Improved anti-ox40l antibodies

Abstract

The present invention relates to antibodies and antibody derivatives against OX40L, which have improved effects compared to prior art antibodies.

Description

Technical Field

[0001] This invention relates to antibodies and antibody derivatives targeting OX40L, which have improved effects compared to antibodies of the prior art. Background Technology

[0002] The immune system is composed of various cell types, divided into the innate immune system and the adaptive immune system. At the cellular level, the innate immune system is composed of cells such as macrophages, dendritic cells, monocytes, and granulocytes, while the adaptive immune system is composed of T cells and B cells. Cells of the innate immune system play a crucial role in connecting the innate and adaptive immune responses.

[0003] T cell activation is achieved through two cell-mediated signaling mechanisms: first, the interaction between MHC (metaphorically presenting antigens) and the TCR; and second, the interaction through co-stimulatory molecules (OX40 / OX40L, CD40L / CD40, etc.). The second signaling mechanism is essential for T cell activation. Third, soluble factors such as cytokines determine the final T cell response.

[0004] Human OX40L (gp34, CD252, SwissProt P23510) belongs to the tumor necrosis factor superfamily (TNFSF) and can be expressed on B cells, dendritic cells, macrophages, monocytes, endothelial cells, and fibroblasts (Review: Weinberg, AD, Trends Immunol. 23 (2002) 102-109). Its associated receptor OX40 is expressed on T cells. OX40 / OX40L is a co-stimulatory pathway. It is not constitutively expressed, but rather inducibly expressed approximately 2-3 days after T cell activation. The interaction between OX40 and OX40L has been studied. In its crystallizable form, OX40L exists as a trimer and interacts with OX40 in this trimer form (Compaqueous). et Hymowitz. (2006).

[0005] Yoshioka, A. et al. (2000) demonstrated the therapeutic potential of neutralizing anti-mOX40LmAb in a mouse model of rheumatoid arthritis. Administration of this antibody significantly reduced the severity of the disease. The antibody also showed similar activity in other related disease models, such as inflammatory skin disease, experimental autoimmune disease (EAE), GvHD, and inflammatory bowel disease.

[0006] Unhealthy T-cell activation is a pathological driver of many diseases, such as autoimmune diseases (e.g., diabetes, multiple sclerosis, SLE, rheumatoid arthritis), inflammatory diseases (asthma, atopic dermatitis, chronic sinusitis, inflammatory bowel disease), and transplant-related diseases (graft-versus-host disease, allogeneic transplant rejection). In numerous cases, inhibiting the activation and proliferation of pathogenic T cells has been demonstrated to alleviate various diseases (Chatenoud et al, 1994; Haile et al, 2020).

[0007] Monoclonal antibodies can undergo various post-translational modifications, including glycosylation. Monoclonal antibodies, such as IgG, have an N-linked glycosylation site at position 297 (Asn297) of each heavy chain (two per complete antibody). The glycans linked to Asn297 on antibodies are typically complex biantennary structures with very low or no di-G1cNAc content, low terminal sialic acid content, and varying galactose content. These glycans also often exhibit high levels of core fucosylation. Studies have shown that reducing core fucosylation in antibodies alters Fc receptor affinity, thereby changing Fc-mediated effector function. In particular, the absence of core fucosylation on Fc N-glycans has been shown to increase the binding affinity of IgG1 Fc to FcγRIIIa on the surface of immune effector cells (e.g., natural killer cells), resulting in enhanced ADCC activity. This discovery has sparked interest in producing antibodies with reduced core fucosylation.

[0008] Other anti-OX40L antibodies

[0009] Antibodies against human OX40L are known to exist. One example is Oxelumab (clone R4930), which was previously developed for the treatment of asthma. A description of this antibody can be found in US 7501496 B1.

[0010] Amlitelimab (also known as KY1005 / SAR445229) is another anti-OX40L antibody used to treat atopic dermatitis. Its description can be found in US9139653B1 and WO2015 / 132580 A1. Amlitelimab is a non-depleting IgG4 isotype antibody. WO 2018 / 083248 demonstrates the efficacy of Amlitelimab in a primate GVHD model. Invention Overview

[0012] This invention provides antibodies, antibody derivatives, and methods for preparing anti-OX40L antibodies and antibody derivatives with reduced core fucosylation. The antibodies of this invention are Fc engineered to improve their affinity for multiple Fcγ receptors.

