Fc binding fragment containing the OX40 antigen binding site

Abstract

The present application relates to specific binding members that bind to OX40. The specific binding members comprise an OX40 antigen binding site located in the constant domain of the specific binding member and can be used, for example, in the treatment of cancer and infectious diseases.

Description

Invention Field

[0001] This invention relates to a specific binding member for OX40. The specific binding member comprises an OX40 antigen-binding site located in a constant domain of the specific binding member, and can be used, for example, in the treatment of cancer and infectious diseases. Background of the Invention

[0003] Cellular signaling is a vital component of all life and typically involves cell surface receptors that interact with soluble or cell-surface-expressed ligands. This interaction leads to changes in the receptor, ligand, or both. For example, ligand binding can induce conformational changes in receptors, causing them to cluster into dimers or oligomers. This clustering effect then leads to activation of intracellular signaling pathways. Many receptors are activated in this manner, including members of the tumor necrosis factor receptor superfamily (TNFRSF), such as OX40.

[0004] OX40 (also known as CD134 and TNFRSF4) is primarily expressed on activated T cells, including CD4+ T cells, CD8+ T cells, type 1 and type 2 helper T cells (Th1 and Th2), and regulatory T (Treg) cells, and is also expressed on activated natural killer (NK) cells. The interaction between OX40 and its ligand OX40L, expressed on antigen-presenting cells (APCs), leads to OX40 receptor clustering. OX40L, like most ligands of other tumor necrosis factor (TNF) receptors, is expressed on the cell surface as a trimer. A hypothetical model of OX40 activation is that the interaction of OX40 with the cell-surface-expressed trimer OX40L induces the clustering of OX40 receptors, which are present on the cell surface either as monomers or pre-formed trimers. This clustering of OX40 receptors activates the NF-κB signaling pathway (Croft, 2010). Activation of the NF-κB signaling pathway, in turn, increases T cell activation, T cell clonal expansion, T cell differentiation and survival, and enhances the generation of memory T cells. The primary role of the OX40 / OX40L interaction is to regulate the accumulation of effector (protective or pathogenic) T cells in the late stages of the primary immune response, thereby increasing the number of memory T cells available for use in a secondary immune response upon re-encounter with the antigen (Croft, 2010). OX40 can mediate its effects on T cells directly, as described above, or indirectly, by enhancing the production of inflammatory cytokines such as IL2 and IFNγ. OX40 signaling can also modulate the function of Treg cells to eliminate their immunosuppressive activity (Croft, 2010).

[0005] The therapeutic efficacy of OX40 agonists has been demonstrated in mouse tumor models. Specifically, OX40 agonists (OX40L-Ig and anti-OX40 mAb OX86) have been shown to be effective in mouse tumor models of melanoma, glioma, breast and colon cancer, sarcoma, renal cell carcinoma, and prostate cancer (Weinberg et al., 2000; Morris et al., 2001; Ali et al., 2004; Sadun et al., 2008; Redmond et al., 2009). The efficacy of OX40 agonist monotherapy appears to be related to tumor immunogenicity (Kjaergaard et al., 2000), suggesting that OX40 expression on tumor-specific T cells requires adequate activation by tumor antigens, while poorly immunogenic tumors fail to provide sufficient activation.

[0006] The efficacy of anti-OX40 agonist antibodies is also being studied in clinical trials, and they can be used as a monotherapy or in combination with other monoclonal antibodies (mAbs).

[0007] Clinical trials of anti-OX40 mAb as a monotherapy include a Phase I study of mouse monoclonal anti-OX40 mAb in patients with advanced cancer, which showed acceptable toxicity issues and regression of at least one metastatic lesion in 12 of 30 patients (Curti et al., 2013). Preliminary results from a Phase I study of humanized anti-OX40 mAb (MEDI0562; MedImmune) in patients with advanced solid tumors showed no dose-limiting toxicities (DLT), with one in 32 patients demonstrating an objective response (Glisson et al., 2016).

[0008] As mentioned above, anti-OX40 mAbs in combination with other mAbs are also being tested for cancer treatment. For example, combinations of anti-OX40 mAbs with anti-PD-L1 mAbs (durvalumab) or anti-CTLA4 mAbs (tremelimumab) (clinical trials, government identifier: NCT02705482) are being tested in advanced solid tumors. These combinations have been tested in preclinical models and have shown improved tumor regression and survival (Guo et al., 2014; Redmond et al., 2014).

[0009] Clinical trials are underway to test anti-OX40 mAb (MOXR0916; Genentech) as monotherapy (NCT02219724) and in combination with anti-PD-L1 mAb (atezolizumab) (NCT03029832) for the treatment of locally advanced or metastatic solid tumors. The combination of humanized anti-OX40 mAb (GSK3174998; GlaxoSmithKline) and anti-PD-1 mAb (pembrolizumab) is currently being evaluated for the treatment of selected advanced or recurrent solid tumors (NCT02528357). Human anti-OX40 mAb (PF-04518600; Pfizer) has been tested in clinical trials for the treatment of locally advanced or metastatic cancers and has been shown to be well-tolerated, with 27 out of 48 patients achieving partial remission (2 patients) or stable disease (25 patients) (NCT02315066; El-Khoueiry et al., 2017). This mAb has also been tested in combination with an anti-4-1BB agonist mAb (PF-05082566 / utomilumab) (NCT02315066) and an anti-PD-L1 mAb (avelumab) (NCT02554812) for the treatment of locally advanced or metastatic solid tumors. Human IgG1 anti-OX40 mAb (BMS-986178; Bristol-Myers Squibb) is being clinically tested in combination with anti-PD-1 mAb (nivolumab) or anti-CTLA-4 mAb (ipilimumab), or a combination of both, for the treatment of advanced or metastatic solid tumors (NCT02737475).

[0010] Invention Description

[0011] The inventors conducted extensive selection and affinity maturation procedures to isolate a set of antibody Fc region fragments (Fcabs). TM It contains an OX40 antigen binding site engineered into its CH3 domain.

[0012] The Fcab molecule consists of two identical polypeptide chains, each containing a truncated hinge region, a CH2 domain, and a CH3 domain. The two polypeptide chains are linked together by multiple disulfide bonds in the hinge region and a hydrophobic region present in the CH3 domain. As described above, the initial binding of the OX40 ligand to its receptor OX40 triggers a series of events leading to OX40 receptor clustering, which subsequently activates the NF-κB intracellular signaling pathway and subsequently induces potent T cell activity. For therapeutic agents to effectively achieve activation, several OX40 monomers need to be bridged together in a manner mimicking the surface expression of trimer ligands. The inventors isolated a subset of anti-OX40 Fcab based on its ability to bind OX40, demonstrating its ability to drive OX40 clustering and activation on the T cell surface. This is surprising, given the rigid structure and small molecular distance between the constant domains of the Fcab molecule (especially the two CH3 domains), which contrasts with the known flexibility of antibody molecules in the hinge region, allowing the Fab arm of the anti-OX40 antibody molecule to move and bind to its target. Given the tight geometry of the binding sites of the constant domains of the Fcab molecule, it was expected that these binding sites would not be able to induce the aggregation and activation of OX40 molecules, which may not initially be near the T cell surface. However, contrary to expectations, the results obtained by the inventors described herein clearly demonstrate that anti-OX40 Fcab can induce the aggregation and activation of OX40 in vitro and in vivo.

[0013] Fcab was chosen to bind to the dimer OX40 with high affinity, which is the expected outcome. High affinity for the dimer OX40 is thought to favor inducing OX40 clustering and activation.

[0014] As this article refers to, "affinity" can refer to the strength of the binding interaction between an antibody molecule and its homologous antigen, measured by K... D Assay. It will be apparent to those skilled in the art that the antibody molecule is capable of forming multiple binding interactions with the antigen (e.g., the antibody molecule is capable of binding divalently to the antigen and optionally the antigen is a dimer), such as via K... D The measured affinity may also be affected by avidity, which refers to the overall strength of the antibody-antigen complex.

[0015] The inventors identified two groups of Fcabs capable of inducing OX40 clustering and activation. The first group of Fcabs (FS20-11 lineage) depends on cross-linking, such as an anti-CH2 domain antibody for OX40 clustering and activation, while the second group (FS20-22 and FS20-31 lineages) exhibits low levels of OX40 clustering and activation even in the absence of cross-linking. OX40 agonist antibodies have not shown any clinically significant lesions (DLTs). Therefore, OX40 agonist activity is not expected to be a problem for clinical treatment in the absence of cross-linking. Conversely, depending on the condition to be treated, the low levels of OX40 agonist activity of the Fcab in the absence of cross-linking may be advantageous. Not wishing to be bound by theory, it is believed that anti-OX40 Fcabs with this property can be used, for example, in cancer treatment by inducing limited activation and expansion of tumor-reactive T cells in the absence of cross-linking, leading to a larger pool of tumor-reactive T cells, which can then be further activated by cross-linked Fcab molecules in the tumor microenvironment.

[0016] Conventional antibodies specific to the TNF receptor (e.g., OX40) typically possess little or no intrinsic agonistic activity and require secondary cross-linking with external cross-linking agents, such as proteins A or G or secondary antibodies, or binding the antibody to a plasma membrane-localized Fcγ receptor to induce higher levels of TNF receptor member aggregation and activation (Wajant, 2015). The low levels or lack of agonist activity of TNF receptor-specific antibodies in the absence of cross-linking can be explained by the fact that normal bivalent antibodies maximally cross-link two monomeric TNF receptors, which is insufficient for TNF receptor activation. Therefore, for in vivo efficacy, monospecific antibodies targeting OX40 require the presence of Fcγ receptor-expressing cells near OX40-expressing T cells to facilitate OX40-specific antibody cross-linking and subsequent OX40 receptor aggregation and activation. However, Fcγ receptor-mediated cross-linking is considered inefficient. Furthermore, Fcγ receptor-expressing cells are distributed throughout the body, meaning that antibody cross-linking and activation of OX40-expressing T cells are not limited to specific sites, such as the tumor microenvironment. Furthermore, it is necessary to select the isotype of this type of OX40 antibody to mediate efficient binding to the Fcγ receptor for cross-linking. However, this may cause the antibody to trigger effector functions mediated by the Fcγ receptor (e.g., ADCC), thereby eliminating T cells that would otherwise be activated by the antibody.

[0017] The inventors have studied the cross-linked Fcab-OX40 complex (with mAb) 2 Mass spectrometry analysis of the Fcab in its form showed that 17% of the complex contained two OX40 moieties, indicating that the anti-OX40 Fcab of the present invention can bind OX40 divalently.

[0018] The inventors recognize that the anti-OX40 Fcab of the present invention can be used to prepare multispecific molecules, such as bispecific molecules, which bind a second antigen, such as a tumor antigen, in addition to binding OX40. Preferably, the multispecific molecule can also bind a second antigen bivalently, although the second antigen is expected to be a cell-bound tumor antigen; monovalent binding of the antigen will be sufficient to crosslink the specific binding member / antibody molecule and induce OX40 clustering and activation.

[0019] The inventors have prepared an antibody molecule comprising the anti-OX40 Fcab of the present invention, which can bivalently bind a second antigen through its Fab region. The inventors have demonstrated that this bispecific antibody molecule can conditionally activate OX40 in the presence of the second antigen without requiring activation via cross-linking to a conventional antibody molecule, such as an Fcγ receptor. The same effect was observed regardless of whether the second antigen was a cell surface receptor or a multimeric soluble factor. It is believed that the binding of the antibody molecule to the second antigen causes cross-linking of the antibody molecule at the antigen site, which in turn leads to the clustering and activation of OX40 on the T cell surface. Therefore, the agonistic activity of the antibody molecule depends on the presence of both the second antigen and OX40, or is enhanced when both are present. In other words, the agonistic activity is conditional. Furthermore, when the second antigen is present, it is believed that the cross-linking of the antibody contributes to the clustering of OX40 bound via the constant domain antigen-binding site of the antibody molecule, because when an increase in the agonistic activity of the antibody molecule is observed, both binding sites of the antibody molecule bind to their respective targets, but there is no increase when only one binding site is bound. Therefore, it is anticipated that the multispecific molecules containing the anti-OX40 Fcab of the present invention can effectively activate immune cells in a disease-dependent manner, such as in the tumor microenvironment.

[0020] The inventors have demonstrated that bispecific antibody molecules containing the anti-OX40 Fcab of the present invention can inhibit tumor growth in vivo. Furthermore, more effective tumor growth inhibition has been observed with these bispecific antibody molecules compared to a combination of two monospecific antibody molecules, one containing the same constant domain as the bispecific molecule and the other having the same variable domain binding site, indicating enhanced OX40 clustering and signaling. Therefore, when both binding sites are present in the same molecule, T cell activation and corresponding antitumor effects can be observed.

[0021] As described above, unlike conventional antibodies, antibody molecules containing the anti-OX40 Fcab of the present invention do not depend on Fcγ receptor cross-linking to drive OX40 clustering and activation. Mutations for eliminating Fcγ receptor binding are known in the art and can be included in the molecules of the present invention. However, in certain situations, such as cancer therapy, preserving Fcγ receptor binding may be beneficial. For example, if the antibody molecule binds to a tumor antigen via its Fab region and the OX40 antigen binding site is not bound, antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells will be induced. In addition to T cell activation and subsequent T cell-mediated tumor cell killing, the antibody molecule also induces ADCC activity.

[0022] The antibody molecule comprising the anti-OX40 Fcab of the present invention and a Fab region specific to a second antigen is preferably bivalently bound to both OX40 and the second antigen. This is advantageous because the bivalent binding of the two targets is expected to stabilize the bridging between OX40-expressing T cells and the second antigen, thereby prolonging the time T cells spend localizing to specific sites, such as the tumor microenvironment, and thus enabling them to act on diseases, such as tumors. This differs from the vast majority of conventional bispecific antibody forms, which are heterodimers and monovalently bind to each target antigen via a single Fab arm. Such monovalent interactions are not only expected to be unstable but, in many cases, insufficient to induce clustering of TNFRSF receptors such as OX40.

[0023] Another feature of the antibody molecules containing the anti-OX40 Fcab of the present invention is that both antigen-binding sites of OX40 and the second antigen are contained within the antibody structure itself. In particular, the antibody molecules do not require the fusion of other proteins to the antibody molecules via linkers or other means to produce molecules that bind bivalently to both of their targets. This has several advantages. Specifically, since the antibody molecules do not contain any additional fusion portions, they can be produced using methods similar to those used to produce standard antibodies. This structure also promises to improve antibody stability, as linkers can degrade over time, leading to a heterogeneous population of antibody molecules. Antibodies in populations containing only one fusion protein will not induce conditional activation of the TNFRSF receptor (e.g., OX40) as effectively as antibodies containing two fusion proteins. Cleavage or degradation of the linker can occur before or after administration of the therapeutic agent to a patient (e.g., by enzymatic cleavage or in vivo pH), resulting in reduced efficacy during circulation within the individual patient. Since the antibody molecules identified in the invention do not contain linkers, it is expected that the antibody molecules will retain the same number of binding sites before and after administration. Furthermore, from the perspective of molecular immunogenicity, the structure of the antibody molecule of the present invention is also preferred, because when the antibody molecule is administered to a patient, the introduction of the fusion protein or the linker or both can induce immunogenicity, leading to a reduction in the therapeutic effect.

[0024] Therefore, the present invention provides:

[0025] [1] A specific binding member for OX40, comprising an OX40 antigen binding site located in the CH3 domain of the specific binding member, wherein the OX40 antigen binding site comprises a first, second, and / or third, preferably first and third, sequence of the specific binding member FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, more preferably first, second, and third sequences, wherein the first, second, and third sequences of the specific binding member are:

[0026] (i)FS20-22-49 is listed in SEQ ID NO: 43, 54 and 71 respectively;

[0027] (ii) FS20-22-41 is listed in SEQ ID NO:43, 54 and 45 respectively;

[0028] (iii) FS20-22-47 is listed in SEQ ID NO:43, 54 and 62 respectively;

[0029] (iv) FS20-22-85 is listed in SEQ ID NO:43, 54 and 80 respectively; and

[0030] (V)FS20-22-38 is listed in SEQ ID NO:43, 44 and 45 respectively; and

[0031] The first, second, and third sequences are located in the AB, CD, and EF structural loops of the CH3 domain of the specific binding member, respectively.

[0032] [2] A specific binding member for OX40, comprising an OX40 antigen binding site located in the CH3 domain of the specific binding member, wherein the OX40 antigen binding site comprises a first, second, and / or third, preferably first and third sequences, of FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, wherein the first, second, and third sequences of the specific binding member are:

[0033] (i)FS20-31-115 is listed in SEQ ID NO: 122, 142 and 133 respectively;

[0034] (ii) FS20-31-108 is listed in SEQ ID NO:122, 132 and 133 respectively;

[0035] (iii) FS20-31-58 is listed in SEQ ID NO:91, 92 and 93 respectively;

[0036] (iv)FS20-31-94 is listed in SEQ ID NO:111, 112 and 113 respectively;

[0037] (v)FS20-31-102 is listed in SEQ ID NO:122, 123 and 102 respectively; and

[0038] (vi)FS20-31-66 is listed in SEQ ID NO:91, 92 and 102 respectively; and

[0039] The first, second, and third sequences are located in the AB, CD, and EF structural loops of the CH3 domain of the specific binding member, respectively.

[0040] [3] A specific binding member for OX40, comprising an OX40 antigen binding site located in the CH3 domain of the specific binding member, wherein the OX40 antigen binding site comprises a first, second, and / or third, preferably first and third, sequence of the specific binding member FS20-11-131, FS20-11-127, or FS20-11-134, more preferably first, second, and third sequences, wherein the first, second, and third sequences of the specific binding member are:

[0041] (i)FS20-11-131 is listed in SEQ ID NO:12, 13 and 23 respectively;

[0042] (ii) FS20-11-127 is listed in SEQ ID NO:12, 13 and 14 respectively; and

[0043] (iii) FS20-11-134 is listed in SEQ ID NO: 12, 13 and 32 respectively; and

[0044] The first, second, and third sequences are located in the AB, CD, and EF structural loops of the CH3 domain of the specific binding member, respectively.

[0045] [4] Based on the specific binding member of [1], the third sequence is located between positions 92 and 102 of the CH3 domain, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0046] [5] Based on the specific binding member of [2], the third sequence is located between positions 91 and 102 of the CH3 domain, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0047] [6] Based on the specific binding member of [3], the third sequence is located between positions 96 and 102 of the CH3 domain, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0048] [7] According to the specific binding member of [3] or [6], wherein the specific binding member contains an amino acid deletion at position 14, 15, 16, 17 or 18 of the CH3 domain, wherein the amino acid residues are numbered according to the IMGT numbering scheme.

[0049] [8] According to any one of [1] to [7], the specific binding member, wherein the first sequence is located between positions 13 and 19 of the CH3 domain, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0050] [9] According to any one of [1] to [8], the specific binding member is located between positions 45 and 78 of the CH3 domain, wherein the amino acid residues are numbered according to the ImMunoGeneTics (IMGT) numbering scheme.

[0051]

[10] According to the specific binding member of any one of [1] to [9], wherein the CH3 domain is the human IgG1 CH3 domain.

[0052]

[11] The specific binding member according to any one of [1], [4] and [8] to

[10] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38 listed in SEQ ID NO:72, 55, 63, 81 and 46 respectively.

[0053]

[12] According to the specific binding member of

[11] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding member FS20-22-49 shown in SEQ ID NO: 72.

[0054]

[13] A specific binding member according to any one of [2], [5] and [8] through

[10] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66 listed in SEQ ID NO:143, 134, 94, 114, 124 and 103, respectively.

[0055]

[14] A specific binding member according to any one of [3], [6], [7] and [8] to

[10] , wherein the specific binding member comprises the CH3 domain sequence of the specific binding members FS20-11-131, FS20-11-127 or FS20-11-134 listed in SEQ ID NO:24, 15 and 33 respectively.

[0056]

[15] A specific binding member according to any one of [1] to

[14] , wherein the specific binding member further comprises a CH2 domain, preferably a CH2 domain of human IgG1.

[0057]

[16] According to the specific binding member of

[15] , wherein the CH2 domain has the sequence listed in SEQ ID NO: 5, 6 or 7.

[0058]

[17] According to any one of

[15] to

[16] , the specific binding member further comprises an immunoglobulin hinge region or a portion thereof at the N-terminus of the CH2 domain.

[0059]

[18] According to the specific binding member of [1], the hinge region or a portion thereof is the human IgG1 hinge region or a portion thereof.

[0060]

[19] According to the specific binding member of

[18] , the hinge region has the sequence or a fragment thereof listed in SEQ ID NO: 170.

[0061]

[20] According to the specific binding member of

[18] or

[19] , the hinge region has the sequence listed in SEQ ID NO: 171.

[0062]

[21] A specific binding member according to any one of [1], [4], [8] to

[10] ,

[11] to

[12] and

[15] to

[20] , wherein the specific binding member comprises a sequence of the following specific binding member:

[0063] (i) FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38 listed in SEQ ID NO:74, 57, 65, 83 and 48 respectively; or

[0064] (ii) FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38 listed in SEQ ID NO:76, 59, 67, 85 and 50 respectively.

[0065]

[22] According to the specific binding member of

[21] , wherein the specific binding member comprises the sequence of the specific binding member FS20-22-49 listed in SEQ ID NO: 74 or SEQ ID NO: 76.

[0066]

[23] A specific binding member according to any one of [2], [5], [8] to

[10] ,

[13] , and

[15] to

[20] , wherein the specific binding member comprises a sequence of the following specific binding member:

[0067] (i) FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66 listed in SEQ ID NO: 145, 136, 96, 116, 126 and 105 respectively; or

[0068] (ii) FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66 listed in SEQ ID NO:147, 138, 98, 118, 128 and 107 respectively.

[0069]

[24] A specific binding member according to any one of [3], [6], [7] to

[10] , and

[14] to

[20] , wherein the specific binding member comprises a sequence of the following specific binding member:

[0070] (i) FS20-11-131, FS20-11-127 or FS20-11-134 listed in SEQ ID NO:26, 17 and 35 respectively; or

[0071] (ii) FS20-11-131, FS20-11-127 or FS20-11-134 listed in SEQ ID NO:28, 19 and 37 respectively.

[0072]

[25] The specific binding member according to any one of [1] to

[24] , wherein the specific binding member binds to human OX40.

[0073]

[26] According to the specific binding member of

[25] , wherein the human OX40 has, comprises or consists of the sequence listed in SEQ ID NO: 161.

[0074]

[27] According to [1], [2], [4], [5], [8] to

[13] ,

[15] to

[23] and

[25] to

[26] , the specific binding member binds to cynomolgus monkey OX40.

[0075]

[28] According to the specific binding member or antibody molecule of

[27] , wherein the cynomolgus monkey OX40 has, comprises or consists of the sequence listed in SEQ ID NO: 166.

[0076]

[29] The specific binding member according to any one of [1] to

[28] , wherein the specific binding member is a multispecific molecule.

[0077]

[30] According to

[29] , the specific binding member is a bispecific, trispecific or tetraspecific molecule.

[0078]

[31] According to the specific binding member of

[30] , wherein the specific binding member is a bispecific molecule.

[0079]

[32] According to any one of [1] to

[31] , wherein the specific binding member further comprises a second antigen binding site.

[0080]

[33] According to the specific binding member of

[32] , the second antigen binding site is an antigen binding site based on CDR.

[0081]

[34] According to the specific binding members of

[33] , the second antigen binding site includes heavy chain variable domains CDR1, CDR2 and CDR3, and light chain variable domains CDR1, CDR2 and CDR3.

[0082]

[35] According to the specific binding members of

[33] to

[34] , wherein the second antigen binding site comprises a heavy chain variable region and a light chain variable region.

[0083]

[36] The specific binding member according to any one of

[32] to

[35] , wherein the specific binding member is an antibody molecule.

[0084]

[37] The antibody molecule according to

[36] , wherein the antibody molecule is a human IgG1 molecule.

[0085]

[38] An antibody molecule according to any one of

[33] to

[37] , wherein the antibody molecule binds a second antigen at a CDR-based antigen-binding site, the second antigen being selected from: immune cell antigens and disease antigens.

[0086]

[39] According to the antibody molecule of

[38] , wherein the disease antigen is a tumor antigen or a pathogen antigen.

[0087]

[40] According to the antibody molecule of

[38] , wherein the immune cell antigen is a member of the tumor necrosis factor receptor superfamily (TNFRSF).

[0088]

[41] According to the antibody molecule of

[39] , wherein the tumor antigen is a tumor-associated antigen (TAA).

[0089]

[42] According to the antibody molecule of

[39] , wherein the pathogen antigen is a bacterial or viral antigen.

[0090]

[43] An antibody molecule according to any one of

[32] to

[42] , wherein the antibody molecule is capable of activating OX40 present on T cells in the presence of a second antigen.

[0091]

[44] An antibody molecule according to any one of

[32] to

[43] , wherein the binding of the antibody molecule to OX40 and the second antigen causes OX40 to cluster on immune cells.

[0092]

[45] According to the antibody molecule of

[43] or

[44] , wherein the immune cell is a T cell.

[0093]

[46] An antibody molecule according to any one of

[38] ,

[41] and

[43] to

[45] , wherein the tumor antigen is a cell surface antigen on a cancer cell.

[0094]

[47] An antibody molecule according to any one of

[38] ,

[41] and

[43] to

[45] , wherein the tumor antigen is a soluble polymer.

[0095]

[48] ​​According to the antibody molecule of

[47] , the soluble polymer is at least a dimer.

[0096]

[49] According to the antibody molecule of

[48] , the soluble polymer is at least a trimer.

[0097]

[50] A specific binding member or antibody molecule according to any one of [1] to

[49] , wherein the specific binding member or antibody molecule is modified to reduce or eliminate the binding of the CH2 domain of the specific binding member or antibody molecule to one or more Fcγ receptors.

[0098]

[51] A specific binding member or antibody molecule according to any one of [1] to

[50] , wherein the specific binding member or antibody molecule does not bind to the Fcγ receptor.

[0099]

[52] According to the specific binding member or antibody molecule of

[50] or

[51] , wherein the Fcγ receptor is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb and FcγRIII.

[0100]

[53] A specific binding member or antibody molecule according to any one of [1] to

[52] , wherein the specific binding member or antibody molecule is conjugated to a biologically active molecule.

[0101]

[54] A specific binding member or antibody molecule according to any one of [1] to

[52] , wherein the specific binding member or antibody molecule is conjugated to a detectable label.

[0102]

[55] A nucleic acid molecule that encodes a specific binding member or antibody molecule as described in any one of [1] to

[52] .

[0103]

[56] The nucleic acid molecule according to

[55] , wherein said one or more nucleic acid molecules comprise:

[0104] (i) Nucleic acid sequences that specifically bind to the CH3 domain of members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38, as listed in SEQ ID NO: 73, 56, 64, 82 and 47, respectively;

[0105] (ii) Nucleic acid sequences that specifically bind to the CH3 domain of members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, as listed in SEQ ID NO: 144, 135, 95, 115, 125, and 104, respectively; or

[0106] (iii) Nucleic acid sequences that specifically bind to the CH3 domain of members FS20-11-131, FS20-11-127 or FS20-11-134, as listed in SEQ ID NO: 25, 16 and 34, respectively.

[0107]

[57] The nucleic acid molecule according to

[55] or

[56] , wherein the nucleic acid molecule comprises a nucleic acid sequence of the following specific binding member:

[0108] (i) FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38 as listed in SEQ ID No: 75, 58, 66, a84 and 49 respectively;

[0109] (ii) FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66 respectively, as listed in SEQ ID Nos: 146, 137, 97, 117, 127 and 106; or

[0110] (iii) FS20-11-131, FS20-11-127 or FS20-11-134 as listed in SEQ ID NO: 27, 18 and 36 respectively.

[0111]

[58] The nucleic acid molecule according to

[55] or

[56] , wherein the nucleic acid molecule comprises a nucleic acid sequence of the following specific binding member:

[0112] (i) FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38 as listed in SEQ ID NO: 77, 60, 68, 86 and 51 respectively;

[0113] (ii) FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66 listed in 148, 139, 99, 119, 129 and 108 respectively.

[0114] (ii) FS20-11-131, FS20-11-127 or FS20-11-134 as listed in SEQ ID NO: 29, 20 and 38 respectively.

[0115]

[59] A vector comprising any one of the nucleic acids in

[55] to

[58] .

[0116]

[60] A recombinant host cell comprising any one of the nucleic acids of

[55] to

[58] , or a vector of

[59] .

[0117]

[61] A method for preparing a specific binding member or antibody molecule according to any one of [1] to

[52] , comprising culturing

[60] recombinant host cells under conditions for preparing a specific binding member or antibody molecule.

[0118]

[62] The method as described in

[61] further includes isolating and / or purifying the specific binding member or antibody molecule.

[0119]

[63] A pharmaceutical composition comprising a specific binding member or antibody molecule according to any one of [1] to

[54] and a pharmaceutically acceptable excipient.

[0120]

[64] A treatment method for treating humans or animals that specifically binds to a member or antibody molecule according to any one of [1] to

[54] .

[0121]

[65] A method of treating an individual’s disease or condition, comprising administering to the individual a therapeutically effective amount of a specific binding member or antibody molecule according to any one of [1] to

[54] .

[0122]

[66] The specific binding member or antibody molecule used according to

[64] , or the method described in

[65] , wherein the treatment is for an individual cancer or infectious disease.

[0123]

[67] The specific binding member or antibody molecule used according to

[64] or

[66] , or the method according to

[65] or

[66] , wherein the treatment method comprises administering the specific binding member or antibody molecule in combination with a second therapeutic agent to an individual.

[0124] Brief description of the attached figures

[0125] Figure 1A -C shows the sequence alignment of Fcab FS20-11, FS20-11-127, FS20-11-131, FS20-11-134, FS20-22, FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, and FS20-31-115, as well as the CH3 domain of wild-type (WT) Fcab. The positions of the AB, CD, and EF loops in the CH3 domain of Fcab compared to the WT sequence are indicated, along with any amino acid substitutions or deletions (marked with tilde "+"). "" indicates or is inserted. Residue numbers are displayed according to the IMGT, IMGT exon (sequential numbering), EU, and Kabat numbering systems.

[0126] Figure 2This shows the release of IL-2 in the T cell activation assay in the presence of anti-human OX40 Fcabs. Representative plots of one anti-human OX40 Fcab from each lineage (FS20-11, FS20-22, and FS20-31) are shown in [images / plots]. Figure 2 Displayed in A, 2B, and 2C. Simulated (4420LALA)mAb 2 The anti-human OX40 Fcab assay was performed. IL-2 release was tested in the presence and absence of a crosslinking agent (Xlink). Anti-FITC antibody 4420 and anti-OX40 antibody 11D4, respectively, in the human IgG1 backbone (G1 / 4420 and G1 / 11D4), served as negative and positive controls, respectively. The effect of increasing concentrations of anti-human OX40 Fcab and control antibodies on IL-2 release was tested. Increased IL-2 release was observed in the crosslinked positive control mAb (G1 / 11D4) and anti-human OX40 Fcab, but no increase in IL-2 release was observed in the non-crosslinked positive control mAb or negative control mAb (G1 / 4420), demonstrating a concentration-dependent increase in T cell activation. A simulated (4420LALA) mAb was also tested. 2 The form FS20-11-131Fcab is inactive without cross-linking. Simulated (4420LALA) mAb 2 The forms FS20-22-49 and FS20-31-115Fcab exhibit some activity without crosslinking, and this activity increases with crosslinking.

[0127] Figure 3 The simulated (HEL D1.3 LALA) mAb in the CT26 tumor model is shown. 2 In vivo antitumor activity of the anti-mouse OX40 Fcab form. Tumor growth curves of the CT26 syngeneic model in Balb / c mouse groups are shown. (OX40 / mammalian mAb) 2 LALA) and not having (OX40 / analog mAb) 2 Simulation of LALA mutation (HEL D1.3) mAb 2 The in vivo antitumor activity of the anti-mouse OX40 Fcab was compared with that of the positive control anti-mouse OX40 mAb (OX86 in the human IgG1 backbone; none / OX40mAb) and the negative control antibody (4420 antibody in the human IgG1 backbone; none / FITC). Different molecules were administered at a dose of 1 mg / kg on days 10, 12, and 14 post-tumor inoculation. A graph was plotted showing the mean tumor volume plus or minus the mean standard error.

