RU2793167C2 - Monospecific and bispecific proteins with immune checkpoint regulation for cancer treatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody or its antigen-binding portion that binds to OX40 (CD134) is proposed. Also a bispecific antibody that binds to OX40 and a programmed cell death protein 1 (PD-L1) ligand is provided. Nucleic acids encoding the said antibodies are also disclosed. In addition, the use of these antibodies, including as part of a pharmaceutical composition, for the treatment of cancer selected from prostate cancer, lung cancer, non-small cell lung cancer, melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma and ovarian cancer is proposed.

EFFECT: invention provides improved agonistic activity of antibodies in T-cell activation.

14 cl, 36 dwg, 2 tbl, 7 ex

Description

Background of the Invention

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to an antibody. More specifically, the present invention relates to an antibody for the treatment of cancer.

Description of the Prior Art

[0002] The two main types of lymphocytes in humans are T (derived from the thymus) and B (derived from the bone marrow). These cells are derived from hematopoietic stem cells in the fetal bone marrow and liver, which are involved in the lymphoid pathway of development. The progeny of these stem cells follow different pathways, maturing into B- or T-lymphocytes. The development of human B-lymphocytes occurs exclusively in the bone marrow. On the other hand, T cells develop from immature progenitors that leave the bone marrow and travel through the bloodstream to the thymus, where they proliferate and differentiate into mature T lymphocytes.

[0003] T cells

[0004] T cells are the most abundant (approximately 75% of blood lymphocytes) and strong immune killer cells. The role of effector T cells in the antitumor immune response is fully supported by in vitro studies and the observation that high infiltration of CD8+ T cells in several tumor types correlates with a favorable clinical prognosis (Fridman et al., 2012). Activation of effector naive T cells requires at least three additional signals: (i) interaction of TCR-CD3/Ag-MHC via co-receptors (CD4 or CD8), (ii) binding of co-stimulatory molecules such as CD80 or CD86 to CD28, CD40/CD40L, and (iii) accessory molecules such as cytokines.

[0005] Costimulation or provision of two different signals to T cells is a widely accepted model for the activation of resting T lymphocytes by antigen presenting cells (APC) (Lafferty and Cunningham, 1975). This model further provides for auto-non-auto discrimination and immune tolerance (Bretscher and Cohn, 1970; Bretscher, 1999; Jenkins and Schwartz, 1987). The primary signal, or antigen-specific signal, is transmitted through the T cell receptor (TCR) upon recognition of a foreign antigenic peptide presented in the context of a major histocompatibility complex (MHC). The second or costimulatory signal is delivered to T cells by costimulatory molecules expressed on antigen presenting cells (APC) and induces T cells to activate clonal expansion, cytokine secretion and effector function (Lenschow et al., 1996). In the absence of costimulation, T cells may become unresponsive to antigen challenge, fail to elicit an effective immune response, and furthermore may lead to depletion or tolerance to foreign antigens.

[0006] Immune checkpoint protein: PD-L1 and OX40

[0007] Immune checkpoints refer to a group of inhibitory and stimulatory pathways primarily initiated by ligand-receptor interactions that bind the immune system, in particular T-cell mediated immunity, to maintain self-tolerance and modulate the duration and amplitude of physiological responses in peripheral tissues to in order to minimize damage to collateral tissues in normal conditions (Pardoll, 2012). Tumor cells choose certain checkpoint pathways as the main mechanism of immune resistance. For example, the programmed cell death protein 1 ligand, PD-L1, is typically activated on the surface of human cancer tumor cells. Interaction of PD-L1 with its receptor, PD-1, expressed on tumor infiltrating lymphocytes (TILs), specifically T cells, inhibits the local T cell-mediated response, avoiding immune surveillance (Liang et al. al., 2006; Sznol and Chen, 2013). Thus, inhibition of immunosuppressive signals on cancer cells or direct agonistic stimulation of T cells results in and/or induces a strong sustained antitumor immune response. Recent clinical studies strongly suggest that blocking immune checkpoint proteins via antibodies or modulating with soluble ligands or receptors is the most promising approach to activate therapeutic antitumor immunity (Topalian et al., 2014). Currently, antibodies to PD-1 and to CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) have been approved by the FDA for the treatment of diseases such as melanomas.

[0008] Another stimulator molecule is the OX40 receptor (CD134), a member of the TNFR superfamily, which is membrane-bound and is predominantly expressed on activated CD4+ T cells (Paterson et al., 1987). Signaling through the OX40 receptor (hereinafter "OX40") is costimulatory for effector T cells and causes T cell proliferation (Watts, 2005; Weinberg et al., 1994). Research on OX40 suggests that its primary role is to determine the number of effector T cells that accumulate in primary immune responses and ultimately determine the number of memory T cells that subsequently develop and survive (Croft, 2003). A number of in vitro studies have shown that OX40 provides a costimulatory signal leading to increased T cell proliferation and cytokine production.

[0009] Bispecific/bifunctional antibody

[0010] The idea of using bispecific antibodies to effectively retarget effector immune cells to tumor cells appeared in the 1980s (Karpovsky et al., 1984; Perez et al., 1985; Staerz et al., 1985). Bispecific scaffolds are generally classified into two main groups with different pharmacokinetic properties based on the absence or presence of an Fc moiety, IgG-like molecules, and small recombinant bispecific formats, most of which are derived from the single chain variable fragment (scFv). Due to their compact size, antibody fragments tend to enter tumors more efficiently than IgG-like molecules, but this advantage is mitigated by a short serum half-life (several hours), limiting overall uptake and residence time in the tumor (Goldenberg et al. ., 2007). In contrast, the presence of an Fc fragment that binds to neonatal Fc receptors provides a long serum half-life (>10 days) for IgG-like formats, promoting tumor uptake and retention but limiting tumor entry.

[0011] Recent studies have identified the therapeutic efficacy of immunotherapy, a class of cancer treatment that uses the patient's own immune system to destroy cancer cells. The presence within a tumor of a family of negative regulatory molecules collectively known as "checkpoint inhibitors" can inhibit the function of T cells to suppress antitumor immunity. Checkpoint inhibitors such as CTLA-4 and PD-1 impair T cell proliferation and cytokine production. Targeted blockade of CTLA-4 or PD-1 with monoclonal antagonist antibodies (mAbs) releases the brakes on T cells to enhance antitumor immunity. Optimal CD8 T cell kill responses also require T cell receptor activation plus costimulation, which can be achieved by ligation of members of the tumor necrosis factor receptor family, including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with an agonistic anti-OX40 mAb increases T cell differentiation and cytolytic function leading to enhanced antitumor immunity against various tumors. When used alone, these drugs can induce strong clinical and immunological responses in patients with metastatic disease (Linch et al., 2015).

Brief summary of the present invention

[0012] The present disclosure is intended to investigate a bispecific immunomodulatory antibody for the treatment of a patient with cancers such as prostate cancer, lung cancer, NSCLC, melanoma, lymphoma, breast cancer, head and neck cancer, RCC, or ovarian cancer.

[0013] The present invention relates to an antibody or antigen-binding portion thereof that binds to OX40 (CD134) comprising: a heavy chain variable region containing an amino acid sequence that is characterized by a sequence homology of at least about 80% with respect to the amino acid sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 8, amino acids 128-246 of SEQ ID NO. 10 and amino acids 124-241 of SEQ ID NO. 13 and a light chain variable region comprising an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-108 of SEQ ID NO. 5, 1-108 of SEQ ID NO. 7, 1-112 of SEQ ID NO. 10 and 1-108 of SEQ ID NO. 13. [0014] In one embodiment, the antibody or antigen-binding portion thereof is a single chain variable fragment (scFv) sequence selected from the group consisting of SEQ ID NO. 10, 11, 12 and 13.

[0015] In one embodiment, the antibody, or antigen-binding portion thereof, is a bispecific antibody.

[0016] In one embodiment, the bispecific antibody is an immune checkpoint protein binding site.

[0017] In one embodiment, the immune checkpoint protein binding site includes a programmed cell death protein 1 (PD-L1) ligand binding site, a PD-1 binding site, an epidermal growth factor receptor (EGFR) binding site, an epidermal human growth factor 2 (HER2), cytotoxic T-lymphocyte-associated antigen binding site 4 (CTLA-4), or lymphocyte activation gene binding site 3 (LAG3).

[0018] The present invention also provides an antibody, or an antigen-binding portion thereof, that binds to PD-L1, comprising a heavy chain variable domain comprising an amino acid sequence that has a sequence homology of at least about 80% with respect to an amino acid sequence selected from group consisting of SEQ ID NO. 2 and SEQ ID NO. 4 and a light chain variable domain comprising an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-111 of SEQ ID NO. 1 and 1-110 of SEQ ID NO. 3.

