20 cell analyzer (BD Biosciences). Data were analyzed using Kaluza analysis software. Absolute counts were calculated using the 123 count eBead method as described above.

[0571] Table 24: Flow cytometry measurements used for immunophenotypes

[0572]

[0573] Table 25: Flow Cytometry Measurements for T Cell Characterization

[0574]

[0575] Plasma samples were obtained from mice in PD Part 1 and Part 2 on days 0, 2, and 5. 50 μL of blood was aspirated from each mouse 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). Before centrifugation (8000 RPM, 4°C for 5 min), the tubes were thoroughly mixed to ensure complete contact of the blood sample with the anticoagulant. After centrifugation, the supernatant was transferred to 1.5 mL microcentrifuge tubes.

[0576] The levels of mouse cytokines (IFNγ, IL-2, IL-4, IL-10, TNFα, IP-10, MCP-1, IL-27p28) in plasma samples were determined using a multi-task ECLIA with the V-PLEX Pro-inflammatory Group 1 Mouse Kit (MSD LLC, catalog number K15048D-2) on a MESO QuickPlex SQ 120 instrument (MSD, LLC., catalog number AI0AA-0) according to the manufacturer's instructions. Cytokine concentrations were calculated using a standard curve fitted with a four-parameter logic (4PL) and plotted using MSDWorkbench software and GraphPad Prism software.

[0577] Treatment was well tolerated, and no clinical signs of disease or weight loss in response to antibody therapy were reported in any of the mice in these studies. Treatment with IgG1-CD134-003-HC6LC2-RR at 5 mg / kg significantly delayed tumor outgrowth compared to IgG1-b12-FEAL treatment (Manwhitney, P=0.0011), and all treatment groups remained intact on the last day as measured at 14 days after treatment initiation, achieving a TGI-based intermediate response (50.0%). Figure 39 A, B; Table 26). Compared with the control group, 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 on day 14 (Mantel-Cox, P=0.3527 and P=0.0753, respectively). Mice treated with IgG1-CD134-003-HC6LC2-RR (5 mg / kg or 20 mg / kg) had significantly longer progression-free survival compared with the control group treated with IgG1-b12-FEAL (Mantel-Cox, P<0.001 and P=0.031, respectively). Figure 39 C and Table 27).

[0578] Table 26: Man Whitney analysis of tumor volume in hOX40 KI mice after treatment. Tumor volume in the IgG1-CD134-003-HC6LC2-RR treatment group was compared with tumor volume in the IgG1-b12-FEAL control group on the last day (day 14) after all groups were completed, using Man Whitney analysis. TGI values ​​for each treatment group were calculated based on tumor volume on day 14. = P<0.01.

[0579]

[0580] Table 27: Mantel-Cox analysis of progression-free survival in hOX40 KI mice after treatment. Progression-free survival (based on 500 mm) 3 The tumor volume cutoff value was used to identify the overall difference in survival between groups using Mantel-Cox analysis, followed by paired comparisons between groups (SPSS). = P<0.05, and = P<0.001.

[0581]

[0582] The percentage of tumor-specific CD8+ T cells after treatment was determined using MHC class I ADPGK tetramer.

[0583] Compared with mice treated with IgG1-b12-FEAL at 20 mg / kg, significantly increased CD4 counts were observed in the peripheral blood of hOX40 KI mice carrying MC38 tumors after treatment with IgG1-CD134-003-HC6LC2-RR at 1, 5, and 20 mg / kg. + percentage of T cells ( Figure 40 A). CD4 + The absolute number of T cells remained unchanged, which may indicate an increase in CD4. + Cell frequency may be attributed to reduced CD8. + T cells ( Figure 40 B, D). Peripheral blood CD4+ was induced in hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR. + T cell proliferation and activation are significantly enhanced by the increased expression of proliferation marker Ki67 and activation markers CD25, IA / IE, PD-1, and 4-1BB CD4.+ The percentage of T cells is shown in the figure. Figure 41 (A to E). Furthermore, compared to treatment with IgG1-b12-FEAL, IgG1-CD134-003-HC6LC2-RR treatment resulted in a significant reduction in CD4+ expression of OX40. + The percentage of T cells, possibly due to OX40 shedding (data not shown). All observed against CD4 + The effects of T cells were all dose-independent, except for the upregulation of 4-1BB, which showed a dose-dependent effect. Figure 41 E).