[0128] Figure 4Figures A through D show representative diagrams of IL-2 release in a T-cell activation assay in the presence of HPAC cells. In this assay, increasing concentrations of mAb / mAb were used. 2 The samples were labeled according to their Fcab / Fab clone names. Results showed that mAbs / mAbs targeting OX40 were targeted when cross-linked with a cross-linking agent (anti-human CH2 antibody or FITC-glucan) or via TAA+HPAC cell cross-linking. 2 The concentration-dependent effect on T cell activation increases.

[0129] Figure 5 A and B show representative graphs of IL-2 release in a T cell activation assay in the presence of HPAC cells. In this assay, increasing concentrations of mAb / mAb were used. 2 The samples were labeled according to their Fcab / Fab clone names. Results showed that mAbs / mAbs targeting OX40 were targeted when cross-linked with a cross-linking agent (anti-human CH2 antibody or FITC-glucan) or via TAA+HPAC cell cross-linking. 2 The concentration-dependent effect on T cell activation increases.

[0130] Figure 6 A shows that when mAbs targeting OX40 and EphA2 are present... 2 In the presence of EphA2-expressing cells (HPAC), T cell activation increased, while in the presence of other antibodies targeting OX40 but not cross-linked, T cell activation did not increase. This indicates that mAbs... 2 Cross-linking occurs through binding to two targets, OX40 and EphA2. Figure 6 B shows antibodies targeting OX40-activated T cells in the presence of non-physiological cross-linking agents (anti-Fc antibody or FITC-dextran). As expected, anti-EphA2 and anti-FITC antibodies did not induce T cell activation in the presence of cross-linking agents. When cross-linked with anti-Fc antibody, anti-mouse OX40 antibody (G1 / OX86) induced some T cell activation. When anti-mouse OX40 Fcab was cross-linked with a mimic mAb... 2 When anti-FITC Fab in (FS20m-232-91AA / 4420) is paired with anti-OX40 antibody G1 / OX86, anti-mouse OX40 Fcab exhibits lower ECG activation in T cells via FITC-dextran cross-linking compared to anti-OX40 antibody G1 / OX86. 50 And a higher maximum response. When the same anti-OX40 Fcab is used with mAb 2 When paired with the anti-EphA2 Fab in (FS20m-232-91AA / E2A), it is compatible with the anti-OX40 / anti-FITC mAb. 2 In contrast, activated T cells exhibited lower ECGs in the presence of HPAC cells. 50And a comparable maximum response.

[0131] Figure 7 Tumor growth curves of the CT26 syngeneic model in Balb / c mice treated with G1 / 4420, FS20m-232-91AA / 4420, FS20m-232-91AA / 4420+G1 / E2A, and FS20m-232-91AA / E2A are shown. A plot of mean tumor volume plus or minus the mean standard error is presented. A two-tailed t-test is used to compare tumor volumes on the last day between different groups. The groups treated with anti-mOX40 / anti-EphA2 mAb are compared to those treated with the control antibody (G1 / 4420). 2 The group treated with (FS20m-232-91AA / E2A) showed a statistically significant reduction in tumor volume.

[0132] Figure 8 This diagram shows a representative graph of IL-2 release in a T cell activation assay in the presence of soluble VEGF. In this assay, mAb / mAb concentrations are incremented. 2 The tests were conducted and tagged according to their Fcab / Fab clone names. Results showed that when targeted with mAb / mAb... 2 When crosslinked with crosslinking agents (anti-hCH2, FITC-glucan, or VEGF), mAb / mAb targeting OX40 2 Concentration-dependent increase in T cell activation.

[0133] Figure 9 It shows the existence of various mAbs / mAbs 2 A representative graph of IL-2 release in a T-cell activation assay. Five different mAb / mAb ratios were tested in this assay at increasing concentrations. 2 They were named according to their Fcab / Fab clones (G1 / 4420, G1 / R84, G1 / OX86, FS20m-232-91AA / 4420, and FS20m-232-91AA / R84). The results showed that, in the presence of VEGF, anti-mouse OX40 / anti-VEGF mAb... 2 (FS20m-232-91AA / R84) enhanced T cell activation. This result demonstrates that antibodies targeting OX40 require cross-linking to increase T cell activation, and that anti-OX40 / anti-VEGF mAb... 2 It can be cross-linked with Fabs that target VEGF.

[0134] Figure 10Tumor growth curves of the CT26 syngeneic model in Balb / c mice treated with G1 / 4420, G1 / R84, FS20m-232-91AA / 4420+G1 / R84, and FS20m-232-91AA / R84 are shown. The mean tumor volume is plotted with or without the standard error mean, and a two-tailed t-test is used to compare the tumor volume on the last day between different groups. Compared with the control group (antibody-free / FITC treatment group), the anti-mOX40 / anti-VEGF mAb... 2 The tumor volume in the antibody treatment group was statistically significantly reduced. This result indicates that, compared with the simulated mAb... 2 Compared to combinations of anti-mOX40 Fcab and anti-VEGF antibodies, anti-mOX40 / anti-VEGF mAb... 2 The antibody exhibits better in vivo anti-tumor effects, reportedly due to increased VEGF concentration in its microenvironment, suggesting that this is achieved through the interaction of anti-mOX40 / anti-VEGF mAb. 2 In vivo OX40 cross-linking, mediated by the bispecific binding of OX40 and VEGF, can effectively control tumor growth.

[0135] Figure 11 A through C show representative graphs of IL-2 release used in the T cell activation assay. In this assay, antibodies were used at increasing concentrations and labeled according to their Fcab / Fab clone names. Results showed that the concentration of the OX40-targeting antibody increased in a concentration-dependent manner with respect to T cell activation when cross-linked with its cross-linking agent (anti-hCH2; FITC-dextran), and the concentration of anti-OX40 / anti-ICOS mAb increased in a concentration-dependent manner. 2 (FS20-22-49AA / ICOSj), Anti-OX40 / Anti-CD27 mAb 2 (FS20-22-49AA / 695) and anti-OX40 / anti-GITR mAb 2 (FS20-22-49AA / 6C8) exhibits agonistic activity in the absence of other crosslinking agents, which is superior to that in simulated mAbs. 2 The non-crosslinked form of the anti-OX40 Fcab (FS20-22-49AA / 4420) exhibits agonistic activity.

[0136] Figure 12 A representative graph of IL-2 release for a T cell activation assay is shown. In this assay, antibodies were used at incremental concentrations and labeled according to their Fcab / Fab clone names. The results showed that T cell activation increased in a concentration-dependent manner when the antibody targeting OX40 was cross-linked with its cross-linking agent (anti-hCH2; FITC-dextran), and that the anti-OX40 / anti-PD1 mAb without other cross-linking agents was also effective. 2The agonistic activity of (FS20-22-49AA / 5C4) is comparable to that of the cross-linked anti-OX40 Fcab (FS20-22-49AA / 4420Xlink).

[0137] Figure 13 A representative graph of IL-2 release for a T cell activation assay is shown. In this assay, antibodies were used at incremental concentrations and labeled according to their Fcab / Fab clone names. The results showed that T cell activation increased in a concentration-dependent manner when the antibody targeting OX40 was cross-linked with its cross-linking agent (anti-hCH2; FITC-dextran), and that the anti-OX40 / anti-LAG3 mAb without other cross-linking agents was also effective. 2 The agonistic activity of (FS20-22-41AA / 25F7) is comparable to that of the cross-linked anti-OX40 Fcab (FS20-22-41AA / 4420Xlink).

[0138] Figure 14 A representative graph of IL-2 release for a T cell activation assay is shown. In this assay, antibodies were used at incremental concentrations and labeled according to their Fcab / Fab clone names. The results showed that T cell activation increased in a concentration-dependent manner when the antibody targeting OX40 was cross-linked with its cross-linking agent (anti-hCH2; FITC-dextran), and that the anti-OX40 / anti-LAG3 mAb without other cross-linking agents was also effective. 2 The agonistic activity of (FS20m-232-91AA / C9B7W) was higher than that of the non-crosslinked anti-OX40 Fcab (FS20m-232-91AA / 4420). This indicates that the Fab crosslinked anti-OX40 Fcab bound to LAG3 activates T cells.

[0139] Figure 15 The use of anti-mOX40 / anti-LAG3 mAb was demonstrated. 2 Tumor growth curves of the CT26 syngeneic model in Balb / c mice treated with PBS were plotted. The mean tumor volume was added to or subtracted from the standard error mean, and a two-tailed t-test was used to compare tumor volumes on the last day between different groups. Tumor volumes treated with anti-OX40 / anti-LAG3 mAb were compared with the control group treated with PBS. 2 The treated group showed a statistically significant reduction in tumor volume. This result indicates that anti-OX40 / anti-LAG3 mAb... 2 The antibody exhibits anti-tumor activity in vivo and is described as containing tumor-infiltrating lymphocytes (TILs), including T cells expressing OX40 and LAG3, suggesting efficacy through anti-OX40 / anti-LAG3 mAb. 2 In vivo OX40 clusters, mediated by the bispecific binding of OX40 and LAG3, can effectively control tumor growth.

[0140] Detailed description

[0141] This invention relates to a specific binding member for OX40. OX40 is also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4) or CD134. Specifically, the specific binding member includes an OX40 antigen-binding site located in a constant domain of the specific binding member. The specific binding member is preferably capable of bivalent binding to OX40. The specific binding member preferably binds to human OX40, more preferably to human and cynomolgus monkey OX40. The portion of OX40 bound by the specific binding member is preferably the extracellular domain of OX40. The extracellular domains of human and cynomolgus monkey OX40 may contain or consist of the sequences shown in SEQ ID NO: 161 and SEQ ID NO: 162, respectively. The specific binding member is preferably capable of binding to OX40 expressed on the cell surface, preferably T cells, such as CD4+ T cells, CD8+ T cells, type 1 helper T cells (Th1), type 2 helper T cells (Th2), or regulatory T (Treg) cells, or tumor-infiltrating T cells or natural killer (NK) cells. Tumor-infiltrating T cells are a subset of tumor-infiltrating lymphocytes (TILs) found in many cancers.

[0142] The specific binding member preferably specifically binds to OX40. The term "specific" can refer to a situation where the specific binding member will not show significant binding to any molecule other than one or more of its specific binding partners. The term "specific" also applies to situations where the specific binding member molecule is specific to a particular antigenic determinant, such as the antigenic determinant on OX40, which is carried by a variety of antigens; in this case, the specific binding member will be able to bind to a variety of antigens carrying that epitope. The specific binding member preferably does not bind to CD40, TNFRI, TNFRII, NGFR, and / or CD137, or does not show any significant binding.

[0143] The specific binding members of this invention are selected based on their ability to bind to the dimer OX40. Specific binding members can bind to the dimer OX40 with a higher affinity than those binding to the monomeric OX40. High affinity for the dimer OX40 is considered beneficial for inducing OX40 clustering and T cell activation. Antibodies binding to the TNF receptor Fas with high affinity have been shown to have reduced agonist activity. Like OX40, Fas requires trimerization to activate. It is believed that bivalent agonists such as IgG antibodies must be able to bind to Fas and then partially dissociate to recruit more Fas monomers and form an active signaling complex. Antibodies binding to Fas monomers with high affinity are thought to be locked in a non-signaling state (Chodorge et al., 2012).

[0144] Specific binding members preferably have an affinity (K0) of 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or higher. D () binds to dimeric human OX40. Preferably, the specific binding member has an affinity (K) of 1 nM. D (or higher affinity for human OX40)

[0145] Specific binding members of the FS20-22 and FS20-31 lineages have also been shown to bind to the dimer cynomolgus OX40. Binding to both cynomolgus OX40 and human OX40 is beneficial because it allows for testing the efficacy and toxicity of the specific binding members in cynomolgus monkeys before administration to humans. Specific binding members of the FS20-11 lineage have shown binding to the dimer cynomolgus OX40, but with lower affinity, suggesting they are less suitable for preclinical testing in cynomolgus monkeys.

[0146] In a preferred embodiment, the specifically binding member preferably has an affinity (K0.05) of 150 nM, 140 nM, 120 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, or 2 nM or higher. D () binds to the dimeric cynomolgus monkey OX40. Preferably, the specific binding member has an affinity of 2 nM (K). D (or higher affinity) combined with cynomolgus monkey OX40.

[0147] As described in this embodiment, the similarity to human and cynomolgus monkey antigens is considered potentially advantageous, as it is hoped that mAbs in cynomolgus monkey studies will be beneficial. 2 The behavior can be extrapolated to humans. Studies of the efficacy and toxicity of antibody molecules in cynomolgus monkeys are considered beneficial, as they could predict the efficacy and toxicity of specific binding members in humans.

[0148] Therefore, in a preferred embodiment, the affinity of the specific binding member to dimeric cynomolgus monkey OX40 is no more than 10 times, preferably no more than 5 times, lower or higher than the affinity to dimeric human OX40.

[0149] For example, the binding affinity of antibody molecules to associated antigens such as human or cynomolgus monkey OX40 can be determined by surface plasmon resonance (SPR), such as Biacore.

[0150] Specific binding members may block the interaction between OX40 and its ligand OX40L, preferably between human OX40 and human OX40L. The ability of specific binding members to block the binding of OX40L to OX40 can be determined using enzyme-linked immunosorbent assay (ELISA).

[0151] The term "specific binding member" describes an immunoglobulin or fragment thereof containing a constant domain of an OX40 antigen-binding site. Therefore, as used herein, the term "specific binding member" includes an antigen-binding fragment containing an OX40 antigen-binding site located within the constant domain of the specific binding member. The constant domain may be a CL, CH1, CH2, CH3, or CH4 domain, preferably a CH1, CH2, or CH3 domain, more preferably a CH2 or CH3 domain, and most preferably a CH3 domain. Specific binding members may be partially or wholly synthesized.

[0152] Preferably, the specific binding member comprises CH2 and CH3 domains, wherein the CH2 or CH3 domain, preferably the CH3 domain, contains the OX40 antigen binding site. The specific binding member is preferably a dimer of two (identical) polypeptide chains, each containing both the CH2 and CH3 domains. In a preferred embodiment, the specific binding member further comprises an immunoglobulin hinge region or a portion thereof at the N-terminus of the CH2 domain. Such a molecule is also referred to herein as an antigen-binding Fc fragment, or Fcab. TM The hinge region may consist of, or contain, the sequence listed in SEQ ID NO: 170 or a fragment thereof. Preferably, the fragment is a C-terminal fragment of the sequence listed in SEQ ID NO: 170. The length of the fragment may be at most 20, at most 10, at most 8, or at most 6 amino acids. The length of the fragment may be at least 3, at least 4, at least 5, or at least 6 amino acids. In a preferred embodiment, the hinge region has the sequence listed in SEQ ID NO: 171.

[0153] In a preferred embodiment, the specifically binding member is an antibody molecule, preferably a monoclonal antibody or a fragment thereof. The antibody molecule is preferably human or humanized. The antibody molecule can be an immunoglobulin G molecule, such as IgG1, IgG2, IgG3, or IgG4 molecules, preferably IgG1, IgG2, or IgG4 molecules, more preferably IgG1 molecules or fragments thereof.

[0154] Because antibodies can be modified in many ways, the term "antibody molecule" should be interpreted to encompass antibody fragments, derivatives, functional equivalents, and homologs, whether natural or wholly or partially synthetic. An example of an antibody fragment containing a CH3 domain is the Fc domain of an antibody. An example of an antibody fragment containing both a CDR sequence and a CH3 domain is a microantibody containing an scFv linked to the CH3 domain (Hu et al., 1996).

[0155] The specific binding member contains an OX40 antigen binding site. The OX40 antigen binding site is located within a constant domain of the specific binding member, preferably the CH3 domain. Within the constant domain of the specific binding member, the OX40 antigen binding site contains one or more modified structural loops. The engineering of antibody constant domain structural loops to generate antigen binding sites for target antigens is known in the art and described, for example, in Wozniak-Knopp et al., 2010, and patent publications WO2006 / 072620 and WO2009 / 132876.

[0156] Preferably, the OX40 antigen binding site comprises modified AB, CD, and / or EF loop structures. The inventors have recognized that, in certain cases, the AB and EF loop sequences play a greater role in antigen binding than the CD loop sequence. This is evident from the fact that Fcabs FS20-22 and FS20-11 bind OX40 but do not contain any modifications in their CD loop sequences (see Figure 1). Therefore, in a preferred embodiment, the OX40 antigen binding site comprises modified AB and EF loop structures. In another preferred embodiment, the OX40 antigen binding site comprises modified AB, CD, and EF loop structures.

[0157] In a preferred embodiment, the residues at positions 95 and 96 of the CH3 domain of the specifically binding member are wild-type, i.e., preferably arginine (R) and tryptophan (W), respectively. Both residues are located within the EF ring. Unless otherwise stated, the amino acid residue positions herein are numbered according to the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., 2005.

[0158] Therefore, the OX40 antigen binding site of the specific binding member may contain a first, second, and / or a third sequence, preferably the first and third sequences, or the first, second, and third sequences, wherein the first, second, and third sequences are located in the constant structural domains of the specific binding member, preferably the AB, CD, and EF structural loops of the CH3 domain.

[0159] The first, second, and third sequences can be the first, second, and third sequences of the following CH3 domains: specifically binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, more preferably specifically binding members FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85, even more preferably specifically binding members FS20-22-49, FS20-22-41, or FS20-22-47, and most preferably specifically binding member FS20-22-49.

[0160] Optionally, the first, second, and third sequences can be the first, second, and third sequences of the CH3 domain: specifically binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, more preferably specifically binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, or FS20-31-102, more preferably specifically binding members FS20-31-115 or FS20-31-108, and even more preferably specifically binding member FS20-31-115.

[0161] As a further option, the first, second and third sequences can be the first, second and third sequences of the CH3 domain: specifically binding members FS20-11-131, FS20-11-127 or FS20-11-134, more preferably specifically binding member FS20-11-131.

[0162] The CH3 domain sequences of members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, FS20-31-115, FS20-11-127, FS20-11-131 and FS20-11-134 that specifically bind are shown in SEQ ID NO:46, 55, 63, 72, 81, 94, 103, 114, 124, 134, 143, 15, 24 and 33, respectively.

[0163] The first, second, and third sequences that specifically bind to members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, and FS20-22-38 can be sequences at the following positions within the CH3 domains of FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, and FS20-22-38, respectively:

[0164] (i) 14 to 18, 45.1 to 77 and 97 to 101; or

[0165] (ii) 14 to 18, 45.1 to 77 and 93 to 101.

[0166] After FS20-31 lineage affinity maturation for OX40 binding, all 76 specific binding members were identified. All of these members contained an aspartic acid (D) residue at position 77 of the CH3 domain, suggesting that this residue may be important for OX40 binding in these molecules. Similarly, all specific binding members in FS20-31 retained a wild-type glutamic acid (E) residue at position 45.3 of the CH3 domain, indicating that this residue may also be important for OX40 binding in these molecules. Therefore, the specific binding member may comprise a first sequence and a third sequence of the specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66, wherein the first sequence is the sequence located at positions 14 to 18 of the CH3 domain of the specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66, the second sequence is the sequence located at positions 92 to 101, or 97 to 101, and wherein the specific binding member also comprises an aspartic acid (D) residue at position 77 of the CH3 domain and optionally a glutamic acid (E) residue at position 45.3 of the CH3 domain. The specific binding member may further optionally contain amino acid substitutions at positions 45.1, 45.2 and / or 45.4 of the CH3 domain.

[0167] Optionally, the first, second, and third sequences that specifically bind to members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, and FS20-31-66 can be sequences located at the following positions within the CH3 domain of FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, and FS20-31-66, respectively:

[0168] (i) 14 to 18, 45.1 to 77 and 97 to 101; or

[0169] (ii) 14 to 18, 45.1 to 77 and 92 to 101.

[0170] The first, second, and third sequences of specifically binding members FS20-11-127, FS20-11-131, and FS20-11-134 can be sequences between the following positions in the CH3 domains of FS20-11-127, FS20-11-131, and FS20-11-134, respectively:

[0171] (i) 13 and 19, 45.2 and 78, 96 and 102;

[0172] (ii) 11 and 19, 45.2 and 78, 96 and 102;

[0173] (iii) 13 and 19, 45 and 78, and 96 and 102; or

[0174] (iv) 11 and 19, 45 and 78, and 96 and 102.

[0175] The first, second, and third sequences can be the complete AB, CD, and EF loop sequences in the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, FS20-22-38, FS20-31-115, FS20-31-10, FS20-31-58, FS20-31-94, FS20-31-102, FS20-31-66, FS20-11-131, FS20-11-127, or FS20-11-134, respectively. The positions of the AB, CD, and EF loops in the CH3 domain sequence are determined, for example, by the IMGT, IMGT exon, EU, or Kabat numbering system, which is within the capabilities of those skilled in the art and is described in Hasenhindl et al. (2013). In a preferred embodiment, the AB, CD, and EF structural loops according to the IMGT numbering system are located between positions 10 and 19, 42 and 79, and 91 and 102 of the CH3 domain of the specifically binding member, respectively. Therefore, in a preferred embodiment, the first, second, and third sequences are the sequences between positions 10 and 19, 42 and 79, and 91 and 102 of the CH3 domain of the specifically binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, FS20-22-38, FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, FS20-31-66, FS20-11-131, FS20-11-127, or FS20-11-134.

[0176] In a preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0177] (i)FS20-22-49, listed in SEQ ID NO:43, 54 and 71 respectively;

[0178] (ii)FS20-22-41, listed in SEQ ID NO:43, 54 and 45 respectively;

[0179] (iii)FS20-22-47, listed in SEQ ID NO:43, 54 and 62 respectively;

[0180] (iv)FS20-22-85, listed in SEQ ID NO:43, 54 and 80 respectively; or

[0181] (v)FS20-22-38, listed in SEQ ID NO:43, 44 and 45 respectively;

[0182] The first, second, and third sequences are preferably located at positions 14 to 18, 45.1 to 77, and 93 to 101, respectively, within the CH3 domain of the specific binding member.

[0183] In a more preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0184] (i)FS20-22-49, listed in SEQ ID NO:43, 54 and 71 respectively;

[0185] (ii)FS20-22-41, listed in SEQ ID NO:43, 54 and 45 respectively;

[0186] (iii)FS20-22-47, listed in SEQ ID NO:43, 54 and 62 respectively; or

[0187] (iv)FS20-22-85, listed in SEQ ID NO:43, 54 and 80 respectively.

[0188] In a more preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0189] (i)FS20-22-49, listed in SEQ ID NO:43, 54 and 71 respectively;

[0190] (ii)FS20-22-41, listed in SEQ ID NO:43, 54 and 45 respectively; or

[0191] (iii)FS20-22-47, listed in SEQ ID NO:43, 54 and 62 respectively.

[0192] In another preferred embodiment, the OX40 antigen binding site of the specific binding member comprises the first, second, and third sequences of the specific binding member FS20-22-49, respectively, listed in SEQ ID NO:43, 54, and 71.

[0193] The specific binding member may further include a leucine (L) at position 91 of the CH3 domain of the specific binding member. In particular, the specific binding member containing the first, second, and third sequences of FS20-22-85 of the OX40 antigen binding site may include a leucine at position 91 of the CH3 domain of the specific binding member.

[0194] In an alternative preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0195] (i)FS20-31-115, listed in SEQ ID NO:122, 142 and 133 respectively;

[0196] (ii)FS20-31-108, listed in SEQ ID NO:122, 132 and 133 respectively;

[0197] (iii)FS20-31-58, listed in SEQ ID NO:91, 92 and 93 respectively;

[0198] (iv)FS20-31-94, listed in SEQ ID NO:111, 112 and 113 respectively;

[0199] (v)FS20-31-102, listed in SEQ ID NO:122, 123 and 102 respectively; or

[0200] (vi)FS20-31-66, listed in SEQ ID NO: 91, 92 and 102 respectively;

[0201] The first, second, and third sequences are preferably located at positions 14 to 18, 45.1 to 77, and 92 to 101, respectively, of the CH3 domain of the specific binding member.

[0202] In a more preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0203] (i)FS20-31-115, listed in SEQ ID NO:122, 142 and 133 respectively;

[0204] (ii)FS20-31-108, listed in SEQ ID NO:122, 132 and 133 respectively;

[0205] (iii)FS20-31-58, listed in SEQ ID NO:91, 92 and 93 respectively;

[0206] (iv)FS20-31-94, listed in SEQ ID NO:111, 112 and 113 respectively; or

[0207] (v)FS20-31-102, listed in SEQ ID NO:122, 123 and 102 respectively.

[0208] In a more preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0209] (i)FS20-31-115, listed in SEQ ID NO:122, 142 and 133 respectively; or

[0210] (ii)FS20-31-108, listed in SEQ ID NO:122, 132 and 133 respectively.

[0211] In another preferred embodiment, the OX40 antigen binding sites of the specific binding member comprise the first, second, and third sequences of the specific binding member FS20-31-115, as shown in SEQ ID NO: 122, 142, and 133, respectively.

[0212] In another alternative preferred embodiment, the OX40 antigen binding site for the specific binding member comprises the first, second, and third sequences of the specific binding member:

[0213] (i)FS20-11-131, listed in SEQ ID NO:12, 13 and 23 respectively;

[0214] (ii)FS20-11-127, listed in SEQ ID NO:12, 13 and 14 respectively; or

[0215] (iii)FS20-11-134, listed in SEQ ID NO:12, 13 and 32 respectively;

[0216] The first, second, and third sequences are preferably located between positions 13 and 19, 45 and 78, and 96 and 102 of the CH3 domain of the specific binding member.

[0217] In a more preferred embodiment, the OX40 antigen binding sites of the specific binding member comprise the first, second, and third sequences of the specific binding member FS20-11-131, as listed in SEQ ID NO:12, 13, and 23, respectively.

[0218] The specific binding member may further include glutamic acid (E) at position 12, asparagine (N) at position 94, and / or leucine (L) at position 103 of its CH3 domain. Specifically, a specific binding member containing the first, second, and third sequences of FS20-11-131, FS20-11-127, or FS20-11-134 at an OX40 antigen-binding site may include glutamic acid at position 12 and leucine at position 103 of its CH3 domain. Additionally, a specific binding member containing the first, second, and third sequences of FS20-11-131 at an OX40 antigen-binding site may include asparagine at position 94 of its CH3 domain.

[0219] The specific binding member containing the AB, CD, and EF loop sequences of the specific binding member FS20-11-131, FS20-11-127, or FS20-11-134 may contain a leucine residue at position 103 of the CH3 domain of the specific binding member.

[0220] If the OX40 antigen-binding site of a specific binding member contains the first, second, and third sequences or AB, CD, and EF loop sequences of the specific binding member FS20-11-131, FS20-11-127, or FS20-11-134, the specific binding member may contain an amino acid deletion between positions 13 and 19 of the CH3 domain of that specific binding member, for example, at positions 14, 15, 16, 17, or 18. Deletions present in these specific binding members are considered to occur due to primer errors, therefore the exact location of the deletion is unknown. Figure 1A In the text, the missing information is shown at bit 18 of the CH3 structure domain, but it could also be located at bit 14, 15, 16, or 17.

[0221] The positions of amino acid residues, including the amino acid sequences, substitutions, deletions, and insertions described herein, can be numbered according to IMGT exon numbering (also known as sequential numbering), EU numbering, or Kabat numbering as an alternative to IMGT numbering. The consistency between IMGT, IMGT exon, EU, and Kabat numbers for the CH3 domain residue positions is shown in Figure 1. Thus, for example, in this application, the first, second, and third sequences are located at positions 14 to 18, 45.1 to 77, and 93 to 101 of the CH3 domain of a specific binding member, respectively, where the residue positions are numbered according to the IMGT numbering scheme; the first, second, and third sequences are located at positions 18 to 22, 46 to 50, and 74 to 82 of the CH3 domain, where the residue positions are numbered according to the IMGT exon numbering scheme, as shown in Figure 1. Optionally, as described herein, the positions of amino acid residues in the CH3 domain, including the positions of amino acid sequences, substitutions, deletions, and insertions, can be defined with reference to their positions in the wild-type CH3 domain, the sequence of which is listed in SEQ ID NO: 4. The consistency between the IMGT number and the wild-type CH3 domain sequence is also shown in Figure 1.

[0222] In a preferred embodiment, the specific binding member comprises a CH3 domain, which includes or consists of a CH3 domain sequence having or composed of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, preferably a CH3 structure of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85. The CH3 domain sequence, more preferably a CH3 domain sequence that specifically binds to members FS20-22-49, FS20-22-41 or FS20-22-47, and most preferably a CH3 domain sequence that specifically binds to member FS20-22-49, wherein the CH3 domain sequences that specifically bind to members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49 and FS20-22-85 are shown in SEQ ID NO:46, 55, 63, 72 and 81, respectively.

[0223] In an alternative preferred embodiment, the specific binding member comprises a CH3 domain, which includes, or consists of, a CH3 domain sequence having or composed of specific binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, preferably specific binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, or FS20-31-66. The CH3 domain sequence of -102 is preferred, more preferably the CH3 domain sequence of member FS20-31-115 or FS20-31-108 is preferred, and the CH3 domain sequence of member FS20-31-115 is most preferred. The CH3 domain sequences of members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115 are listed in SEQ ID NO: 94, 103, 114, 124, 134 and 143, respectively.

[0224] In another alternative preferred embodiment, the specific binding member comprises a CH3 domain, which includes, or consists of, a CH3 domain sequence having or composed of the specific binding members FS20-11-131, FS20-11-127, or FS20-11-134, more preferably, the CH3 domain sequence of the specific binding member FS20-11-131, wherein the CH3 domain sequences of the specific binding members FS20-11-127, FS20-11-131, and FS20-11-134 are listed in SEQ ID NO: 15, 24, and 33.

[0225] The CH3 domain of a specific binding member may optionally contain an additional lysine residue (K) at the direct C-terminus of the CH3 domain sequence.

[0226] Monoclonal antibodies and other antibodies, along with recombinant DNA technology, can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technique involves introducing a CDR or variable region into different immunoglobulins. Methods for introducing the CDR of one immunoglobulin into another immunoglobulin are described, for example, in EP-A-184187, GB 2188638A, and EP-A-239400. A similar technique can be used to introduce a constant domain sequence of the OX40 antigen-binding site constituting a specific binding member of the present invention into a constant domain, such as the CH3 domain of another specific binding member, thereby resulting in a specific binding member containing the OX40 antigen-binding site within its constant domain. Alternatively, the entire constant domain sequence of a specific binding member can be replaced with a constant domain sequence of a specific binding member of the present invention to prepare a specific binding member containing the OX40 antigen-binding site within its constant domain. Similarly, a corresponding fragment of the constant domain sequence of a specific binding member containing the OX40 antigen-binding site according to the present invention can be replaced with a fragment of the constant domain sequence of the specific binding member.

[0227] Additionally, the specific binding member may contain the CH2 domain of an immunoglobulin G molecule, such as the CH2 domain of IgG1, IgG2, IgG3, or IgG4 molecules. Preferably, the specific binding member contains the CH2 domain of an IgG1 molecule. The CH2 domain may have the sequence listed in SEQ ID NO: 5. The CH2 domain is known to bind to the Fcγ receptor and complement. Binding of the CH2 domain to the Fcγ receptor is essential for antibody-dependent cell-mediated cytotoxicity (ADCC), while binding to complement is essential for complement-dependent cytotoxicity (CDC). In some embodiments, the specific binding member induces ADCC and / or CDC. This is preferred when the specific binding member contains a second antigen-binding site for the tumor antigen. It is not desirable to be bound by theory that the binding of the specific binding member to tumor cells will induce ADCC or CDC-mediated killing of tumor cells when the specific binding member does not bind to OX40. This action will be additional T cell-mediated killing of tumor cells, in which the specific binding member binds to both the tumor antigen and OX40, thereby leading to T cell activation.