[0019] The present invention also provides a bispecific antibody comprising at least one polypeptide chain, wherein the polypeptide chain contains an OX40 binding site and a PD-L1 binding site. The OX40 binding site contains a heavy chain variable region containing an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 8, amino acids 128-246 of SEQ ID NO. 10 and amino acids 124-241 of SEQ ID NO. 13 and a light chain variable region comprising an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-108 of SEQ ID NO. 5, 1-108 of SEQ ID NO. 7, 1-112 of SEQ ID NO. 10 and 1-108 of SEQ ID NO. 13. The PD-L1 binding site contains a heavy chain variable domain containing an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 4 and a light chain variable domain comprising an amino acid sequence that has at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-111 of SEQ ID NO. 1 and 1-110 of SEQ ID NO. 3.

[0020] In one embodiment, the polypeptide chain further comprises an Fc domain, a Fab fragment, and a scFv. The Fab fragment is linked to the N-terminus of the Fc domain and the Fab fragment contains the PD-L1 binding site. scFv is linked to the C-terminus of the Fc domain, and scFv contains an OX40 binding site.

[0021] In one embodiment, the polypeptide chain further comprises a linker between the Fc domain and the scFv.

[0022] In one embodiment, the scFv comprises an amino acid sequence selected from the group consisting of amino acids 455-707 of SEQ ID NO. 18, 455-708 of SEQ ID NO. 19, 455-701 of SEQ ID NO. 20, 455-706 of SEQ ID NO. 21, 455-706 of SEQ ID NO. 22, 455-706 of SEQ ID NO. 23, 455-706 of SEQ ID NO. 24, 455-706 of SEQ ID NO. 25, 455-706 of SEQ ID NO. 26, 455-706 of SEQ ID NO. 27, 455-706 of SEQ ID NO. 28 and 455-706 of SEQ ID NO. 29.

[0023] In one embodiment, the bispecific antibody comprises one pair of polypeptide chains.

[0024] In one embodiment, the bispecific antibody is an IgG, IgE, IgM, IgD, IgA, or IgY antibody.

[0025] In one embodiment, the bispecific antibody is an IgG antibody.

[0026] In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0027] The present disclosure also provides an antibody-drug conjugate comprising a therapeutic agent and an antibody or antigen-binding portion that binds PD-L1 and/or OX40, wherein the therapeutic agent is covalently conjugated to the antibody or antigen-binding portion via a linker.

[0028] In one embodiment, the antibody or antigen-binding portion thereof is selected from the above antibody or antigen-binding portion.

[0029] The present disclosure also provides a pharmaceutical composition comprising an antibody, an antigen-binding portion thereof, or a bispecific antibody as defined above, and at least one pharmaceutically acceptable carrier.

[0030] The present disclosure also provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of an antibody, an antigen-binding moiety thereof, or a bispecific antibody as defined above.

[0031] In one embodiment, the cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), and cancer ovaries.

[0032] The present disclosure also provides a nucleic acid molecule encoding an antibody, an antigen-binding portion thereof, or a bispecific antibody as defined above.

[0033] It is to be understood that both the following general description and the following detailed description are provided by way of example and are intended to provide further explanation of the present invention as claimed.

Brief description of graphic materials

[0034] The present invention can be more fully understood by reading the following detailed description of the embodiment with reference to the accompanying drawings as follows:

[0035] Figure 1 shows immune checkpoints modulating T-cell mediated immunity. Antibody, either agonistic or antagonistic for checkpoints, such as ICOS, CD28, OX40 and CD27, or PD-1 , to CTLA4, to LAG3, to BTLA can be used to construct a bifunctional fusion protein, depending on the application.

[0036] FIG. 2A and 2B show direct ELISA screening of a phage clone for HEK293 cell-expressed PD-L1.

[0037] FIG. 3A and 3B show a screening of a phage clone by direct cell-based ELISA with expressed HEK293 OX40 cells.

[0038] FIG. 4 shows purified prototype antibodies specific for PD-L1 using SDS-PAGE with a non-reducing reagent for integrity and purity.

[0039] FIG. 5 shows purified prototype antibodies specific for OX40 using SDS-PAGE with a non-reducing or reducing reagent for integrity and purity.

[0040] FIG. 6 shows examples of direct ligand binding activity of purified antibody prototypes to immune checkpoint proteins and to PD-L1 against PD-L1. Wells precoated with ligand were first incubated with various concentrations of the antibody prototypes as indicated. Bound proteins were then detected with an HRP-conjugated goat anti-human Fab IgG specific antibody and OD450 readings plotted.

[0041] In FIG. 7 shows examples of direct ligand binding activity of purified prototype antibodies to immune checkpoint proteins and anti-OX40 OX40 antibodies. First, the pre-liganded wells were incubated with various concentrations of the prototype antibodies as indicated. Proteins were then detected with an HRP-conjugated goat anti-human Fab IgG specific antibody and OD450 readings were plotted.

[0042] FIG. 8 shows a flow analysis using PD-L1 expressing 293 cells. PD-L1 expressing HEK293 cells were first incubated with purified prototype antibodies and bound antibodies were detected with Alexa-488 conjugated goat anti-human IgG (H+L) followed by analysis. by means of a fluorescence activated cell sorter (FACS).

[0043] FIG. 9 shows a flow analysis using OX40-expressing 293 cells. OX40-expressing HEK293 cells were first incubated with purified anti-OX40 antibody prototypes, and bound antibodies were detected with Alexa-488-conjugated goat anti-human IgG (H+L) followed by FACS analysis. NS: no staining.

[0044] FIG. 10 shows blockage of PD-1/PD-L1 interaction with purified anti-PD-L1 antibodies. Purified antibodies, as indicated, were applied to biotinylated PD-L1-Fc and recombinant human PD-1/His (hPD-1/His) to assess the activity of inhibiting the PD-1/PD-L1 interaction. Binding of recombinant PD-L1-Fc and hPD-1/His was detected with streptavidin-HRP and analyzed by ELISA.

[0045] FIG. 11A and 11B show that prototype anti-PD-L1 antibodies at 1 or 10 μg/mL stimulate T cell proliferation and induce IL-2 and/or IFN-γ production in a mixed lymphocyte response (MLR) assay after 3 days. (FIG. 11A) or 5 days (FIG. 11B) after antibody exposure.

[0046] FIG. 12A shows the ability of an anti-OX40 antibody to increase CD3+ T cell activation by dose response as well as a reference antibody. In FIG. 12B shows the concentration of human IL-2 and IFN-α present in cell culture medium after 3 days of human T cell stimulation with plate-bound anti-CD3 antibodies and several concentrations of prototypical anti-OX40 antibodies.

[0047] FIG. 13A and 13B show the concentration of human IL-2 (FIG. 13A) and IFN-γ (FIG. 13B) present in cell culture media after 3 days of human T cell stimulation with plate-bound anti-CD3 antibody and several concentrations of OX40-specific antibody prototypes.

[0048] FIG. 14 shows the structure of an Fc heavy chain of an antibody fused to an OX40-specific scFv domain.

[0049] FIG. 15 shows exemplary PAGE gel analysis of anti-human OX40 immune checkpoint antibody fusion proteins. Purified fusion proteins, anti-PD-L1-OX40 scFv fusion proteins have been shown to have a molecular weight of approximately 220 kDa (non-reducing) and a heavy chain fusion of approximately 85 kDa and a light chain of approximately 25 kDa (reducing) when both antibodies are fused .

[0050] FIG. 16A and 16B show synergistic bispecific antibodies stimulating T cell activation to produce IL-2 and IFN-γ in a mixed lymphocyte reaction (MLR) assay after 3 days (FIG. 16A) or 5 days (FIG. 16B) with mono -, combined or anti-PD-L1-OX40 scFv bispecific antibodies.

[0051] FIG. 17A-17E respectively show aggregation and purity determination of bispecific antibodies, anti-PD-L1-OX40 Ab and anti-PD-L1-OX40 Ab V1 to V4, with 5 different linkers in OX40 scFv.

[0052] FIG. 18 shows sequence variants among scFv clone B17 OX40 anti-PD-L1-OX40 Ab V4 to V12 (SEQ ID NO. 30-38).

[0053] FIG. 19 shows exemplary PAGE gel analysis of anti-human OX40 immune checkpoint antibody fusion proteins. The purified fusion proteins, anti-PD-L1-OX40 Ab-V5 fusion proteins, have been shown to have a molecular weight of approximately 220 kDa (non-reducing) and a heavy chain fusion of approximately 80 kDa and a light chain of approximately 30 kDa (reducing) in both antibody fusions.

[0054] FIG. 20 is a flowchart illustrating an ELISA method for evaluating the binding activity of bispecific antibody variants.

[0055] FIG. 21 shows the binding activity of human PD-L1 to variants of the bispecific antibody and its EC50.

[0056] FIG. 22 shows the binding activity of human OX40 to variants of the bispecific antibody and its EC50.

[0057] FIG. 23 shows the ex vivo serum stability of the bispecific antibody variant, anti-PD-L1-OX40 Ab-V5.

[0058] FIG. 24A and 24B show IL-2 production for 3 days (FIG. 24A) and IFN-γ production for 5 days (FIG. 24B) after T cell modulation with anti-PD-L1-mono-, combined, or bispecific antibodies. OX40 Ab-V5.

[0059] FIG. 25 is a graph showing the effect of exposure to anti-PD-L1-OX40 Ab-V5 bispecific antibodies and exposure to monoclonal antibodies on PC-3 tumor growth in Fox Chase SCID® Beige mice.