[0013] Currently, clinical drugs used to treat autoimmune and inflammatory diseases often cause serious adverse reactions and safety issues due to their non-specific immunomodulatory effects. In fact, widespread depletion of T cells without differentiating between pathological populations can lead to a variety of adverse consequences, such as increased cancer risk, higher transplant failure rates, disease relapse, drug toxicity, and decreased immunity to post-transplant infections (such as cytomegalovirus infection), ultimately limiting the long-term use of these drugs.

[0014] The antibodies, antibody derivatives, and methods described herein are partly based on unexpected results presented in the examples: antibodies and antibody derivatives with higher affinity for both OX40L and Fc receptors can be prepared through affinity maturation and Fc region optimization to reduce core fucosylation. Compared to antibodies or antibody derivatives in the prior art, these antibodies and antibody derivatives exhibit higher effector function (ADCC), lower T cell activation, a richer regulatory T cell (T-Reg) population, and the ability to maintain a naïve immune cell population. These improved properties have been demonstrated both in vitro and in vivo.

[0015] In fact, the antibodies, antibody derivatives, and methods of the present invention exhibit higher affinity for the human OX40L antigen compared to known anti-OX40L antibodies (such as Oxelumab). Furthermore, compared to prior art antibodies and other binding proteins known to manipulate T cell activation via APC targeting (such as Belatacept, Oxelumab, and Amlitelimab), the antibodies, antibody derivatives, and methods of the present invention unexpectedly and significantly reduce the proliferation of CD4+ and CD8+ T cells. Moreover, compared to prior art antibodies and other binding proteins known to manipulate T cell activation via APC targeting (such as Belatacept, Oxelumab, and Amlitelimab), the antibodies, antibody derivatives, and methods of the present invention induce a significant increase in T-Reg and naive CD4+ T cell populations within the proliferating cell population. These data are unexpected because there have been no previous reports on the level of T cell control and the induction of regulatory T cell phenotypes in proliferating cells. Meanwhile, compared to controls and another antibody targeting OX40L, the antibodies, antibody derivatives, and methods of the present invention reduce the levels of TNFα and IL-4. Surprisingly, compared to previously published non-Fc region-optimized anti-OX40L antibodies (Tripathi et al., 2019), the antibodies, antibody derivatives, and methods of the present invention exhibit a 3-4 fold increase in in vivo control of chimerism / graft proliferation. Interestingly, compared to known Fc-active (Oxelumab) and Fc-silencing (Amlitelimab) anti-OX40L antibodies in the art, the antibodies, antibody derivatives, and methods of the present invention demonstrate superior efficacy, highlighting the unexpected effect of the core fucosylation reduction of the antibodies, antibody derivatives, and methods of the present invention.

[0016] Based on the embodiments, the antibodies, antibody derivatives, and methods of the present invention are superior to prior art anti-OX40L antibodies. The present invention provides an alternative strategy for controlling T cells in a non-depleting manner while maintaining the naive phenotype of the immune cell population (i.e., naive T cells and Tregs). T cells are indirectly controlled by blocking OX40-OX40L. The important role of effector T cells in the pathogenesis of many autoimmune and inflammatory diseases, and the preferential localization of OX40-OX40L at sites of inflammation by co-stimulatory receptor-ligands, validate this strategy. Accordingly, this strategy avoids the complex downstream effects of directly targeting T cells and preserves the self-regulatory capacity of the adaptive immune system.

[0017] The antibodies and antibody derivatives described herein possess novel and innovative properties, and therefore offer significant benefits to patients requiring antibody therapy against OX40L, particularly those with inflammatory diseases and autoimmune diseases, including rheumatoid arthritis, allergic asthma, and post-transplant GvHD.

[0018] On one hand, the present invention provides a polypeptide comprising the amino acid sequence (VL) of SEQ ID NO: 1.

[0019] On the other hand, the present invention provides an anti-OX40L antibody having a variable light chain and a variable heavy chain, wherein the variable light chain comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; And the variable heavy chain mentioned therein includes the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8.