[0228] The CH2 domain of a specific binding member may contain one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIII), and / or complement. The inventors hypothesize that reducing or eliminating binding to Fc receptor γ will reduce or eliminate ADCC mediated by the specific binding member. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate CDC mediated by the specific binding member. Mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). These mutations include the LALA mutation described in Bruhns et al., 2009 and Hezareh et al., 2001, which involves replacing leucine residues at positions 1.3 and 1.2 of the IMGT site of the CH2 domain with alanine residues (L1.3A and L1.2A). Alternatively, glycosylation at the conserved N-chain glycosylation site can be mutated to produce glycosyl antibodies by replacing asparagine (N) at position 84.4 of the IMGT site in the CH2 domain with alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), which is known to reduce IgG1 effector function (Wang et al., 2018). As another alternative, complement activation (C1q binding) and ADCC are known to be reduced by replacing proline at position 114 of the IMGT site in the CH2 domain with alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce specific binding members with further reduced or absent ADCC or CDC activity.

[0229] Therefore, the specific binding member may contain a CH2 domain, wherein the CH2 domain contains:

[0230] (i) alanine residues at positions 1, 3 and 1, 2; and / or

[0231] (ii) alanine or glycine at position 114; and / or

[0232] (iii) Alanine, glutamine, or glycine at position 84.4;

[0233] The amino acid residues are numbered according to the IMGT numbering scheme.

[0234] In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain preferably comprises:

[0235] (i) alanine residues at positions 1, 3 and 1, 2; and / or

[0236] (ii) Alanine or glycine at position 114;

[0237] The amino acid residues are numbered according to the IMGT numbering scheme.

[0238] In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain comprises:

[0239] (i) the alanine residue at position 1 and 3; and

[0240] (ii) alanine residues at positions 1 and 2;

[0241] The amino acid residues are numbered according to the IMGT numbering scheme.

[0242] For example, the CH2 domain can have a sequence as listed in SEQ ID NO: 6.

[0243] In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain comprises:

[0244] (i) Alanine residue at position 1 and 3;

[0245] (ii) alanine residues at positions 1 and 2; and

[0246] (iii) Alanine at position 114;

[0247] The amino acid residues are numbered according to the IMGT numbering scheme.

[0248] For example, the CH2 domain can have a sequence as listed in SEQ ID NO: 7.

[0249] In a preferred embodiment, the specific binding member comprises a CH2 and CH3 domain sequence having or consisting of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, preferably a CH2 and CH3 domain sequence of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85, more preferably... The CH2 and CH3 domain sequences that specifically bind to members FS20-22-49, FS20-22-41, or FS20-22-47 are selected, with the CH2 and CH3 domain sequences of member FS20-22-49 being the most preferred. The CH2 and CH3 domain sequences of members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, and FS20-22-85 are shown in SEQ ID NO:48, 57, 65, 74, and 83, respectively, starting from the 7th amino acid.

[0250] In an alternative preferred embodiment, the specific binding member comprises a CH2 and CH3 domain sequence having or consisting of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, preferably a CH2 and CH3 domain sequence of a specific binding member FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85. The CH2 and CH3 domain sequences that specifically bind to members FS20-22-49, FS20-22-41, or FS20-22-47 are preferred, and the CH2 and CH3 domain sequences that specifically bind to member FS20-22-49 are most preferred. The CH2 and CH3 domain sequences that specifically bind to members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, and FS20-22-85 are shown in SEQ ID NO:50, 59, 67, 76, and 85, respectively, starting from the 7th amino acid.

[0251] In another alternative preferred embodiment, the specific binding member comprises a CH2 and CH3 domain sequence having or consisting of a specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, preferably a CH2 domain of a specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, or FS20-31-102. The CH2 and CH3 domain sequences, more preferably the CH2 and CH3 domain sequences of members FS20-31-115 or FS20-31-108, and most preferably the CH2 and CH3 domain sequences of members FS20-31-115, wherein the CH2 and CH3 domain sequences of members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115 are shown in SEQ ID NO:96, 105, 116, 126 and 145, respectively, starting from the 7th amino acid.

[0252] In another alternative preferred embodiment, the specific binding member comprises a CH2 and CH3 domain sequence having or consisting of a specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, preferably a CH2 domain of a specific binding member FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, or FS20-31-102. The CH2 and CH3 domain sequences, more preferably the CH2 and CH3 domain sequences of members FS20-31-115 or FS20-31-108, and most preferably the CH2 and CH3 domain sequences of members FS20-31-115, wherein the CH2 and CH3 domain sequences of members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115 are shown in SEQ ID NO:98, 107, 118, 128 and 147, respectively, starting from the 7th amino acid.

[0253] In another alternative preferred embodiment, the specific binding member comprises, having or consisting of, the CH2 and CH3 domain sequences of the specific binding members FS20-11-131, FS20-11-127, or FS20-11-134, preferably the CH2 and CH3 domain sequences of the specific binding member FS20-11-131, wherein the CH2 and CH3 domain sequences of the specific binding members FS20-11-127, FS20-11-131, or FS20-11-134 are shown in SEQ ID NO:17, 26, and 35, respectively, starting from the 7th amino acid.

[0254] In another alternative preferred embodiment, the specific binding member comprises, or is composed of, a CH2 and CH3 domain sequence having or consisting of a specific binding member FS20-11-131, FS20-11-127, or FS20-11-134, preferably a CH2 and CH3 domain sequence of a specific binding member FS20-11-131, wherein the CH2 and CH3 domain sequences of specific binding members FS20-11-127, FS20-11-131, or FS20-11-134 are shown in SEQ ID NO:19, 28, and 37, respectively, starting from the 7th amino acid.

[0255] In a preferred embodiment, the specific binding member includes a sequence having or consisting of the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, preferably the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85, more preferably the specific binding members FS20-22-49, FS20-22-41, or FS20-22-47, and most preferably the specific binding member FS20-22-49, wherein the sequences of the specific binding members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, and FS20-22-85 are listed in SEQ ID NO:48, 57, 65, 74, and 83, respectively.

[0256] In an alternative preferred embodiment, the specific binding member comprises a sequence having or consisting of the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, or FS20-22-38, preferably the specific binding members FS20-22-49, FS20-22-41, FS20-22-47, or FS20-22-85, more preferably the specific binding members FS20-22-49, FS20-22-41, or FS20-22-47, and most preferably the specific binding member FS20-22-49, wherein the sequences of the specific binding members FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, and FS20-22-85 are respectively in SEQ ID NO. Listed in NO:50, 59, 67, 76 and 85.

[0257] In another alternative preferred embodiment, the specific binding member includes a sequence having or consisting of the specific binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, preferably the specific binding members FS20-31-115, FS20-31-108, FS20-31-58, or FS20-31-94. The sequence of 4 or FS20-31-102, more preferably the sequence of the specific binding member FS20-31-115 or FS20-31-108, most preferably the sequence of the specific binding member FS20-31-115, wherein the sequences of the specific binding members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115 are listed in SEQ ID NO:96, 105, 116, 126, 136 and 145, respectively.

[0258] In another alternative preferred embodiment, the specific binding member includes a sequence having or consisting of the specific binding members FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, or FS20-31-66, preferably the specific binding members FS20-31-115, FS20-31-108, FS20-31-58, or FS20-31-94. The sequence of 4 or FS20-31-102, more preferably the sequence of the specific binding member FS20-31-115 or FS20-31-108, most preferably the sequence of the specific binding member FS20-31-115, wherein the sequences of the specific binding members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115 are listed in SEQ ID NO:98, 107, 118, 128 and 147, respectively.

[0259] In another alternative preferred embodiment, the specific binding member includes a sequence having or consisting of the specific binding member FS20-11-131, FS20-11-127 or FS20-11-134, more preferably the sequence of the specific binding member FS20-11-131, wherein the sequences of the specific binding members FS20-11-127, FS20-11-131 and FS20-11-134 are listed in SEQ ID NO: 17, 26 and 35, respectively.

[0260] In another alternative preferred embodiment, the specific binding member includes sequences FS20-11-131, FS20-11-127, or FS20-11-134 of the specific binding member, more preferably, the sequence of the specific binding member FS20-11-131, wherein the sequences of the specific binding members FS20-11-127, FS20-11-131, and FS20-11-134 are listed in SEQ ID NO: 19, 28, and 37, respectively.

[0261] In a preferred embodiment, in addition to the OX40 antigen-binding site located in the constant domain of the specific binding member, the specific binding member also includes one or more additional antigen-binding sites that bind to one or more other antigens. These one or more additional antigen-binding sites preferably specifically bind their homologous antigens.

[0262] One or more additional antigen-binding sites may bind OX40 or another antigen. Therefore, the specific binding member can be a multispecific molecule, such as a bispecific, trispecific, or tetraspecific molecule, preferably a bispecific molecule. In a preferred embodiment, the specific binding member is capable of binding both OX40 and one or more other antigens simultaneously.

[0263] Antibody molecules are known to have modular structures comprising discrete domains that can be combined in various ways to produce multispecific, such as bispecific, trispecific, or tetraspecific antibody forms. Exemplary multispecific antibody forms are described in Spiess et al. (2015) and Kontermann (2012). The specific binding member of the present invention can be used in such a multispecific antibody form. This has the added advantage of introducing additional antigen-binding sites into such a multispecific antibody form by the presence of antigen-binding sites in constant domains, such as the CH3 domain of the specific binding member.

[0264] For example, the specific binding member of the present invention can be a heterodimeric antibody molecule, such as a heterodimeric intact immunoglobulin molecule or a fragment thereof. In this case, a portion of the antibody molecule will have one or more sequences as described herein. For example, in the case where the specific binding member of the present invention is a bispecific heterodimeric antibody molecule, the specific binding member may comprise a heavy chain containing a CH3 domain as described herein, paired with a heavy chain that binds to an antigen other than OX40. Techniques for preparing heterodimeric antibodies are known in the art, including knob-in-pore (KIH) techniques, which involve modifying the CH3 domain of an antibody molecule to create a “knob” or “pore” to facilitate chain heterodimerization. Alternatively, heterodimeric antibodies can be prepared by introducing charge pairs into the antibody molecule to avoid homodimerization of the CH3 domain due to electrostatic repulsion and to guide heterodimerization by electrostatic attraction. Examples of heterodimeric antibody forms include CrossMab, mAb-Fv, SEED antibodies, and KIH IgG.

[0265] Optionally, the multispecific binding member of the present invention may comprise an intact immunoglobulin molecule or a fragment thereof, and one or more additional antigen-binding moieties. The antigen-binding moieties may be, for example, Fv, scFv, or single-domain antibodies, and may be fused to an intact immunoglobulin molecule or a fragment thereof. Examples of multispecific antibody molecules comprising other antigen-binding moieties fused to an intact immunoglobulin molecule include DVD-IgG, DVI-IgG, scFv4-IgG, IgG-scFv, and scFv-IgG molecules (Spiess et al., 2015; Figure 1). Examples of multispecific antibody molecules comprising other antigen-binding moieties fused to immunoglobulin fragments containing a CH3 domain include, for example, scDiabody-CH3, Diabody-CH3, and scFv-CH3 KIH (Spiess et al., 2015; Figure 1).

[0266] Other suitable multispecific forms will be obvious to those skilled in the art.

[0267] In a preferred embodiment, the specific binding member includes a second antigen binding site that binds to a second antigen, wherein the second antigen binding site is preferably a CDR-based antigen binding site. A CDR-based antigen binding site is an antigen binding site within the antibody variable region. A CDR-based antigen binding site consists of six CDRs: three light chain variable domain (VL) CDRs and three heavy chain variable domain (VH) CDRs.

[0268] The preparation of antibody molecules against a given antigen and the determination of the CDR sequence of such antibody molecules are well known, and many suitable techniques are known in the art. The CDR sequence can be determined according to, for example, Kabat et al., 1991 or the International ImMunoGeneTics Information System (IMGT) (Lefranc et al., 2015).

[0269] For example, the specific binding member could be mAb. 2 ( TM Bispecific antibodies. As referred to in this article, mAbs 2 Bispecific antibodies are IgG immunoglobulins that include a CDR-based antigen-binding site in each of their variable regions and at least one antigen-binding site in their constant domain. When the specific binding member of the present invention is a mAb... 2 In this form, in addition to the OX40 antigen-binding site in the constant domain of its specific binding member, the specific binding member also contains a CDR-based antigen-binding site in each of its variable regions.

[0270] The three VH domain CDRs of the antigen-binding site can be located within the immunoglobulin VH domain, and the three VL domain CDRs can be located within the immunoglobulin VL domain. For example, the CDR-based antigen-binding site can be located in the antibody variable region.

[0271] The specific binding member may have one or more, preferably two, CDR-based antigen-binding sites for the first antigen. Therefore, the specific binding member may contain one VH and one VL domain, but preferably two VH and two VL domains, i.e., two VH / VL domain pairs, just as naturally occurring IgG molecules do.

[0272] In some preferred embodiments, the specific binding member may be an immunoglobulin containing two variable regions, each variable region containing a CDR-based antigen binding site for a second antigen.

[0273] In a preferred embodiment, the antibody is therefore an antibody that binds to OX40 and a second antigen, and the antibody molecule comprises:

[0274] (i) The two antigen-binding sites of OX40 are located in the two CH3 domains of the antibody molecule; and

[0275] (ii) The second antigen has two CDR-based antigen-binding sites, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain.

[0276] In a more preferred embodiment, the antibody is a complete immunoglobulin molecule, such as a complete IgG1 molecule that binds to OX40 and a second antigen, the antibody molecule comprising:

[0277] (i) The two antigen-binding sites of OX40 are located in the two CH3 domains of the antibody molecule; and

[0278] (ii) The second antigen has two CDR-based antigen-binding sites, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain; and

[0279] The immunoglobulin molecule further includes CH1, CH2 and CL domains.

[0280] Activation of OX40 requires its aggregation on the surface of T cells, which in turn stimulates intracellular signaling pathways and T cell activation. In the absence of specific binding members cross-linking, the binding of specific binding members to OX40 on the surface of T cells may not lead to OX40 clustering, or may only induce limited aggregation of OX40, and therefore may not lead to T cell activation, or may only lead to limited T cell activation.

[0281] The inventors have demonstrated that, in the absence of cross-linking of the specific binding members, the specific binding members FS20-11-131, FS20-11-127, and FS20-11-134 do not activate T cells. Conversely, in the absence of cross-linking, FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85, FS20-22-38, FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102, and FS20-31-66 induce limited T cell activation. The OX40 agonistic effects of these specific binding members are induced or increased in the presence of cross-linking of the specific binding members (see Example 5).

[0282] As mentioned above, cross-linking antibody molecules by binding to the Fcγ receptor is both inefficient and fails to target specific sites, such as disease sites, because cells expressing the Fcγ receptor are present throughout the human body. Therefore, the secondary antigen bound by the secondary antigen binding site is preferably not the Fcγ receptor.

[0283] Therefore, in a preferred embodiment, the specific binding member of the present invention includes a second antigen binding site that binds to a second antigen, wherein the second antigen is capable of binding and crosslinking multiple specific binding members.

[0284] For example, the inventors have shown that the second antigen is a surface antigen, such as a cell surface antigen, which may be monomeric or polymeric and aggregated at high concentrations on a surface, such as a cell surface, and that the binding of a specific binding member to the second antigen leads to or enhances T cell activation. Not wishing to be bound by theory, such as the notion that the binding of a specific binding member to an abundant cell surface antigen results in a high concentration of the specific binding member binding to the cell surface, such that the specific binding members are close enough to drive OX40 aggregation and T cell activation, the inventors have indicated a high concentration of the second antigen on a surface, such as a cell surface.

[0285] The inventors also indicate that the second antigen is a multimeric soluble molecule, such as a multimeric soluble factor, and the binding of a specific binding member to the second antigen leads to or enhances T cell activation. In a preferred embodiment, when the second antigen is a soluble molecule, the second antigen is therefore a multimeric antigen, such as a dimer, trimer, or a higher-order multimer, and is thus capable of crosslinking several specific binding members.

[0286] As described herein, a specific binding member contains a second antigen binding site that binds to a second antigen. This specific binding member activates T cells only upon binding to the second antigen, or its T cell activation activity is enhanced upon binding to the second antigen. Specific binding members are also referred to as conditional agonists. This T cell activation activity upon binding to the second antigen is independent of the binding of the specific binding member to the Fcγ receptor and / or external cross-linking agents (such as protein A or G or secondary antibodies), thus allowing the conditional agonist activity of the specific binding member to target the site where the second antigen is present. For example, in the case where the second antigen is a disease antigen, the specific binding member can selectively activate T cells at the disease site rather than at other sites in the individual, or selectively enhance T cell activation at the disease site rather than at other sites in the individual.

[0287] In addition, compared with specific binding members that rely on cross-linking through other mechanisms (e.g., external cross-linking agents, or cross-linking through Fcγ receptor interaction), specific binding members activate T cells only when bound to a second antigen, or their T cell activation activity is enhanced when bound to a second antigen, preferably enhancing T cell activation activity, because OX40 activation is more effective. Therefore, compared with other specific binding members, the specific binding members described herein can achieve T cell activation at lower concentrations.

[0288] Therefore, when the specific binding member is cross-linked, for example by binding to a second antigen, the specific binding of the present invention preferably induces an increase in T cell activation, compared to when the specific binding member is not cross-linked.

[0289] The ability of antibody molecules or specifically binding members to activate T cells can be measured using a T cell activation assay. T cells release IL-2 upon activation. Therefore, a T cell activation assay can measure IL-2 release to determine the level of T cell activation induced by antibody molecules or specifically binding members.

[0290] For example, the ability of an antibody molecule or specific binding member to activate T cells can be determined by measuring the concentration of the antibody molecule or specific binding member required for T cells to achieve half-maximal release of IL-2 when the specific binding member or antibody molecule cross-links in a T cell activation assay. This will be referred to below as the EC50 of the antibody molecule or specific binding member. 50 Lower EC 50 This indicates that in T cell activation assays, lower concentrations of antibody molecules or specific binding members are required to achieve the half-maximal release of IL-2; therefore, antibody molecules or specific binding members exhibit high T cell activation activity. Specific binding members or antibody molecules can be cross-linked using, for example, anti-CH2 antibodies.

[0291] In a preferred embodiment, the antibody molecule or specifically binding member in the EC of the T cell activation assay 50 It is FS20-22-49 / 4420 (containing LALA mutation) in the same trial in EC 50 Within 50 times, 40 times, 30 times, 20 times, 10 times or 5 times, wherein FS20-22-49 / 4420 (LALA) consists of or contains the heavy chain listed in SEQ ID No: 78 and the light chain listed in SEQ ID No: 156.

[0292] In a preferred embodiment, in the presence of crosslinking of antibody molecules or specific binding members, the EC50 of antibody molecules or specific binding members in the T cell activation assay... 50 It is 10, 20, 30 or 40 times lower than when there is no cross-linking.

[0293] For example, in T cell activation assays, antibody molecules or specific binding members of EC2 cells... 50 It can be less than or equal to 5nM, less than or equal to 4nM, less than or equal to 3nM, less than or equal to 2nM, less than or equal to 1nM, less than or equal to 0.5nM, less than or equal to 0.3nM, less than or equal to 0.2nM or less than or equal to 0.1nM, with less than or equal to 0.1nM being preferred.

[0294] Alternatively, the ability of an antibody molecule or specific binding member to activate T cells can be determined by measuring the maximum concentration of IL-2 released by T cells in a T cell activation assay in the presence of the antibody molecule or specific binding member, wherein the antibody molecule or specific binding member is cross-linked.

[0295] In a preferred embodiment, in the presence of an antibody molecule or a specific binding member, in the case of crosslinking, the maximum concentration of IL-2 released by T cells in a T cell activation assay is within 20% or 10% of the maximum concentration of IL-2 released by T cells in the presence of FS20-22-49 / 4420 (containing the LALA mutation) in the same assay, wherein FS20-22-49 / 4420 (LALA) consists of, or contains, the heavy chain listed in SEQ ID NO: 78 and the light chain listed in SEQ ID NO: 156.

[0296] T cell activation assays can be the T cell assays described herein, such as the pan-T cell assay described in this embodiment.

[0297] For example, a T-cell activation assay can be based on an IL-2 release assay from T cells isolated from human peripheral blood mononuclear cells (PBMCs). For example, a T-cell activation assay may include the isolation of human PBMCs from a leukocyte depletion cone. Methods for isolating PBMCs are known in the art and are described in this embodiment. T cells can then be isolated from the PBMCs. Methods for isolating T cells from PBMCs are known in the art and are described in this embodiment.

[0298] T cell activation assays can involve preparing the required number of T cells, for example, in a suitable culture medium such as T cell culture medium. This can be done at a concentration of 1.0 x 10⁻⁶. 6 Prepare the desired number of T cells at a concentration of cells / ml. The T cells can then be stimulated using a suitable T cell activation reagent, which provides the signal required for T cell activation. For example, a T cell activation reagent can be a reagent containing CD3 and CD28, such as beads containing CD3 and CD28. The isolated T cells can be incubated overnight with the T cell activation reagent to activate the T cells. Afterwards, the activated T cells can be washed to separate the T cells from the T cell activation reagent and then prepared at a suitable concentration, such as 2.0 × 10⁻⁶ cells / ml. 6 Cells / ml were resuspended in T cell culture medium. The activated T cells could then be added to a plate coated with anti-human CD3 antibody.

[0299] Appropriate dilutions of each test antibody molecule or specific binding member can be prepared and added to the wells. T cells can then be incubated with the test antibody at 37°C and 5% CO2 for 24 hours. The supernatant can be collected and measured to determine the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in solution are known in the art and are described in this embodiment. The concentration of human IL-2 can be plotted against the logarithmic concentration of the antibody molecule or specific binding member. The resulting curve can be fitted to a logarithmic (agonist) response equation.

[0300] The second antigen that binds to the second antigen binding site of a specific binding member can be an immune cell antigen or a disease antigen. Disease antigens include pathogenic antigens and tumor antigens.

[0301] In a preferred embodiment, the second antigen binding site of the specific binding member binds to the immune cell antigen.

[0302] Immune cell antigens bound by specific binding members can be present on the same immune cells or different immune cells as OX40.

[0303] Immune cell antigens can be members of the tumor necrosis factor receptor superfamily (TNFRSF) other than OX40. TNFRSF receptors are membrane-bound cytokine receptors containing an extracellular cysteine-rich domain that binds one or more ligands of the tumor necrosis factor superfamily (TNFSF).

[0304] TNFRSF receptors can be located on the surface of immune cells. Upon binding to a TNFRSF ligand, TNFRSF receptors form clusters on the surface of immune cells, thereby activating them. For example, ligand-bound TNFRSF receptors can form multimers, such as trimers or multimer clusters. The presence of ligand-bound TNFRSF receptor clusters stimulates intracellular signaling pathways that activate immune cells.

[0305] To avoid being bound by theory, it is believed that by binding both OX40 and the second TNFRSF receptor to the surface of immune cells, the specifically binding member will cause both OX40 and the second TNFRSF receptor to cluster and activate the immune cells. In other words, when both targets are bound, the specifically binding member will act as a TNFRSF receptor agonist.

[0306] TNFRSF receptors include CD27, CD40, EDA2R, EDAR, FAS, LTBR, ​​RELT, TNFRSF1A, TNFRSF1B, TNFRSF6B, TNFRSF8, TNFRSF9, TNFRSF10A-10D, TNFRSF11A, TNFRSF19B, TNFRSFF12C, TNFRSFF12C, and TNFRSF25.

[0307] In a preferred embodiment, the TNFRSF receptor is TNFRSF9 (CD137;4-1BB).

[0308] CD27 (TNFRSF7: gene ID 939) has a reference amino acid sequence of NP_001233.1 and can be encoded by a reference nucleotide sequence of NM_001242.4. CD40 (TNFRSF5: gene ID 958) has a reference amino acid sequence of NP_001241.1 and can be encoded by a reference nucleotide sequence of NM_001250.5. EDA2R (TNFRSF27: gene ID 60401) has a reference amino acid sequence of NP_001186616.1 and can be encoded by a reference nucleotide sequence of NM_001199687.2. EDAR (gene ID 10913) has a reference amino acid sequence of NP_071731.1 and can be encoded by a reference nucleotide sequence of NM_022336.3. FAS (TNFRSF6: gene ID 355) has a reference amino acid sequence of NP_000034.1 and can be encoded by a reference nucleotide sequence of NM_000043.5. LTBR (TNFRSF3: gene ID 4055) has a reference amino acid sequence of NP_001257916.1 and can be encoded by a reference nucleotide sequence of NM_001270987.1. RELT (TNFRSF19L: gene ID 84957) has a reference amino acid sequence of NP_116260.2 and can be encoded by a reference nucleotide sequence of NM_032871.3. TNFRSF1A (gene ID 7132) has a reference amino acid sequence of NP_001056.1 and can be encoded by a reference nucleotide sequence of NM_001065.3. TNFRSF1B (gene ID 7133) has a reference amino acid sequence of NP_001057.1 and can be encoded by a reference nucleotide sequence of NM_001066.2. TNFRSF6B (gene ID 8771) has a reference amino acid sequence of NP_003814.1 and can be encoded by a reference nucleotide sequence of NM_003823.3. TNFRSF8 (gene ID 943) has a reference amino acid sequence of NP_001234.3 and can be encoded by a reference nucleotide sequence of NM_001243.4. TNFRSF9 (gene ID 3604) has a reference amino acid sequence of NP_001552 and can be encoded by a reference nucleotide sequence of NM001561. TNFRSF10A (gene ID 8797) has a reference amino acid sequence of NP_003835.3 and can be encoded by a reference nucleotide sequence of NM_003844.3. TNFRSF10B (gene ID 8795) has a reference amino acid sequence NP_003833.4 and can be encoded by a reference nucleotide sequence NM_003842.4.TNFRSF10C (gene ID 8794) has a reference amino acid sequence of NP_003832.2 and is encoded by a reference nucleotide sequence of NM_003841.4. TNFRSF10D (gene ID 8793) has a reference amino acid sequence of NP_003831.2 and is encoded by a reference nucleotide sequence of NM_003840.4. TNFRSF11A (gene ID 8792) has a reference amino acid sequence of XP_011524547.1 and is encoded by a reference nucleotide sequence of XM_11526245.2. TNFRSF11B (gene ID 4982) has a reference amino acid sequence of NP_002537.3 and is encoded by a reference nucleotide sequence of NM_002546.3. TNFRSF12A (gene ID 51330) has a reference amino acid sequence of NP_057723.1 and is encoded by a reference nucleotide sequence of NM_016639.2. TNFRSF13B (gene ID 23495) has a reference amino acid sequence of NP_0036584.1 and is encoded by a reference nucleotide sequence of NM_012452.2. TNFRSF13C (gene ID 115650) has a reference amino acid sequence of NP_443177.1 and is encoded by a reference nucleotide sequence of NM_052945.3. TNFRSF14 (gene ID 8764) has a reference amino acid sequence of NP_001284534.1 and is encoded by a reference nucleotide sequence of NM_001297605.1. TNFRSF17 (gene ID 608) has a reference amino acid sequence of NP_001183.2 and can be encoded by a reference nucleotide sequence of NM_001192.2. TNFRSF18 (gene ID 8784) has a reference amino acid sequence of NP_004195.2 and can be encoded by a reference nucleotide sequence of NM_004186.1. TNFRSF19 (gene ID 55504) has a reference amino acid sequence of NP_001191387.1 and can be encoded by a reference nucleotide sequence of NM_001204585.1. TNFRSF21 (gene ID 27242) has a reference amino acid sequence of NP_055267.1 and can be encoded by a reference nucleotide sequence of NM_014452.4. TNFRSF25 (DR3: gene ID 8718) binds to the ligand TNFSF15 (TL1A), which has the reference amino acid sequence NP_001034753.1 and can be encoded by the reference nucleotide sequence NM_001039664.1.

[0309] Alternatively, the immune cell antigen that binds to the second antigen binding site can be a molecule that has a regulatory function in the immune system other than TNFRSF members, such as an immune co-stimulatory molecule or an inhibitory checkpoint molecule. Examples of such immunomodulatory molecules include ICOS (CD278), LAG3, PD1, PD-L1, PD-L2, B7H3, B7H4, CTLA4, TIGIT, BTLA, HVEM, T-cell immunoglobulin, mucin-containing domain 3 (TIM-3), CD47, CD73, A2aR, CD200, CD200R, colony-stimulating factor 1 receptor (CSF-1R), VISTA CD28, CD80, LLT1, galactagogue-9, NKG2A, NKG2D, and KIR.

[0310] Immune cells containing immune cell antigens can belong to any immune cell subset, including T cells, tumor-infiltrating leukocytes (TILs), bone marrow lineage cells (e.g., antigen-presenting cells (APCs), NK cells, and / or B cells). When the immune cell antigen is a TNFRSF receptor, the immune cells containing the TNFRSF receptor are preferably T cells.

[0311] Alternatively, the second antigen-binding site may bind to the disease antigen as described above. Not wishing to be bound by theory, it is believed that the binding of a specific binding member to OX40 and the disease antigen will lead to the activation of T cells near the disease site. Subsequently, the activated T cells can initiate, promote, or participate in immune responses, such as those against pathogens or cancer cells. The role of the immune system in recognizing and eliminating cancer cells is outlined in Chen and Mellman, 2013.

[0312] The second antigen-binding site of a specific binding member can bind tumor antigens. Tumor antigens are antigens that are primarily present in the tumor environment rather than being ubiquitous in other environments within the individual. For example, tumor antigens can be present on the surface of tumor cells, on other stromal cells in the tumor microenvironment, or in the biological fluids near the tumor. Therefore, tumor antigens serve as markers of the location of tumor cells within an individual.

[0313] In some implementations, tumor antigens can be antigens located on the surface of cancer cells. Tumor antigens may be upregulated or overexpressed on tumor cells, while they may not be expressed in large quantities in corresponding normal somatic cells of the same tissue without a tumor.

[0314] In some implementations, tumor antigens are upregulated or overexpressed on stromal cells in the tumor microenvironment compared to stromal cells in corresponding normal tissues where no tumor is present.

[0315] Tumor antigens can exist on the cell surface and may not be internalized quickly.

[0316] Tumor antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, specific binding members targeting OX40 receptors and tumor antigens can be used in experiments co-culturing OX40-expressing cells with tumor antigen-expressing cells, and the activation of OX40-expressing cells can be measured, for example by T-cell activation assays, proliferation assays, or cytotoxicity assays.

[0317] Cell surface tumor antigens can be tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0318] Tumor antigens expressed by cancer cells can include, for example, cancer-testis (CT) antigens encoded by cancer germline genes, such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE- Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 / CT7, MAGE-C2, NY-ESO-I, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and XAGE and their immunogenic fragments or variants (Simpson et al., 2005; Gure et al., 2005; Velazquez et al., 2007; Andrade et al., 2008; Tinguely et al., 2008; Napoletano et al., 2008).

[0319] Other cell surface tumor antigens include, for example, AFP, α v β3 (polinecin receptor), α vβ6, B cell maturation agent (BCMA), CA125 (MUC16), CD4, CD20, CD22, CD33, CD52, CD56, CD66e, CD80, CD140b, CD227 (MUC1), EGFR (HER1), EpCAM, GD3 ganglioside, HER2, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), CD5, CD19, CD21, CD25, CD37, CD30 CD33, CD45, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA) (e.g., CEA-associated cell adhesion molecule 5 (CEACAM5)), TAG-72, folate-binding protein, A33, G250, ferritin, glycolipids (e.g., gangliosides), carbohydrates (e.g., CA-125), IL-2 receptor, fibroblast activating protein (FAP), IGF1R, B7H3, B7H4, PDL1, CD200, EphA2, and mesothelin or variants thereof. These and other cell surface tumor antigens are described in Carter et al., 2004; Scott and Renner, 2001; Cheever et al., 2009; Tai and Anderson, 2015; and Podojil and Miller, 2017.

[0320] Other tumor antigens include extraframework peptide-MHC complexes produced by “stressed” cancer cells using non-AUG translation initiation mechanisms (Malarkannan et al., 1999).

[0321] Other tumor antigens include peptide-MHC complexes on the surface of tumor cells or tumor microenvironment cells, wherein the peptide-MHC complex contains a tumor-specific neoantigen peptide fragment of a mutated intracellular tumor antigen, and the peptide neoantigen contains one or more tumor-specific mutations (Gubin et al., 2015). Other tumor antigens are well known in the art (see, for example, the examples in WO00 / 20581; Cancer Vaccines and Immunotherapy (2000), Eds Stern, Beverley, and Carroll, Cambridge University Press, Cambridge). Sequences of these tumor antigens are readily available from public databases, but can also be found in WO1992 / 020356 A1, WO1994 / 005304 A1, WO1994 / 023031 A1, WO1995 / 020974 A1, WO1995 / 023874 A1, and WO1996 / 026214 A1.