[0584] Reduced CD8 levels were observed in peripheral blood after treatment with IgG1-CD134-003-HC6LC2-RR at doses of 1, 5, and 20 mg / kg. + The percentage and absolute number of T cells, along with the increased proliferation of CD8 cells. + Ki67 + T cell percentage (1, 5, and 20 mg / kg) and tumor-specific (ADPGK+) CD8 + T cell percentage and absolute number (1 and 5 mg / kg); Figure 40 C, D, Figure 42 ).

[0585] IgG1-CD134-003-HC6LC2-RR treatment induces CD8 in peripheral blood + T cell activation, such as through significantly increased expression of CD25 and CD8. + T cells (all doses of IgG1-CD134-003-HC6LC2-RR), IA / IE (1 and 5 mg / kg), PD-1 (all doses of IgG1-CD134-003-HC6LC2-RR), and 4-1BB (5 and 20 mg / kg). Fi...

Claims

1. A method for treating a disease in a subject, preferably a method for reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject i) an antibody capable of binding to human OX40; and ii) a PD-1 inhibitor.

2. The method of claim 1, wherein the antibody comprises an antigen-binding region comprising a heavy chain variable (VH) region, wherein CDR1, CDR2 and CDR3 comprise sequences as listed in SEQ ID NO: 16, 17 and 18, respectively, and a light chain variable (VL) region, wherein CDR1, CDR2 and CDR3 comprise sequences as listed in SEQ ID NO: 20, DAS and 21, respectively.

3. The method of claim 1 or 2, wherein the antibody is capable of binding to human OX40 having the sequence SEQ ID NO:

52.

4. The method as described in any of the preceding claims, wherein the antibody is capable of binding to the CRD1 domain of human OX40.

5. The method as described in any of the preceding claims, wherein the antibody has a 3 x 10⁻⁶ Ω·cm ratio to human OX40. -9 M to 4x 10 -9 Binding affinity of M to K D .

6. The method as described in any of the preceding claims, wherein the antibody has a 3.4 x 10⁻⁶ phosphate density against human OX40. -9 Binding affinity of M to K D .

7. The method as described in any of the preceding claims, wherein the antibody is capable of binding to cynomolgus monkey OX40 having the sequence SEQ ID NO:

51.

8. The method of any of the preceding claims, wherein the antibody comprises a VH region having the sequence listed in SEQ ID NO: 15 and a VL region having the sequence listed in SEQ ID NO:

19.

9. The method of any of the preceding claims, wherein the antibody is a humanized or chimeric antibody.

10. The method of any of the preceding claims, wherein the antibody further comprises an Fc region, which is a human IgG isotype, optionally a modified human IgG comprising one or more amino acid substitutions.

11. The method of claim 10, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1.

12. The method of claim 10 or 11, wherein the Fc region is a human IgG1 Fc region containing the P329R mutation and the E345R mutation, wherein the amino acid positions are numbered according to the Eu number.

13. The method of any one of claims 10 to 12, wherein the Fc region is an allotype of human IgG1mf, human IgG1ma, human IgG1mx, or human IgG1mz.

14. The method of any one of claims 10 to 13, wherein the Fc region is a human IgG1mf allotype.

15. The method of any one of claims 10 to 14, wherein the Fc region comprises the sequence listed in SEQ ID NO:

3.

16. The method as described in any of the preceding claims, wherein the antibody is bivalent.

17. The method of any of the preceding claims, wherein the antibody is a monospecific, bispecific, or multispecific antibody.

18. The method of any of the preceding claims, wherein the antibody is a full-length antibody.

19. The method of any of the preceding claims, wherein the antibody has a heavy chain constant region comprising a sequence selected from the group comprising SEQ ID NO: 58, 59, 60 and 61.

20. The method of any of the preceding claims, wherein the antibody has a heavy chain constant region comprising the sequence listed in SEQ ID NO:

58.

21. The method of any of the preceding claims, wherein the antibody comprises a heavy chain (HC) as listed in SEQ ID NO: 13 and a light chain (LC) as listed in SEQ ID NO:

14.