[0020] On the other hand, the present invention provides an anti-OX40L antibody comprising a heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; Furthermore, the heavy chain described herein comprises a constant region and a VH, wherein the VH comprises the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8, and, The core fucosylation of the heavy chain constant region is less than 80%, expressed as a percentage of fucosylated glycans to total glycans.

[0021] In some aspects, the core fucosylation of the heavy chain constant region is less than 80%, which is determined by capillary electrophoresis, LC-MS, high-performance liquid chromatography with fluorescence detection (HPLC-FD), high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), mass spectrometry, hydrazine hydrolysis and / or enzymatic hydrolysis.

[0022] On the other hand, the present invention provides an anti-OX40L antibody comprising an IgG1 heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; And the IgG1 heavy chain contains the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8, and, The core fucosylation of the IgG1 heavy chain is less than 80%.

[0023] On the other hand, the present invention provides an antibody comprising a light chain comprising the VL sequence of SEQ ID NO:1, wherein the core fucosylation of the antibody is less than 80%.

[0024] On the other hand, the present invention provides an anti-OX40L antibody comprising a heavy chain and a light chain, wherein the light chain comprises the VL sequence of SEQ ID NO: 1, and wherein the core fucosylation of the heavy chain is less than 80%.

[0025] In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the antibody comprises an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region is glycosylated.

[0026] On the other hand, the present invention provides an antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing a VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0027] On the other hand, the present invention provides an anti-OX40L antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0028] In some embodiments, the antibody comprises a variable heavy chain of SEQ ID NO: 5 and a variable light chain of SEQ ID NO: 1. In another embodiment, the antibody comprises a heavy chain of SEQ ID NO: 14 and a light chain of SEQ ID NO: 13.

[0029] The antibody described in this invention is preferably characterized as a fully human anti-OX40L antibody with mature affinity for the IgG1 subclass.

[0030] On one hand, the present invention provides a method for producing anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing inhibitors of glucosidase I and II, and subsequently purifying the antibody.

[0031] On one hand, the present invention provides a method for producing anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing a fucose analogue, and the antibody is subsequently purified.

[0032] On one hand, the present invention provides a method for producing anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells to prevent core fucosylation.

[0033] On one hand, the present invention provides a method for producing anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO: 13 and SEQ ID NO: 14 in cells, subsequently purifying the antibody, and optionally folding the antibody.

[0034] On one hand, the present invention provides a method for testing defucosylated anti-OX40L antibody or antibody derivative, including testing the antibody in a T cell activation assay and verifying whether T cell activation is reduced compared to the fucosylated anti-OX40L antibody. Attached Figure Description

[0035] Figure 1 Inhibitory effect of 2-fluorofucose (2-FF) on fucosylation. The X-axis represents the concentration of 2-FF (µM). The Y-axis represents the relative fucosylation (%). Treatment with 200 µM 2-FF reduced fucosylation to less than 40%.

[0036] Figure 2Affinities of OX118 to various Fc receptors. The X-axis represents the degree of fucosylation. The Y-axis represents the affinity for Fc receptors. Decreased fucosylation leads to increased affinity for CD16a (FcRgIIIa / CD16a 158V, FcRgIIIa / CD16a 158F), while having no effect on binding to FcRn, C1q, or CD32a.

[0037] Figure 3 CD3+ T cell activation was reduced after OX118 treatment.

[0038] The X-axis represents antibody concentration (ng / mL), and the Y-axis represents T cell activation level (CD3+). Two variants of OX118 were compared: one with reduced fucosylation (34%) and the other with complete fucosylation (97%). The reduced fucosylation level led to decreased T cell activation.

[0039] Figure 4 OX118 reduced T cell proliferation compared to different anti-OX40L antibodies and CTLA-4 Ig. (A) CD4+ and (B) CD8+ T cells were treated with different anti-OX40L antibodies; anti-OX40L (10 µg / mL) and CTLA-4 Ig (15 µg / mL). The X-axis represents the treatment conditions. The Y-axis represents the percentage of proliferating T cells (low CFSE) after 7 days of incubation under bidirectional MLR conditions. OX118 (10 µg / mL) resulted in lower proliferation of both CD4+ and CD8+ T cells compared to control and other forms of OX40L targeting (10 µg / mL) and CTLA-4 Ig (15 µg / mL).