[0322] Exemplary tumor antigens include HER2, FAP, EpCAM, CEACAM5, CD20, CD73, PSMA, mesothelin, EphA2, IGF1R, CD200, and α.v β6, BCMA, PD-L1, B7H3, B7H4 and EGFR.

[0323] For example, a tumor antigen could be mesothelin (MSLN).

[0324] HER2 (ERBB2; gene ID 2064) can have a reference amino acid sequence of NP_001005862.1 and can be encoded by a reference nucleotide sequence of NM_001005862.2. FAP (gene ID 2191) can have a reference amino acid sequence of NP_001278736.1 and can be encoded by a reference nucleotide sequence of NM_001291807.1. EpCAM (gene ID 4072) can have a reference amino acid sequence of NP_002345.2 and can be encoded by a reference nucleotide sequence of NM_002354.2.

[0325] CEACAM5 (gene ID 1048) can have a reference amino acid sequence of NP_001278413.1 and can be encoded by a reference nucleotide sequence of NM_001291484.2. CD20 (MS4A1; gene ID 931) can have a reference amino acid sequence of NP_068769.2 and can be encoded by a reference nucleotide sequence of NM_021950.3. CD73 (NT5E; gene ID 4907) can have a reference amino acid sequence of NP_001191742.1 and can be encoded by a reference nucleotide sequence of NM_001204813.1. PSMA (FOLH1; gene ID 2346) can have a reference amino acid sequence of NP_001014986.1 and can be encoded by a reference nucleotide sequence of NM_001014986.1. Mesothelin (MSLN; gene ID 10232) can have a reference amino acid sequence of NP_001170826.1 and can be encoded by a reference nucleotide sequence of NM_001177355.2. EphA2 (gene ID 1969) can have a reference amino acid sequence of NP_001316019.1 and can be encoded by a reference nucleotide sequence of NM_001329090.1. IGF1R (gene ID 3480) can have a reference amino acid sequence of NP_000866.1 and can be encoded by a reference nucleotide sequence of NM_000875.4. CD200 (gene ID 4345) can have a reference amino acid sequence of NP_001004196.2 and can be encoded by a reference nucleotide sequence of NM_001004196.3. αvβ6 is a heterodimer composed of the integrin subunit αV and the integrin subunit β6. Integrin subunit αV (ITGAV; gene ID 3685) can have a reference amino acid sequence of NP_001138471.1 and can be encoded by a reference nucleotide sequence of NM_001144999.2. Integrin subunit β6 (ITGB6; gene ID 3694) can have a reference amino acid sequence of NP_000879.2 and can be encoded by a reference nucleotide sequence of NM_000888.4. BCMA (TNFRSF17; gene ID 608) can have a reference amino acid sequence of NP_001183.2 and can be encoded by a reference nucleotide sequence of NM_001192.2. PD-L1 (CD274; gene ID 29126) can have a reference amino acid sequence of NP_001254635.1 and can be encoded by a reference nucleotide sequence of NM_001267706.1.B7H3 (CD276; gene ID 80381) can have a reference amino acid sequence of NP_001019907.1 and can be encoded by a reference nucleotide sequence of NM_001024736.1. B7H4 (VTCN1; gene ID 79679) can have a reference amino acid sequence of NP_001240778.1 and can be encoded by a reference nucleotide sequence of NM_001253849.1. EGFR (gene ID 1956) can have a reference amino acid sequence of NP_001333826.1 and can be encoded by a reference nucleotide sequence of NM_001346897.1.

[0326] In other embodiments, the tumor antigen may be a soluble tumor antigen, such as one produced by cancer cells or in response to cancer cell growth factors. Soluble factors may be upregulated or overexpressed in the biological fluids surrounding the tumor. Soluble tumor antigens may be polymers, such as dimers or trimers. The concentration of soluble tumor antigens at the tumor site or tumor microenvironment may be higher than at other sites in the individual. The tumor microenvironment and associated soluble tumor antigens are described in more detail in Bhome et al., (2015).

[0327] Suitable soluble tumor antigens include VEGF, HGF, SDF1, and TGF-β, such as TGF-β-1, TGF-β-2, TGF-β-3, and TGF-β-4.

[0328] VEGF (VEGFA; gene ID 7422) has a reference amino acid sequence of NP_001020537.2 and can be encoded by a reference nucleotide sequence of NM_001025366.2. HGF (gene ID 3082) has a reference amino acid sequence of NP_000592.3 and can be encoded by a reference nucleotide sequence of NM_000601.5. SDF1 (CXCL12; gene ID 6387) has a reference amino acid sequence of NP_000600.1 and can be encoded by a reference nucleotide sequence of NM_000609.6. TGF-β-1 (TGFB1; gene ID 7040) can have a reference amino acid sequence of NP_000651.3 and can be encoded by a reference nucleotide sequence of NM_000660.6. TGF-β-2 (TGFB2; gene ID 7042) can have a reference amino acid sequence of NP_001129071.1 and can be encoded by a reference nucleotide sequence of NM_001135599.3. TGF-β-3 (TGFB3; gene ID 7043) can have a reference amino acid sequence of NP_001316867.1 and can be encoded by a reference nucleotide sequence of NM_001329938.1. TGF-β-4 (LEFTY2; gene ID 7044) can have a reference amino acid sequence of NP_001165896.1 and can be encoded by a reference nucleotide sequence of NM_001172425.2.

[0329] In another preferred embodiment, the disease antigen is a pathogenic antigen.

[0330] Near the site of an infectious disease, it is anticipated that T cells activated by specific binding members can be used to treat the infectious disease. The infectious disease can be acute or persistent, but persistent infectious diseases are preferred.

[0331] The pathogenic antigen is preferably an antigen expressed by a human pathogen, such as a viral, bacterial, fungal, or parasitic antigen (e.g., protozoan antigen), with viral or bacterial antigens being more preferred. The pathogenic antigen is one that is primarily present on the pathogen or near the site of infection, rather than being ubiquitous in other parts of the individual.

[0332] For example, a pathogenic antigen can be an antigen present on the surface of a virus, bacteria, fungus, or parasite, or a soluble antigen expressed by a virus, bacteria, fungus, or parasite. A virus, bacteria, fungus, or parasite can be any virus, bacteria, fungus, or parasite mentioned elsewhere in this document.

[0333] When a pathogenic antigen is a soluble antigen, it may be upregulated or overexpressed in bodily fluids near the site of infection. For example, soluble pathogen antigens may be present at or near the site of infection at higher concentrations than elsewhere in the individual's body. Soluble pathogen antigens can be polymers, such as dimers or trimers.

[0334] Pathogen antigens suitable for targeting by specific binding members can be identified using methods known in the art. For example, specific binding members targeting OX40 receptors and pathogenic antigens can be used in experiments where OX40-expressing cells are co-cultured with pathogens or pathogenic antigens, and the activation of OX40-expressing cells can be measured, for example by T-cell activation assays, proliferation assays, or cytotoxicity assays.

[0335] Many pathogen antigens suitable for targeting by specific binding members are further known in the art, and those skilled in the art can select them according to the infectious disease to be treated. Examples of viral antigens include proteins p24, gp120, and gp41 expressed by human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg) expressed by hepatitis B virus (HBV), and hemagglutinin and neuraminidase expressed by influenza virus. Examples of bacterial antigens include Rv1733, Rv2389, and Rv2435n expressed by Mycobacterium tuberculosis.

[0336] The specific binding member may also include a first, second, or third sequence, an AB, CD, or EF loop sequence, a CH3 domain, a CH2 domain, CH2 and CH3 domains, Fcab, CDR, VH domain, VL domain, a variant of the light chain, and / or a variant of the heavy chain sequence disclosed herein. Suitable variants can be obtained by sequence alteration or mutation and screening. In a preferred embodiment, the specific binding member containing one or more variant sequences retains one or more functional characteristics of the parental specific binding member, such as binding specificity and / or binding affinity for OX40. For example, the specific binding member containing one or more variant sequences preferably has the same or higher affinity for OX40 as the (parental) specific binding member. The parental specific binding member is a specific binding member that does not contain amino acid substitutions, deletions, and / or insertions that have been incorporated into the variant specific binding member.

[0337] For example, the specifically binding member may include a first, second, or third sequence, an AB, CD, or EF loop sequence, a CH3 domain, a CH2 domain, CH2 and CH3 domains, Fcab, CDR, VH domain, VL domain, light chain, and / or heavy chain sequence, which are similar to the first, second, or third sequence, AB, CD, or EF loop sequence, CH3 domain, CH2 domain, CH2 and CH3 domains, Fcab, CDR, etc. disclosed herein. The VH domain, VL domain, light chain, and / or heavy chain sequence have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

[0338] The CH3 domain sequence of the specifically binding member FS20-22-49 shares at least 95% sequence identity with the CH3 domains of the specifically binding members FS20-22-38, FS20-22-41, FS20-22-47, and FS20-22-85. The CH3 domain sequence of the specifically binding member FS20-31-115 shares at least 92% sequence identity with the CH3 domains of the specifically binding members FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, and FS20-31-108. The CH3 domain sequence of the specifically binding member FS20-11-131 shares at least 97% sequence identity with the CH3 domains of the specifically binding members FS20-11-127 and FS20-11-134.

[0339] Therefore, in a preferred embodiment, the specific binding member has or includes a CH3 domain sequence, which is consistent with SEQ ID NO. The CH3 domain sequences listed in NO: 15, 24, 33, 46, 55, 63, 72, 81, 94, 103, 114, 124, 134, or 143 have at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity, preferably at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%.

[0340] In a preferred embodiment, the specific binding member of the invention comprises a CH2 domain sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the CH2 domain sequence listed in SEQ ID NO: 5, 6, or 7.

[0341] In a preferred embodiment, the specific binding member of the invention comprises a CH2 domain sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the CH2 domain sequences listed in SEQ ID NO: 17, 19, 26, 28, 35, 37, 48, 50, 57, 59, 65, 67, 74, 76, 83, 85, 96, 98, 105, 107, 116, 118, 126, 128, 136, 138, 145, or 147.

[0342] Sequence identity is typically defined using the GAP algorithm (GCG software package, Accelerys Ltd., San Diego, USA). GAP uses the Needleman and Wunsch algorithms to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. The default parameters are typically a gap creation penalty of 12 and a gap extension penalty of 4. GAP is preferred, but other algorithms can also be used, such as BLAST (using the method of Altschul et al., 1990), FASTA (using the method of Pearson and Lipman, 1988), or the Smith-Waterman algorithm (Smith and Waterman, 1981) or the TBLASTN procedure, see Altschul et al., 1990, as above, usually with the default parameters. In particular, the psi-Blast algorithm (Altschul et al., 1997) can be used.

[0343] The specific binding member may include a first, second, or third sequence, an AB, CD, or EF loop sequence, a CH3 domain, a CH2 domain, a CH2 and CH3 domain, an Fcab, CDR, VH domain, a VL domain, a light chain, or a heavy chain sequence, having one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues) compared to the first, second, or third sequence, the AB, CD, or EF loop sequence, the CH3 domain, the CH2 domain, the CH2 and CH3 domain, the Fcab, CDR, VH domain, VL domain, light chain, or heavy chain sequence disclosed herein, preferably less than or equal to 20 alterations, less than or equal to 15 alterations, less than or equal to 10 alterations, less than or equal to 5 alterations, less than or equal to 4 alterations, less than or equal to 3 alterations, less than or equal to 2 alterations, or less than or equal to 1 alteration.

[0344] In a preferred embodiment, the specific binding member may comprise a CH3 domain sequence having one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), preferably less than or equal to 20 alterations, less than or equal to 15 alterations, less than or equal to 10 alterations, less than or equal to 5 alterations, less than or equal to 4 alterations, less than or equal to 3 alterations, less than or equal to 2 alterations, or less than or equal to 1 alteration, compared to the CH3 domain sequences listed in SEQ ID NO: 15, 24, 33, 46, 55, 63, 72, 81, 94, 103, 114, 124, 134, or 143.

[0345] In a preferred embodiment, the specific binding member may comprise a CH2 domain sequence having one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), preferably less than or equal to 20 alterations, less than or equal to 15 alterations, less than or equal to 10 alterations, less than or equal to 5 alterations, less than or equal to 4 alterations, less than or equal to 3 alterations, less than or equal to 2 alterations, or less than or equal to 1 alteration, compared to the CH2 domain sequence listed in SEQ ID NO: 5, 6, or 7.

[0346] In a preferred embodiment, the specific binding member may include or have a sequence with one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), preferably less than or equal to 40 alterations, less than or equal to 30 alterations, less than or equal to 20 alterations, less than or equal to 15 alterations, less than or equal to 10 alterations, less than or equal to 5 alterations, less than or equal to 4 alterations, less than or equal to 3 alterations, less than or equal to 2 alterations, or less than or equal to 1 alteration, compared to the Fcab domain sequence listed in SEQ ID NO: 17, 19, 26, 28, 35, 37, 48, 50, 57, 59, 65, 67, 74, 76, 83, 85, 96, 98, 105, 107, 116, 118, 126, 128, 136, 138, 145, or 147.

[0347] When the specific binding member comprises a variant of the CH3 domain, CH2 and CH3 domains, Fcab, light chain, or heavy chain sequence disclosed herein, the variant preferably does not contain any amino acid alterations in the first, second, and third sequences located in the AB, CD, and EF rings of the CH3 domain of the specific binding member. For example, the variant may not contain any amino acid alterations in the AB, CD, and EF rings of the CH3 domain of the specific binding member.

[0348] In a preferred embodiment where one or more amino acids are substituted by another amino acid, the substitution can be a conservative substitution, such as as shown in the table below. In some embodiments, amino acids of the same class in the middle column are substituted for each other, i.e., a nonpolar amino acid is substituted for another nonpolar amino acid. In some embodiments, amino acids in the same row of the rightmost column are substituted.

[0349]

[0350] In some implementations, the substitution may be functionally conservative. That is, in some implementations, the substitution may not affect (or may substantially not affect) one or more functional properties (e.g., binding affinity) of the substituted specific binding member compared to an equivalent unsubstituted specific binding member.

[0351] A specific binding member is also anticipated, comprising a constant domain, preferably the CH3 domain, of the OX40 antigen binding site, and competing with the specific binding member of the present invention for binding to OX40, or having the same epitope on OX40 as the specific binding member of the present invention. Methods for determining which two specific binding members are competing for antigen are known in the art. For example, surface plasmon resonance, such as Biacore, mapping methods can be used to determine the competitive binding of two specific binding members to an antigen.

[0352] In some implementations, the specific binding member may not contain a CDR-based antigen binding site.

[0353] In particular, specific binding members may not contain a CDR-based antigen-binding site that binds to CD137.

[0354] Alternatively, the specific binding member may not contain a CDR-based antigen-binding site that binds mesothelin (MSLN).

[0355] For example, a specific binding member may not contain a CDR-based antigen binding site that binds to CD137 or MSLN, wherein the specific binding member contains the first, second, and third sequences in the AB, CD, and EF loops of the CH3 domain of the specific binding member FS20-22-49, the AB, CD, and EF loop sequences of the CH3 domain of the specific binding member FS20-22-49, and / or the CH3 domain sequence of the specific binding member FS20-22-49.

[0356] For example, the specific binding member may not contain the CDR of anti-CD137mAb FS30-10-16 listed below, and / or the VH and / or VL domains.

[0357] Heavy chain CDR of FS30-10-16mAb

[0358] CDR1(IMGT)GFTFSSYD

[0359] CDR1(Kabat)SYDMS

[0360] CDR2(IMGT)IDPTGSKT

[0361] CDR2(Kabat)DIDPTGSKTDYADSVKG

[0362] CDR3(IMGT)ARDLLVYGFDY

[0363] CDR3(Kabat)DLLVYGFDY

[0364] VH domain of FS30-10-16mAb

[0365] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVYGFDYWGQGTLVTVSS

[0366] Light chain CDR of FS30-10-16mAb

[0367] CDR1(IMGT)QSVSSSY

[0368] CDR1(Kabat)RASQSVSSSYLA

[0369] CDR2(IMGT)GAS

[0370] CDR2(Kabat)GASSRAT

[0371] CDR3(IMGT)QQSYSYPVT

[0372] CDR3(Kabat)QQSYSYPVT

[0373] VL domain of FS30-10-16mAb

[0374] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSYSYPVTFGQGTKVEIK

[0375] In particular, the specific binding member may not contain, or not consist of, the heavy and light chain sequences of FS20-22-49-AA / FS30-10-16 as shown in SEQ ID NO:172 and 173, respectively.

[0376] Specific binding members can conjugate to bioactive molecules or detectable markers. In this case, the specific binding member can be referred to as a conjugate. Such conjugates can be used to treat the diseases described herein.

[0377] For example, bioactive molecules can be immune system modulators, such as cytokines, preferably human cytokines. For instance, cytokines can be those that stimulate T cell activation and / or proliferation. Examples of cytokines conjugated to specific binding members include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-γ.

[0378] Alternatively, bioactive molecules can be ligand traps, such as ligand traps for cytokines, such as TGF-β or IL-6.

[0379] Suitable detection markers that can be conjugated to specific binding members are known in the art, including radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111, and technetium-99; and fluorescent dyes such as fluorescein, rhodamine, phycoerythrin, Texas red, and cyanine dye derivatives such as Cy7 and Alexa750; chromophores such as diaminobenzidine; latex beadase markers such as horseradish peroxidase; fluorescent powders or laser dyes with spectrally isolated absorption or emission properties; and chemical moieties such as biotin that can be detected by binding to a specific homologous detectable moiety, such as avidin markers.

[0380] Specific binding members can be conjugated to biologically active molecules or detectable tags via any suitable covalent or non-covalent bond, such as a disulfide bond or peptide bond. When the biologically active molecule is a cytokine, the cytokine can be linked to the specific binding member via a peptide linker. Suitable peptide linkers are known in the art and can be 5 to 25, 5 to 20, 5 to 15, 10 to 25, 10 to 20, or 10 to 15 amino acids in length.

[0381] In some embodiments, the bioactive molecule can be conjugated to a specific binding member via a cleavable linker. The linker allows for the release of the bioactive molecule from the specific binding member at the treatment site. The linker may include an amide bond (e.g., a peptide linker), a disulfide bond, or a hydrazone. Peptide linkers can be cleaved, for example, by site-specific proteases; disulfide bonds can be cleaved by a reducing environment in the cytosol; and hydrazones can be cleaved by acid-mediated hydrolysis.

[0382] Conjugates can be fusion proteins comprising a specific binding member and a bioactive molecule. In this case, the bioactive molecule can be conjugated to the specific binding member via a peptide linker or peptide bond. This is especially true when the specific binding member is a multi-chain molecule, for example, when the specific binding member is or contains Fcab or mAb. 2 At this time, bioactive molecules can conjugate with one or more chains of a specific binding member. For example, bioactive molecules can bind with mAbs. 2 Fusion proteins are formed by the fusion of one or two heavy chains of a molecule. Their advantages include ease of production and purification, which facilitates the production of clinical-grade materials.

[0383] This invention also provides isolated nucleic acid molecules or molecules encoding the specific binding members of this invention. Those skilled in the art will have no difficulty preparing such nucleic acid molecules using methods well-known in the art.

[0384] In a preferred embodiment, the nucleic acid molecule encodes the CH3 domain of the following specific binding member: FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38, preferably FS20-22-49, FS20-22-41, FS20-22-47 or FS20-22-85, more preferably FS20-22-49, FS20-22-41 or FS20-22-47, and most preferably FS20-22-49.

[0385] In an alternative preferred embodiment, the nucleic acid molecule encodes the CH3 domain of the following specific binding member: FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66, preferably FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94 or FS20-31-102, more preferably FS20-31-115 or FS20-31-108, and most preferably FS20-31-115.

[0386] In another alternative preferred embodiment, the nucleic acid molecule encodes the CH3 domain of a member that specifically binds to the nucleic acid: FS20-11-131, FS20-11-127 or FS20-11-134, with FS20-11-131 being the most preferred.

[0387] The CH3 domain sequences of these specific binding members are described in this paper.

[0388] For example, nucleic acid molecules that encode the CH3 domain of the following specific binding members:

[0389] (i)FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49 or FS20-22-85, respectively, are listed in SEQ ID NO: 47, 56, 64, 73 and 82;

[0390] (ii) FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115, respectively, listed in SEQ ID NO: 95, 104, 115, 125, 135, and 144; or

[0391] (iii)FS20-11-127, FS20-11-131 or FS20-11-134 are listed in SEQ ID NO: 16, 25 and 34 respectively.

[0392] In a preferred embodiment, the nucleic acid molecule encodes the following specific binding member: FS20-22-49, FS20-22-41, FS20-22-47, FS20-22-85 or FS20-22-38, preferably FS20-22-49, FS20-22-41, FS20-22-47 or FS20-22-85, more preferably FS20-22-49, FS20-22-41 or FS20-22-47, and most preferably FS20-22-49.

[0393] In an alternative preferred embodiment, the nucleic acid molecule encodes the following specific binding member: FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94, FS20-31-102 or FS20-31-66, preferably FS20-31-115, FS20-31-108, FS20-31-58, FS20-31-94 or FS20-31-102, more preferably FS20-31-115 or FS20-31-108, and most preferably FS20-31-115.

[0394] In another alternative preferred embodiment, the nucleic acid molecule encodes the following specific binding member: FS20-11-131, FS20-11-127 or FS20-11-134, with FS20-11-131 being the most preferred.

[0395] For example, nucleic acid molecules that encode the following specific binding member sequences:

[0396] (i)FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49 and FS20-22-85, respectively, are listed in SEQ ID NO: 49, 58, 66, 75 and 84; and

[0397] (ii)FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49 and FS20-22-85 are listed in SEQ ID NO: 51, 60, 68, 77 and 86 respectively.

[0398] Nucleic acid molecules encoding the sequences of the specific binding members of the following specific binding members:

[0399] (i)FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, and FS20-31-115, listed in SEQ ID NO: 97, 106, 117, 127, 137, and 146, respectively; and (ii)FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, and FS20-31-115, listed in SEQ ID NO: 99, 108, 119, 129, 139, and 148, respectively.

[0400] Nucleic acid molecules that encode the following specific binding member sequences:

[0401] (i)FS20-11-127, FS20-11-131, or FS20-11-134, listed in SEQ ID NO: 18, 27, and 36, respectively; and

[0402] (ii)FS20-11-127, FS20-11-131 or FS20-11-134, listed in SEQ ID NO: 20, 29 and 38 respectively.

[0403] The isolated nucleic acid molecules can be used to express the specific binding members of the present invention. Nucleic acids are typically provided in the form of recombinant vectors for expression. Therefore, another aspect of the present invention provides a vector comprising the nucleic acid as described above. Suitable vectors can be selected or constructed that contain suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylated sequences, enhancer sequences, marker genes, and other suitable sequences. Preferably, the vector contains suitable regulatory sequences to drive the expression of the nucleic acid in host cells. The vector can be a plasmid, a virus, such as a bacteriophage or phage particle, as appropriate.

[0404] Nucleic acid molecules or vectors as described herein can be introduced into host cells. Techniques for introducing nucleic acids or vectors into host cells are established in the art, and any suitable technique can be used. A range of host cells suitable for generating recombinant-specific binding members are known in the art, including bacterial, yeast, insect, or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NSO, or HEK cells, such as HEK293 cells. The most preferred host cell is CHO cells.

[0405] Another aspect of the invention provides a method for generating the specific binding member of the invention, the method comprising expressing a nucleic acid encoding the specific binding member in a host cell and optionally isolating and / or purifying the resulting specific binding member. Methods of culturing host cells are well known in the art. The method may further include isolating and / or purifying the specific binding member. Techniques for purifying recombinant specific binding members are well known in the art, including, for example, HPLC, FPLC, or affinity chromatography, such as HPLC. In some embodiments, purification may be performed using an affinity tag on the specific binding member. The method may also include formulating the specific binding member into a pharmaceutical composition, optionally together with pharmaceutically acceptable excipients or other substances as described below.

[0406] As mentioned above, OX40 is expressed on cells of the immune system, including activated T cells, particularly CD4+ T cells, CD8+ T cells, type 1 helper T (Th1) cells, type 2 helper T (Th2) cells, regulatory T (Treg) cells, tumor-infiltrating T cells, and activated natural killer (NK) cells. OX40 activation has been shown to play a role in enhancing T cell activation, T cell clonal expansion, T cell differentiation and survival, and the generation of memory T cells. Given the immune-enhancing activity of OX40, OX40 agonist molecules have been investigated in the context of cancer therapy.

[0407] Therefore, the specific binding members described herein can be used for therapeutic applications, particularly in the treatment of cancer. Additionally, the specific binding members are expected to be used to treat infectious diseases, such as persistent infections.

[0408] The specific binding members described herein can be used in methods for treating human or animal bodies. Related aspects of the invention are provided as follows:

[0409] (i) The specific binding members described herein are used as drugs.

[0410] (ii) The methods described herein for the specific binding of members for the treatment of diseases or conditions.

[0411] (iii) The use of the specific binding members described herein in the preparation of medicaments for treating diseases or conditions; and,

[0412] (iv) A method of treating an individual’s disease or condition, wherein the method comprises administering to the individual a therapeutically effective amount of the specific binding member described herein.

[0413] Individuals can be patients, preferably human patients.

[0414] Treatment can be any treatment or therapy that achieves a desired therapeutic effect, such as inhibiting or delaying the progression of a disease, and includes slowing the rate of progression, stopping the rate of progression, improving, treating or alleviating (partial or complete) the condition, preventing, improving, delaying, reducing or inhibiting one or more symptoms and / or conditions, or prolonging the life of an individual or patient without treatment.

[0415] This also includes treatment as a preventative measure (i.e., preventative measures). For example, as described herein, it is possible to treat individuals susceptible to diseases or diseases such as cancer that may occur or recur. Such treatment can prevent or delay the onset or recurrence of disease in an individual.

[0416] The described treatment methods may include administering at least one other treatment to an individual besides the specific binding member. Therefore, the specific binding member described herein may be administered to an individual alone or in combination with one or more other treatments. When the specific binding member is administered to an individual in combination with another treatment, an adjunct therapy may be given to the patient simultaneously, sequentially, or separately from the specific binding member. If the adjunct therapy is administered with the specific binding member, the specific binding member and the adjunct therapy may be given to the individual as a combined formulation. For example, the adjunct therapy may be a known therapy or treatment agent for the disease to be treated.

[0417] While specific binding members can be administered alone, they are typically administered in the form of pharmaceutical compositions that may include at least one component other than the specific binding member. Therefore, another aspect of the invention provides a pharmaceutical composition comprising the specific binding member as described herein. A method comprising formulating the specific binding member into a pharmaceutical composition is also provided.

[0418] In addition to specific binding members, pharmaceutical compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" means a compound, material, component, composition, and / or dosage form that, to a reasonable medical judgment, is suitable for use in contact with the tissues of a relevant subject (e.g., a human), without excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be "acceptable" in the sense of compatibility with other components of the formulation. The exact nature of the carrier or other substance will depend on the route of administration, which may be infusion, injection, or any other suitable route, as described below.

[0419] For parenteral administration, such as subcutaneous or intravenous administration, such as by injection, pharmaceutical compositions containing specific binding members can be in parenteral acceptable aqueous solutions that are pyrogen-free and have suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's solution, lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight... Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0420] In some implementations, the specific binding member can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized specific binding member can be reconstituted in sterile water and mixed with saline before administration to the individual.

[0421] The dosage administered may be a “therapeutic effective dose” that is sufficient to demonstrate benefit to the individual. The actual dosage, rate of administration, and timing will depend on the nature and severity of the drug being treated, the specific individual being treated, the individual’s clinical condition, the cause of the disease, the composition of the delivery site, the type of specific binding member, the method of administration, the dosing schedule, and other factors known to the physician. Treatment prescriptions, such as the determination of dosage, are the responsibility of general practitioners and other physicians and may depend on the severity of symptoms and / or the progression of the disease being treated. Appropriate doses of immunoglobulins are well known in the art (Ledermann et al. (1991) Int. J. Cancer 47:659-664; and Bagshawe et al. (1991) Antibodies, Immunoconjugates, and Radiopharmaceuticals 4:915-922). The specific doses described herein or indicated in the Physician’s Handbook (2003) may be used for the administered antibody molecule. For antibody molecules, the therapeutically effective amount or appropriate dose of the specific binding member can be determined by comparing in vitro and in vivo activities in animal models. Methods for extrapolating effective doses from mice and other experimental animals to humans are known. The precise dosage will depend on many factors, including the size and location of the area to be treated and the precise nature of the specific binding member.

[0422] For systemic application, the typical immunoglobulin dose range is 100 μg to 1 g, and for topical application, it ranges from 1 μg to 1 mg. An initial higher loading dose may be administered, followed by one or more lower doses. This is the dose for a single treatment in an adult individual; the dose may be adjusted proportionally for children and infants, or adjusted by molecular weight for other specific binding member forms.

[0423] Treatment may be repeated at intervals of daily, twice weekly, weekly, or monthly, depending on the physician's judgment. Individual treatment regimens may depend on the pharmacokinetic and pharmacodynamic properties of the specific combination of ingredients, the route of administration, and the nature of the disease being treated.

[0424] Treatment can be periodic, and the time between two doses can be approximately two weeks or longer, for example, approximately three weeks or longer, approximately four weeks or longer, approximately once a month or longer, approximately five weeks or longer, or approximately six weeks or longer. For example, treatment can be every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are as described above.

[0425] In a preferred embodiment, the specific binding member described herein can be used in methods for treating cancer.

[0426] Cancer can be characterized by the abnormal proliferation of malignant cancer cells. When referring to a specific type of cancer, such as breast cancer, this refers to the abnormal proliferation of malignant cells in a related tissue (such as breast tissue). Secondary cancer resulting from the abnormal proliferation of malignant cells in another tissue (such as ovarian tissue) but located in the breast is not breast cancer as discussed in this article, but rather ovarian cancer.

[0427] The cancer can be primary or secondary. Therefore, the specific binding members described herein can be used to treat individual cancers, wherein the cancer is a primary tumor and / or a metastatic tumor.

[0428] Tumors of cancer treated with the specific binding member described herein may contain tumor-infiltrating T cells expressing OX40, for example, expressed on their cell surface. In one embodiment, the tumor may have been identified as containing tumor-infiltrating T cells expressing OX40. Methods for determining antigen expression on the cell surface are known in the art and include, for example, flow cytometry.

[0429] For example, cancers treated with the specific binding members described herein can be selected from leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma; solid cancers such as sarcomas (e.g., soft tissue sarcoma), skin cancers (e.g., Merkel cell carcinoma), melanoma, bladder cancers (e.g., urothelial carcinoma), and brain cancers (e.g., glioblastoma). Breast cancer, uterine / endometrial cancer, ovarian cancer (e.g., ovarian serous cystadenoma), prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), colorectal cancer (e.g., colorectal cancer), cervical cancer (e.g., cervical squamous cell carcinoma and cervical endometrial adenocarcinoma), liver cancer (e.g., hepatocellular carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), adrenal cancer, stomach cancer, testicular cancer, gallbladder cancer and bile duct cancer (e.g., bile duct cancer), thyroid cancer, thymic cancer, bone cancer, and brain cancer.

[0430] In a preferred embodiment, the cancer treated with the specific binding member described herein is a solid cancer. More preferably, the cancer treated with the specific binding member described herein is a solid cancer selected from the following: sarcoma, melanoma, bladder cancer, brain cancer, breast cancer, uterine / endometrial cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and stomach cancer.

[0431] In the case of cancer, treatment can include suppressing cancer growth, including complete cancer remission, and / or suppressing cancer metastasis, as well as suppressing cancer recurrence. Cancer growth is generally defined by any of a number of indicators that suggest the state within the cancer has evolved into a more advanced form. Therefore, indicators used to measure the suppression of cancer growth include reduced cancer cell survival, reduced tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, disruption of the tumor vascular system, improved performance of delayed-type hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and reduced levels of tumor-specific antigens. Activating or enhancing the immune response to cancerous tumors in an individual can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject and / or reduce the tendency for cancer to grow in the individual.