22. The method as described in any of the preceding claims, wherein the antibody is agonistic.

23. The method as described in any of the preceding claims, wherein the antibody has increased agonistic activity compared to a wild-type parent antibody that does not have the P329R and E345R mutations.

24. The method of any of the preceding claims, wherein the antibody induces increased T cell proliferation.

25. The method as described in any of the preceding claims, wherein the antibody induces increased T cell proliferation compared to a parental antibody that does not have the P329R and E345R mutations.

26. The method of any of the preceding claims, wherein when measured as described in Example 7 herein, the antibody is capable of inducing the proliferation of human T cells, such as CD4. + and CD8 + T cells, such as helper T cells and cytotoxic T cells.

27. The method of any of the preceding claims, wherein the antibody comprises a modified heavy chain constant region such that the antibody induces one or more Fc-mediated effector functions to a lesser extent relative to a parent antibody without the P329R and E345R mutations.

28. The method of claim 27, wherein the one or more Fc-mediated effector functions are reduced by at least 20%, such as at least 30% or at least 40%, or at least 50% or at least 60% or at least 70%, or at least 80% or at least 90%.

29. The method of claim 27 or 28, wherein the antibody does not induce one or more Fc-mediated effector functions.

30. The method of any one of claims 27 to 29, wherein the one or more Fc-mediated effector functions are selected from the group consisting of complement-dependent cytotoxicity (CDC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding.

31. The method of any one of claims 29 or 30, wherein the antibody does not induce C1q binding.

32. The method of any of the preceding claims, wherein the PD-1 inhibitor is an antibody that binds to PD-1 or PD-L1, preferably an antibody that is an antagonist of PD-1 / PD-L1 interaction and / or an antibody that is a PD-1 or PD-L1 blocking antibody.

33. The method as described in any of the preceding claims, wherein PD-L1 is human PD-L1, particularly human PD-L1 contained in the sequence listed in SEQ ID NO:

97.

34. The method of any of the preceding claims, wherein PD-1 is human PD-1, preferably said PD-1 has or comprises an amino acid sequence as listed in SEQ ID NO: 98 or SEQ ID NO: 99, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence listed in SEQ ID NO: 98 or SEQ ID NO: 99, or is an immune fragment thereof.

35. The method of any of the preceding claims, wherein the PD-1 inhibitor is an isotype antibody selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, such as an IgG1 isotype antibody.

36. The method of any of the preceding claims, wherein the PD-1 inhibitor is a full-length antibody or antibody fragment, such as a full-length IgG1 antibody.

37. The method of any of the preceding claims, wherein the PD-1 inhibitor is a monospecific antibody.

38. The method of any of the preceding claims, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences listed in SEQ ID NO: 79, 80 and 81 respectively; and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences listed in SEQ ID NO: 82, LAS and SEQ ID NO: 83 respectively.

39. The method of any of the preceding claims, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 84, and a VL region comprising the amino acid sequence of SEQ ID NO:

85.

40. The method of any of the preceding claims, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 86, and a light chain comprising the amino acid sequence of SEQ ID NO:

87.

41. The method of any one of claims 1 to 37, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences listed in SEQ ID NO: 89, 90 and 91 respectively; and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences listed in SEQ ID NO: 93, QAS and SEQ ID NO: 94 respectively.

42. The method of claim 41, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 88, and a VL region comprising the amino acid sequence of SEQ ID NO:

92.

43. The method of claim 41 or 42, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a light chain comprising the amino acid sequence of SEQ ID NO:

96.

44. The method of any one of claims 1 to 37, wherein the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiprimab, dostalimumab, rivanilimab, toripalimab, vopalimumab, spartazumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680), axolizumab, or biosimilars thereof; preferably, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiprimab, dostalimumab, rivanilimab, toripalimab, vopalimumab, spartazumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-514 (MEDI0680), axolizumab or biosimilars thereof; more preferably, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cimiprizumab, dostalimab, riverimab, toripalimab or biosimilars thereof.

45. The method of any one of claims 1 to 37, wherein the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab, KN035, cocibelimab, AUNP12, CA-170, BMS-986189 or biosimilars thereof; preferably, the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab, KN035, cocibelimab or biosimilars thereof; more preferably, the PD-1 inhibitor is a PD-L1 inhibitor selected from the group consisting of: atezolizumab, avelumab, durvalumab or biosimilars thereof.