[0040] Figure 5 OX118 increases the proportion of regulatory T cells (T-Reg) in proliferating CD4+ T cells. (A) Addition of OX118 (10 µg / mL) to a two-way MLR resulted in an increase in regulatory phenotypes (Treg, CD25hi, CD4+, FoxP3+) in proliferating CD4+ cells compared to control, other forms of anti-OX40L antibody (10 µg / mL), and CTLA-4 Ig (15 µg / mL). (B) A concentration-dependent increase in the T-reg population in proliferating CD4+ cells compared to control, other forms of anti-OX40L antibody, and CTLA-4 Ig. The X-axis represents the concentration of the test substance (ng / mL).

[0041] Figure 6OX118 increases the proportion of naive CD4+ T cells in proliferating CD4+ T cells. The X-axis represents the treatment condition. The Y-axis represents the percentage of naive (CD3+, CD4+, CD45RA+, CCR7+, low CFSE) CD4+ T cells in proliferating cells. Adding OX118 at a concentration of 10 µg / mL to a two-way MLR resulted in an increase in naive T cells in proliferating CD4+ T cells compared to control and anti-OX40L IgG1 ref (10 µg / mL) and CTLA-4 Ig (15 µg / mL).

[0042] Figure 7 OX118 inhibited the induction of pro-inflammatory phenotypes. The X-axis represents treatment conditions. The Y-axis represents cytokine concentrations (ng / mL); (A) IL-10, (B) IL-4, and (C) TNFα in the supernatant. Adding OX118 (10 µg / mL) to a two-way MLR resulted in lower IL-4 and TNFα levels compared to the control. IL-10 levels were comparable to the control.

[0043] Figure 8 OX118 treatment prevented GvHD in mice transplanted with human peripheral blood mononuclear cells (PBMCs). The Y-axis represents the frequency of circulating human CD45+ leukocytes (hCD45+) in mice. The X-axis represents the number of days after GvHD induction (days 7, 14, and 21). Peripheral blood chimera or graft proliferation. Flow cytometry-assisted immunophenotyping was performed on days 7, 14, and 21. hCD45+ cells were counted and compared between the treatment and control groups.

[0044] Figure 9 OX118 reduces the activation of T cells in peripheral blood of mice after GvHD induction. The X-axis represents the treatment. The Y-axis represents the percentage of activated CD4+, CD8+ T cells, and CD14+ monocytes in peripheral blood.

[0045] Figure 10 OX118 increased the proportion of regulatory T cells (T-Reg) in proliferating CD4+ T cells after GvHD induction in mice. The X-axis represents treatment. The Y-axis represents the percentage of regulatory T cells in CD4+ cells. Regulatory T cells were defined as CD3+, CD4+, CD25+, and FoxP3+, and were detected by FACS. Data were obtained 22 days after GvHD induction.

[0046] Figure 11Treatment A effectively suppressed the GvHD response in NSG mice. A) Clinical scores, with mice treated with treatment A scoring lower than those treated with treatment B. B) Weight changes during the study period. C) Chimerism (defined as the percentage of hCD45-positive cells), with significantly reduced chimerism in animals treated with treatment A. D) Tissue-infiltrating hCD45+ cells in the skin, lungs, and colon, expressed as positivity rate (positivity rate / total cell count). 100).

[0047] Figure 12 Three batches of OX118 were compared with trastuzumab using ADCC assays. The X-axis represents treatment concentration. The Y-axis represents luminescence intensity (corresponding to ADCC signal in reporter cell lines). Each batch of OX118 showed reproducible results with high batch-to-batch consistency. OX118's high affinity for FcYRIIIa compared to the control antibody (trastuzumab) resulted in a lower EC50 value in ADCC signal assays. Invention Details

[0049] definition

[0050] As used herein, the singular forms “a,” “an,” and “the” all include plural references unless the context clearly specifies otherwise. Thus, for example, “an antibody” includes multiple such constructs.

[0051] The term "some implementation schemes" can include one or more implementation schemes.