[0432] In the context of cancer treatment, the specific binding member described herein may be administered to an individual in combination with another anticancer therapeutic agent or treatment agent (e.g., one that has proven to be suitable or is intended to treat the cancer in question). For example, the specific binding member may be administered to an individual in combination with chemotherapy agents, radiation therapy, immunotherapy agents, antitumor vaccines, oncolytic viruses, adoptive cell transfer (ACT) therapies (e.g., adoptive NK cell therapy or chimeric antigen receptor (CAR) T cell therapy, autologous tumor-infiltrating lymph nodes (TILs), or γ / δ T cell therapy), or hormone therapy.

[0433] Not wanting to be bound by theory, the specific binding members described in this article are thought to serve as adjuvants in cancer therapy. Specifically, for example, administering specific binding members in combination with chemotherapy and / or radiation therapy, or in combination with anti-tumor vaccines, to an individual is thought to elicit a stronger immune response against cancer compared to using chemotherapy and / or radiation therapy alone, or anti-tumor vaccines.

[0434] One or more chemotherapeutic agents that are used in combination with the specific binding members described herein may be selected from: taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, β-Raf enzyme inhibitors, MEK inhibitors, c-MET inhibitors, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogs, nucleoside analogs, antifolate agents, thalidomide derivatives, antitumor chemotherapeutic agents, etc. Taxanes include docetaxel, paclitaxel, and nalbutaxel; cytotoxic antibiotics include actinomycin, bleomycin, and anthracyclines such as doxorubicin, mitoxantrone, and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobemitinib, and trametinib; PARP inhibitors include piraparib; and β-Raf enzyme inhibitors include vemurafenib and dabradex. Dabrafenib; alkylating agents include dacarbazine, cyclophosphamide, and temozolomide; platinum analogs include carboplatin, cisplatin, and oxaliplatin; nucleoside analogs include azacitidine, capecitabine, fludarabine, fluorouracil, and gemcitabine; and antifolate drugs include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in this invention include defactinib, entinostat, eribulin, irinotecan, and vinblastine.

[0435] Preferred therapeutic agents to be administered in conjunction with the antibody molecules described herein are doxorubicin, mitoxantrone, cyclophosphamide, cisplatin, and oxaliplatin.

[0436] Radiotherapy administered in combination with the specific binding members described herein can be external beam radiotherapy or brachytherapy.

[0437] Immunotherapy agents co-administered with the specific binding members described herein can be therapeutic antibody molecules, nucleic acid cytokines, or cytokine-based therapies. For example, therapeutic antibody molecules can bind to immunomodulatory molecules, such as inhibitory checkpoint molecules or immune co-stimulatory molecules, or tumor antigens, such as cell surface tumor antigens or soluble tumor antigens. Examples of immunomodulatory molecules that therapeutically specific binding members can bind include CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, PD-L1, PD-1, CD47, CD73, CSF-1R, KIR, CD40, HVEM, IL-10, and CSF-1. Examples of receptors of the innate immune system that therapeutic antibody molecules can bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING. Examples of tumor antigens that therapeutic antibody molecules can bind include HER2, EGFR, CD20, and TGF-β.

[0438] The nucleic acid that can be co-administered with the specific binding members described herein can be siRNA.

[0439] Cytokine-based or cytokine-based therapies may be selected from the following group: IL-2, conjugated IL-2 prodrugs, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferon.

[0440] Anti-tumor vaccines for cancer treatment have been implemented in clinical trials and discussed in detail in the scientific literature (e.g., Rosenberg, 2000). This primarily involves strategies that enhance the immune system's response to different cellular markers expressed by autologous or allogeneic cancer cells, using autologous or allogeneic cancer cells as a vaccination method, regardless of whether they carry granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF elicits a strong response in antigen presentation and is highly effective when used in conjunction with the aforementioned strategies.

[0441] Chemotherapy agents, radiotherapy, immunotherapy agents, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy agents are preferred. These include chemotherapy agents, radiotherapy, immunotherapy agents, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy agents used for the cancer in question, i.e., chemotherapy, radiotherapy, immunotherapy agents, antitumor vaccines, oncolytic viruses, ACT therapy, or hormone therapy agents that have been proven effective for the cancer in question. The selection of a suitable chemotherapy agent, radiotherapy, immunotherapy, antitumor vaccine, oncolytic virus, ACT therapy, or hormone therapy agent that has been shown to be effective for the aforementioned cancer is entirely within the capabilities of a skilled technician.

[0442] Given the immune-enhancing activity of OX40, OX40 agonist molecules hold promise for the treatment of infectious diseases. Therefore, in another preferred embodiment, the specific binding members described herein can be used to treat infectious diseases, such as acute or persistent infectious diseases.

[0443] Not wishing to be bound by theory, the specific binding members of this invention are thought to potentially enhance the immune response against acute infectious diseases caused by pathogens by inducing rapid infiltration and activation of innate immune cells such as neutrophils and monocytes, thereby contributing to the clearance of pathogens causing acute infectious diseases. Therefore, in another embodiment, the specific binding members described herein can be used in methods for treating acute infectious diseases, such as acute bacterial diseases. In a preferred embodiment, the acute infectious disease is an acute bacterial disease caused by infection with Gram-positive bacteria such as Listeria, Streptococcus pneumoniae, or Staphylococcus aureus.

[0444] Infectious diseases are usually cleared by the immune system, but some infections can persist for a long time, such as months or years, and cannot be effectively fought off by the immune system. Such infections are also called persistent or chronic infections.

[0445] Preferably, the specific binding member described herein is used to treat persistent infectious diseases, such as persistent viral, bacterial, fungal, or parasitic infections, preferably persistent viral or bacterial infections.

[0446] In a preferred embodiment, the persistent viral infection for which the specific binding member treatment described herein is used is a persistent infection of the following individuals: human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus, hepatitis B virus, hepatitis C virus, or varicella-zoster virus.

[0447] In a preferred embodiment, the persistent bacterial infection treated with the specific binding member described herein is a persistent infection of the following: Staphylococcus aureus, Haemophilus influenzae, Mycobacterium tuberculosis, Mycobacterium leprae, Escherichia coli, Salmonella typhi, Helicobacter pylori, Pseudomonas aeruginosa, Treponema pallidum, Enterococcus faecalis, or Streptococcus pneumoniae.

[0448] In a preferred embodiment, the persistent fungal infection treated with the specific binding member described herein is a persistent infection of the following: Candida (e.g., Candida albicans), Cryptococcus (e.g., Cryptococcus gattii or Cryptococcus neoformans), Penicillium (e.g., Talaromyces marneffe), Microsporum (e.g., Microsporum audouinii), or Trichophyton tonsurans.

[0449] In a preferred embodiment, the persistent parasitic infection treated with the specific binding member described herein is a persistent infection of Plasmodium, such as Plasmodium falciparum, or Leishmania, such as Leishmania donovani.

[0450] In the treatment of persistent infectious diseases, treatment may include eliminating the infection, reducing the individual's pathogenic burden, and preventing recurrence of the infection. For example, treatment may include preventing, improving, delaying, alleviating, or stopping one or more symptoms and / or signs of persistent infection. Alternatively, treatment may include preventing infectious diseases.

[0451] In the context of treating infectious diseases, the specific binding members described herein can be administered to an individual in combination with another therapeutic agent used to treat the infectious disease (e.g., an agent that has proven to be suitable or is expected to be suitable for treating the infectious disease in question). For example, the specific binding member can be administered to an individual in combination with an immunotherapeutic agent. The immunotherapeutic agent administered in combination with the antibody molecules described herein can be a therapeutic antibody molecule. For example, a therapeutic antibody molecule can bind to a receptor of the innate immune system. Examples of receptors of the innate immune system that a therapeutic antibody molecule can bind to include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING.

[0452] When specific binding members are used to prevent infectious diseases, they can be administered in combination with vaccines for said pathogens. Not wishing to be bound by theory, the specific binding members described herein are considered to act as adjuvants in vaccination. In particular, individuals given specific binding members in combination with vaccines are thought to elicit a greater immune response against pathogens than those given vaccines alone.

[0453] Other aspects and embodiments of the invention will be apparent to those skilled in the art from this disclosure, which includes the following experimental examples.

[0454] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0455] As used herein, “and / or” should be considered as a specific disclosure of each of the two specified features or components having or not having the other. For example, “A and / or B” will be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as they are listed separately herein.

[0456] Unless the context otherwise indicates, the description and definition of the features set forth above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0457] Unless the context otherwise indicates, other aspects and embodiments of the invention provide for the above aspects and embodiments in which the term “comprising” is replaced with the term “consisting of” or “substantially consisting of”.

[0458] Some aspects and embodiments of the invention will now be described by way of example and with reference to the accompanying drawings. Example

[0459] Example 1 - Antigen Selection and Characterization

[0460] The Fcab used to select Fcab specific to human and mouse OX40, and the OX40 antigen used to test the cross-reactivity of the selected Fcab with cynomolgus monkey OX40, can be prepared in-house or obtained from commercial sources as described below.

[0461] 1.1 Self-made antigens

[0462] We developed a self-made recombinant, soluble, dimer OX40 antigen and a cell line expressing OX40.

[0463] 1.1.1 Preparation of recombinant, soluble human, cynomolgus monkey, and mouse OX40 antigens

[0464] To prepare recombinant soluble dimer OX40 antigen, the extracellular domain of OX40 was fused with mouse Fc, thereby improving the solubility and stability of the antigen. Specifically, the extracellular domain of the relevant OX40 (human, cynomolgus monkey, or mouse) was cloned into the pFUSE-mIgG2aFc2 vector (Invivogen product number pfuse-mg2afc2) using EcoRI-HF and BgIII restriction endonucleases to generate an antigen with a mouse IgG2a Fc domain at its C-terminus. The recombinant OX40 antigen was then transiently expressed in HEK293-6E cells (National Research Council of Canada) and purified using a mAb-selective SuRe protein A column (Genetic Medical Systems, 11003494), followed by size exclusion chromatography (SEC) to ensure that the generated antigen was a single species and free of aggregates.

[0465] To prepare the biotinylated form of the recombinant OX40 antigen, EZ-Link was used according to the manufacturer's experimental method. TMThe antigen was biotinylated using the Sulfo-NHS-SS-Biotin Kit (Thermo Fisher Scientific, catalog number 21331). The biotinylated OX40 antigen was used for the selection assays described below, but not for binding affinity measurements. Purification of the biotinylated OX40 antigen was performed in two steps using a PD10 desalting column (GE Healthcare, 17-0851-01) followed by an Amicon 30k spinning column (Millipore, UFC903024), following the manufacturer's instructions. The biophysical properties of the recombinant antigen were characterized by SE-HPLC analysis to ensure the absence of aggregates, and the molecular size was verified by PAGE. The size determined by PAGE indicated that the soluble antigens were dimers, as their estimated molecular weight was twice the predicted monomer molecular weight. The recombinant antigen was also analyzed by gel shift assay, which showed a biotinylation degree of over 90%. Biotinylated recombinant human (hOX40-mFc), mouse (mOX40-mFc), and cynomolgus monkey (cOX40-mFc) OX40 antigens were confirmed by ELISA and surface plasmon resonance (SPR) assays to bind to OX40-specific antibodies (antibody 11D4 against human and cynomolgus monkey OX40 [European Patent 2242771]; polyclonal sheep anti-human OX40 antibody against cynomolgus monkey OX40 [Andy Biotech catalog number AF3388]; antibody ACT35 against human OX40 [Bio-Tech catalog number 35002]; and antibody OX86 against mouse OX40 [Bio-Tech catalog number 119408]). These antigens are listed in Table 2 below.

[0466] 1.1.2 Preparation of cell lines expressing human, cynomolgus monkey and mouse OX40

[0467] Human, cynomolgus monkey, and mouse OX40 (sequences shown in Table 1) were cloned into the vector pLVX-EF1a-IRES-puro (Clontech, catalog number 631253) using SpeI-HF and NotI-HF restriction enzymes. The vector was then transformed into the Lenti-X 293T cell line (Clontech, catalog number 632180) and lentivirus was generated along with the Lenti-X HTX packaging mixture (Clontech, catalog number 631249). The lentivirus was then transduced into DO11.10 cells (National Jewish Health Service). Cells overexpressing OX40 were selected by incubating the cells with 5 μg / ml puromycin (Lifetech Corporation, catalog number A11113803) for approximately 2 weeks, followed by serial dilution of the cloned cell lines. The expression of OX40 in cell lines was tested by flow cytometry using fluorescently labeled OX40-specific antibodies (OX86; ACT35; and polyclonal sheep anti-human OX40, as described in Example 1.1.1 and Table 2). Cell lines expressing human (DO11.10-hOX40), mouse (DO11.10-mOX40), or cynomolgus monkey (DO11.10-cOX40) OX40 were selected, in which all cells showed fluorescence values ​​at least 10-fold higher than those of non-transduced cells in flow cytometry analysis. These cell lines are listed in Table 2 below.

[0468] Table 1: OX40 sequence

[0469]

[0470]

[0471] 1.2 Commercially available OX40 antigen

[0472] Several commercially available OX40 antigens were tested.

[0473] The recombinant His-tagged human OX40 extracellular domain was obtained from Sino Biosciences (catalog number 10481-H08H-50). However, SE-HPLC analysis of this antigen showed that less than 50% of the antigen was in a non-aggregated monomeric form. Therefore, this antigen was not used in subsequent analyses.

[0474] Recombinant human OX40 / human Fc (hOX40-hFc) and recombinant mouse OX40 / human Fc (mOX40-hFc) contain a human IgG1 Fc domain at their C-terminus and are available from Andy Biotech (hOX40-hFc: catalog number 3388-OX-050; mOX40-hFc: catalog number 1256-OX-050) and are internally biotinylated. The biophysical properties of these soluble antigens were characterized by SE-HPLC analysis to ensure the absence of aggregates, and the molecular size was verified by PAGE. The size determined by PAGE indicated that the soluble antigens are dimers, as their estimated molecular weight is twice the predicted molecular weight of the monomeric antigens. The soluble antigens were also analyzed by gel shift assay, which showed a biotinylation degree of over 90%. ELISA and SPR were used to confirm that biotinylated recombinant human (hOX40-hFc) and mouse (mOX40-hFc) OX40 antigens can be bound by OX40-specific antibodies (11D4; ACT35; and OX86), as described in Example 1.1.1 and Table 2 below.

[0475] Table 2: OX40 antigen

[0476]

[0477] Example 2 – Anti-human OX40 Selection and characterization of Fcab

[0478] 2.1 Initial selection of anti-human OX40 antibody

[0479] To select Fcab specific to human OX40 from an initial phage library, recombinant biotinylated soluble dimer human OX40 (hOX40-mFc; see Table 2) and cell-expressed human OX40 (DO11.10-hOX40) were used as antigens. In some selection protocols, in addition to recombinant biotinylated soluble dimer human OX40, cells expressing human OX40 were used to ensure that the selected Fcab could bind to OX40 in its native conformation and bind on the cell surface.

[0480] Six initial phage libraries were generated, showing the CH3 domain (IMGT numbers 1.4–130), including partial random AB loops (residues 14–18 according to the IMGT numbering scheme) and EF loops (residues 92–101 according to the IMGT numbering scheme) within the CH3 domain. One of the six libraries also contained clones with two or four amino acids (encoded by two or four NNK codons) inserted at position 101 of the EF loop within the CH3 domain (residues numbered 101.1–101.4 according to the IMGT numbering scheme).

[0481] All six libraries underwent three rounds of selection using recombinant biotinylated soluble dimer human OX40 (hOX40-mFc; see Table 2). Specifically, the libraries were analyzed in three rounds, with hOX40-mFc captured in streptavidin-coated (Thermo Fisher Scientific, 11206D) or neutral avidin-coated (Thermo Fisher Scientific, 14203 and A2666) immunomagnetic beads (in round 1 and 3) or (in round 2).

[0482] In the first round of selection, all six libraries were further selected using hOX40-mFc, followed by cell-expressed human OX40 (DO11.10-hOX40 in the other two rounds of selection; see Table 2).

[0483] Three rounds of selection were performed on the two libraries using DO11.10-hOX40 antigen-expressing cells.

[0484] Following a third round of selection from six libraries using ELISA, 2133 clones binding to human OX40 were identified. This led to the identification of 32 unique positive binders in HEK Expi293 cells (Fcab cloned into pTT5 vector [National Research Council of Canada], transfected into Expi293F cells [Lifetech, A14524] via ExpiFectamine 293 transfection kit [Lifetech, A14527]), which were subcloned and expressed as soluble Fcab (consisting of truncated hinge [SEQ ID NO: 171], CH2 and CH3 domains).

[0485] The ability of 32 unique Fcab to bind to human OX40 (DO11.10-hOX40) expressed in cells was tested. Of the 32 screened Fcab, 15 showed binding to DO11.10-hOX40 in interacting ECs. 50 The range was 0.1 to 62 nM. Fifteen Fcabs (described in Example 4.4 below) showing binding to DO11.10-hOX40 were tested in a human NF-κB reporter gene assay. In the human NF-κB reporter gene assay, six of the 15 Fcabs showed increased activity upon crosslinking with an anti-human Fc antibody. The three Fcabs, FS20-11, FS20-22, and FS20-31, showed the highest activity levels in this assay, and their activity increased when the Fcabs were crosslinked with anti-human CH2 mAb (cloned MK1A6 (Jefferis et al., 1985 and Jefferis et al., 1992), self-made). These three Fcabs were selected for affinity maturation.

[0486] 2.2 Affinity maturation of anti-human OX40 antibody

[0487] Based on the three Fcabs (FS20-11, FS20-22, and FS20-31) selected in Example 2.1, nine phage-displayed affinity maturation libraries (three for each Fcab) were constructed using ELLA Biotechnology's random primers to achieve an equimolar distribution of amino acids (excluding cysteine). Five residues (residues 14 to 18) in the AB ring or five residues (residues 45.1 to 77) in the CD ring of the CH3 domain were randomized, or by randomizing portions of the EF ring of the CH3 domain (residues 92 to 94 and 97 to 101 for FS20-22 and FS20-31, and residues 97 to 100 and 101.1 to 101.4 for FS20-11 [see Example 2.1 above]). (All residues were numbered according to the IMGT numbering scheme.) Affinity maturation of the fourth Fcab clone, FS20-10, was also attempted, but its progeny exhibited poor binding properties and functional activity compared to the other three Fcab lineages, so no further work was conducted on this lineage.

[0488] Three rounds of selection were performed on affinity-mature libraries using recombinant human biotinylated hOX40-mFc alternately captured on streptavidin-coated (Thermo Fisher Scientific, 11206D) and neutral avidin-coated (Thermo Fisher Scientific, 14203 and A2666) immunomagnetic beads (in rounds 1 and 3) and in round 2. High-affinity binders were identified using decreasing antigen concentrations from 50 nM (round 1) to 10 nM (round 2) to 1 nM (round 3) (for FS20-11 and FS20-22 lineages), or from 100 nM (round 1) to 50 nM (round 2) to 10 nM or 1 nM (round 3) (for FS20-31 lineage). For the two outputs from the third round—the FS20-11 library with randomized residues 14 to 18 in the AB loop and the FS20-22 library with randomized residues 45.1 to 77 in the CD loop—a fourth round of selection was performed using immunomagnetic beads coated with the same antigen and streptavidin at a concentration of 1 nM. As described in Example 2.1 above, 1410 Fcab samples from the outputs of the third and fourth rounds of selection were screened by ELISA for binding to human OX40, and 204 unique positive binders were identified. Subclones were then expressed in HEK Expi293 cells as soluble Fcab samples.

[0489] In the absence and presence of anti-CH2 crosslinking, the dissociation rate of soluble Fcab bound to hOX40-mFc was measured using the anti-human CH2 mAb clone MK1A6 (see Example 2.1) with a Biacore 3000 (GE Healthcare). Fcab with relatively higher dissociation rates were further screened against the relevant parental Fcab for binding to cell-expressed human OX40 and activity in the human T cell activation assay (see Example 5.1 below). All Fcab bound cell-expressed human OX40. The 20Fcab from the FS20-11 lineage, the 10Fcab from the FS20-22 lineage, and the 18Fcab from the FS20-31 lineage, which exhibited the highest activity in the human T cell activation assay, were selected for shuffling as described below.

[0490] For the FS20-11 lineage, two ring reconfiguration libraries were generated. One was generated by reconfiguring 9 AB rings with 10 EF rings and WT CD rings, while the other was generated by reconfiguring AB and EF rings with affinity-mature CD rings. For the FS20-22 lineage, two ring reconfiguration libraries were generated by reconfiguring three CD rings, six EF rings, and either parental AB rings or affinity-mature AB rings. For the FS20-31 lineage, one ring reconfiguration library was generated, containing four AB rings, seven CD rings, and seven EF rings.

[0491] As described in Example 2.1 above, the revised sequence was expressed in HEK Expi293 cells in the form of soluble Fcab, and it was effective against biotinylated hOX40-mFc antigen in Octet QK cells. e Using Dip and Read on the system (PallFortéBio) TM Screening was performed using a streptavidin biosensor (PallFortéBio, 18-5050). Fcabs exhibiting increased dissociation rates compared to parental Fcabs bound to hOX40-mFc were sequenced, yielding 66 unique Fcab sequences from the FS20-11 lineage, 35 unique Fcab sequences from the FS20-22 lineage, and 62 unique Fcab sequences from the FS20-31 lineage. For binding to the hOX40-mFc antigen, the identified unique Fcabs were tested using the anti-human CH2 mAb clone MK1A6 with and without CH2 crosslinking using a Biacore 3000 instrument (General Medical).

[0492] For the FS20-11 lineage, the 18 Fcabs that bind most strongly to hOX40-mFc were selected (determined by yielding the highest response on a Biacore instrument at a fixed concentration) as simulated (4420LALA) mAbs as described below. 2 Formal expression and further characterization. For the FS20-22 lineage, 18 Fcabs were selected to simulate (4420LALA)mAb. 2 The data are formally expressed and further characterized based on the following: the slowest dissociation rate when bound to hOX40-mFc under CH2 crosslinking conditions; the maximum difference in dissociation rate between the uncrosslinked and CH2 crosslinked dissociation rates when bound to hOX40-mFc; and the binding strength with hOX40-mFc as described above. For the FS20-31 series, nine Fcabs with the slowest dissociation rates when bound to hOX40-mFc under CH2 crosslinking conditions and nine Fcabs with the slowest dissociation rates when bound to hOX40-mFc without CH2 crosslinking conditions were selected to simulate (4420LALA) mAbs. 2 Formal expression and further characterization. Since there are several Fcabs in these nine groups, other Fcabs from the FS20-31 lineage that exhibit slow dissociation rates when bound to hOX40-mFc in the absence of CH2 crosslinking were selected to simulate mAbs. 2 The total number of Fcabs from this lineage expressed and further characterized was 18. Using data from a T-cell activation assay, six additional Fcabs from the FS20-22 lineage and eight Fcabs from the FS20-31 lineage were identified, exhibiting high activity in the assay and thus expressed as a simulated (4420LALA) mAb. 2 It is expressed in form and further characterized.

[0493] Example 3 - Anti-mouse OX40 Selection and characterization of Fcab

[0494] 3.1 Initial selection of anti-mouse OX40 Fcab

[0495] Screening was performed using an initial yeast library displaying the human IgG1 CH1 to CH3 domains, containing randomization of the AB loop (residues 11-18 according to the IMGT numbering scheme) and EF loop (residues 92-101 according to the IMGT numbering scheme) within the CH3 domain, with a random insertion of 5 residues between residues 16 and 17 of the AB loop (according to the IMGT numbering scheme). Yeast was incubated with biotinylated recombinant mouse OX40 fused to the human IgG Fc domain (mOX40-hFc; Table 2), and beads coated with streptavidin were sorted by MACS. Subsequently, three rounds of FACS screening were performed using decreasing concentrations of biotinylated mOX40-hFc in the presence of a five-fold excess molar concentration of hFc. Cells were stained with streptavidin-aluminophytic phycocyanin (APC) (BD Biosciences, 349024) or avidin-APC (Mitentech, 130-090-856) and sorted using a FACSAria (BD Biosciences) cell sorter. As previously described in Example 2.1, 182 individual Fcab from the rich cluster were screened based on antigen binding, and two distinct positive binders were subcloned and expressed as soluble Fcab. Fcab binding to mOX40-hFc was characterized by ELISA, and activity was characterized in a mouse NF-κB reporter assay 1 (see Example 4.5 below). In the NF-κB reporter assay, only one Fcab, FS20m-232, was active and showed binding to cells expressing mouse OX40; therefore, this Fcab was screened for affinity maturation.

[0496] 3.2mOX40 Fcab's affinity maturity

[0497] Three phage display affinity maturation libraries were constructed using randomized primers from ELLA Biotechnology with equimolarly distributed amino acids, excluding cysteine, for seven residues in the AB loop of FS20m-232Fcab (residues 15–16.5 according to the IMGT numbering scheme) (library 1), six residues in the CD loop (residues 45.1–78 according to the IMGT numbering scheme) (library 2), or five residues in the EF loop (residues 92–94 and 97–98 according to the IMGT numbering scheme) (library 3).

[0498] Three rounds of screening were performed on an affinity-matured library using recombinant biotinylated mOX40-mFc to optionally capture streptavidin-coated (Thermo Fisher Scientific, 11205D) and neutral avidin-coated (Thermo Fisher Scientific, 14203 and A2666) immunomagnetic beads. High-affinity binders were identified using antigen concentrations decreasing from 50 nM (round 1) to 10 nM (round 2) to 1 nM (round 3). Phage ELISA was used to screen 1655 individual phages from the third round of screening that bound to mOX40-mFc, and 98 unique positive binders, subclones, and soluble Fcab expressed in HEKExpi293 cells were identified, as described in Example 2.1. Fcab were further screened for cell binding and activity in mouse NF-κB reporter gene assay 2 (see Example 4.6 below). The most active Fcab was selected for loop shuffling.

[0499] A loop shuffle library was generated containing 27 CD loops (all 26 unique sequences and WT sequences identified by affinity maturation), shuffled with 37 EF loops (sequences exhibiting optimal binding to mouse OX40 in phage ELISA and WT sequences), where all shuffled clones contained the AB loop of FS20m-232Fcab. As described above, 750 shuffled sequences were expressed in HEK Expi293 cells as soluble Fcabs (containing truncated hinges). This was achieved via Octet QK... e The binding of Fcab to biotinylated mOX40-mFc (Table 2) was measured using the Dip and Read streptavidin biosensors (PallFortéBio, 18-5050) on the system (PallFortéBio) to improve desorption rates and screen HEK supernatants containing Fcab. As described above, 11 unique AB-ring randomized Fcab subclones and 60 unique EF-ring randomized Fcab subclones were expressed as soluble Fcab in HEKExpi293 cells. These Fcabs were further screened for cell binding and activity in mouse T cell activation assays (see Example 5.2 below) along with 43 Fcab shuffles with the slowest dissociation rates. When crosslinked with the anti-human CH2 mAb clone MK1A6, FS20m-232-91Fcab exhibits the slowest dissociation rate when bound to biotinylated mOX40-mFc and the highest activity in mouse T cell activation assays. Therefore, it was selected as the mouse (alternative) Fcab for subsequent experiments.

[0500] Example 4 – Simulated mAb 2 Construction, expression and representation

[0501] 4.1 Simulating mAb 2 Construction and expression

[0502] Preparation of “mimetic” mAbs containing the identified anti-human OX40 and anti-mouse OX40 Fcab 2 In order to use mAb 2 Forms characterize these Fcabs. These pattern mAbs 2 The anti-OX40 Fcab antibody was prepared by replacing the CH3 domain of the anti-FITC and anti-HEL antibodies with the CH3 domain of the anti-OX40 Fcab antibody in the XhoI and BamHI sites within the unmodified CH3 domain of human IgG1, using the variable regions of anti-OX40 Fcab and anti-FITC antibody 4420 (Bedzyk et al., 1989; Bedzyk et al., 1990) in the human IgG1 backbone (see SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 156) or anti-chicken oocyte lysozyme (HEL) antibody D1.3 in the human IgG1 backbone (Braden et al., 1996) (see SEQ ID NO: 169 and 157 for details). The simulated mAb was prepared by replacing the CH3 domain of the anti-OX40 Fcab antibody with the CH3 domain of the anti-FITC and anti-HEL antibodies within the XhoI and BamHI sites in the unmodified CH3 domain sequence of human IgG1. 2 These mAbs contain either a light chain (SEQ ID NO: 156) anti-FITC mAb 4420 or an anti-HEL mAb D1.3 (SEQ ID NO: 157), and also contain an LALA mutation (Hezareh et al., 2001 and Bruhns et al., 2009) in the CH2 domain of the heavy chain to reduce Fc-γ receptor interactions and potential Fc-γ receptor-induced crosslinking. The pattern mAbs mentioned in these examples... 2 and mAb 2 The LALA mutation exists, and it is indicated by the suffix "AA" at the end of the Fcab part of its clone name.

[0503] By transiently expressing a mimicry mAb in HEK293-6E cells 2 The protein was purified using a mAb Select SuRe protein A column.

[0504] 4.2 Simulated mAb 2 The binding affinity of the anti-human OX40 form of Fcab to human and cynomolgus monkey OX40 expressed in cells.

[0505] Simulation (4420LALA)mAb 2 The anti-human OX40 Fcab form was measured by flow cytometry with cells expressing human or cynomolgus monkey OX40 (DO11.10 cells expressing human [DO11.10-hOX40] or cynomolgus monkey OX40 [DO11.10-cOX40]; see Table 2). Non-specific binding was also assessed by flow cytometry by testing binding to HEK cells that do not express OX40.

[0506] Simulated (4420 LALA) mAbs were prepared in triplicate in 1xDPBS (Gibco, 14190-094). 2 And control mAb dilution (2x final concentration). Prepare DO11.10-hOX40 or DO11.10-cOX40 or HEK cell suspensions in PBS + 2% BSA (Sigma, A7906) and seed 4 x 10 cells at 50 L / well in V-bottom 96-well plates (Costar, 3897). 6 Cells / ml. Add 50 μl of simulated (4420 LALA) mAb. 2 Alternatively, add the control mAb (anti-human OX40 mAb, 11D4) dilution to the wells containing cells (final volume 100 μl) and incubate at 4°C for 1 hour. Wash the plate, then dilute 100 μl / well of the secondary antibody (anti-human Fc-488 antibody, Jackson Immunological Research, 109-546-098) 1:1000 in PBS + 2% BSA, and incubate in the dark at 4°C for 30 minutes. Wash the plate and resuspend it at a concentration of 1 μg / ml in 100 μl of PBS containing DAPI (Biotium, catalog number 40043). Read the plate using a Canto II flow cytometer (BD Biosciences). Exclude dead cells and measure fluorescence in the FITC channels (488nm / 530 / 30). Fit the data using the log (agonist) ratio in GraphPad Prism software.

[0507] Fcab (all simulated [4420LALA]mAb) 2 The anti-human OX40 mAb (11D4), a positive control and a form of test, is located in the human IgG1 backbone and contains an LALA mutation in the CH2 domain of the heavy chain, binding to human OX40 with a certain affinity. In further characterization of the selected clones in the form of a simulated mAb2 as described in Example 2.2, 14 Fcabs (three from the FS20-11 lineage, five from the FS20-22 lineage, and six from the FS20-31 lineage) exhibited significantly higher affinity for human OX40 compared to other Fcabs. Table 3 lists the binding affinity of these 14 Fcab clones to cellularly expressed human and cynomolgus monkey OX40.

[0508] Fcabs from the FS20-22 and FS20-31 lineages bind to macaque OX40 with an affinity comparable to human OX40. This may be advantageous because, after demonstrating appropriate activation of cynomolgus OX40 in vitro, these Fcabs can be used for toxicological studies in cynomolgus monkeys, with results that can predict toxicological effects in humans. Fcabs from the FS20-11 lineage also bind to cynomolgus OX40, but with lower affinity, making them unsuitable for testing in cynomolgus monkeys. Neither the tested Fcabs nor the positive control mAb showed any nonspecific binding to HEK cells.

[0509] Table 3: Simulated (4420LALA) mAb 2 The binding affinity of the anti-OX40 form of Fcab to human or cynomolgus monkey OX40 expressed in cells.