Detailed description of the present invention

[0060] Detailed reference will now be made to the present embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same identifiers are used in the graphics and in the description to refer to the same or similar parts.

[0061] The present invention describes the expression, purification and characterization of bifunctional proteins with isolated functional agonist scFv OX40 fused to the C-terminus of the Fc domain of antibodies to immune checkpoint proteins. These proteins interact with their respective checkpoint target and must transmit an inhibitory or stimulatory signal to modulate T cell-associated immunity. The Fc fusion protein components of the present invention are all of human origin and thus are expected to be non-immunogenic and useful as human therapeutics.

[0062] Bispecific molecules, such as bispecific antibodies (BsAbs), provide a means of simultaneously targeting multiple epitopes to the same molecular target or different targets with a single therapeutic agent. As anti-cancer therapeutics, they have the potential to impart new or stronger activities, reduce product costs, and facilitate the development of new treatment regimens, as opposed to a mixture of two mAbs (Chames and Baty, 2009; Hollander, 2009; Thakur and Lum, 2010). Recently, catumaxomab, a trifunctional bispecific antibody targeting human epithelial cell adhesion molecule (EpCAM) and CD3, has demonstrated a clear clinical benefit in patients with peritoneal carcinomatosis among epithelial cancers (Heiss et al., 2010), and the bispecific antibody is a T cell activator (BiTE) with dual specificity for CD19 and CD3 has also demonstrated promising clinical activity in patients with CD19-expressing hematologic malignancies (Bargou et al., 2008). Despite strong interest in the development of bispecific molecules as cancer therapeutics, technical challenges in the production of stable and active bispecific molecules have in the past made clinical evaluation of most bispecific formats difficult. Many engineered antibody formats, including IgG-like bispecific antibodies, have been characterized by impaired stability or solubility (Bargou et al., 2008; Demarest and Glaser, 2008; Lu et al., 2005). Several strategies have been undertaken to improve product quality and in vivo stability of the bispecific molecules, including PEGylation, human serum albumin conjugation, and Fc construction (Muller et al., 2007; Ridgway et al., 1996). The bispecific single chain antibodies of the general form described above have the advantage that the nucleotide sequence encoding four V domains, two linkers and one spacer can be incorporated into a suitable expression host organism under the control of a single promoter. This increases both the flexibility with which these designs can be designed and the degree of experimenter control during their production. In addition, IgG Fc represents another attractive scaffold for the development of new therapeutic agents, since it contains all the functions of antibodies, except for the ability to bind. Fc construction is important for improving the performance of bispecific antibodies. Thus, the IgG-based conformation is used in the present invention for two independent targets on immune cells or target cells in immunotherapy.

[0063] Targeting immune checkpoint proteins is a promising approach for activating antitumor immunity. Immune checkpoint proteins such as PD-1, PD-L1, CTLA-4, LAG3, etc. are currently being evaluated clinically (FIG. 1). Preliminary data with immune checkpoint protein blockers have been shown to be able to enhance antitumor immunity with the potential to induce long-term clinical responses. However, despite the remarkable clinical efficacy of these agents in a number of malignant neoplasms, it has become clear that they are not sufficiently active for many patients. Numerous additional immunomodulatory pathways, as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumor microenvironment, are potential targets for immune checkpoint synergy. Thus, combined anticancer or bispecific antibody therapy is required to achieve complete remission and cure cancer patients.

[0064] The present invention describes the construction, expression and characterization of an Fc antibody to an immune checkpoint protein fused to another immune checkpoint protein specific scFv protein. scFv OX40 located at the C-terminus in fusion constructs should allow the power of fusion proteins to be extended beyond the approach to OX40 activation if the fusion partner is an immune system potentiator such as, for example, an antibody to EGFR, to HER2, or to CTLA- 4.

[0065] Obtaining antibodies from the OmniMab library

[0066] To obtain therapeutic antibodies against PD-L1 or OX40, selection was made using the OmniMab phagemid library. The phagemid library is manufactured by AP Biosciences Inc. (APBio Inc.) from a collection of B cells from over one hundred healthy donors. Phages for the 1st round of panning were prepared by Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany). Solid phase panning and cell panning against PD-L1 or OX40 was used to select specific PD-L1 or OX40 binders and isolate from OmniMab. Solid phase panning was performed using recombinant human PD-L1-Fc or OX40-Fc (APBio Inc.) in the first round of selection, and then HEK293 cells expressing PD-L1 or OX40 were used for two and three rounds of enrichment. After three rounds of selection, specific binders for PD-L1 or OX40 were screened and isolated by direct ELISA or cell-based ELISA with the appropriate recombinant protein (FIGS. 2A, 2B, 3A and 3B). PD-L1-Fc pre-coated recombinant proteins or OX40-expressing 293 cells were blotted with the supernatant containing the detected phages for 1 hour and washed three times with PBS containing 0.1% Tween-20. Bound phages were detected with an HRP-conjugated anti-M13 antibody (Roche) and TMB substrate was used for signal development. OD450 readings were recorded. Positive binders were isolated and sent for sequencing to confirm the sequence and diversity of the heavy chain and light chain. The heavy chain and light chain variable region specific for PD-L1 or OX40 was described from SEQ ID NO. 1 to SEQ ID NO. 8: SEQ ID NO. 1 is the light chain of clone 6 PD-L1, SEQ ID NO. 2 is the heavy chain variable region of clone 6 PD-L1, SEQ ID NO. 3 is a light chain of clone 32PD-L1, SEQ ID NO. 4 is the heavy chain variable region of clone 32 PD-L1, SEQ ID NO. 5 is the light chain of clone B17 OX40, SEQ ID NO. 6 is the heavy chain variable region of clone B17 OX40, SEQ ID NO. 7 is the light chain of clone B19 OX40, SEQ ID NO. 8 is the heavy chain variable region of clone B19 OX40. As shown in FIG. 2A, 2B, 3A and 3B, several clones have been isolated and are known to be specifically recognized by the respective antigen compared to the negative control.

[0067] Subcloning and expression/purification of the selected specific binder for PD-L1 or OX40 in IgG format

[0068] To facilitate rapid screening of a specific binder with T cell activation functionality, the heavy chains and light chains of positive binders against PD-L1 or OX40 by ELISA were then amplified, digested, and subcloned into an APGio-specialized IgG expression vector carrying an IgG4 constant region. (SEQ ID NO. 9). After sequence confirmation, plasmids were then prepared and transfected into HEK293 cells for antibody expression using fectin transfection reagent 293 (Invitrogen). After 4 days of culture, the antibody secreted into serum-free medium is affinity purified from the culture supernatant by protein G binding chromatography. The purified antibody is then concentrated, followed by dialysis in PBS buffer. The final dialyzed protein concentration was determined using a NanoDrop2000 spectrophotometer, and purity and integrity were determined using SDS-PAGE with or without reducing reagent as shown in FIG. 4 and 5. The integrity of various purified prototype antibodies specific for PD-L1 or OX40 is normal in HEK293 cells, as is the reference antibody, MPDL3280A for PD-L1 or GSK3174998 for OX40.

[0069] In one embodiment, provided herein is an OX40 (CD134) binding antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region contains an amino acid sequence having at least about 80% sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 8, amino acids 128-246 of SEQ ID NO. 10 and amino acids 124-241 SEQ ID NO. 13. In some examples, the heavy chain variable region contains an amino acid sequence having at least about 85%, 90%, or 95% sequence homology to the amino acid sequence as defined above. The light chain variable region contains an amino acid sequence having at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-108 of SEQ ID NO. 5, 1-108 of SEQ ID NO. 7, 1-112 of SEQ ID NO. 10 and 1-108 of SEQ ID NO. 13. In some examples, the light chain variable region contains an amino acid sequence having at least about 85%, 90%, or 95% sequence homology to the amino acid sequence as defined above.

[0070] In one embodiment, provided herein is an antibody or antigen-binding portion thereof that binds to PD-L1, comprising a heavy chain variable domain and a light chain variable domain. The heavy chain variable domain contains an amino acid sequence having at least about 80% sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 4. In some examples, the heavy chain variable region contains an amino acid sequence having at least about 85%, 90%, or 95% sequence homology to the amino acid sequence as defined above. The light chain variable domain contains an amino acid sequence having at least about 80% sequence homology to an amino acid sequence selected from the group consisting of amino acids 1-111 of SEQ ID NO. 1 and 1-110 of SEQ ID NO. 3. In some examples, the light chain variable region contains an amino acid sequence having at least about 85%, 90%, or 95% sequence homology to the amino acid sequence as defined above.

[0071] Determination of Binding Activity for PD-L1 Specific, OX40 IgG Prototypes by Direct ELISA

[0072] Purified prototype anti-PD-L1 or OX40 antibodies (prototype anti-PD-L1 antibody or prototype anti-OX40 antibody) are then used for binding characterization by ELISA on human PD-L1-Fc or OX40-Fc in a direct coating formulation. In FIG. 6 and 7 show the result of ELISA binding for anti-PD-L1 and anti-OX40 antibodies, respectively. For antibodies specific for PD-L1, most of the prototypes showed similar or better binding activity compared to the reference antibody (Ref Ab, MPDL3280A, Roche).