46. ​​The method of any of the preceding claims, wherein the method comprises administering to the subject one or more additional therapeutic agents, such as one or more chemotherapeutic agents, such as platinum-based compounds (such as cisplatin, carboplatin), taxane-based compounds (such as paclitaxel, nab-paclitaxel), nucleoside analogs (such as gemcitabine), antifolate agents (such as pemetrexed), and any combination thereof.

47. The method as described in any of the preceding claims, wherein the subject is a human subject.

48. The method as claimed in any of the preceding claims, wherein the disease, tumor, or cancer is a solid tumor, such as colorectal cancer.

49. A kit comprising (i) an antibody capable of binding to human OX40, (ii) a PD-1 inhibitor, and optionally (iii) one or more additional therapeutic agents, such as one or more chemotherapeutic agents.

50. The kit of claim 49, wherein the antibody is as defined in any one of claims 1 to 48 and / or the PD-1 inhibitor is as defined in any one of claims 1 to 48.

51. The kit of claim 49 or 50, wherein the antibody, the PD-1 inhibitor, and one or more additional therapeutic agents, if present, are for systemic administration, particularly for injection or infusion, such as intravenous injection or infusion.

52. The kit according to any one of claims 49 to 51, for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating the subject's cancer.

53. The kit for use as claimed in claim 52, wherein the subject is a human subject, and / or the disease, tumor, or cancer is a solid tumor, such as colorectal cancer.

54. A pharmaceutical composition comprising (i) an antibody capable of binding to human OX40, (ii) a PD-1 inhibitor, and (iii) optionally a pharmaceutically acceptable carrier.

55. The pharmaceutical composition of claim 54, wherein the antibody is as defined in any one of claims 1 to 48 and / or the PD-1 inhibitor is as defined in any one of claims 1 to 48.

56. The pharmaceutical composition of claim 54 or 55, used in a method of treating a subject's disease, preferably used in a method of reducing or preventing tumor progression in the subject or treating the subject's cancer.

57. The pharmaceutical composition for use as claimed in claim 56, wherein the subject is a human subject, and / or the disease, tumor, or cancer is a solid tumor, such as colorectal cancer.

58. An antibody capable of binding to human OX40, used in a method of treating a subject's disease, preferably used in a method of reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering (i) the antibody to the subject; and (ii) a PD-1 inhibitor.

59. The antibody for use as claimed in claim 58, wherein the method is as defined in any one of claims 1 to 48, and / or the antibody is as defined in any one of claims 1 to 48, and / or the PD-1 inhibitor is as defined in any one of claims 1 to 48.

60. A PD-1 inhibitor for use in a method of treating a subject's disease, preferably for use in a method of reducing or preventing tumor progression in the subject or treating cancer in the subject, the method comprising administering to the subject (i) an antibody capable of binding to human OX40; and (ii) the PD-1 inhibitor.

61. The PD-1 inhibitor for use as claimed in claim 60, wherein the method is as defined in any one of claims 1 to 48, and / or the antibody is as defined in any one of claims 1 to 48, and / or the PD-1 inhibitor is as defined in any one of claims 1 to 48.

62. Use of an antibody capable of binding to human OX40 in the preparation of a medicament, preferably, said medicament in combination with a PD-1 inhibitor for reducing or preventing tumor progression in a subject or for treating cancer in a subject.

63. Use of a PD-1 inhibitor in the preparation of a medicament, preferably, the medicament is combined with an antibody capable of binding to human OX40 for the purpose of reducing or preventing tumor progression in a subject or treating cancer in a subject.

64. (i) the use of an antibody capable of binding to human OX40 and (ii) a PD-1 inhibitor in the preparation of a medicament, preferably, said medicament is used to reduce or prevent tumor progression in a subject or to treat cancer in a subject.

65. The use as claimed in any one of claims 62 to 64, wherein the subject is a human subject, and / or the tumor or cancer is a solid tumor, such as colorectal cancer.

66. A medical preparation comprising an antibody and a PD-1 inhibitor as defined in any one of claims 1 to 65.

67. The medical preparation of claim 66, for use in the method of any one of claims 1 to 48.

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