[0510]

[0511]

[0512] 4.3 Simulation of mAb 2 The binding affinity of the anti-mouse OX40 form of Fcab to mouse OX40 expressed in cells.

[0513] Flow cytometry was used to measure simulated mAbs. 2 The affinity of the anti-mouse OX40 Fcab in the form (4420LALA) for mouse OX40 expressed in cells (DO11.10-mOX40; see Table 2). Non-specific binding was also assessed by flow cytometry by testing binding to HEK cells that do not express OX40.

[0514] Simulated (4420LALA) mAb was prepared in 1xDPBS (Gibco, 14190-094). 2 And control mAb dilution (2x final concentration). Prepare DO11.10 mOX40 or HEK cell suspensions in PBS + 2% BSA (Sigma, A7906) and seed 4 x 10 cells at 50 L / well in V-bottom 96-well plates (Costar, 3897). 6 Cells / ml. Add 50 μl of simulated (4420 LALA) mAb. 2Alternatively, control mAb (anti-mouse OX40 mAb, OX86 dilution) was added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plate was washed, and then 100 μl / well of secondary antibody (anti-human Fc-488 antibody, Jackson Immunological Research, 109-546-098) was diluted 1:1000 in PBS + 2% BSA, followed by incubation in the dark at 4°C for 30 minutes. The plate was washed and resuspended at 1 μg / ml in 100 μl of PBS containing DAPI (Biotium, 40043). The plate was read using a Canto II flow cytometer (BD Biosciences). Dead cells were excluded, and fluorescence in the FITC channels (488 nm / 530 / 30) was measured. Data were fitted using the log(agonist) ratio response in GraphPad Prism software.

[0515] Using simulation (4420LALA)mAb 2 The Fcab and positive control, in the human IgG1 backbone, specifically bound to mouse OX40 mAb and OX86 (SEQ ID NO: 175 and 176) with the LALA mutation, with the affinity listed in Table 4. The affinity of the anti-mouse OX40 Fcab to cell-expressed mouse OX40 was comparable to that of the anti-mouse OX40 positive control mAb. Neither the tested Fcab nor the positive control mAb showed any nonspecific binding to HEK cells.

[0516] Table 4: Simulated (4420LALA) mAb 2 The binding affinity of the anti-mouse OX40 form of Fcab to mouse OX40 expressed in cells.

[0517] <![CDATA[Simulated (4420LALA) mAb 2 / mAb]]> <![CDATA[Bound to DO11.10-mOX40 EC 50 (nM)]]> mFS20-232-91AA / 4420 1.006 G1AA / OX86 3.099

[0518] 4.4 Human NF-κB Reporting Experiment

[0519] A assay is needed to simply and rapidly isolate the OX40 agonist activity of Fcab from the initial selection process, so as to quickly determine which Fcab to continue with. As described below, the development of such an assay is technically challenging.

[0520] The binding of OX40 to its ligands leads to OX40 clustering and activation of the NF-κB signaling pathway (Arch and Thompson, 1998). Anti-OX40 Fcab with agonist activity mimics the OX40 ligand by inducing OX40 clustering and emitting signals. Therefore, a detection method was designed to assess the activity of anti-OX40 Fcab by detecting the activation of the NF-κB signaling pathway following OX40 clustering.

[0521] Flp-In T-REx 293HEK cell line (LifeTech, R780-07) was transduced with Qiagen Cignal Lenti NFkBReporter (luc) (Qiagen catalog number 336851) lentivirus, which contains an NF-κB-sensitive promoter controlling luciferase expression. Cells were then selected by culturing them for approximately 2 weeks in the presence of 5 μg / ml puromycin (LifeTech catalog number A11113803), and the cell line was cloned using a serial dilution method. The presence of the luciferase reporter construct was tested according to the manufacturer's instructions by incubating cells with 10 ng / ml TNFα (Andy Biotech catalog number 210-TA-005) for 24 hours and measuring luminescence after 15 minutes of treatment with the Promega Bio-Glo luciferase assay system (Promega catalog number G7941). Luminescence was measured using a BioTek microplate reader with Gen5 software (integration time 0.5 seconds).

[0522] Human OX40 was subcloned into the vector pcDNA5FRT (Life Science Catalog No. V6010-20) using EcoRI-HF and NotI-HF restriction enzymes. The vector was then transformed into the Flp-In T-REx 293HEK cell line (Life Science, R780-07) using Lipofectamine 2000 (Life Science, 11668-019). The transformed Flp-In T-REx 293 cells (designated HEK.FRT.luc cell line) were grown in DMEM (Life Science, 61965-026) containing 10% FBS (Life Science, 10270-1-6), 100 μg / ml hygromycin B (Merford Laboratories, Z2475), and 15 μg / ml blasticidin (Merford Laboratories, B1105) for 3–4 weeks until stable transformed cell colonies appeared. These colonies were amplified in the presence of 1 μg / ml deoxyoxygenated oxytetracycline (Sigma, D9891), and the expression of human OX40 was detected by flow cytometry using anti-OX40 mAb ACT35 (Thermo Fisher Scientific, catalog number 17-1347-42).

[0523] In the newly constructed HEK.FRT.luc cell line, expression of human OX40 unexpectedly led to constitutive activation of the NF-κB signaling pathway and high levels of luciferase expression. Therefore, differential activation of this signaling pathway could not be detected by OX40 agonist antibodies. To reduce the constitutive activation of the NF-κB signaling pathway, an attempt was made to reduce OX40 expression by replacing the highly expressed CMV promoter with another promoter (from the Invivogen PromTest plasmid). However, contrary to expectations, this strategy did not alter OX40 expression levels, and the NF-κB signaling pathway remained constitutively active.

[0524] Another attempt was made to reduce the constitutive activity of the NF-κB signaling pathway by fusing the extracellular domain of human OX40 with the intracellular domain of one of the members of the TNFR family (CD40, GITR, TNFRII, CD27, CD30, CD137, and HVEM) (Song et al., 2014), and the resulting chimeric protein was expressed in the HEK.FRT.luc cell line as described above. OX40 surface expression was determined by flow cytometry using an OX40-specific antibody. Surprisingly, HEK.FRT.luc cells expressing the hEK40-hCD137 chimera showed reduced background activation of the NF-κB signaling pathway and a concentration-dependent response to OX40 agonist antibodies. Other chimeric receptors tested did not lead to a reduction in the constitutive activation of the NF-κB signaling pathway. Only the cell line expressing the hOX40-hCD137 chimera (HEK.FRT.luc.hOX40hCD137) can therefore be used to test and grade anti-human OX40 Fcab identified after initial selection in terms of its agonistic activity against human OX40 Fcab, and for this purpose.

[0525] As described in Example 2.1, anti-OX40 Fcab was expressed as a soluble protein, and OX40 agonist activity was tested as follows. HEK.FRT.luc.hOX40hCD137 cells were cultured at 1x10⁻¹ under 37°C and 5% CO₂. 5Cells were seeded at a concentration per well in 96-well white transparent flat plates and incubated overnight. On the second day, 2 μM dilutions of each Fcab to be tested or control mAb (11D4 in human IgG1 form) were prepared in DPBS (Gibco) and further diluted 1:10 in reporter cell culture medium (DMEM (Gibco catalog number 61965-026)); 10% FCS (Gibco catalog number 10270-106); 1x penicillin-streptomycin (Gibco catalog number 15140-122); hygromycin B 100 μg / ml (Merford Laboratories, Z2475); blastomycin 15 μg / ml (Merford Laboratories, B1105); puromycin 5 μg / ml (Life Technology catalog number A11113803) and doxycycline 1 μg / ml (Sigma catalog number D9891) (30 μl + 270 μl) to obtain 200 nM dilutions. When needed, the cross-linking agent (anti-human CH2mAb clone MK1A6) was added to the wells at a 1:1 molar ratio to the test Fcab or control mAb. In a 96-well plate, serial dilutions of the Fcab or control mAb were prepared with or without the cross-linking agent, and 100 μl of each dilution was added to the cells on the culture plate.

[0526] Cells were incubated at 37°C and 5% CO2 for 24 hours, and luminescence was measured after 15 minutes of treatment with a Promega Bio-Glo luciferase assay system (Promega catalog number G7941) according to the manufacturer's instructions. Fluorescence was measured using a BioTek microplate reader with Gen5 software (integration time 0.5 seconds) as a measure of the luciferase produced in response to activation of the NF-κB signaling pathway, which induces the aggregation of human OX40 via the binding of cross-linked agonist Fcab or positive control monoclonal antibody to OX40. Fluorescence values ​​were plotted relative to the logarithmic concentration of Fcab / mAb, and the resulting curve was fitted to the logarithmic (agonist) response equation using GraphPad Prism.

[0527] 4.5 Mouse NF-κB Reporter Experiment 1

[0528] To enable rapid and simple testing of the activity of OX40 agonists using isolated anti-mouse OX40 Fcab, a mouse NF-κB reporter assay was developed.

[0529] Initially, a method similar to the human NF-κB reporter gene assay described in Example 4.4 was followed, using a mouse OX40 sequence. However, similar problems were encountered, leading to constitutive activation of the NF-κB signaling pathway.

[0530] To reduce constitutive activation of the NF-κB signaling pathway, the extracellular domain of mouse OX40 was fused with the intracellular domain of the human CD40 receptor (Song et al., 2014), and expressed in the HEK.FRT.luc cell line described above. OX40 surface expression was determined by flow cytometry using an OX40-specific antibody to test the presence of the chimeric receptor. Compared to cells expressing full-length mouse OX40, the chimeric HEK.FRT.luc cells expressing the mOX40-hCD40 receptor (HEK.FRT.luc.mOX40hCD40 cells) showed reduced background activation of the NF-κB signaling pathway and exhibited a concentration-dependent response to the OX40 agonist antibody. Therefore, this cell line was selected to test the OX40 agonist activity against the anti-mouse OX40Fcab identified after initial selection. The assay protocol is essentially as described in Example 4.4, but these HEK.FRT.luc.mOX40hCD40 cells were used to test mouse OX40 binding to Fcab, and OX86 in the form of human IgG1 was used as a positive control.

[0531] 4.6 Mouse NF-κB Reporter Experiment 2

[0532] To develop an improved mouse OX40 NF-κB reporter assay, the strategy used for the human OX40 NF-κB reporter assay was employed. As described in Example 4.4, the extracellular domain of mouse OX40 was fused with the intracellular domain of the human CD137 receptor and expressed in the HEK.FRT.luc cell line. OX40 surface expression was determined by flow cytometry using an OX40-specific antibody to detect the presence of the chimeric receptor. Compared to the background activation observed in mouse NF-κB reporter assay 1, HEK.FRT.luc cells expressing the chimeric mOX40-hCD137 receptor (HEK.FRT.luc.mOX40hCD137 cells) showed reduced background activation of the NF-κB signaling pathway and exhibited a concentration-dependent response to the anti-mouse OX40 agonist antibody. Therefore, this cell line allows for improved testing and grading of anti-mouse OX40 Fcab and can be used to test and grade anti-mouse OX40 Fcab identified after affinity maturation of mouse OX40 agonist activity. The assay protocol using these cells is essentially as described in Example 4.5.

[0533] 4.7 Binding affinity of Fcab to human and cynomolgus monkey OX40

[0534] Anti-human OX40 Fcabs (in OX40 / EGFR mAb) was measured by SPR. 2 (Form) Affinity to human and cynomolgus monkey OX40. Due to the presence of mAb or mAb as the antigen passes over the flow cell.2 Molecular orientation affects binding kinetics, therefore an attempt was made to measure mAb affinity by adjusting the orientation of mAb molecules during Fcab affinity measurements. 2 The Fab portion is positioned on the bonding surface far from the Biacore chip, and mAb is used to measure the affinity of the Fab. 2 The Fab portion is positioned far from the bonding surface of the Biacore chip. To achieve this, a target acquisition method is used to position the mAb... 2 The molecules are oriented as needed. EGFR is used to capture OX40 / EGFR mAb. 2 The antigen (see Table 5) is used in the structure with the variable region of anti-human OX40 Fcabs and anti-EGFR antibody cetuximab (US Patent No. 6217866; indicated by “Cx” in Table 5), and the mimic mAb described in Example 4.1. 2 Constructed in the same manner. To compare the affinity of anti-OX40 Fcab with that of anti-OX40 mAb, EGFR-bound Fcab (patent publication number WO 2018 / 015448 A1) was paired with the Fab of anti-OX40 mAb 11D4 (EP2242771 B1), and EGFR was used to capture the resulting EGFR / OX40 mAb. 2 The antigen. This causes both Fcab and the positive control mAb to be away from the Biacore chip surface and oriented towards the OX40 antigen flowing above the Biacore chip.

[0535] Following the manufacturer's instructions for the BIAcore T200 instrument, EGFR (AndyBio catalog number 344-ER) was immobilized on the SCM5 series chip (General Medical, BR-1005-30) using amine coupling (GE Healthcare, BR-1000-50) to a surface density of 5000 RU. mAbs diluted in HBS-EP+ buffer (GE catalog number BR100669) were injected at a rate of 10 μl / min. 2 A 1 μg / ml solution was applied for 40 seconds, and mAb was added. 2 Samples were captured to approximately 150 RU. Different concentrations of OX40 antigen (homemade unbiotinylated hOX40-mFc or unbiotinylated cOX40-mFc; see Table 2) in HBS-EP+ buffer (GE catalog number BR100669) were then flowed over the chip at 70 μl / min for 3 min, followed by dissociation for 6 min. After each antigen concentration, the chip was regenerated by injecting 30 mM sodium hydroxide (NaOH) at a flow rate of 30 μl / min for 10 seconds. For reference subtraction purposes, HBS-EP+ buffer was injected before the highest concentration of antigen and after the lowest concentration.

[0536] The binding kinetics were fitted using a 1:1 Langmuir model to generate the equilibrium binding constant (K) for each sample. D Data analysis was performed using BiaEvaluation software version 3.2. The results are shown in Table 5.

[0537] Table 5: Binding affinity to human and cynomolgus monkey OX40 determined by SPR

[0538]

[0539]

[0540] OX40 Fcab was observed to have a certain affinity for human OX40 (see Table 5). The binding affinity of Fcab from the FS20-11 lineage to cynomolgus monkey OX40 was below the detection threshold, indicating that these Fcab have a low affinity for cynomolgus monkey OX40, as observed in the cell binding assay described in Example 4.2 above. The affinity of anti-human OX40 Fcab from the FS20-22 and FS20-31 lineages for cynomolgus monkey OX40 was comparable to that for human OX40.

[0541] 4.8 Binding affinity of anti-mouse OX40 Fcab to mouse OX40

[0542] The same target capture and EGFR immobilization method as described in Example 4.7 was used, but the difference was that mAbs diluted in HBS-EP+ buffer (GE catalog number BR100669) were injected at a rate of 10 μl / min. 2 Infuse the 5 nM solution for 1 minute, then add mAb. 2 Samples were captured to approximately 200 RU, and the affinity of FS20m-232-91 anti-mouse OX40 Fcab for mouse OX40 was measured by SPR. OX40 / EGFR mAbs were constructed using the variable regions of FS20m-232-91 anti-mouse OX40 Fcab (with the LALA mutation) and the anti-EGFR antibody cetuximab (patent US 6,217,866B1; indicated by Cx in Table 6). 2Subsequently, OX40 antigen (self-made mOX40-mFc; see Table 2) at different concentrations in HBS-EP+ buffer (GE catalog number BR100669) was flowed over the chip at 70 μl / min for 5 minutes, followed by dissociation for 10 minutes. After each antigen concentration, the chip was regenerated by injecting 30 mM sodium hydroxide (NaOH) at a flow rate of 30 μl / min for 10 seconds. For reference subtraction purposes, HBS-EP+ buffer was injected before the highest concentration of antigen and after the lowest concentration of antigen.

[0543] The binding kinetics were fitted using a 1:1 Langmuir model to generate the equilibrium binding constant (K) of the sample. D Data analysis was performed using BiaEvaluation software version 3.2. Table 6 shows that the affinity of anti-mouse OX40 Fcab FS20m-232-91 is approximately 0.7 nM.

[0544] Table 6: mAb determination by SPR 2 Binding affinity to mouse OX40

[0545] <![CDATA[mAb 2 ]]> KD(nM) FS20m-232-91AA / Cx 0.681

[0546] 4.9 Specificity of anti-human OX40 antibody

[0547] To simulate mAb 2The specificity of anti-human OX40 Fcab against human OX40 was tested formally, and the binding of Fcab to other human TNFR family receptors (CD40, TNFRI, TNFRII, NGFR, and CD137) was tested using SPR in a Biacore T200. Human CD40, TNFRI, TNFRII, NGFR, and CD137 receptors (all from AndyBio) were coated to approximately 1000 RU in a Biacore CM5 chip (General Medical, catalog number 29149603) using an amine conjugation kit (amine conjugation kit, GE Healthcare, BR-1000-50). Dilutes of anti-human OX40 Fcab in a mimicry mAb form (see Table 7) starting at 1 μM were prepared in HBS-EP+ buffer (BR100669) and injected at a rate of 30 μl / min for 3 minutes, followed by dissociation in buffer for 4 minutes. The chip was regenerated by injecting 10 mM glycine at pH 2.5 at a rate of 30 μl / min for 12 s. Antibodies specific to different TNFR family members were used as positive controls to validate the Biacore chip coating. Data were subjected to double reference subtraction and analyzed using BIAevaluation 3.2 software. The Fcab listed in Table 7 did not bind to any of the tested TNFR family receptors, indicating its specificity for human OX40.

[0548] Table 7: Simulation of mAb through SPR testing 2 The specificity of the form of anti-human OX40 antibody.

[0549] <![CDATA[Tested mock mAb 2 > FS20-11-127AA / 4420 FS20-11-131AA / 4420 FS20-11-134AA / 4420 FS20-22-38AA / 4420 FS20-22-41AA / 4420 FS20-22-47AA / 4420 FS20-22-49AA / 4420 FS20-22-85AA / 4420 FS20-31-58AA / 4420 FS20-31-66AA / 4420 FS20-31-94AA / 4420 FS20-31-102AA / 4420 FS20-31-108AA / 4420 FS20-31-115AA / 4420

[0550] 4.10 Specificity of anti-mouse OX40 Fcab

[0551] By testing binding to other mouse TNFR family receptors (mouse CD40, TNFRI, TNFRII, NGFR, and CD137 receptors), simulated (HEL D1.3 LALA) mAbs were measured using SPR in a Biacore T200 instrument. 2 Specificity of the anti-mouse OX40Fcab (FS20m-232-91) form was determined. Amine conjugation (Amine Conjugation Kit, GE Healthcare, BR-1000-50) was used to coat mouse CD40, TNFRI, TNFRII, NGFR, and CD137 receptors (all obtained from AndyBio) onto a Biacore CM5 chip (GE Healthcare, catalog number 29149603) to approximately 1000 RU. Simulated mAbs starting from 1 μM were prepared in HBS-EP+ buffer (BR100669). 2(FS20m-232-91AA / HEL D1.3) dilution buffer was injected at a rate of 30 μl / min for 3 minutes, followed by dissociation in buffer for 6 minutes. The chip was regenerated by injecting 10 mM glycine at pH 2.5 at a rate of 30 μl / min for 20 seconds. Antibodies specific to different TNFR family members were used as positive controls to validate the Biacore chip coating.

[0552] The data underwent double reference subtraction and was analyzed using BIAevaluation 3.2 software. Simulation (HELD1.3 LALA) mAb 2 The anti-mouse OX40 form of Fcab FS20m-232-91 does not bind to any of the tested relevant TNFR family members, demonstrating the specificity of this Fcab for mouse OX40.

[0553] Examples 5 to 8 – Functional activity of Fcab inducing OX40 activity in vitro and in vivo through different cross-linking mechanisms

[0554] In previous embodiments, Fcab that binds to human OX40 or mouse OX40 was identified. These simulated (4420LALA) mAbs were subsequently tested in NF-κB assays. 2 The ability of Fcab in its form to activate OX40 clustering and signal transduction. The following examples demonstrate the ability of mimicking mAb when crosslinking to another target via its Fc region or via Fab binding. 2 and mAb 2 The ability of Fcab in vitro and in vivo to activate OX40. Because Fcab can induce mAbs containing multiple Fabs... 2 The OX40 clusters are activated in various forms, thus they hold promise for treating a wide range of different diseases.

[0555] Example 5 – Simulated mAb 2 Anti-OX40 Fcab activates OX40 in vitro and in vivo.

[0556] Activated T cells express OX40 on their cell surface. Binding of the trimerized OX40 ligand to OX40 leads to receptor trimerization. Since OX40 ligands are expressed in clusters on the cell surface of antigen-presenting cells, the interaction between OX40 ligands and OX40 results in OX40 clustering, which is crucial for OX40 signaling and further T cell activation. Antibodies activating OX40 must mimic the clustering activity of the OX40 ligand. For monospecific anti-OX40 antibodies, the Fcγ receptor binds to and cross-links the Fc domain of the antibody, leading to OX40 clustering.

[0557] In the presence of the cross-linking agent, the ability of the simulated (4420) LALA form of anti-human OX40 and anti-mouse OX40 Fcab described in Example 4 to activate OX40 expressed on T cells was tested in a T cell activation assay. Simulated (HEL D1.3) mAb was also used. 2 The ability of FS20m-232-91 to inhibit tumor growth in vivo against mouse OX40Fcab was tested in a form (see Example 4.1) by activating tumor-infiltrating lymphocytes expressing OX40 in a CT26 syngeneic mouse tumor growth model.

[0558] 5.1 Using simulated mAb 2 Assay for human T cell activation using anti-human OX40 Fcab

[0559] Activated human T cells express human OX40 on their cell surface. The aggregation of OX40 is known to be crucial for inducing receptor signaling and further T cell activation. In a simulated (4420LALA) mAb... 2 In the presence of mAb molecules, OX40 clustering and signaling were assessed using a T-cell activation assay, as detailed in Table 8 below. T-cell activation was detected by measuring IL-2 release.

[0560] 5.1.1 Isolation and activation of human T cells

[0561] To isolate T cells, peripheral blood mononuclear cells (PBMCs), a byproduct of platelet donation, were isolated from leukocyte-depleted cones (NHS Blood and Transplant Services). Briefly, the contents of the leukocyte cones were washed with PBS and centrifuged at density using a Ficoll gradient (GE Life Sciences Catalog No. 17144002). PBMCs were isolated by centrifugation and recovery of cells that did not pass through the Ficoll gradient. The PBMCs were further washed with PBS, and the remaining red blood cells were lysed by adding 10 ml of erythrocyte lysis buffer (eBioSciences) according to the manufacturer's instructions. T cells were then isolated from the PBMCs present in the eluent using the Pan T Cell Isolation Kit II (Mitentech Biotechnology) according to the manufacturer's instructions.

[0562] Human T-activator CD3 / CD28 immunomagnetic beads (Lifetech 11452D) were vortexed for resuspension. The beads were washed twice with T-cell culture medium (RPMI medium (Lifetech) containing 10% FBS (Lifetech), 1x penicillin-streptomycin (Lifetech), sodium pyruvate (Gibco), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol (Gibco)).

[0563] In a T-25 flask (Sigma), the required number of T cells is 1.0 x 10⁻⁶. 6 T cells were eluted with human T-activator CD3 / CD28 immunomagnetic beads at a cell-to-bead ratio of 2:1 in T cell culture medium and incubated overnight at 37°C in 5% CO2 to activate T cells. Activated T cells were eluted from the immunomagnetic beads and incubated at 2.0 × 10⁻⁶ cells / ml. 6 Cells were resuspended in T cell culture medium at a concentration of 2.5 μg / ml. The bottom of 96-well plates was coated with 2.5 μg / ml anti-human CD3 antibody (Andy Bioclone UHCT1) diluted in PBS and incubated at 37°C, 5% CO2 for 2 h, followed by washing twice with PBS. Activated T cells were then sputtered at 2 x 10⁻⁶ cells / ml. 5 Cell / well concentration added to the plate. 2 μM simulated (4420 LAL) mAb 2 Molecules, dilutions of the positive control 11D4 mAb (in the human IgG1 backbone and containing the LALA mutation) and the negative control 4420 mAb (in the human IgG1 backbone and containing the LALA mutation), were prepared in DPBS (Gibco) and subsequently diluted 1:10 in T cell culture medium (30 μl + 270 μl) to obtain a 200 nM dilution. The anti-human CH2 mAb clone MK1A6 was cross-linked with the positive control mAb via Fc or FITC-glucan (Sigma) and bound via Fab to mimic the (4420LALA) mAb. 2 Formal crosslinking of Fcab (see Table 8), compared with simulated mAb 2 Or a positive control mAb was added to the well at a 1:1 molar ratio. In a 96-well plate, (1) a positive control or simulated mAb with the relevant crosslinking agent was prepared. 2 Or (2) positive control or simulated mAb 2 A series of dilutions. 100 μl of simulated mAb was diluted... 2 / Positive control mAb or diluted simulated mAb 2 Alternatively, a positive control mAb and a cross-linking agent can be added to activated T cells on a plate.

[0564] T cells were incubated at 37°C and 5% CO2 for 72 hours. The supernatant was collected and analyzed using a human IL-2 ELISA kit (eBioScience or AndyBio) according to the manufacturer's instructions. The plate was read at 450 nm using a BioTek microplate reader equipped with Gen5 software. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (correction). A standard curve for cytokine concentrations was calculated based on a four-parameter logarithmic curve fit (Gen5 software, BioTek). The concentrations of human IL-2 (hIL-2) were compared with a simulated mAb. 2Logarithmic concentrations of the positive control mAb were plotted, and the resulting curves were fitted to the logarithmic (agonist) versus response equation in GraphPad Prism. Table 8 shows the results with and without the simulated mAb. 2 EC was observed in the T cell activation assay in the case of a positive control mAb and the test. 50 The maximum response value and IL-2 release. Figure 2 Representative plots of IL-2 release from T cell activation assays of representative clones from each lineage (lineages FS20-11, FS20-22, and FS20-31) are shown.

[0565] Table 8: Using Simulated (4420LALA) mAb 2 Forms of anti-human OX40 Fcab activate T cells

[0566]

[0567]

[0568] *In the uncrosslinked case, these simulated mAbs 2 The control mAb showed no activity in the T cell activation assay.

[0569] As shown in Table 8, the simulated (4420LALA)mAb 2 When anti-human OX40 Fcab forms are cross-linked with the Fab target (FITC-glucan), they exhibit a range of activities. In the presence of anti-CD3 antibody, all Fcab forms possess the ability to co-stimulate T cells and induce human IL2 production.

[0570] The FS20-11 lineage of Fcabs only co-stimulates T cells upon cross-linking and is inactive in the absence of cross-linking. This activity, present only upon cross-linking, means that when administered to patients, these Fcabs are expected to activate T cells only in the presence of the Fab target or other cross-linking mechanisms. FS20-11-131 exhibits a lower EC50 compared to other clones in this lineage. 50 However, due to the low cross-reactivity between the FS20-11 lineage clone and cynomolgus monkey OX40, further improvements in affinity for cynomolgus monkey OX40 are needed in toxicological studies of this species.

[0571] Fcabs from the FS20-22 and FS20-31 lineages exhibited activity in both cross-linked and uncross-linked states. Specifically, Fcabs from these lineages were active in the absence of a cross-linking agent, which increased upon cross-linking. Due to the high cross-reactivity of these Fcabs with cynomolgus monkey OX40 (compared to human OX40), toxicological studies in this species are feasible. EC50 data were obtained for cross-linking agonist activity of clones FS20-22-41, FS20-22-47, FS20-22-49, and FS20-22-85 from the FS20-22 lineage. 50 The agonist activity was lowest in this lineage, therefore cloning from this lineage was the preferred method. Among them, clone FS20-22-49 showed the greatest increase in agonist activity upon crosslinking and had the lowest EC50 value for agonist activity in the presence of crosslinking. 50 Therefore, these are the preferred clones. Among the clones of the FS20-31 lineage, clones FS20-31-108, FS20-31-108, and FS20-31-115 showed the highest maximum response, while also exhibiting low EC50. 50 It has a value, and therefore has good efficacy.

[0572] The fact that clones in the FS20-22 and FS20-31 lineages exhibit limited T-cell activation in the absence of crosslinking is not expected to pose a safety risk, as molecules targeting OX40 have not shown adverse effects clinically. Instead, the limited T-cell activation activity of these clones in the absence of crosslinking is thought to be potentially beneficial, as they may activate OX40-expressing memory T cells without crosslinking, thereby inducing their proliferation and thus generating a larger T-cell population, which could subsequently be further activated by OX40 clustering driven by binding to crosslinked anti-OX40 Fcab.

[0573] 5.2 Using simulated mAb 2 Assay of mouse T cell activation using anti-mouse OX40 Fcab

[0574] To assess the activity of Fcab bound to mouse OX40, the simulated (4420LALA) mAbs are detailed in Table 9 below. 2 In the presence of mAb molecules, mouse OX40 clustering and signal transduction were assessed using a T-cell activation assay. As in the human assay, T-cell activation was detected by measuring IL-2 release.

[0575] 5.2.1 Isolation and activation of mouse T cells

[0576] To isolate T cells, spleens were collected from 4-8 week old female Balb / C mice (Charles River). Mice were humanely euthanized and the spleens were dissected. Spleen cells were isolated by pushing the spleen into a 5 ml plastic syringe and passing it through a 70 μm cell filter (Corning). The cell filter was washed 10 times with 1 ml Dulbecco phosphate-buffered saline (DPBS) (Gibco), and the eluent was collected in a 50 ml tube. Red blood cells present in the eluent were lysed by adding 10 ml of erythrocyte lysis buffer (eBioscience) according to the manufacturer's instructions. T cells were isolated from the spleen cells present in the eluent using the pan T Cell Isolation Kit II (Mitentech Biotechnology) according to the manufacturer's instructions.

[0577] Mouse T-activator CD3 / CD28 immunomagnetic beads (Lifetech, 11452D) were vortex-resuspended. The beads were washed twice with T-cell culture medium (RPMI medium (Lifetech) containing 10% FBS (Lifetech), 1x penicillin-streptomycin (Lifetech), sodium pyruvate (Gibco), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol (Gibco)).

[0578] The required concentration of T cells in the T cell culture medium is 1.0 x 10⁻⁶. 6 Cells / ml, T cells were stimulated in T-25 flasks (Sigma) with eluted mouse T-activator CD3 / CD28 immunomagnetic beads at a cell-to-bead ratio of 2:1 and incubated overnight at 37°C and 5% CO2 to activate T cells.

[0579] After overnight incubation, activated T cells were eluted from the immunomagnetic beads and then... 6 The cells were resuspended in T cell culture medium at a concentration of [cells / ml]. 96-well plates were coated with anti-mouse CD3 antibody (Biolegend clone 145-2C11) diluted in PBS for 2 hours at 37°C and 5% CO2, followed by elution twice with PBS. Activated T cells were then cultured at 2 x 10⁻⁶ cells / ml. 5 Cell / well concentrations were added to the plates. 2 μM of the mimic (4420LALA) mAb was prepared in DPBS (Gibco). 2The positive control anti-mouse OX40 OX86 mAb dilution (in the human IgG1 backbone with LALA mutation; SEQ ID NO: 175 and 176) (see Table 9 for details) was then further diluted 1:10 in T cell culture medium (30 μl + 270 μl) to obtain a 200 nM dilution. Anti-human CH2 mAb clone MK1A6 was used for cross-linking via the Fc of the OX86 positive control mAb, and FITC-glucan (Sigma) was used for cross-linking via a simulated (4420 LALA) mAb. 2 The Fab binding of Fcab in the form is cross-linked to simulate mAb. 2 Or a positive control mAb was added to the well at a 1:1 molar ratio. In a 96-well plate, (1) a positive control or simulated mAb with the relevant crosslinking agent was prepared. 2 Or (2) positive control or simulated mAb 2 A series of dilutions were prepared. 100 μl of the diluted simulant (4420LALA) mAb was added. 2 / Control mAb or analog (4420LALA) mAb 2 Alternatively, a mixture of positive control mAb and cross-linked antibody can be added to activated T cells on a plate.