[0073] Purified human PD-L1 or OX40 IgG1 Fc chimera (PD-L1-Fc or OX40-Fc, APBio) was dialyzed in phosphate buffered saline (PBS), adjusted to 1 mg/mL and then diluted with PBS to final concentration 1 µg/ml. Nunc-Immuno Maxisorp 96-well plates were coated with 0.1 ml per well of recombinant PD-L1-Fc or OX40-Fc chimera, leaving empty wells to monitor for non-specific binding, and incubated at 4° C. overnight. The PD-L1-Fc or OX40-Fc chimera solution was removed and the plates were washed three times with 0.4 ml wash buffer (0.1% Tween-20 in PBS). 0.4 ml blocking buffer (5% low fat milk powder in PBS) was added to all wells and incubated at room temperature for 1 hour with shaking. The blocking buffer was removed and the plates were washed three times with 0.4 ml wash buffer. Serial dilutions of test antibodies to PD-L1 or OX40 were prepared in PBS and 0.1 ml of the diluted antibody was added per well. The plates were incubated for 1 hour at room temperature. The antibody solution was removed and the plates were washed three times with 0.4 ml of wash buffer per well. Horseradish peroxidase labeled F(ab') ₂ goat anti-F(ab') ₂ human IgG (Jackson Immunoresearch #109-036-097) was diluted 1:2000 with PBS and 0.1 ml per well was added. The plates were incubated 1 hour at room temperature and washed with 0.4 ml per well of wash buffer. 0.1 ml of TMB reagent (Invitrogen) was added and incubated for 1 to 5 minutes at room temperature. The reaction was stopped by adding 0.05 ml of 1N. HCl and absorbance was read at 450 nm on a Bio-Tek spectrometer. The calculated EC50 for prototype PD-L1 antibodies to PD-L1 indicated that most of the prototypes had good binding activity, as did MPDL3280A (Ref Ab) by direct ELISA analysis (FIG. 6). In contrast, most of the prototype OX40 antibodies showed much lower binding activity compared to the reference antibody (Ref Ab, GSK3174998) (FIG. 7).

[0074] Determination of Binding Activity for PD-L1 and OX40 Specific IgG Prototypes by FACS

[0075] Purified prototype antibodies (anti-PD-L1 antibody prototypes or anti-OX40 antibody prototypes) were also used for flow cytometry to determine and compare binding activity to expressed PD-L1 or OX40 HEK293 cells. In FIG. 8 and 9 show the binding activities of the respective prototype antibodies, as shown by FACS, to stably expressed HEK293 PD-L1 or OX40 cells.

[0076] FACS analysis of stable PD-L1 expressing 293 cells stained with anti-PD-L1 antibody prototypes results in PD-L1 binding activity, cells with stable expression were incubated with 1 μg/mL of purified anti-PD-L1 antibody prototypes, reference antibody (Ref Ab MPDL3280A) or isotype antibody as a negative control on ice for 1 h. Cells were washed three times with 1X PBS and then incubated with Alexa-488 conjugated goat anti-human IgG (H+L) (Invitrogen Inc. .) on ice for an additional 1 hour. After staining, cells were washed three times with 1x PBS, resuspended in 1x PBS/2% FBS before analysis in FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC) . In the same scenario, the binding activity of prototype anti-OX40 antibodies for stably expressed OX40 cells in HEK293 in FIG. 9 was also performed with a similar strategy and analyzed as described above. As shown in FIG. 8, most of the prototype anti-PD-L1 antibodies have good binding activity as well as the reference antibodies. This indicates that the phage clones selected from the OmniMab library indeed recognize native PD-L1 in the cells.

[0077] This phenomenon is also observed for prototype anti-OX40 antibodies, as shown in FIG. 9. FACS analysis of 293 clone 2D5 stably expressing OX40 cells stained with purified OX40 antibody prototypes to examine OX40 binding activity was performed as follows: stably expressing cells were incubated with 2 μg/mL of OX40 reference antibody (OX40 ref.) or reference anti-OX40 antibody. CD137 (CD137 ref.) as control antibody on ice for 1 h. Cells were washed three times with 1X PBS and then incubated with Alexa-488 conjugated goat anti-human IgG (H+L) (Invitrogen Inc.) on ice for an additional 1 hour. After staining, cells were washed three times with 1x PBS, resuspended in 1x PBS/2%) FBS before analysis with FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC).

[0078] Competitive ligand binding (ELISA)

[0079] Prototype antibody binding selectivity and binding affinity assays have been shown to be used to evaluate the anti-PD-L1 prototype antibodies of the present invention for their ability to block PD-L1 binding to PD-1. [0080] The antibodies were tested for their ability to block the binding of human PD-L1-Fc chimera (PD-L1-Fc) to recombinant human PD-1/His (hPD-1/His) by ELISA. Purified recombinant hPD-1/His (APBio) was dialyzed to 1 mg/ml in PBS and then conjugated with biotin (Abcam). A 96-well Nunc Maxisorp plate was coated with 250 ng hPD-1/His per well in PBS overnight. The hPD-1/His solution was removed and the plates were washed three times with 0.4 ml wash buffer (0.1% Tween-20 in PBS). 0.4 ml blocking buffer (5% skimmed milk powder in PBS) was added to all wells and incubated at room temperature for 1 hour with agitation. During the blocking step, the antibody stock solution was diluted in the range of 200 nM to 0 nM in PBS with a 2-fold serial dilution. Purified recombinant biotinylated PD-L1-Fc chimera was diluted to 4 μg/ml in PBS. PD-1/His coated plates were washed three times with 0.2 ml wash buffer (0.1% Tween-20 in PBS). Dilutions 60 µl of antibodies (prototype anti-PD-L1 antibodies or MPDL3280A reference antibody) were added separately with 60 µl of biotinylated-PD-L1-Fc chimera and incubated at room temperature for 1 hour. Plates were washed as described previously. Streptavidin-HRP was diluted 1:2000 in PBS, 100 μl of the resulting solution was added to the wells of the washed plates and dried at room temperature for 1 hour. The plates were washed as previously described, 100 μl of TMB substrate solution was added to each well and mixed for 10 minutes. The reaction was stopped with 50 µl of 1N. HCl and absorbance at 450 nm were read using a Bio-Tek reader and presented in FIG. 10. Partial antibody prototypes have been shown to inhibit the interaction between PD-PD-L1 by competitive ELISA. Most of the prototype antibodies showed similar blocking activity compared to the reference antibody (Ref Ab MPDL3280A).

[0081] Enhanced Stimulation of T Cell Activation by Inhibiting PD-1:PD-L1 Ligand Interaction for Anti-PD-L1 Antibody

[0082] The PD-1 signaling pathway inhibits mild costimulatory TCR/CD28 signals, with cytokine production initially decreasing without reducing T cell proliferation. As TCR/CD28 costimulatory signals are attenuated, the PD-1 pathway dominates with a large decrease in cytokine production followed by a decrease in proliferation. Accordingly, to confirm that inhibition of PD-1 through inhibition of interaction with PD-L1 by human antibodies of the present invention enhances T cell activation, mixed lymphocyte tests (MLRs) are performed.

[0083] Human whole blood monocytes were enriched with RosetteSep™ Human Monocyte Enrichment Cocktail (Cat. No. 15068) and cultured in differentiation medium, RPMI 1640 with 10% FBS, 100 ng/mL (1000 U/mL) GM- CSF, 100 ng/ml (500 U/ml) for 6 days. Differentiated dendritic cells (DC) from monocytes were tested with DC-SIGN-PE, FITC-conjugated anti-CD14 antibody, PE-conjugated anti-CD83 antibody, or FITC-conjugated anti-CD80 antibody to confirm differentiation and used as APC in MLR.

[0084] Allogeneic CD4+ T cells from human whole blood were isolated using RosetteSep™ human CD4 ⁺ T cell enrichment cocktail (Cat. No. 15062). The purity of CD4+ T cells was checked with APC-conjugated anti-CD4 antibody to ensure purity above 95% and then labeled with 1 μM CFSE (CFSE CellTrace™ Cell Proliferation Kit, Life Technologies, Cat # C34554) for T cell proliferation assay . Labeled CD4+ T cells were co-cultured with immature DCs with different antibody prototypes as indicated for 3 and 5 days to see if antibody prototypes could restore T cell activation by blocking the interaction between PD-1 and PD-L1 . After the 3rd 5 day incubation, the supernatant was collected for cytokine determination, such as quantification of IL-2 and IFN-γ by ELISA. Addition of prototype anti-PD-L1 antibodies (clones 6, 32, 28, 51, 64, 27 and 37) to cultures of immature dendritic cells plus allogeneic T cells is expected to result in increased T cell proliferation and cytokine production compared to with cultures treated with the IgG isotype (iso#1, #2) and are shown in FIG. 11A and 11B. IL-2 and IFN-γ production is significantly increased in MLR compared to isotype antibody exposure after 3 days (FIG. 11A) or 5 days (FIG. 11B) of antibody exposure, especially for anti-PD-L1 antibody clone 6. The cytokine increment remains evident after 5 days of antibody exposure and is similar to the reference antibody (ref), MPDL3280A. This indicates that anti-PD-L1 clone 6 should be one of the potential prototypes for a bispecific antibody composite.