[0052] The term "antibody" (immunoglobulin) as used herein can refer to oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies), camelified antibodies, chimeric antibodies, CDR transplanted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, anti-idiotype antibodies, and antibodies that can be labeled in soluble or bound forms, as well as fragments, variants, or derivatives thereof, which may exist alone or in combination with other amino acid sequences available by known technologies. Antibodies can be derived from any species. An antibody comprises one or a group of polypeptides containing at least one binding domain formed by a polypeptide chain folded in a three-dimensional binding space, the internal surface shape and charge distribution of which are complementary to the characteristics of the antigenic determinants of the antigen. Antibodies are typically in tetrameric form, comprising two pairs of identical polypeptide chains, each pair containing a "light chain" and a "heavy chain." The variable region of each light / heavy chain pair constitutes the antibody binding site. Natural antibodies are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). Each light chain is linked to one heavy chain by a covalent disulfide bond; the number of disulfide bonds on the heavy chain varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly arranged intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable domains of the light chain are aligned with the variable domains of the heavy chain. Based on the amino acid sequence of the constant regions of the light chains, they can be divided into λ chains and κ chains. The variable domain of the κ light chain can also be abbreviated as VK. The term "variable region" can also be used to describe the variable domains of either the heavy or light chains. Specific amino acid residues are considered to form interfaces between the variable domains of the light and heavy chains. The variable regions of each light / heavy chain pair constitute the antibody binding site.

[0053] The term "antibody derivative" refers to an antibody (including antibody fragments) as defined above, or the Fc domain or region of an antibody containing a complex N-glycoside-linked glycan chain, modified by covalently linking a heterologous molecule, such as by linking a heteropeptide (e.g., a ligand-binding domain of a heteroprotein), or by glycosylation (other than core fucosylation), deglycosylation (other than non-core fucosylation), acetylation, phosphorylation, or other modifications generally unrelated to the antibody or the Fc domain or region.

[0054] The term "antibody-dependent cytotoxicity (ADCC)" refers to the function mediated by Fc receptor binding, which means that in the presence of effector cells, the antibody of the present invention cleaves target cells expressing OX40L.

[0055] The term "monoclonal antibody" refers to an antibody derived from a single-cell clone (including any eukaryotic or prokaryotic cell clone, or a bacteriophage clone), not its production method. Therefore, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology.

[0056] The term "Fc region" refers to the constant region of an antibody, such as the Cnl-hinge-CH2-CH3 domain, optionally having a CH4 domain, or a conserved substituted derivative of such an Fc region.

[0057] The term "glycan" or "N-glycan" refers to an oligosaccharide containing the core pentasaccharide Man3GlcNAc2. N-glycans can be linked to proteins (glycoproteins) via the nitrogen atom of an asparagine (or occasionally arginine) residue and can also exist freely in solution. In this disclosure, unless otherwise stated, the term "glycan" refers to N-glycan. The term "oligosaccharide" refers to a glycan that is not covalently bound to a protein. Helpful references on the nomenclature of glycans, glycoproteins, and oligosaccharides can be found at https: / / iupac.qmul.ac.uk / misc / glycp.html. N-glycans exhibit great variability, but functionally important differences are generally limited to mannose content and the degree of fucylation (Reusch et al, 2015).

[0058] The term "glycosylation pattern" or "glycosylation profile" refers to the observed glycosylation of one or more given glycoproteins. Glycoproteins with a greater number of covalently linked sugar residues in their oligosaccharide chains are considered to have an increased or richer glycosylation pattern and / or increased total glycosylation. Conversely, glycoproteins with a smaller number of covalently linked sugar residues in their oligosaccharide chains are considered to have a decreased or less rich glycosylation pattern and / or decreased total glycosylation. The glycosylation pattern of monoclonal antibodies can vary depending on the synthetic or manufacturing method. As used herein, the term "glycosylation pattern" also refers to the characteristic distribution of several different glycosylation patterns on a single glycoprotein. In this sense, "increased glycosylation pattern" means an increase in the characteristic distribution of glycosylation patterns on the expressed glycoprotein.

[0059] As used herein, the term “total glycosylation” or “total polysaccharides” refers to the total amount of individual polysaccharides that are quantitatively analyzed by methods known in the art, such as capillary electrophoresis, high-performance liquid chromatography with fluorescence detection (HPLC-FD), high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), and mass spectrometry.