[0580] T cells were incubated at 37°C and 5% CO2 for 72 hours. The supernatant was collected and analyzed using a mouse IL-2 ELISA kit (eBioScience or AndyBio) according to the manufacturer's instructions. The plate was read at 450 nm using a BioTek microplate reader equipped with Gen5 software. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (correction). A standard curve for cytokine concentrations was calculated based on a four-parameter logarithmic curve fit (Gen5 software, BioTek). The concentrations of mouse IL-2 (mIL-2) were compared with those of a simulated mAb. 2 Logarithmic concentrations of the positive control mAbs were plotted, and the resulting curves were fitted to the logarithmic (agonist) versus response equation in GraphPad Prism. Table 9 shows the simulated mAbs in and out of the test. 2 EC2g observed in T cell activation assays in the presence of a positive control mAb 50 The maximum response value and IL-2 release.

[0581] Table 9: Using Simulated (4420LALA) mAb 2 Anti-mouse OX40 Fcab activated T cells

[0582]

[0583] *In the uncrosslinked case, these simulated mAbs 2The control mAb showed no activity in the T cell activation assay.

[0584] As shown in Table 9, in the T cell activation assay, when cross-linked with the Fab target (FITC-glucan), the simulated (4420LALA) mAb 2 The activity of the anti-mouse OX40 Fcab form was comparable to that of the positive control anti-mouse OX40 mAb OX86, which was cross-linked in the human IgG1 backbone by the anti-human CH2 mAb clone MK1A6. For the simulated (4420LALA) mAb... 2 The anti-mouse OX40 Fcab or anti-mouse OX40 mAb positive control antibody, in the absence of cross-linking, did not show T cell activation. These results indicate that the anti-mouse OX40 Fcab has similar agonistic activity to the positive control anti-mouse OX40 mAb and suggests that the Fcab form can mediate OX40 clustering and activation upon cross-linking. The activity of the anti-mouse OX40 Fcab is similar to that of the FS20-11 lineage of anti-human OX40 Fcab, and activity was also observed only upon cross-linking. The FS20-22 and FS20-31 lineages of Fcab showed agonist activity in the absence of cross-linking, with further enhancement of activity in the presence of cross-linking. As mentioned above, the background agonist activity of these Fcabs in the absence of cross-linking is expected to make them clinically more potent than Fcabs without this background agonist activity. Therefore, the clinical activity of anti-human OX40 Fcab from the FS20-22 and FS20-31 lineages may be greater than the in vivo activity observed with anti-mouse OX40 Fcab.

[0585] 5.3 Simulation of mAb 2 In vivo antitumor efficacy of anti-mouse OX40 Fcab

[0586] Simulated mAbs were detected using the CT26 syngeneic tumor model. 2 Antitumor activity of the anti-mouse OX40 form Fcab FS20m-232-91 in vivo. Previously, the CT26 syngeneic tumor model has been shown to be sensitive to OX40 agonist antibodies (Sadun et al., 2008), and tumor-infiltrating lymphocytes (TILs) isolated from CT26 tumors are expected to express OX40.

[0587] As demonstrated by the T-cell activation assay, anti-mouse OX40 Fcab FS20m-232-91 and anti-human OX40 Fcab are potent agonists of T-cell activation. For the simulated (4420LALA)mAb... 2The anti-mouse OX40 Fcab in its uncrosslinked form did not show T cell activation (see Example 5.2). Since the FS20-11 lineage exhibits T cell activity only upon crosslinking, similar to the FS20m-232-91 anti-mouse OX40 Fcab, in vivo studies using the FS20m-232-91 anti-mouse OX40 Fcab are expected to achieve the anticipated clinical efficacy of this FS20-11 lineage anti-human OX40 Fcab in human patients (see Example 5.1). Anti-human OX40 Fcabs from the FS20-22 and FS20-31 lineages showed OX40 agonist activity without crosslinking (see Example 5.1). As explained in Example 5.2.1, Fcabs from the FS20-22 and FS20-31 lineages are expected to show higher clinical efficacy than Fcabs without OX40 agonist activity in their uncrosslinked form. Therefore, the clinical efficacy of anti-human OX40 Fcab from the FS20-22 and FS20-31 lineages may be greater than the in vivo results observed with FS20m-232-91 anti-mouse OX40 Fcab.

[0588] Simulated (HEL D1.3) mAbs with or without LALA mutations 2 The tumor growth inhibition ability of the FS20m-232-91 anti-mouse OX40 Fcab form was compared with that of the positive control anti-mouse OX40 mAb (OX86 in the human IgG1 backbone) and the negative control anti-FITC antibody (4420 in the human IgG1 backbone).

[0589] Before the study began, 8-10 week old female BALB / c mice (Charles River) (approximately 20g each) were acclimatized for one week. All animals were microchipped and assigned a unique identifier. Each group consisted of 12 mice. The CT26 colon cancer cell line (ATCC, CRL-2638) was first expanded and stockpiled, then pre-screened for pathogens using the IMPACT I protocol via IDEXX Biosciences, which showed the absence of pathogens. CT26 cells (approximately 3-5 x 10⁻⁶ cells) were then... 6 Thaw at -150°C and add to 20 ml DMEM (Gibco, 61965-026) T175 tissue culture flasks containing 10% FCS (Gibco, 10270-106). Anesthetize mice with isoflurane (Abbott Laboratories) and subcutaneously inject 1 x 10⁻⁶ dextrose solution into the left flank of each animal. 6 Cells. On day 10 after tumor cell inoculation, the health and tumor growth of mice were monitored, and they were classified and randomly assigned to study groups. Mice without tumors were then removed from the study.

[0590] Simulation (HEL D1.3) mAb 2 Molecular and control mAbs were analyzed by SEC-HPLC, and impurities were checked within 24 hours prior to injection. Simulated (HEL D1.3) mAbs were prepared in PBS to a final concentration of 0.1 mg / ml. 2 The drug was administered via intraperitoneal (IP) injection at a dose of 200 μl / mouse on days 10, 12, and 14 post-tumor inoculation, with a final dose of 1 mg / ml for 20g mice. Animals under anesthesia under blinded control underwent health checks three times weekly, during which the tumor was precisely measured. Tumor volume was measured using calipers to determine the longest and shortest axes of the tumor. The following formula was used to calculate the tumor volume:

[0591] Lx(S 2 ) / 2

[0592] (Where L = longest axis; S = shortest axis)

[0593] The experiment was stopped on day 22 when the tumor burden was considered to be nearing its limit, and all mice were humanely euthanized. Results were as follows: Figure 3 As shown. Statistical analysis of the final tumor volume was performed using the two-tailed Student's t-test from the GraphPad Prism software package.

[0594] In inhibiting tumor growth, there was a statistically significant difference between the positive control anti-mouse OX40 mAb (G1 / OX86) and the negative control anti-FITC antibody (normal growth). (HEL D1.3) mAb 2 Compared with the simulated (HEL D1.3) mAb with LALA mutation 2 There were also statistically significant differences in their ability to inhibit tumor growth.

[0595] The CT26 tumor model is an aggressive, rapidly growing tumor model in mice, which are inherently prone to intestinal metastasis, thus having a very limited therapeutic window. In this tumor model, the clustering and activation of OX40 expressed on tumor-infiltrating T cells, through FcγR-mediated mimicry of mAbs... 2 Crosslinking in the form of FS20m-232-91Fcab and the G1 / OX86 positive control (neither containing LALA mutations) inhibited tumor growth. However, as with mimicry mAbs... 2 As observed with the LALA-mutant form of FS20m-232-91AA Fcab, no inhibition of tumor growth was observed when there was little or no FcγR-mediated Fcab crosslinking, and therefore no OX40 clustering and activation. Therefore, it can be concluded that anti-mouse OX40 Fcab only exhibits tumor-reducing activity upon crosslinking.

[0596] Based on these results, it is expected that anti-human OX40 Fcab, when cross-linked, will similarly be able to inhibit tumor growth in human patients containing tumor-infiltrating T cells expressing OX40.

[0597] Example 6 - mAbs that bind to cell surface receptors 2 Crosslinking

[0598] Activated T cells express OX40 on their cell surface. Binding of the trimerized OX40 ligand to OX40 leads to receptor trimerization. Since OX40 ligands are expressed in clusters on the cell surface of antigen-presenting cells, the interaction between OX40 ligands and OX40 results in OX40 clustering, which is crucial for OX40 signaling and further T cell activation. Antibodies activating OX40 must mimic the clustering activity of the OX40 ligand. For monospecific anti-OX40 antibodies, the Fcγ receptor binds to and cross-links the Fc domain of the antibody, leading to OX40 clustering. Bispecific antibodies can bind to a second cell surface-expressed receptor via their second antigen-binding site, resulting in antibody cross-linking and OX40 clustering. The second cell surface-expressed receptor bound by the bispecific antibody can be a tumor-associated antigen (TAA). Bispecific antibodies have an advantage in cross-linking at the tumor site, leading to OX40 clustering and T cell activation at the tumor site. Therefore, the use of bispecific antibodies has the potential to activate the immune system at the tumor site, thereby eliminating or controlling the tumor.

[0599] Therefore, as described below, tumor cells expressing TAA on their surface were used in a T-cell activation assay to evaluate mAbs containing the anti-OX40 Fcab of the present invention and a TAA antigen-binding site in the variable region. 2 Does combining TAA lead to mAb? 2 Cross-linking, thus inducing the clustering and signal transduction of OX40.

[0600] 6.1 Use mAbs containing anti-human OX40 Fcab and anti-EGFR, anti-EphA2, anti-CEACAM5, or anti-EpCAM Fab. 2 Human T cell activation assay performed

[0601] As described in Example 4.1, antibody molecules with LALA mutations listed in Tables 10 and 11 below were prepared for testing in a T-cell activation assay. Antibody molecules were constructed using anti-FITC antibody 4420 (SEQ ID NO: 167, 168, and 156), anti-EGFR antibody cetuximab (US Patent No. 6217866; referred to as 'Cx'), EphA2 antibody E2A (WO 2004 / 014292 A2), EpCAM antibody MOC31 (US Patent No. 8637017), OX40 antibody 11D4 (EP 2 242 771 B1), or anti-CEACAM5 antibody CEA (US 8,771,690 B2, clone hMN15), in conjunction with the constructed mAbs described in Example 4.1. 2 The method is the same. In these assays, tumor cells act as mAbs. 2 The antibody contains a cross-linking agent and includes a variable region specific to TAA (EGFR, EphA2, CEACAM5, or EpCAM in the case of anti-human OX40 Fcab FS20-22-49AA in Table 10, and EphA2 or CEACAM5 in the case of anti-human OX40 Fcab FS20-11-131AA in Table 11), which binds to TAA through the variable region binding site. T cell activation is determined by the release of IL-2.

[0602] Table 10. Detected mAbs and mAbs 2

[0603] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC n / a hIgG1 none FITC-glucan G1 / 11D4 OX40 n / a hIgG1 none Anti-hCH2 FS20-22-49AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20-22-49AA / Cx EGFR OX40 hIgG1 have HPAC cells FS20-22-49AA / E2A EphA2 OX40 hIgG1 have HPAC cells FS20-22-49AA / CEA CEACAM5 OX40 hIgG1 have HPAC cells FS20-22-49AA / MOC31 EpCAM OX40 hIgG1 have HPAC cells

[0604] Table 11. Detected mAbs and mAbs 2

[0605] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1AA / 4420 FITC n / a hIgG1 none FITC-glucan G1 / 11D4 OX40 n / a hIgG1 none Anti-hCH2 FS20-11-131AA / 4420 FITC OX40 hIgG1 have none FS20-11-131AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20-11-131AA / E2A EphA2 OX40 hIgG1 have HPAC cells FS20-11-131AA / CEA CEACAM5 OX40 hIgG1 have HPAC cells

[0606] T cells were isolated and activated as described in Example 5.1.1 above.

[0607] HPAC cells (ATCC, CRL-2119) expressing EGFR, EpCAM, EphrinA2, and CEACAM5 on their cell surface were maintained in DMEM medium containing 10% FBS. HPAC cells were washed once in T cell culture medium and, if necessary, injected at 2.5 x 10⁻⁶ cells / mL. 4 The cell / well concentration was added to the plate.

[0608] Prepare 2 μM of various test mAbs / mAbs in DPBS (Gibco). 2The diluent (see Tables 10 and 11 for details) was then diluted 1:10 in T cell culture medium (30 μl + 270 μl) to obtain a 200 nM diluent.

[0609] The cross-linking agent (anti-human CH2 mAb clone MK1A6 or FITC-glucan (Sigma); see Tables 10 and 11) was mixed with mAb / mAb at a 1:1 molar ratio. 2 Add to the wells to be tested. Prepare the test mAb / mAb in a 96-well plate. 2 A series of dilution solutions were prepared, and 100 μl of mAb / mAb was diluted. 2 The mixture was added to activated T cells and HPAC cells on the plate.

[0610] T cells were incubated as described in Example 5.1.1, and the supernatant was collected. The plate was read, and the concentration of human IL-2 (hIL-2) was plotted against the test mAb / mAb ratio. 2 The logarithmic concentration was plotted, and the curve obtained was fitted using the logarithmic (agonist) response equation, as described in Example 5.1.1.

[0611] Table 12 shows the mAbs / mAbs listed in Table 10 with or without cross-linking agents or HPAC cells. 2 Activation of T cells (EC) 50 (Value and maximum IL-2 release). Figure 4 The graph shows IL-2 release for T cell activation assay.

[0612] Table 12: T cell activation in the presence of HPAC tumor cells

[0613]

[0614] *In the absence of cross-linking, this control mAb showed no activity in the T cell activation assay.

[0615] Figure 4 The results show that when OX40 is cross-linked with anti-OX40 mAb / mAb 2 Upon binding, T cell activation increased. As expected, no T cell activation was observed with the cross-linked anti-FITC antibody G1AA / 4420, which served as a negative control. When the anti-human CH2 antibody was cross-linked, the OX40-targeting mAb G1 / 11D4 induced T cell activation in the presence of HPAC cells. As previously seen, the mimicry mAb... 2 The (4420LALA) form of Fcab FS20-22-49AA / 4420, which targets OX40, exhibits agonistic activity in the absence of cross-linking and binds to the mimic mAb in the presence of the cross-linking agent FITC-glucan.2 When the Fab arm is engaged, the agonistic activity is enhanced. When OX40-targeting Fcab is paired with anti-TAA Fab (EGFR is cetuximab, EphrinA2 is E2A, EpCAM is MOC31, and CEACAM5 is CEA), the agonistic activity is enhanced compared to the mimic mAb. 2 Compared to the form of Fcab, the obtained mAb 2 The increased agonistic activity indicates that the binding of TAA Fab to TAA expressed on the cell surface of HPAC cells leads to mAb... 2 Crosslinking leads to the aggregation and activation of OX40 clusters.

[0616] Table 13 shows the mAbs / mAbs listed in Table 11, with or without cross-linking agents or HPAC cells. 2 T cell activation (EC) 50 (Value and maximum IL-2 release). Figure 5 The graph shows IL-2 release for T cell activation assay.

[0617] Table 13: T cell activation assay in the presence of HPAC tumor cells

[0618]

[0619] *In the absence of cross-linking, this control mAb showed no activity in the T cell activation assay.

[0620] Figure 5 The data plotted shows that when OX40 is cross-linked, the anti-OX40 mAb / mAb 2 Upon binding, T cell activation increased. As expected, no T cell activation was observed with the cross-linked anti-FITC antibody G1 / 4420, which served as a negative control. When cross-linked with an anti-human CH2 antibody, the OX40-targeting mAb antibody G1 / 11D4 induced T cell activation in the presence of HPAC cells. As previously seen, the mAb-mimicking antibody... 2 The (4420LALA) form of Fcab FS20-11-131AA / 4420 targeting OX40 does not exhibit agonistic activity and only shows agonistic activity in the presence of the cross-linking agent FITC-glucan, which is similar to that of the mimic mAb. 2 The Fab arm is combined. When the OX40-targeting Fab is paired with the anti-TAA Fab (EphrinA2 is E2A or CEACAM5 is CEA), it is combined with the simulated mAb. 2 Compared to HPAC cells expressing TAA, the form of Fcab yielded mAb. 2The increased agonistic activity indicates that TAA Fab binding to TAA expressed on the cell surface of HPAC cells leads to mAb... 2 Crosslinking leads to the aggregation and activation of OX40 clusters.

[0621] Use anti-OX40 / anti-EGFR, anti-OX40 / anti-CEACAM5, anti-OX40 / anti-EphA2, or anti-OX40 / anti-EpCAM mAb 2 The T-cell activation observed by the antibody suggests that more than one type of cell surface receptor can pair with the anti-OX40 antibody.

[0622] 6.2 Use of mAbs containing anti-mouse OX40 Fcab and anti-EphA2 Fab 2 Mouse T cell activation assay

[0623] The test mAb / mAb listed in Table 14 below are available. 2 In this case, the T cell activation assay was used to assess OX40 clustering and signal transduction. As described in Example 4.1, mAbs with the LALA mutation were prepared. 2 The tumor cells used in the analysis bound to EphA2 via Fab, serving as positive control mAbs and mAbs targeting TAA EphA2. 2 Crosslinking agent.

[0624] Table 14: Tested mAbs and mAbs 2

[0625] <![CDATA[mAb / mAb 2 ]]> Molecular type Fab target Fab clone Fcab target G1 / 4420 monoclonal antibodies FITC 4420 n / a FS20m-232-91AA / 4420 <![CDATA[mAb 2 ]]> FITC 4420 mOX40 G1 / E2A monoclonal antibodies EphA2 E2A n / a FS20m-232-91AA / E2A <![CDATA[mAb 2 ]]> EphA2 E2A mOX40 G1 / OX86 monoclonal antibodies mOX40 OX86 n / a

[0626] Mouse T cells were isolated and activated as described in Example 5.2.1, and HPAC cells were used in essentially the same manner as the human T cell activation assay described in Example 6.1, but with different cross-linking agents and control antibodies as described above and below, and IL-2 production was evaluated.

[0627] Add the cross-linking agent (anti-human Fc(a-hFc), Jackson Immunological Research; or FITC-glucan, Sigma) to the wells at a 1:1 molar ratio, and use a test mAb / mAb if necessary. 2 mAb / mAb were prepared in a 96-well plate. 2 or mAb / mAb 2 Six 5-fold serial dilutions of the cross-linked antibody mixture (60 μl + 240 μl T cell culture medium). Add 100 μl of diluted mAb / mAb. 2 or mAb / mAb 2 The cross-linked antibody mixture was added to the activated T cells on the plate.

[0628] Table 15 shows the mAb / mAb ratios listed in Table 14 with or without a cross-linking agent or with HPAC cells cross-linked. 2 Activation of T cells (EC) 50 (Value and maximum IL-2 release). Figure 6 A representative graph of IL-2 release used in T cell activation assays is shown.

[0629] Table 15: T cell activation in the presence of HPAC tumor cells

[0630]

[0631]

[0632] *These mAbs / mAbs 2 No activity was observed in the T-cell activation assay.

[0633] Figure 6 Displayed in various mAbs / mAbs 2 A representative graph of IL-2 release as measured by T cell activation in HPAC cells expressing EphA2 in the presence of the virus. In this assay, six different mAb / mAb ratios were tested at incremental concentrations. 2 Or a combination thereof, and labeled according to its human IgG isotype / Fab clone or the appropriate Fcab / Fab clone name (G1 / 4420, G1 / E2A, G1 / OX86, FS20m-232-91AA / 4420, FS20m-232-91AA / 4420+G1 / E2A and FS20m-232-91AA / E2A). Figure 6 The results shown in Figure A indicate that, in the presence of HPAC cells expressing EphA2, FS20m-232-91AA / E2AmAb 2 Antibodies can enhance T cell activation. This suggests that antibodies targeting OX40 require cross-linking to increase T cell activation, and FS20m-232-91AA / E2A mAb 2 Antibodies are the only molecules that can be cross-linked in the presence of HPAC (EphA2+) cells without requiring any other non-physiological cross-linking agents. Figure 6 B shows that when targeting OX40 and anti-OX40 mAbs / mAb 2 Cross-linking increases T cell activation. These results suggest that mAb / mAb targeting OX40... 2 Cross-linking is required to increase T cell activation.

[0634] In the presence of HPAC cells expressing EphA2, anti-mouse OX40 / anti-EphA2 mAb was used. 2The observation of T cell activation (FS20m-232-91AA / E2A) indicates that this receptor is bound by mAbs with binding sites for EphA2 and OX40. 2 When targeted, it can also mediate the cross-linking of OX40.

[0635] 6.3 Anti-mOX40 / Anti-EphA2 mAb 2 Inhibit tumor growth in the body

[0636] The CT26 syngeneic tumor model was used in this experiment because CT26 cells express EphA2, and TILs isolated from CT26 tumors include T cells expressing OX40. The anti-mOX40 / anti-EphA2 mAb described in Example 6.2 was tested. 2 In vivo activity of antibody (FS20m-232-91AA / E2A) in a CT26 syngeneic mouse tumor growth model.

[0637] Anti-mOX40 / anti-EphA2 mAb 2 The ability to inhibit tumor growth and the ability to mimic mAbs 2 The resistance to OX40 Fcab in form (FS20m-232-91AA / 4420) was compared, and mAb was simulated. 2 The combination of anti-OX40 Fcab (FS20m-232-91AA / 4420), anti-EphA2 mAb (G1 / E2A), and anti-FITC mAb (G1 / 4420) was used as a control.

[0638] Female BALB / c mice were injected with CT26 cells, and their health status and tumor growth were monitored. They were then classified and randomly assigned to experimental groups as described in Example 5. Mice without tumors were removed from the study at this point.

[0639] For mAb 2 The mAb was analyzed, impurities were checked, and the solution was prepared and administered to mice as described in Example 5. As described in Example 5, the animals underwent health checks, tumor measurements were performed, and tumor volume was calculated.

[0640] When the tumor burden was considered to be nearing its limit, the experiment was stopped on day 20, and all mice were humanely euthanized. Results were as follows: Figure 7 As shown. Statistical analysis of the final tumor volume was performed using the two-tailed Student's t-test from the GraphPad Prism software package.

[0641] In terms of inhibiting tumor growth, anti-OX40 / anti-EphA2 mAb 2There was a statistically significant difference between (FS20m-232-91AA / E2A) and the anti-FITCmAb (G1 / 4420) control (normal growth). No such statistically significant difference was observed in combinations of FS20m-232-91AA / 4420 and G1 / E2A or FS20m-232-91AA / 4420 and the G1 / 4420 control group.

[0642] Surprisingly, compared with the IgG1 control group (G1 / 4420), the antibody combination targeting OX40 and EphA2 did not significantly inhibit tumor growth. However, compared with the IgG1 control, the anti-mOX40 / anti-EphA2 mAb... 2 The (FS20m-232-91AA / E2A) treatment group did indeed show significant growth inhibition. This trial demonstrates that, similar to the observed in vitro results, mAbs targeting EphA2 expressed on tumor cells... 2 OX40 crosslinking on tumor-infiltrating T cells expressing OX40 led to T cell activation and subsequent tumor growth was observed in the control.

[0643] Figure 7 Tumor growth curves of the CT26 syngeneic model in Balb / c mice treated with G1 / 4420, FS20m-232-91AA / 4420, FS20m-232-91AA / 4420+G1 / E2A, and FS20m-232-91AA / E2A are shown. Mean tumor volume is plotted with or without the mean standard error, and a two-tailed t-test is used to compare tumor volumes on the last day between different groups. The groups treated with anti-mOX40 / anti-EphA2 mAb showed significantly higher tumor growth rates compared to those treated with the control antibody (G1 / 4420). 2 The group treated with the antibody (FS20m-232-91AA / E2A) showed a statistically significant reduction in tumor volume. This result indicates that the combination of anti-mOX40 / anti-EphA2 mAb significantly reduced tumor volume compared to the combination of FS20m-232-91AA / 4420 and G1 / E2A antibodies. 2 The antibody exhibits better anti-tumor efficacy against tumors expressing EphA2 in vivo, indicating that the anti-mOX40 / anti-EphA2 mAb... 2 The bispecific binding of OX40 and EphA2 mediated by OX40 and the in vivo cross-linking of OX40 can effectively control tumor growth.

[0644] Example 7-mAb 2 Cross-linking by binding with soluble factors

[0645] Vascular endothelial growth factor (VEGF) is a soluble homodimer molecule expressed in response to hypoxia. It binds to receptors on endothelial cells, leading to the formation of new blood vessels – a process known as angiogenesis. The tumor microenvironment (TME) is hypoxic and VEGF levels are elevated, providing sufficient nutrients for tumor cells to grow. The use of monoclonal antibodies targeting VEGF is an established form of anti-tumor therapy. Because TAAs expressed on the cell surface can mediate OX40-targeting mAbs… 2 The cross-linking of OX40 and VEGF-targeted mAbs using soluble VEGF were also tested. 2 Crosslinking.

[0646] 7.1 Use mAbs containing anti-human OX40 Fcab and anti-VEGF Fab 2 Human T cell activation assay

[0647] In the presence or absence of additional VEGF, T cell activation assays were used to assess OX40 clustering and signaling in the presence of the antibodies listed in Table 16 below. OX40 / VEGF mAbs with the LALA mutation were prepared as described in Example 4.1. 2 and OX40 / FITC analog mAb 2 In this assay, VEGF acts as the OX40 / VEGF mAb 2 The cross-linking agent was constructed using the variable region of OX40 targeting Fcab FS20-22-49AA and the anti-VEGF antibody bevacizumab (EP1325932B9 clone A4.6.1; indicated by “Bev” in Tables 16 and 17).

[0648] Table 16. Tested mAbs and mAbs 2

[0649] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC none hIgG1 none FITC-glucan G1 / 11D4 OX40 none hIgG1 none Anti-hCH2 FS20-22-49AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20-22-49AA / Bev VEGF OX40 hIgG1 have none FS20-22-49AA / Bev VEGF OX40 hIgG1 have VEGF

[0650] T cells were isolated and activated as described in Example 5.1.1 above, and the above-mentioned positive control antibody and mAb were used. 2 The cross-linking agent (e.g., soluble VEGF instead of HPAC cells) was used, following essentially the same protocol as the human T cell activation assay described in Example 6.1. hIL-2 production was determined as previously described. The cross-linking agent (anti-human CH2 mAb clone MK1A6, FITCdex (Sigma), or VEGF (Peprotech, catalog number 100-20; see Table 16) was used with the test mAb / mAb. 2 Add to the desired holes at a 1:1 molar ratio.

[0651] Table 17 shows the mAb / mAb ratios listed in Table 16, with or without crosslinking with a crosslinking agent. 2 T cell activation (EC) 50 (Value and maximum IL-2 release). Figure 8 The graph shows IL-2 release used in the T cell activation assay.

[0652] Table 17: T cell activation in the presence of VEGF

[0653]

[0654] *In the absence of cross-linking, this control mAb showed no activity in the T cell activation assay.

[0655] Figure 8 The results showed that when targeting OX40 and anti-OX40 mAb / mAb 2 Crosslinking increased T cell activation. As expected, no T cell activation was observed with the crosslinked anti-FITC antibody G1 / 4420. When crosslinked with the anti-human CH2 antibody, the OX40-targeting mAb G1 / 11D4 induced T cell activation. (mAb analogue) 2 (4420LALA) form of Fcab targeting OX40, FS20-22-49AA / 4420, in conjunction with simulated mAb 2 The Fab arm-binding cross-linking agent FITC-glucan exhibits agonistic activity in the presence of this agent. When OX40-targeting Fcab is paired with anti-VEGF Fab bevacizumab, the mAb… 2 The antibody exhibits some agonistic activity in the absence of cross-linking, which is likely a product of the agonistic activity observed in the absence of cross-linking in anti-OX40 Fcab FS20-22-49. This activity is further evidenced by the addition of VEGF to the OX40 / bevacizumab mAb. 2 When antibodies are present, agonist activity increases, such as EC. 50 As indicated by the approximately 100-fold reduction, this suggests that anti-VEGF Fab can crosslink mAbs in the presence of VEGF. 2 .

[0656] Using anti-hOX40 / anti-VEGF mAb 2 The observed T cell activation demonstrates that soluble factors can be used as cross-linking agents.

[0657] 7.2 Use mAbs containing anti-mouse OX40 Fcab and anti-VEGF Fab 2 Mouse T cell activation assay

[0658] The mAb / mAb ratios listed in Table 18 below 2In the presence of OX40, T-cell activation assays were used to assess OX40 clustering and signal transduction. Anti-mouse OX40 Fcab FS20m-232-91 was paired with the Fab region of anti-VEGF mAb R84 (patent publication number US2009 / 0175791A1), and all mAbs with the LALA mutation were prepared as described in Example 4.1. 2 In this assay, VEGF acted as a mAb that binds to mouse OX40 and VEGF. 2 Crosslinking agent.

[0659] Table 18: Tested mAbs and mAbs 2

[0660]

[0661]

[0662] Mouse T cells were isolated and activated as described in Example 5.2.1, using essentially the same protocol as in Example 7.1, but with the positive control antibody and mAb described above. 2 And the crosslinking agent described below. The generation of mIL-2 was determined as previously described.

[0663] Add the cross-linking agent (anti-human CH2 mAb clone MK1A6, FITCdex, (Sigma) or VEGF (Peprotech, catalog number 100-20; see Table 18) to the test antibody at a 1:1 molar ratio to the desired location in the well.

[0664] Table 19 shows the mAbs available in the presence of the test. 2 In the case of mAb, EC was observed in T cell activation assays. 50 The maximum response value and IL-2 release. Figure 9 A representative graph of IL2 release used in T cell activation assays is shown.

[0665] Table 19: T cell activation in the presence of VEGF

[0666]

[0667] *In the absence of cross-linking, this control mAb showed no activity in the T cell activation assay.

[0668] Figure 9 The data shows that, in the presence of VEGF, when mAb... 2 T cell activation increases when OX40 and VEGF-soluble factors are present; however, when other mAbs targeting OX40 are present... 2 When mAb is present but not cross-linked, T cell activation does not increase. This indicates that mAb...2 Crosslinking occurs through binding with OX40 and VEGF. Figure 9 The results showed that, in the presence of non-physiological cross-linking agents (anti-Fc antibody or FITC-glucan), the mAb / mAb targeting OX40 was [value missing]. 2 T cell activation. As expected, the anti-VEGF and anti-FITC control antibodies did not induce T cell activation in the presence of the cross-linking agent. The anti-mouse OX40 antibody induced some T cell activation when cross-linked with the anti-Fc antibody (G1 / OX86 cross-linking agent) (see Table 19). The anti-OX40 Fcab antibody, when cross-linked with FITC-glucan (FS20m-232-91AA / 4420Xlink), showed some activation compared to the mAb. 2 When anti-FITC Fab is paired in activated T cells, it has a lower ECG than OX40 antibody G1 / OX86. 50 And a higher maximum response. Same anti-OX40 Fcab in mAb 2 When paired with anti-VEGF Fab in (FS20m-232-91AA / R84), T cells are activated in the absence of other cross-linking agents, likely due to VEGF production by the activated T cells. Additional VEGF is generated through anti-OX40 / anti-VEGF mAb. 2 (FS20m-232-91AA / R84) increased T cell activation, along with anti-OX40 / anti-VEGF mAb. 2 The antibody observed lower levels of EC compared to the antibody without additional VEGF. 50 And a comparable maximum response.

[0669] In the presence of VEGF, use anti-mOX40 / anti-VEGF mAb 2 The observed T cell activation suggests that soluble factors can also mediate OX40 crosslinking, for example, when mAbs containing VEGF and OX40 are bound. 2 When targeted.

[0670] 7.3 Anti-mOX40 / anti-VEGF mAb 2 Inhibit tumor growth in the body

[0671] The anti-mOX40 / anti-VEGF mAb described in Example 7.2 was tested. 2 In vivo activity of antibody (FS20m-232-91AA / R84) in a CT26 syngeneic mouse tumor growth model.

[0672] This experiment used the CT26 syngeneic tumor model because CT26 tumors have been described to have elevated VEGF concentrations (Voron et al., 2015), and TILs isolated from CT26 tumors include T cells expressing OX40.

[0673] Anti-mOX40 / anti-VEGF mAb 2 The ability to inhibit tumor growth was compared with the combination of mAb G1 / 4420, FS20m-232-91AA / 4420 and mAb G1 / R84, as well as mAb G1 / R84 as a control.