[0085] Anti-OX40 antibody agonist activity assay

[0086] To activate costimulation of OX40 T cell proliferation and cytokine production, purified prototype antibodies were functionally screened for their ability to enhance cytokine production, proliferation, and induce proliferation in human CD3+ T cells. Prototypes of anti-CD3 antibody (OCTS, BioLegend, cat. no. 317304) and anti-OX40 antibodies (clones B6, B70, B120, A4, B17, B19 and B30), reference antibody (GSK3174998) or isotopic antibodies (iso#1, #2 ) was used to coat in a 96-well Maxisorp plate. Meanwhile, naïve human CD3+ T cells were isolated from human blood from healthy adult volunteers using the commercially available Rosette Sep™ human T cell enrichment cocktail (STEMCELL, cat. no. 15061), as described by the manufacturer. The isolated CD3+ T cells were then labeled with CFSE (CFSE CellTrace™ cell proliferation kit, Life Technologies, cat. no. C34554) and plated at 1×10 ⁶ cells/ml in an antibody pre-coated well containing RPMI 1640, 10 % fetal bovine serum and 2.5 mM L-glutamine to determine cell proliferation and cytokine production. After 3 days of culture, cells were harvested for proliferation analysis by flow cytometry, and then the medium was analyzed for IL-2 and IFN-γ production by quantitative ELISA.

[0087] Screening of prototype anti-OX40 antibodies with agonist activity in T cell activation is shown in FIG. 12A. All prototype anti-OX40 antibodies showed the ability to enhance CD3+ T cell activation by dose response, as did the reference antibody. Exposure to the antibody at higher doses showed a clearly higher T-cell activation activity. Meanwhile, the production of cytokines (FIG. 12B) such as IL-2 and IFN-γ also revealed a similar response to T cell activation, especially for the prototype anti-OX40 antibody clone B17. The cytokine has a high induction after 3 days of exposure to an antibody to OX40 B17. The amplification is much higher than when exposed to the reference antibody, this putative B17 clone should be one of the candidates for the construction of bispecific antibodies.

[0088] As shown in the data shown in FIG. 13A and 13B, both prototype anti-OX40 antibodies, clones B17 and B19, showed better agonist activity in the assay after 3 days of exposure to the prototype anti-OX40 antibodies (B17 or B19). Both IL-2 production and IFN-γ production show a clear improvement when exposed to antibodies and show a dose-dependent correlation. Higher production of cytokines was recorded when exposed to antibodies at higher doses.

[0089] To assess the agonistic activity of prototype antibodies to OX40, B17 and B19, EC50 was also determined, and the analysis of agonistic activity and production of cytokines was recorded for comparison.

[0090] Construction, expression and purification of anti-PD-L1-OX40 scFv antibody

[0091] Because the bispecific antibody was designed from IgG fused to the scFv format, the Fc structure of the immune checkpoint antibody is fused at the end to the scFv OX40. The antibody may be inhibitory antibodies against immune checkpoints such as anti-PD-L1, anti-PD-1, anti-CTLA4, anti-LAG3, etc. or stimulatory antibodies such as anti-CD28, anti-CD137, anti-CD27, anti- ICOS et al. The linker is positioned between the antibody Fc and the OX40 scFv to generate a bispecific antibody as shown in FIG. 14.

[0092] In some embodiments, clone 6 of the prototype anti-PD-L1 antibody is assigned to be an IgG form, on the other hand, the prototype anti-OX40 antibody is transformed as an scFv format to merge at the C-terminus of the Fc region in clone 6 of the prototype anti-PD-L1 antibody . Transformation from antibody to scFv format can result in reduced binding activity or specificity, so several anti-OX40 antibody prototypes have been used to transform scFv. Construction of an Fc bifunctional anti-PD-L1 antibody fused to full-length scFv OX40 (SEQ ID NO. 10 as clone A4, SEQ ID NO. 11 as clone B17, SEQ ID NO. 12 as clone B19, or SEQ ID NO. 13 as clone B120). A short flexible peptide linker, (GGGGS) ₂ (SEQ ID NO. 14) was placed, for example, between the C-terminus of the anti-PD-L1 antibody heavy chain Fc region and the N-terminal OX40 scFv module to ensure correct folding and minimize steric interference. The coding sequences for the anti-PL-L1-OX40 scFv antibodies were shown in SEQ ID N0. 16 (heavy chain of clone 6 to PD-L1 - scFv of clone B17 to OX40) and NO. 17 (heavy chain of clone 6 to PD-L1 - scFv of clone B19 to OX40). The engineered antibody Fc fusion proteins were derived with a signal peptide (SEQ ID NO. 15) and expressed in mammalian cells and purified from the transfected cell culture supernatant by one-step protein G binding chromatography. As shown in FIG. 15, purity greater than 90% can be obtained in a one-step purification process and indicates that the purified fusion proteins have the correct molecular weight (MM=220 kDa).

[0093] Enhanced stimulation of T cell activation for anti-PD-L1-OX40 scFv bispecific antibody prototypes in MLR

[0094] To determine the synergistic interaction of a bispecific antibody in enhancing T cell activation by inhibiting the interaction between PD-1 and PD-L1 and agonistic activation of OX40 signaling, the prototypical bispecific antibodies, anti-PD-L1-OX40 scFv, were introduced into MLR, as described above. The production of IL-2 and IFN-γ was then recorded 3 or 5 days after antibody exposure. Mono-, combination or bispecific antibody was used as an equal amount or equal mol to compare the synergistic effect in enhancing T-cell activation, and isotype IgG was used as a negative control. As the data shown in FIG. 16A and 16B, the prototype anti-PD-L1 antibodies alone showed significant induction of IL-2 after 3 days of exposure, as did the reference antibody, MPDL3280A, in contrast, the prototype anti-OX40 antibodies were not able to clearly upregulate cytokine production as after 3 days. and 5 days of antibody exposure. It corresponds to the reference antibody GSK3174998. However, the combination of anti-OX40 antibodies and anti-PD-L1 antibodies showed significant upregulation of cytokine production after 3 and 5 days of antibody exposure. A synergistic effect is also observed when exposed to prototypical bispecific antibodies, and the increase in cytokine production is also similar to that seen with combination therapy. This indicates that the prototypical anti-PDF-L1-OX40 scFv bispecific antibodies also function as combined antibody action without losing any binding activity in scFv transformation.

[0095] Determination of aggregation and purity of a bispecific antibody

[0096] Because the purified anti-PD-L1 clone 6-OX40 clone B17 scFv showed lower purity (74.07%) by size exclusion HPLC analysis after purification by single column chromatography based on protein A binding, therefore, it was Several antibody variants have been created to improve purity and reduce aggregation for the bispecific antibody of the present invention. The linkers described above were used to replace the linker in scFv B17 OX40 in the bispecific antibody, anti-PD-L1-OX40 Ab (SEQ ID NO. 16), and produced as anti-PD-L1-OX40 Ab-V1-V4 (SEQ ID NO. 18 - SEQ ID NO. 21) in CHO cells. These variants were then purified and analyzed on an XBridge Protein BEN SEC-HPLC column (Waters cat. no. 186007640). Data summarized as shown in Table 1 below, one of the bispecific antibody variants, anti-PD-L1-OX40 Ab-V4, showed a significant improvement in antibody purity. Purity increased from 74.07 to 92.27%. Therefore, anti-PD-L1-OX40 Ab-V4 was used to further improve antibody purity.

[0097] To characterize the size distribution of bispecific antibodies, samples were loaded onto an XBridge Protein BEN SEC-HPLC column (Waters, cat. no. 186007640) using a Waters Alliance 2695 separation module. The protein peak was detected at 280 nm using a Water 2996 PDA detector. The mobile phase was 25 mM isocratic sodium phosphate (Sigma, cat.#04272 and cat.#04269) with 200 mM NaCl (AMRESCO, cat.#0241), pH 6.8, at a flow rate of 0.4 ml /min The percentage of peaks was determined from peak area plots as shown in FIG. 17A-17E.

[0098] Anti-PD-L1-OX40 Ab-V4 showed a significant improvement in purity (FIG. 17E). The bispecific antibody was further engineered into a B17 OX40 scFv fragment to again improve purity. Several residues in scFv B17 OX40 shown in FIG. 18 were changed to different amino acids, and the heavy chain variants were paired with the light chain of clone 6 to PD-L1 to produce several bispecific antibody variants, from anti-PD-L1-OX40 Ab-V5 to V12 (SEQ ID NO. 22 - SEQ ID NO. 29) and then expressed and purified as above. The purity of the bispecific antibody variants is summarized as shown in Table 2, anti-PD-L1-OX40 scFv-V5 showed the best purity in these antibody variants. Purity increased to 96.46%. This showed excellent purity of the engineered bispecific antibody, and also revealed a good ability to develop this bispecific antibody in the future. As shown in FIG. 19, the integrity of anti-PD-L1-OX40 Ab-V5 was also analyzed by SDS-PAGE and showed good integrity under reducing and non-reducing conditions.