[0674] Female BALB / c mice were injected with CT26 cells, and their health status and tumor growth were monitored. They were then classified and randomly assigned to experimental groups as described in Example 5. Mice without tumors were removed from the study at this point.

[0675] For mAb 2 The mAb was analyzed, impurities were checked, and the solution was prepared and administered to mice as described in Example 5. As described in Example 5, the animals underwent health checks, tumor measurements were performed, and tumor volume was calculated. The experiment was stopped on day 24 when the tumor burden was considered to be near its limit, and all mice were humanely euthanized. Results are as follows... Figure 10 As shown. Statistical analysis of the final tumor volume was performed using the two-tailed Student's t-test from the GraphPad Prism software package.

[0676] In terms of inhibiting tumor growth, anti-mOX40 / anti-VEGF mAb 2 There was a statistically significant difference between (FS20m-232-91AA / R84) and the anti-FITCmAb G1 / 4420 control (normal growth). No such statistically significant difference was observed in combinations of FS20m-232-91AA / 4420 and controls without FS20m-232-91AA / R84, or in combinations of anti-VEGF mAb G1 / R84 and G1 / 4420 controls.

[0677] The CT26 tumor model is an aggressive, rapidly growing tumor model. Surprisingly, the combination of antibodies targeting OX40 and VEGF did not significantly inhibit tumor growth compared to the IgG1 control group (G1 / 4420). However, compared to the IgG1 control, the use of anti-mOX40 / VEGF mAbs... 2 The (FS20m-232-91AA / R84) treated group did indeed show significant growth inhibition. This experiment indicates that, similar to the observed in vitro results, OX40 crosslinking is mediated by mAb. 2 Targeting VEGF, which is overexpressed in tumor cells or the tumor microenvironment, and OX40 expressed in tumor-infiltrating T cells, led to T cell activation and subsequent tumor growth control that was higher than observed in controls.

[0678] Example 8-mAb 2 Cross-linking through binding to co-expressed receptors

[0679] OX40 expression on tumor-infiltrating T cells may be accompanied by the expression of other receptors, including co-stimulatory receptors and immune checkpoint receptors. In cells containing mAbs... 2 In OX40-Fcab, using these co-expressed receptors as Fab targets can also cross-link mAb2, leading to clustering of OX40 and Fab targets, thereby activating both receptors. To test this concept, the following T cell activation assays were performed.

[0680] 8.1 Use mAbs containing anti-human OX40 Fcab paired with anti-ICOS, anti-CD27, or anti-GITR Fabs. 2 Human T cell activation assay

[0681] In this assay, the mAb / mAb ratios listed in Table 20 below were determined by the co-expression of human OX40 and co-stimulatory molecules ICOS, CD27, and GITR. 2 Cross-linking. mAbs with LALA mutations were prepared using the variable regions of anti-FITC antibody 4420 (SEQ ID NO: 167, 168, and 156), anti-OX40 antibody 11D4 (EP 2242771 B1), anti-ICOS antibody ICOSj (US 2016 / 0304610 A1), anti-CD27 antibody 695 (US 2013 / 0243795 A1), or anti-GITR antibody 6C8 (US 7,812,135 B2). 2 The method is the same as that described in Example 4.1.

[0682] Table 20. Detected mAbs and mAbs 2

[0683] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC none hIgG1 none FITC-glucan G1 / 11D4 OX40 none hIgG1 have Anti-hCH2 G1AA / ICOSj ICOS none hIgG1 none Anti-hCH2 G1AA / 695 CD27 none hIgG1 have Anti-hCH2 G1AA / 6C8 GITR none hIgG1 have Anti-hCH2 FS20-22-49AA / 4420 FITC OX40 hIgG1 have none FS20-22-49AA / 4420 crosslinking agent FITC OX40 hIgG1 have FITC-glucan FS20-22-49AA / ICOSj ICOS OX40 hIgG1 have none FS20-22-49AA / 695 CD27 OX40 hIgG1 have none FS20-22-49AA / 6C8 GITR OX40 hIgG1 have none

[0684] T cells were isolated and activated as described in Example 5.1.1 above, and experiments were performed using the positive control antibody and mAb2 described above, as well as the cross-linking agent described below, in essentially the same protocol as the human T cell activation assay described in Example 6.1. The production of human IL-2 was determined as previously described.

[0685] The cross-linking agent (anti-human CH2 mAb clone MK1A6 or FITC-glucan (Sigma); see Table 20) was used with the test mAb / mAb. 2 Add to the desired holes at a 1:1 molar ratio.

[0686] Table 21 shows the EC observed in the T cell activation assay with or without cross-linking with the cross-linking agent. 50 The maximum response value and IL-2 release. Figure 11 The graph shows IL-2 release for T cell activation assay.

[0687] Table 21: T cell activation in the presence of co-expressed receptors

[0688]

[0689] *In the absence of cross-linking, these control mAbs showed no activity in the T cell activation assay.

[0690] Figure 11 The results showed that T cell activation increased when OX40 was targeted and anti-OX40 antibodies were cross-linked. As expected, no T cell activation was observed with the cross-linked anti-FITC antibody G1 / 4420 or the cross-linked anti-ICOS, anti-CD27, or anti-GITR antibodies (G1AA / ICOSj, G1AA / 695, or G1AA / 6C8, respectively). As previously mentioned, the OX40-targeting mAb G1 / 11D4, when cross-linked with an anti-human CH2 antibody, induced T cell activation. As previously observed, the mimicry mAb... 2 The OX40-targeting Fcab FS20-22-49AA / 4420 in the (4420LALA) form exhibits agonistic activity without cross-linking, and this activity is enhanced by the addition of FITC-glucan, a cross-linking agent that binds to the Fab arm. When the OX40-targeting Fcab is paired with anti-ICOS, anti-CD27, or anti-GITR Fab (ICOSj, 695, or 6C8, respectively), the agonistic activity of the Fcab increases, indicating that the mAb... 2 It is cross-linked by binding to co-expressed receptors on the surface of T cells.

[0691] Using anti-OX40 / anti-ICOS, anti-OX40 / anti-CD27 and anti-OX40 / anti-GITR mAb 2 The T cell activation observed by the antibody demonstrates that a receptor co-expressed with human OX40 can be used as a cross-linking agent on the T cell surface.

[0692] 8.2 Use mAbs containing anti-human OX40 Fcab and anti-PD1 Fab 2 Human T cell activation assay

[0693] In this assay, the mAb / mAb ratios listed in Table 22 below were determined using the co-expression of human OX40 and PD1. 2 Cross-linking. mAbs with LALA mutations were prepared using the variable regions of anti-FITC antibody 4420 (SEQ ID NO: 167 and 156), anti-PD1 antibody 5C4 (US 8,008,449B2), or anti-OX40 antibody 11D4 (EP 2242771B1). 2 In the same manner as described in Example 4.1.

[0694] Table 22: Tested mAbs and mAbs 2

[0695] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC none hIgG1 none FITC-glucan G1AA / 5C4 PD1 none hIgG1 have Anti-hCH2 G1 / 11D4 OX40 none hIgG1 none Anti-hCH2 FS20-22-49AA / 4420 FITC OX40 hIgG1 have none FS20-22-49AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20-22-49AA / 5C4 PD1 OX40 hIgG1 have none

[0696] T cells were isolated and activated as described in Example 5.1.1 above, and the T cells were used in essentially the same manner as in Example 8.1, but with the use of the above-described positive control antibody and mAb2, as well as the cross-linking agent described below. The production of hIL-2 was determined as previously described.

[0697] The crosslinking agent (anti-human CH2 mAb clone MK1A6 or FITCdex (Sigma); see Table 22) was used with the test mAb / mAb. 2 Add to the desired holes at a 1:1 molar ratio.

[0698] Table 23 shows the EC observed in the T cell activation assay with or without cross-linking with the cross-linking agent. 50 The maximum response value and IL-2 release. Figure 12 The graph shows IL-2 release for T cell activation assay.

[0699] Table 23: mAbs targeting co-expressed receptors 2 T cell activation assay

[0700]

[0701]

[0702] *In the absence of cross-linking, these control mAbs showed no activity in the T cell activation assay.

[0703] Figure 12 The results showed that T cell activation increased when OX40 was targeted and anti-OX40 antibodies were cross-linked. As expected, no T cell activation was observed with cross-linked anti-FITC antibody G1 / 4420 or cross-linked anti-PD1 antibody G1AA / 5C4. As previously mentioned, cross-linking of the OX40-targeting mAb G1 / 11D4 with an anti-human CH2 antibody induces T cell activation. As previously observed, the mimicry mAb... 2 The (4420LALA) form of Fcab FS20-22-49AA / 4420 targeting OX40 exhibits agonistic activity without cross-linking, and this activity is further enhanced by the addition of binding mAbs. 2 The cross-linking agent FITC-glucan of the Fab arm enhances this activity. When the OX40-targeting Fcab is paired with the anti-PD1 Fab (5C4), the agonistic activity of Fcab increases, indicating that mAb... 2Cross-linking occurs through binding to the receptor PD1 co-expressed on the surface of T cells.

[0704] With anti-OX40 / anti-PD1 mAb 2 The T-cell activation observed by the antibody suggests that the receptor-specific Fab binding site can be used as a cross-linking agent, which is co-expressed with human OX40 on the surface of T cells.

[0705] 8.3 Use mAbs containing anti-human OX40 Fcab and anti-LAG3 Fab 2 Human T cell activation assay

[0706] In this assay, the mAb / mAb ratios listed in Table 24 below were determined using the co-expression of human OX40 and LAG-3. 2 Crosslinking. As described in Example 4.1, mAbs with LALA mutations were prepared. 2 .

[0707] Table 24. Detected mAbs and mAbs 2

[0708] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC none hIgG1 none FITC-glucan G1 / 25F7 LAG3 none hIgG1 have Anti-hCH2 G1 / 11D4 OX40 none hIgG1 none Anti-hCH2 FS20-22-41AA / 4420 FITC OX40 hIgG1 have none FS20-22-41AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20-22-41AA / 25F7 LAG3 OX40 hIgG1 have none

[0709] T cells were isolated and activated as described in Example 5.1.1 above, and the T cells were used in essentially the same manner as in Example 8.1, but with the use of the above-described positive control antibody and mAb2, as well as the cross-linking agent described below. The production of hIL-2 was determined as previously described.

[0710] The cross-linking agent (anti-human CH2 mAb clone MK1A6 or FITC-glucan (Sigma); see Table 24) was used with the test mAb / mAb. 2 Add to the desired holes at a 1:1 molar ratio.

[0711] Table 25: T cell activation in the presence of co-expressed receptor LAG3

[0712]

[0713]

[0714] *In the absence of cross-linking, these control mAbs showed no activity in the T cell activation assay.

[0715] Figure 13The results showed that T cell activation increased when OX40 was targeted and anti-OX40 antibodies were cross-linked. As expected, no T cell activation was observed with cross-linked anti-FITC antibody G1 / 4420 or cross-linked anti-LAG3 antibody G1 / 25F7. As previously described, the OX40-targeting mAb G1 / 11D4 induced T cell activation when cross-linked with an anti-human CH2 antibody. As previously observed, the mimicry mAb... 2 The (4420LALA) form of Fcab FS20-22-41AA / 4420 targeting OX40 exhibits agonistic activity without cross-linking, and this activity is further enhanced by the addition of binding mAbs. 2 The cross-linking agent FITC-glucan of the Fab arm enhances this activity. When the OX40-targeting Fcab is paired with the anti-LAG3 Fab (25F7), the agonistic activity of Fcab increases, indicating that mAb... 2 Cross-linking occurs through binding to receptors co-expressed on the surface of T cells.

[0716] With anti-OX40 / anti-LAG3 mAb 2 The T cell activation observed by the antibody suggests that the receptor-specific Fab binding site co-expressed with human OX40 on the T cell surface can be used as a cross-linking agent.

[0717] 8.4 Use of mAbs containing anti-mouse OX40 Fcab and anti-LAG3 Fab 2 Mouse T cell activation assay

[0718] In this assay, the co-expression of mouse OX40 and LAG-3 receptors was used to determine the cross-linking of the bispecific antibodies listed in Table 26 below. mAbs with the LALA mutation were prepared as described in Example 4.1. 2 .

[0719] Table 26. Tested mAbs and mAbs 2

[0720] <![CDATA[mAb / mAb 2 ]]> Fab integration to Fcab combines to Same type LALA mutation Crosslinking agent G1 / 4420 FITC none hIgG1 none FITC-glucan G1 / C9B7W LAG3 none hIgG1 none Anti-hCH2 G1 / OX86 OX40 none hIgG1 none Anti-hCH2 FS20m-232-91AA / 4420 FITC OX40 hIgG1 have none FS20m-232-91AA / 4420 FITC OX40 hIgG1 have FITC-glucan FS20m-232-91AA / C9B7W LAG3 OX40 hIgG1 have none

[0721] Mouse T cells were isolated and activated as described in Example 5.2.1, using essentially the same protocol as in Example 8.3, but with the positive control antibody and mAb described above. 2 And the crosslinking agent described below. The generation of mIL-2 was determined as previously described.

[0722] The cross-linking agent (anti-human CH2 mAb clone MK1A6 or FITC-glucan (Sigma); see Table 26) was used with the test mAb / mAb. 2 Add to the desired holes at a 1:1 molar ratio.

[0723] Table 27 shows the mAbs available in the presence of the test. 2 In the case of mAb, EC was observed in T cell activation assays. 50 The maximum response value and IL-2 release. Figure 14 A representative graph of IL2 release used in T cell activation assays is shown.

[0724] Table 27: T cell activation in the presence of LAG3

[0725]

[0726]

[0727] *In the absence of cross-linking, this control monoclonal antibody showed no activity in the T-cell activation assay.

[0728] Figure 14 The results showed that T cell activation increased when OX40 was targeted and anti-OX40 antibodies were cross-linked. As expected, no T cell activation was observed with cross-linked anti-FITC antibody G1 / 4420 or cross-linked anti-LAG3 antibody G1 / C9B7W. As previously mentioned, cross-linking the OX40-targeting mAb G1 / OX86 with an anti-human CH2 antibody induced T cell activation. (mimetic mAb) 2 The (4420LALA) form of OX40-targeting Fcab, FS20m-232-91AA / 4420, showed no agonistic activity without cross-linking, but exhibited potent T-cell activation upon the addition of FITC-dextran, a cross-linking agent binding to the Fab arm. When the OX40-targeting Fcab was paired with anti-LAG3 Fab (C9B7W), the resulting mAb… 2 It exhibited T cell activity without any other cross-linking agent. This indicates that mAb 2 It is cross-linked by binding with LAG3.

[0729] With anti-OX40 / anti-LAG3 mAb 2 The T cell activation observed by the antibody suggests that the receptor co-expressed with mouse OX40 on the T cell surface can be used as a cross-linking agent.

[0730] 8.5 Anti-mOX40 / anti-LAG3 mAb capable of producing OX40 agonist activity in vitro 2 Inhibit tumor growth in the body

[0731] Since TILs isolated from CT26 tumors contain T cells expressing OX40 and LAG3 receptors, this experiment used a CT26 syngeneic tumor model.

[0732] The anti-mOX40 / anti-LAG3 mAb of Example 8.4 was tested.2 In vivo activity of antibody (FS20m-232-91AA / C9B7W) in a CT26 syngeneic mouse tumor growth model. The in vivo activity of anti-mOX40 / anti-LAG3 mAb was assessed. 2 The ability to inhibit tumor growth was compared with that of the PBS control.

[0733] Female BALB / c mice were injected with CT26 cells, and their health status and tumor growth were monitored. They were then classified and randomly assigned to experimental groups as described in Example 5. Mice without tumors were removed from the study at this point.

[0734] As described in Example 5, regarding mAb 2 The / mAb was analyzed to check for impurities, and the solution was prepared and administered to mice. As described in Example 5, the animals underwent health checks, tumor measurements were performed, and tumor volume was calculated.

[0735] The experiment was stopped on day 24 when the tumor burden was considered to be nearing its limit, and all mice were humanely euthanized. Results were as follows: Figure 15 As shown. Statistical analysis of the final tumor volume was performed using the two-tailed Student's t-test from the GraphPad Prism software package.

[0736] In terms of inhibiting tumor growth, anti-mOX40 / anti-LAG3 mAb 2 There was a statistically significant difference compared to the PBS control (normal growth).

[0737] Surprisingly, compared to the PBS control, the anti-mOX40 / anti-LAG3 mAb... 2 The treated groups showed significant growth inhibition. This assay demonstrates that, similar to observed in vitro results, growth inhibition can be achieved by targeting mAbs that target LAG3, which is co-expressed with OX40 in tumor-infiltrating T cells. 2 OX40 crosslinking leads to T cell activation and subsequent tumor growth control.

[0738] sequence list

[0739] The amino acid sequence of the CH3 domain and structural ring of WT Fcab

[0740] WT Fcab AB ring–RDELTKNQ (SEQ ID NO:1)

[0741] WT Fcab CD ring–SNGQPENNY(SEQ ID NO:2)

[0742] WT Fcab EF loop–DKSRWQQGNV (SEQ ID NO:3)

[0743] The amino acid sequence of the CH3 domain of WT Fcab (SEQ ID NO:4)

[0744] Underlined at the AB, CD and EF loops

[0745] GQPREPQVYTLPPS RDELTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNY KTTPPVLDSDGSFFLYSKLTV DKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

[0746] Fcab The amino acid sequence of the CH2 domain (SEQ ID NO: 1) ID NO:5)

[0747] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0748] The amino acid sequence of the Fcab CH2 domain with the LALA mutation (SEQ ID NO:6)

[0749] Underlined at LALA mutation

[0750] APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0751] The amino acid sequence of the CH2 domain of Fcab with the LALA-PA mutation (SEQ ID NO:7)

[0752] Underlined at LALA-PA mutation

[0753] APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL A APIEKTISKAK

[0754] The amino acid sequence of WT Fcab with the LALA mutation (SEQ ID NO:8)

[0755] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0756]

[0757] The amino acid sequence of WT Fcab without the LALA mutation (SEQ ID NO:9)

[0758] Hinge region (underlined), CH2 structural domain (bold), and CH3 structural domain (italicized)

[0759]

[0760] The amino acid sequence of Fcab FS20-11 with the LALA mutation (SEQ ID NO:10)

[0761] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0762]

[0763] The amino acid sequence Fcab FS20-11 does not contain the LALA mutation (SEQ ID NO:11).

[0764] Hinge region (underlined), CH2 structural domain (bold), and CH3 structural domain (italicized)

[0765]

[0766] The amino acid sequence of the CH3 domain-ring structure of Fcab FS20-11-127

[0767] FS20-11-127 first sequence – DDND (SEQ ID NO:12)

[0768] FS20-11-127 second sequence – IPIGP (SEQ ID NO:13)

[0769] FS20-11-127 third sequence – WRHYVEEHP (SEQ ID NO:14)

[0770] The amino acid sequence of the CH3 domain of Fcab FS20-11-127 (SEQ ID NO:15)

[0771] The first, second, and third sequences are underlined.

[0772] GQPREPQVYTLPPSREE DDND VSLTCLVKGFYPSDIAVEWESNG IPIGP YKTTPPVLDSDGSFFLYSKLTVDKSRW WRHYVEEHP FLCSVMHEALHNHYTQKSLSLSPG

[0773] Nucleotide sequence of the CH3 domain of Fcab FS20-11-127 (SEQ ID NO:16)

[0774] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAGGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0775] The amino acid sequence Fcab FS20-11-127 contains the LALA mutation (SEQ ID NO:17).

[0776] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0777]

[0778] The nucleotide sequence of Fcab FS20-11-127 with the LALA mutation (SEQ ID NO:18)

[0779] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAGGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0780] The amino acid sequence of Fcab FS20-11-127 without the LALA mutation (SEQ ID NO:19)

[0781] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

[0782]

[0783] Nucleotide sequence of Fcab FS20-11-127 without the LALA mutation (SEQ ID NO:20)

[0784] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAGGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0785] FS20-11-127 / 4420 mimic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:21)

[0786] VH domain (underlined)

[0787] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKSRWWRHYVEEHPFLCSVMHEALHNHYTQKSLSLSPG

[0788] FS20-11-127 / 4420 simulated mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:22)

[0789] VH domain (underlined)

[0790] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKSRWWRHYVEEHPFLCSVMHEALHNHYTQKSLSLSPG

[0791] The amino acid sequence of the CH3 domain-ring structure of Fcab FS20-11-131

[0792] FS20-11-131 First Sequence – DDND (SEQ ID NO:12)

[0793] FS20-11-131 Second Sequence – IPIGP (SEQ ID NO:13)

[0794] FS20-11-131 third sequence – WKHYVDEHP (SEQ ID NO:23)

[0795] The amino acid sequence of the CH3 domain of Fcab FS20-11-131 (SEQ ID NO:24)

[0796] The first, second, and third sequences are underlined.

[0797] GQPREPQVYTLPPSREE DDND VSLTCLVKGFYPSDIAVEWESNG IPIGP YKTTPPVLDSDGSFFLYSKLTVDKNRW WKHYVDEHP FLCSVMHEALHNHYTQKSLSLSPG

[0798] The nucleotide sequence of the CH3 domain of Fcab FS20-11-131 (SEQ ID NO:25)

[0799] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAACAGATGGTGGAAGCATTATGTTGATGAGCATCCGTTCTTGTGCTCCGGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0800] The amino acid sequence of Fcab FS20-11-131 with the LALA mutation (SEQ ID NO:26)

[0801] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0802]

[0803] The nucleotide sequence of Fcab FS20-11-131 with the LALA mutation (SEQ ID NO:27)

[0804] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAACAGATGGTGGAAGCATTATGTTGATGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0805] The amino acid sequence of Fcab FS20-11-131 without the LALA mutation (SEQ ID NO:28)

[0806] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

[0807]

[0808] Nucleotide sequence of Fcab FS20-11-131 without the LALA mutation (SEQ ID NO:29)

[0809] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAACAGATGGTGGAAGCATTATGTTGATGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0810] FS20-11-131 / 4420 simulated mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:30)

[0811] VH domain (underlined)

[0812] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKNRWWKHYVDEHPFLCSVMHEALHNHYTQKSLSLSPG

[0813] FS20-11-131 / 4420 simulated mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:31)

[0814] VH domain (underlined)

[0815] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKNRWWKHYVDEHPFLCSVMHEALHNHYTQKSLSLSPG

[0816] The amino acid sequence of the CH3 domain-ring structure of Fcab FS20-11-134

[0817] FS20-11-134 First Sequence – DDND (SEQ ID NO:12)

[0818] FS20-11-134 Second Sequence – IPIGP (SEQ ID NO:13)

[0819] FS20-11-134 third sequence – WKHYVEEHP (SEQ ID NO:32)

[0820] The amino acid sequence of the CH3 domain of Fcab FS20-11-134 (SEQ ID NO:33)

[0821] The first, second, and third sequences are underlined.

[0822] GQPREPQVYTLPPSREE DDND VSLTCLVKGFYPSDIAVEWESNG IPIGP YKTTPPVLDSDGSFFLYSKLTVDKSRW WKHYVEEHP FLCSVMHEALHNHYTQKSLSLSPG

[0823] The nucleotide sequence of the CH3 domain of Fcab FS20-11-134 (SEQ ID NO:34)

[0824] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAAGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0825] The amino acid sequence of Fcab FS20-11-134 with the LALA mutation (SEQ ID NO:35)

[0826] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0827]

[0828] The nucleotide sequence of Fcab FS20-11-134 with the LALA mutation (SEQ ID NO:36)

[0829] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAAGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0830] The amino acid sequence of Fcab FS20-11-134 without the LALA mutation (SEQ ID NO:37)

[0831] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

[0832]

[0833] Nucleotide sequence of Fcab FS20-11-134 without the LALA mutation (SEQ ID NO:38)

[0834] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAAGATGATAACGATGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCCCAATCGGTCCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGTGGAAGCATTATGTTGAGGAGCATCCGTTCTTGTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

[0835] FS20-11-134 / 4420 mimic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:39)

[0836] VH domain (underlined)

[0837] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKSRWWKHYVEEHPFLCSVMHEALHNHYTQKSLSLSPG

[0838] FS20-11-134 / 4420 simulated mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:40)

[0839] VH domain (underlined)

[0840] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEDDNDVSLTCLVKGFYPSDIAVEWESNGIPIGPYKTTPPVLDSDGSFFLYSKLTVDKSRWWKHYVEEHPFLCSVMHEALHNHYTQKSLSLSPG

[0841] The amino acid sequence of Fcab FS20-22 with the LALA mutation (SEQ ID NO:41)

[0842] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0843]

[0844] The amino acid sequence of Fcab FS20-22 without the LALA mutation (SEQ ID NO:42)

[0845] Hinge region (underlined), CH2 structural domain (bold), and CH3 structural domain (italicized)

[0846]

[0847] The amino acid sequence of the CH3 domain-ring structure of Fcab FS20-22-38

[0848] FS20-22-38 first sequence – YWDQE (SEQ ID NO:43)

[0849] FS20-22-38 second sequence – AEKYQ (SEQ ID NO:44)

[0850] FS20-22-38 third sequence – QYRWNPGDY (SEQ ID NO:45)

[0851] The amino acid sequence of the CH3 domain of Fcab FS20-22-38 (SEQ ID NO:46)

[0852] The first, second, and third sequences are underlined.

[0853] GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG AEKYQ YKTTPPVLDSDGSFFLYSKLTVD QYRWNPGDY FSCSVMHEALHNHYTQKSLSLSPG

[0854] The nucleotide sequence of the CH3 domain of Fcab FS20-22-38 (SEQ ID NO:47)

[0855] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGCAGAAAAATACCAGTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0856] The amino acid sequence of Fcab FS20-22-38 with the LALA mutation (SEQ ID NO:48)

[0857] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), and LALA mutation (bold and italic)

[0858]

[0859] The nucleotide sequence of Fcab FS20-22-38 with the LALA mutation (SEQ ID NO:49)

[0860] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGCAGAAAAATACCAGTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0861] The amino acid sequence of Fcab FS20-22-38 without the LALA mutation (SEQ ID NO:50)

[0862] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

[0863]

[0864]

[0865] Nucleotide sequence of Fcab FS20-22-38 without the LALA mutation (SEQ ID NO:51)

[0866] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGCAGAAAAATACCAGTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

[0867] FS20-22-38 / 4420 mimic mAb with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:52)

[0868] VH domain (underlined)

[0869] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGAEKYQYKTTPPVLDSDGSFFLYSKLTVDQYRWNPGDYFSCSVMHEALHNHYTQKSLSLSPG

[0870] FS20-22-38 / 4420 simulated mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:53)

[0871] VH domain (underlined)

[0872] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGAEKYQYKTTPPVLDSDGSFFLYSKLTVDQYRWNPGDYFSCSVMHEALHNHYTQKSLSLSPG

[0873] The amino acid sequence of the CH3 domain-loop structure of Fcab FS20-22-41

[0874] FS20-22-41 First Sequence – YWDQE (SEQ ID NO:43)

[0875] FS20-;22-41 Second Sequence – DEQFA (SEQ ID NO:54)

[0876] FS20-22-41 third sequence – QYRWNPGDY (SEQ ID NO:45)

[0877] The amino acid sequence of the CH3 domain of Fcab FS20-22-41 (SEQ ID NO:55)

[0878] The first, second, and third sequences are underlined.

[0879] GQPREPQVYTLPPS RDEYWDQE VSLTCLVKGFYPSDIAVEWE SNGDEQFAY KTTPPVLDSDGSFFLYSKLTV DQYRWNPGDY FSCSVMHEALHNHYTQKSLSLSPG

[0880] The nucleotide sequence of the CH3 domain of Fcab FS20-22-41 (SEQ ID NO:56)

[0881] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGA

[0882] The amino acid sequence of Fcab FS20-22-41 with the LALA mutation (SEQ ID NO:57)

[0883] Hinge region (underlined), CH2 domain (bold), CH3 domain (italic), LALA mutation (bold and underlined)

[0884]

[0885] The nucleotide sequence of Fcab FS20-22-41 with the LALA mutation (SEQ ID NO:58)

[0886] ACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGA

[0887] The amino acid sequence of Fcab FS20-22-41 without the LALA mutation (SEQ ID NO:59)

[0888] Hinge region (underlined), CH2 domain (bold), and CH3 domain (italic)

[0889]

[0890] Nucleotide sequence of Fcab FS20-22-41 without LALA mutation (SEQ ID NO:60)

[0891] ACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGA

[0892] FS20-22-41 / 4420 simulated mAbs with LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:61)

[0893] VH domain (underlined)

[0894] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPGDYFSCSVMHEALHNHYTQKSLSLSPG

[0895] FS20-22-41 / 4420 simulated mAb without LALA mutation 2 The amino acid sequence of the heavy chain (SEQ ID NO:174)

[0896] VH domain (underlined)

[0897] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGR FTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPG DY FSCSVMHEALHNHYTQKSLSLSPG

[0898] The amino acid sequence of the CH3 domain-loop structure of Fcab FS20-22-47

[0899] FS20 - 22 - 47 First Sequence – YWDQE (SEQ ID NO:43)

[0900] FS20 - 22 - 47 Second Sequence – DEQFA (SEQ ID NO:54)

[0901] FS20-22-47 third sequence – QYRWSPGDY (SEQ ID NO:62)

[0902] The amino acid sequence of the CH3 domain of Fcab FS20-22-47 (SEQ ID NO:63)

[0903] The first, second, and third sequences are underlined.

[0904] GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG DEQFA YKTTPPVLDSDGSFFLYSKLTVD QYRWSPGDY FSCSVMHEALHNHYTQKSLSLSPG

[0905] Nucleotide sequence of the CH3 domain of Fcab FS20-22-47 (SEQ ID NO:64)

[0906] GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG...

Claims

1. An antibody molecule or an antigen-binding fragment thereof that binds to OX40, comprising an OX40 antigen-binding site located in the CH3 domain of the antibody molecule or the antigen-binding fragment thereof, The antibody molecule or antigen-binding fragment thereof comprises a CH3 domain sequence as shown in SEQ ID NO: 72, 55, 63, 81 or 46.

2. The antibody molecule or antigen-binding fragment thereof according to claim 1, wherein the CH3 domain comprises an additional lysine residue immediately C-terminal to the CH3 domain sequence.

3. The antibody molecule or antigen-binding fragment thereof according to claim 1, wherein the antibody molecule or antigen-binding fragment thereof comprises the following sequence: (i) as shown in SEQ ID NO:74, 57, 65, 83 or 48; or (ii) as shown in SEQ ID NO:76, 59, 67, 85 or 50.

4. An antibody molecule or antigen-binding fragment thereof that binds to OX40, comprising an OX40 antigen-binding site located in the CH3 domain of the antibody molecule or antigen-binding fragment thereof, wherein The antibody molecule or antigen-binding fragment thereof comprises a CH3 domain sequence as described below, as shown in SEQ ID NO: 143, 134, 94, 114, 124 or 103.

5. The antibody molecule or antigen-binding fragment thereof according to claim 4, wherein the CH3 domain comprises an additional lysine residue immediately C-terminal to the CH3 domain sequence.

6. The antibody molecule or antigen-binding fragment thereof according to claim 4, wherein the antibody molecule or antigen-binding fragment thereof comprises the following sequence: (i) as shown in SEQ ID NO: 145, 136, 96, 116, 126 or 105; or (ii) as shown in SEQ ID NO:147, 138, 98, 118, 128 or 107.

7. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule or antigen-binding fragment thereof further comprises an antigen-binding site based on CDRs.

8. The antibody molecule or antigen binding fragment thereof according to claim 7, wherein the CDR-based antigen binding site binds a second antigen selected from the group consisting of an immune cell antigen, a tumor antigen and a pathogen antigen. 9 . The antibody molecule or antigen-binding fragment thereof according to claim 1 , wherein the antibody molecule or antigen-binding fragment thereof does not bind to Fcγ receptors.

10. A nucleic acid molecule encoding the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 9. A recombinant host cell comprising the nucleic acid molecule of claim 10 .

12. A method for producing the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing the recombinant host cell according to claim 11 under conditions where the antibody molecule or antigen-binding fragment thereof is produced.

13. Use of the antibody molecule or antigen binding fragment thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating colon cancer in an individual.

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