[0099] Meanwhile, engineered variants of the bispecific antibodies were also used to evaluate binding activity for human PD-L1 and OX40 by direct ELISA assay, as shown in FIG. 20. All variants of the bispecific antibodies were indicated to exhibit the same binding activity for human PD-L1 (Fig. 21), this binding activity being similar to anti-PD-L1 antibody 6. This phenomenon was also observed in the human OX40 binding assay (Fig. 22). Only anti-PD-L1-OX40 Ab-V10 showed weaker binding activity for human OX40 compared to other variants. It was indicated that the construction of scFv OX40 does not affect the binding activity of OX40. Binding activity is maintained for either PD-L1 or OX40. Because anti-PD-L1-OX40 Ab-V5 showed excellent antibody purity and binding activity for PD-L1 and OX40, therefore, anti-PD-L1-OX40 Ab-V5 was selected for serum stability analysis.

[00100] Ex vivo serum stability

[00101] Stability was assessed in human sera (BioreclamationIVT, cat.#HMSRM), as well as in the sera of the corresponding preclinical species: rhesus monkey (BioreclamationlVT, cat.#RHSSRM) and CD1 mouse (BioreclamationlVT, cat.#MSESRM). Samples were added to serum of different types to a final concentration of 15 μg/ml and incubated in a water bath at 37°C. Serum samples were collected after an incubation period of 0, 1, 2, 3, 7, 10 and 14 days and stored frozen at -80°C until analysis.

[00102] Quantitative ELISA sandwich analysis

[00103] OX40-FC was coated in an ELISA plate (NUNC cat#442404) 100 µl at 1 µg/ml in PBS and left overnight at 4°C. Wash buffer was prepared as PBS with 0.1% Tween-20 (Sigma, cat. no. P2287-500 ml) and blocking buffer was prepared as 1% BSA (UniRegion, cat. no. UR-BSA001-100G) in wash buffer. Serum samples were prepared at 10-fold dilution with 3-fold serial dilution in blocking buffer, and standards were prepared at 10 nM concentration with 3-fold serial dilution in blocking buffer. Biotinylated PD-Ll-Fc was labeled with a rapid biotin conjugation kit (abcam, cat# ab201796) using a standard protocol and prepared at 30 nM in blocking buffer. Streptavidin-HRP (abcam, cat. no. ab7403) was prepared at a concentration of 1 μg/ml in blocking buffer. All samples were added to each well at 100 μl after the plates were washed 3 times with wash buffer and incubated for 1 hour at ambient temperature. The development of TMB was carried out with 100 μl of TMB solution (Invitrogen, Cat. No. 00-2023) for 2 minutes and stop using 100 μl of 1N solution. HC1 (Merck, Cat. No. 1.00317.1000). Absorbance at 450 µm was read using an ELISA reader (Biotek, Powerwave XS).

[00104] Anti-PD-L1-OX40 Ab-V5 was selected for ex vivo serum stability analysis because of its superior purity and binding activity for PD-L1 and OX40. Purified bispecific antibodies were mixed with sera from different species such as human, mouse or monkey. After several days of culture, samples were taken and analyzed by sandwich ELISA to determine the amount of antibodies. As shown in FIG. 23, anti-PD-L1-OX40 Ab-V5 still showed good serum stability after 14 days of culture at 37°C. The antibody concentration is still above 70% in humans, mice or monkeys. The antibody is also said to have good serum stability.

[00105] To measure the ability of anti-PD-L1-OX40 Ab-V5 to modulate T cell reactivity, purified T cells will be cultured with allogeneic dendritic cells obtained by culturing monocytes in GM-CSF and IL-4 for several days. Parallel plates were set up to collect supernatants on day 3 and day 5 to measure IL-2 and IFN-γ, respectively, using a commercial ELISA kit. As the data shown in FIG. 24A and 24B, IL-2 and IFN-γ production is greatly increased with bispecific antibody (V5) exposure as well as with combined exposure after 3 or 5 days of antibody exposure. In addition, the enhancement is clearly superior to treatment with only Ab to PD-L1 or Ab to OX40. This implied that the engineered bispecific antibody, V5, still has agonistic activity as well as combination therapy without loss of functionality and could be developed as a therapeutic antibody for various solid tumors or cancers in the future.

[00106] Antitumor activity of a bispecific antibody (in vivo model)

[00107] The lack of cross-reactivity between PD-L1 and OX40 in rodents in bispecific antibodies precluded the use of standard murine syngeneic or human xenograft tumor models to assess the efficacy of antibodies against human tumors. Accordingly, a novel huPBL-SCID-Bg mouse tumor xenograft model was generated using a SCID-Bg mouse (CB.17/Icr.Cg Pkrdc ^scid Lyst ^bg /CrI) that contains the beige (Bg) mutation lacking mouse T- and B- lymphocytes and functional NK cells. The efficacy of bispecific antibodies against human tumors was evaluated using this model, as described below.

[00108] Human prostate PC-3 was obtained from the American Type Culture Collection and cultured in RPMI-1640 (Invitrogen) with L-glutamine, sodium pyruvate, penicillin/streptomycin, and 10% heat-inactivated fetal bovine serum (FBS, Gibco, cat. no. 10437). Cells were grown to confluence in Falcon T-150 flasks. Cells were then trypsinized (trypsin 0.25%-EDTA; Invitrogen) and growth was scaled up to sufficient cells for inoculation. Peripheral blood lymphocytes (PBMCs) were isolated from heparinized blood using Lymphoprep™ according to the manufacturer's protocol (STEMCELL Technologies Inc.). The counted cell suspensions were pooled such that each mouse received an injection of 0.75×10 ⁶ PBMC and 3×10 ⁶ tumor cells in one 0.1 ml injection bolus in PBS. To facilitate tumor cell growth in the mouse, another 0.1 ml volume of Matrigel was then mixed with the pooled cell suspension and then immediately injected into the prepared mice.

[00109] Each mouse was injected subcutaneously with 0.2 ml volume of the pooled cell suspension in the right flank of the animal. After 14 days of inoculation, a solid tumor is formed reaching a volume of approximately 250-300 mm ³ and a bispecific antibody (3 mg/kg anti-PD-L1-OX40 Ab-V5), a PD-L1 reference antibody (Ref Ab, MPDL3280A) or a control antibody (isotype) is administered twice a week for three weeks by intraperitoneal injection (ip). Tumor measurements are taken with a Preisser caliper twice a week, and the administration of the test sample during the duration of the experiments and body weight are also recorded. Tumor volume was calculated using the following calculation: length × width ² × 0.44 = volume (mm ³ ) and plotted in FIG. 25 Mice were removed from the study until termination of the experiment if the tumor volume reached 2000 mm ³ or the animal lost 20% of body weight. Similar results were observed when tumors were measured on day 7 post-inoculation and animals were randomized according to tumor volume. For animal studies, each group contained 6 mice. As the data shown in Fig. 25, the bispecific antibody exhibited significant anti-tumor efficacy in the PC-3 xenograft mouse model. Tumor size decreased 18 days after tumor inoculation as well as anti-PD-L1 reference antibody and continued to decrease below 100 mm ³ . The mouse model of PC-3 xenograft previously demonstrated the antitumor effect of the bispecific antibody and revealed its potential as a therapeutic potential drug in the future.

[00110] Taken together, these results indicate that the bispecific antibody maintains its blocking of the immune checkpoint in PD-1/PD-L1 signaling and agonist activity for OX40 signaling. Research is ongoing to further investigate the biological activity of these proteins using a suitable animal model such as the PC-3 tumor in the humanized NOD.Cg-Prkdc ^scid Il22rg ^tmIwjI /SzJ (NSG) model.

[00111] The Fc region in the present invention may be from any immunoglobulin isotypes, subclasses, allotypes, or engineered mutants, such as bump-in-hole Fc fragment(s).

[00112] EXAMPLES

[00113] The following example describes the production of monoclonal antibodies suitable for therapeutic purposes, targeting human PD-L1 and OX40. Composite human anti-PD-L1 and anti-human OX40 antibodies were prepared from anti-PD-L1 clone 6 and anti-OX40 clone B17, respectively. The sequence segments of the human V region were obtained from sequence databases of unrelated human antibodies (germline and non-germline).

[00114] Example 1 Preparation of IgG antibodies that bind to PD-L1 and OX40.

[00115] Some of the antibodies provided in accordance with the present invention were originally derived from Fabs that bind to human PD-L1 or OX40. Fabs were selected from the phage display library, the OmniMab phagemid library, after alternating panning on the respective Fc fusion proteins (PD-Ll-Fc or OX40-Fc) and cells expressing the respective human protein (PD-L1 or OX40). After direct ELISA or cell-based ELISA screening, positive clones for the heavy chain and light chain were then sequenced. These Fabs included those labeled "OM-PD-Ll-6", "OM-PDLl-32", etc. for PD-L1; "OM-OH40-A4", "OM-OH40-B17" and "OM-OH40-B19", etc. for OX40 antibodies. PD-L1 clone 3, PD-L1 clone 6 and PD-L1 clone 32 anti-PD-L1 antibodies disclosed in this application were obtained from "OM-PD-L1-6" and "OM-PDL1-32" . Meanwhile, OX40-A4, OX40-B17 and OX40-B19 of the OX40 antibodies disclosed in this application were obtained from "OM-OX40-A4", "OM-OX40-B17" and "OM-OX40-B19" in a HEK293 cell or CHO-S cells. And a bispecific antibody targeting PD-L1 and OX40 simultaneously was developed as an anti-PD-L1 6-OX40 scFv B17 antibody and an anti-PD-L1 6-OX40 scFv B19 antibody. The amino acid sequence of the light chain variable region and the heavy chain variable region of this Fab are identical to the amino acid sequence of the light chain variable region and the heavy chain variable region, respectively.

[00116] Example 2 In vitro binding of anti-PD-L1-OX40 scFv to an appropriate target.

[00117] An anti-PD-L1-OX40 bispecific antibody was constructed as shown in FIG. 13 and expressed in HEK293 cells or CHO-S cell. The media containing the bispecific antibody was affinity purified from the culture supernatant by protein G binding chromatography. The purified antibody was then concentrated followed by dialysis in PBS buffer and analyzed by SDS-PAGE as shown in FIG. 14. To study the direct binding of purified fusion proteins to PD-L1 or OX40 by ELISA, 100 ng/well of recombinant PD-L1 or OX40 was used to coat in a 96-well ELISA plate. Then, various concentrations of purified anti-PD-L1-OX40 scFv were added to each well and incubated for 1 hour. After washing, a 1:5000 dilution of HRP-Fab conjugate (Jackson Immunochemicals) was added to each well and incubated for another hour. After the final wash, TMB substrate (Invitrogen Inc.) was added and the absorbance was measured at 450 nm. The data was analyzed by plotting points on a sigmoid curve using GraphPad Prism 5 and the EC50 was calculated.

[00118] Example 3 Antigen-binding specificity of anti-PD-L1-OX40 scFv by FACS analysis

[00119] To study the binding specificity of anti-PD-L1-OX40 scFv antibody, PD-L1 stably expressing 293 cells stimulated with IFNy A549 or WiDr were stained with 1 μg/ml of anti-PD-L1-OX40 scFv antibody for 1 h on ice before washing three times with 1x PBS. Bound antibody fusion proteins were detected using Alexa-488 conjugated goat anti-human IgG (H+L) followed by FACS analysis. An isotype antibody was used as a negative control for the study. The results showed that the anti-PD-L1-OX40 scFv retained its antigen-binding specificity compared to anti-PD-L1 alone. The binding specificity of the anti-PD-L1-OX40 scFv antibody was also examined using 293 cells stably expressing OX40.

[00120] Example 4: In vitro immunomodulatory effects of bifunctional proteins.

[00121] To measure the ability of anti-PD-L1-OX40 scFv antibodies to modulate T cell reactivity, purified T cells will be cultured with allogeneic dendritic cells obtained by culturing monocytes in GM-CSF and IL-4 for several days. Parallel plates were set up to collect day 3 and day 5 supernatants for IL-2 and IFN-γ measurements, respectively, using a commercial ELISA kit. Genentech/Roche's humanized anti-PD-L1, MPDL3280A, will be produced in-house and used as a positive control. As the data shown in Fig. 16A and 16B, IL-2 and IFN-γ production is greatly increased by bispecific antibody exposure, as well as by combined exposure after 3 or 5 days of antibody treatment. Especially bispecific antibodies composed of anti-PD-L1 clone 6 and anti-OX40 clone B17 (anti-PD-L1-OX40 scFv B17 Ab), or a combination (anti-PD-Ll clone 6 Ab plus anti-OX40 clone B17 Ab ) showed an increase in T cell activation higher than the combination of reference antibodies to PD-L1 and OX40 (PD-L1 Ref Ab plus OX40 Ref Ab). This indicates that the B17 anti-OX40 antibody may have specific epitope binding resulting in better agonist activity compared to the reference anti-OX40 antibody, GSK3174998.

[00122] Example 5 Human leukocyte expansion induced by bispecific antibodies in vivo

[00123] The lack of detectable cross-reactivity of antibodies to PD-L1 or OX40 with mouse PD-L1 or OX40 and the need for human immune cells required the development of models for the functional evaluation of bispecific antibodies in vivo. Mice with a NOD genetic background carrying a severe combined immunodeficiency (SCID) mutation and a deficiency in the common IL-2 receptor gamma chain (commonly referred to as NSG) are able to maintain engraftment of large numbers of human peripheral blood leukocytes (huPBLs) and maintain engraftment for at least least 30 days (King et al., 2008). This mouse model, also known as the huPBL-NSG model, was used to evaluate the functional effect of in vivo systemic administration of antibodies on human immune cells.

[00124] Specifically, 6 million freshly isolated human PBMCs were adopted by intravenous injection into huPBL-NSG mice. Nine days after the PBMC injections, the animals were given a single dose of 1 mg/kg monoantibody, bispecific antibody, or IgG4 isotype control antibody via intraperitoneal injection. On days 24-28 post-engraftment of PBMCs, PBMCs were stained with anti-human and mouse CD45 antibodies assessed by flow cytometry. The forward and side scatter profiles were used to determine the lymphocyte gate. Bispecific antibodies were able to enhance the expansion of human leukocytes, as evidenced by an increase in the proportion of human CD45+ cells in the peripheral blood of inoculated mice. For each group n ≥ 6 mice.

[00125] Example 6 Growth inhibition of PC-3 or A498 tumor cells in huPBL-NSG with anti-PD-L1-OX40 scFv antibody.

[00126] The PD-L1 positive human prostate cancer cell line, PC-3 (ATCC# CRL-1435) or the kidney cancer cell line, A498 (ATCC® HTB-44™) can be used to create xenograft models in huPBL- NSG. For tumor formation, 3×10 ⁶ PC-3 cells (or A498 cells)/mouse will be injected subcutaneously into huPBL-NSG mice as described above. To assess the inhibitory effect on tumor growth, different concentrations of anti-PD-L1-OX40 scFv antibody, reference antibody or isotype antibody at a dose of 0.1-3 mg/kg are administered to mice intravenously twice a week for 4 weeks after 14 days tumor cell implantation. Tumor growth will be measured twice a week for up to 5 weeks as described in the Fox Chase SCLD®Beige mouse model.

[00127] Example 7 Pharmacokinetic evaluation of anti-PD-L1-OX40 scFv in mice and monkeys

[00128] 10-40 mg/kg of bifunctional proteins, anti-PD-L1-OX40 scFv, will be administered to mice or monkeys via subcutaneous injection or intravenous injection. Serum samples will be taken at various time points after injection up to 15 days. Fc fusion protein concentrations in serum samples will be determined using a sandwich ELISA.

[00129] While the present invention has been described in some detail with reference to certain embodiments thereof, other embodiments are possible. Thus, the spirit and scope of the appended claims should not be limited by the description of the embodiments contained herein.

[00130] Those skilled in the art will appreciate that various modifications and changes may be made to the structure of the present invention without departing from the scope or spirit of the present invention. Based on the foregoing, it is intended that the present invention cover modifications and variations of this invention provided that they fall within the scope of the following claims.

SEQUENCE LIST

--->

<110> A.P. BIOSIENCES, INC.

<120> MONOSPECIFIC AND BI-SPECIFIC PROTEINS

WITH REGULATION OF IMMUNE CHECKPOINT FOR CANCER TREATMENT

<130> 9022-1808584

<140> PCT/US2018/067868

<141> 2018-12-28

<150> US 62/611,543

<151> 2017-12-29

<160> 43

<170> PatentIn version 3.5

<210> 1

<211> 217

<212> PRT

<213> Homo sapiens

<400> 1

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Asn Ala Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 2

<211> 118

<212> PRT

<213> Homo sapiens

<400> 2

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 3

<211> 216

<212> PRT

<213> Homo sapiens

<400> 3

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val Gly Ser Tyr

20 25 30

Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Val Ile Tyr Glu Val Ala Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 4

<211> 121

<212> PRT

<213> Homo sapiens

<400> 4

Gln Val Gln Leu Val Gln Ser Gly Ala Lys Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Pro Gly Tyr Ser Tyr Gly Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 5

<211> 214

<212> PRT

<213> Homo sapiens

<400> 5

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Thr Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe

35 40 45

Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 6

<211> 126

<212> PRT

<213> Homo sapiens

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala

100 105 110

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 214

<212> PRT

<213> Homo sapiens

<400> 7

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Thr Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe

35 40 45

Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Ala Glu Cys Ser

210

<210> 8

<211> 118

<212> PRT

<213> Homo sapiens

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Asn Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 9

<211> 327

<212> PRT

<213> Homo sapiens

<400> 9

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 10

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> OX40 scFv (clone A4)

<400> 10

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser

85 90 95

Asn Asn Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Glu

145 150 155 160

Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

165 170 175

Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Gly Gly Val Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

225 230 235 240

Val Thr Val Ser Ser Ala

245

<210> 11

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> OX40 scFv (clone B17)

<400> 11

Tyr Val Leu Thr Gln Pro Ser Val Ser Val Thr Pro Gly Lys Thr

1 5 10 15

Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr

35 40 45

Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn

50 55 60

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val

85 90 95

Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Leu Ile Ser Trp

165 170 175

Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp

210 215 220

Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala Phe Asp Ile Trp Gly

225 230 235 240

Gln Gly Thr Met Val Thr Val Ser Ser Ala

245 250

<210> 12

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<223> OX40 scFv (clone B19)

<400> 12

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Thr Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe

35 40 45

Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser

165 170 175

Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

195 200 205

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Asn

210 215 220

Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser Ala

<210> 13

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> OX40 scFv (clone B120)

<400> 13

Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Thr Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Arg Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ser Tyr

85 90 95

Val Phe Gly Thr Gly Thr Lys Val Thr Val Ser Ser Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

115 120 125

Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr

130 135 140

Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile

145 150 155 160

Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His

165 170 175

Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile

180 185 190

Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val

195 200 205

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Trp Gly

210 215 220

Gly Ser Phe Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

225 230 235 240

Ala

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 14

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

15

<210> 15

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> signal peptide

<400> 15

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 16

<211> 704

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody clone 6 heavy chain-OX40 clone B17 scFv

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

565 570 575

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly

580 585 590

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

595 600 605

Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

610 615 620

Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

625 630 635 640

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu

645 650 655

Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr

660 665 670

Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp

675 680 685

Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala

690 695 700

<210> 17

<211> 696

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody clone 6 heavy chain-OX40 clone B19 scFv

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

565 570 575

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly

580 585 590

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

595 600 605

Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

610 615 620

Val Ser Ser Ile Ser Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser

625 630 635 640

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

645 650 655

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

660 665 670

Cys Ala Arg Gln Asn Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly

675 680 685

Thr Met Val Thr Val Ser Ser Ala

690 695

<210> 18

<211> 707

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V1

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser

565 570 575

Arg Ser Ser Leu Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val

580 585 590

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

595 600 605

Phe Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

610 615 620

Leu Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr

625 630 635 640

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

645 650 655

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala

660 665 670

Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser

675 680 685

Ala Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val

690 695 700

Ser Ser Ala

705

<210> 19

<211> 708

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V2

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Gly Lys Gly Ser Gly Gly Lys Gly Thr Gly Gly Lys Gly Ser

565 570 575

Gly Gly Lys Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Val Val

580 585 590

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

595 600 605

Thr Phe Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys

610 615 620

Gly Leu Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr

625 630 635 640

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

645 650 655

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr

660 665 670

Ala Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg

675 680 685

Ser Ala Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

690 695 700

Val Ser Ser Ala

705

<210> 20

<211> 701

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V3

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Glu Val

565 570 575

Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu

580 585 590

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met

595 600 605

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Leu

610 615 620

Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly

625 630 635 640

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln

645 650 655

Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ser

660 665 670

Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala Phe Asp

675 680 685

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala

690 695 700

<210> 21

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V4

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 22

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V5

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 23

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V6

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Glu Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 24

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V7

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 25

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V8

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Ala Pro Gly Lys Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Ile Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 26

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V9

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Ala Pro Gly Lys Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 27

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V10

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 28

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V11

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Glu Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 29

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V12

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr

20 25 30

Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

450 455 460

Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu

465 470 475 480

Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu

500 505 510

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

515 520 525

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

530 535 540

Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val

545 550 555 560

Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

565 570 575

Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

645 650 655

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu

660 665 670

Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala

675 680 685

Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

690 695 700

Ser Ala

705

<210> 30

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V4

<400> 30

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Ser Val Ser Val Thr Pro Gly Lys Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 31

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V5

<400> 31

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Glu

1 5 10 15

Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 32

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V6

<400> 32

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Glu Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 33

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V7

<400> 33

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 34

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V8

<400> 34

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys Thr Ala Arg

20 25 30

Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Ile Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 35

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V9

<400> 35

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys Thr Ala Arg

20 25 30

Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 36

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V10

<400> 36

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg

20 25 30

Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 37

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V11

<400> 37

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Glu Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 38

<211> 176

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1-OX40 Ab-V12

<400> 38

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val

1 5 10 15

Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg

20 25 30

Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr

50 55 60

Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly

65 70 75 80

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe

100 105 110

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly

115 120 125

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val

165 170 175

<210> 39

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 39

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 40

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 40

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg

1 5 10 15

Ser Ser Leu

<210> 41

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 41

Gly Gly Lys Gly Ser Gly Gly Lys Gly Thr Gly Gly Lys Gly Ser Gly

1 5 10 15

Gly Lys Gly Ser

20

<210> 42

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 42

Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser

1 5 10

<210> 43

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 43

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<---

Claims (30)

1. An antibody or antigen-binding portion thereof that binds to OX40 (CD134), containing at least one polypeptide chain that contains an OX40 (CD134) binding site, containing: a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 6 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 5; a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 8 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 7; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with an amino acid sequence consisting of amino acids 128-246 of SEQ ID NO. 10 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-112 of SEQ ID NO. 10; or a heavy chain variable region comprising an amino acid sequence having at least 90% identity with an amino acid sequence consisting of amino acids 124-241 of SEQ ID NO. 13 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 13, where at least 90% identity with respect to the amino acid sequence excludes complementarity determining regions (CDRs). 2. An antibody or antigen-binding portion thereof according to claim 1, wherein the OX40 binding site is a single chain variable fragment (scFv) sequence selected from the group consisting of SEQ ID NO. 10, 11, 12 and 13. 3. A bispecific antibody that binds to OX40 and a ligand of programmed cell death protein 1 (PD-L1), containing a polypeptide chain, the polypeptide chain containing an OX40 binding site and a PD-L1 binding site, where the OX40 binding site contains: a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 6 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 5; a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 8 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 7; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with an amino acid sequence consisting of amino acids 128-246 of SEQ ID NO. 10 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-112 of SEQ ID NO. 10; or a heavy chain variable region comprising an amino acid sequence having at least 90% identity with an amino acid sequence consisting of amino acids 124-241 of SEQ ID NO. 13 and a light chain variable region comprising an amino acid sequence having at least about 90% identity with an amino acid sequence consisting of amino acids 1-108 of SEQ ID NO. 13, where the PD-L1 binding site contains: a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 2 and a light chain variable domain comprising an amino acid sequence having at least 90% identity with an amino acid sequence consisting of amino acids 1-111 of SEQ ID NO. 1, and a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 4 and a light chain variable domain comprising an amino acid sequence having at least 90% identity to an amino acid sequence of at least about 90% with respect to the amino acid sequence of amino acids 1-110 of SEQ ID NO. 3, where at least 90% identity with respect to the amino acid sequence excludes complementarity determining regions (CDRs). 4. The bispecific antibody of claim 3, wherein the polypeptide chain further comprises: Fc domain; a Fab fragment linked to the N-terminus of an Fc domain and a Fab fragment containing a PD-L1 binding site, and scFv associated with the C-terminus of the Fc domain, and scFv containing the OX40 binding site. 5. The bispecific antibody of claim 4, wherein the polypeptide chain further comprises a linker between the Fc domain and the scFv. 6. The bispecific antibody of claim 5, wherein the amino acid sequence of the linker is SEQ ID NO. 14. 7. The bispecific antibody of claim 4, wherein the scFv comprises an amino acid sequence selected from the group consisting of amino acids 455-707 of SEQ ID NO. 18, 455-708 of SEQ ID NO. 19, 455-701 of SEQ. ID NO. 20, 455-706 of SEQ ID NO. 21, 455-706 of SEQ ID NO. 22, 455-706 of SEQ ID NO. 23, 455-706 of SEQ ID NO. 24, 455-706 of SEQ ID NO. 25, 455-706 of SEQ ID NO. 26, 455-706 of SEQ ID NO. 27, 455-706 of SEQ ID NO. 28 and 455-706 of SEQ ID NO. 29. 8. The bispecific antibody of claim 3, wherein the bispecific antibody contains one pair of polypeptide chains. 9. The bispecific antibody of claim 8, wherein the bispecific antibody is an IgG, IgE, IgM, IgD, IgA, or IgY antibody. 10. The bispecific antibody of claim 9, wherein the bispecific antibody is an IgG antibody. 11. The bispecific antibody of claim 10, wherein the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. 12. Pharmaceutical composition for the treatment of cancer, containing the antibody or its antigennegative part according to claim 1 or 2, or a bispecific antibody according to any one of paragraphs. 3-11 and at least one pharmaceutically acceptable carrier, wherein the cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC) and ovarian cancer. 13. The use of an antibody or antigennegative part according to claim 1 or 2, or a bispecific antibody according to any one of paragraphs. 3-11 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC) and ovarian cancer. 14. A nucleic acid molecule encoding an antibody or its antigen-binding portion according to claim 1 or 2, or encoding a bispecific antibody according to any one of paragraphs. 3-11.

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