[0060] The term "Fc domain" refers to the constant region domain of an antibody, such as the CH1, hinge, CH2, CH3, or CH4 domain, or a conserved substituted derivative of such Fc domain.

[0061] As used herein, the term "activated T cell" refers to a T cell that expresses antigens indicating T cell activation (i.e., T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40, HLA-DR, and 4-1BB. The expression of activation markers can be detected using techniques known to those skilled in the art, such as Western blot analysis, Northern blotting analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorting (FACS) analysis.

[0062] Regulatory T cells, or Tregs, are a type of immune cell characterized by the expression of three biomarkers: CD4, FOXP3, and CD25. Sometimes referred to as suppressor T cells, Tregs are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs have immunosuppressive effects, typically inhibiting or downregulating the induction and proliferation of effector T cells (Teffs). Tregs can be generated in the thymus (i.e., so-called CD4+Foxp3+ "natural" Tregs) or differentiated from naive CD4+ T cells in the periphery, for example, after exposure to TGFβ or retinoic acid. Tregs can express GARP-proTGFβ1 on their cell surface.

[0063] As used in this article, the term "OX40L" refers to the associated ligand of the tumor necrosis factor receptor OX40 (CD134). OX40L functions as a T-cell co-stimulatory molecule. The OX40-OX40L interaction has been identified as a potential therapeutic target for treating autoimmune diseases.

[0064] Anti-OX40L antibody

[0065] On one hand, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (VL).

[0066] On the other hand, the present invention provides an anti-OX40L antibody having a variable light chain and a variable heavy chain, wherein the variable light chain comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; And the variable heavy chain mentioned therein includes the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8.

[0067] On the other hand, the present invention provides an anti-OX40L antibody, the antibody comprising a heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; Furthermore, the heavy chain described herein comprises a constant region and a VH, wherein the VH comprises the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8, and, The core fucosylation of the heavy chain constant region is less than 80%.

[0068] On the other hand, the present invention provides an anti-OX40L antibody comprising an IgG1 heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; And the IgG1 heavy chain contains the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8, and, The core fucosylation of the IgG1 heavy chain is less than 80%.

[0069] In some aspects, the core fucosylation of the heavy chain constant region is less than 80%, which is determined by capillary electrophoresis, LC-MS, high performance liquid chromatography with fluorescence detection (HPLC-FD), high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), mass spectrometry, hydrazine hydrolysis and / or enzymatic hydrolysis.

[0070] On the other hand, the present invention provides an antibody comprising a light chain comprising the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the antibody is less than 80%.

[0071] On the other hand, the present invention provides an anti-OX40L antibody comprising a heavy chain and a light chain, wherein the light chain comprises the VL sequence of SEQ ID NO: 1, and wherein the core fucosylation of the heavy chain is less than 80%.

[0072] In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the antibody comprises an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region is glycosylated.

[0073] On the other hand, the present invention provides an antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing a VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0074] On the other hand, the present invention provides an anti-OX40L antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0075] In some embodiments, the antibody comprises a variable heavy chain of SEQ ID NO: 5 and a variable light chain of SEQ ID NO: 1. In another embodiment, the antibody comprises a heavy chain of SEQ ID NO: 14 and a light chain of SEQ ID NO: 13.

[0076] Glycosylation mode

[0077] Fc glycosylation confers structural integrity to antibodies, but alterations in glycosylation patterns due to variations in manufacturing conditions have been reported to affect antibody sensitivity to proteolysis, in vivo clearance, Fcγ receptor binding and activation, antibody-dependent cytotoxicity (ADCC), and C1q component-mediated complement activation. Therefore, changes in glycosylation may impair antibody effector functions, including biological activity, clinical efficacy, pharmacokinetics, safety, stability, and antigenicity.

[0078] Those skilled in the art will foresee that due to differences in production methods, glycosylation patterns will exhibit heterogeneity. This can lead to variations in the glycosylation pattern, the relative abundance of each type of glycan, or the total amount of glycans, thereby reducing reproducibility, increasing batch-to-batch variability, and causing fluctuations in product quality. In fact, cell expression systems, culture conditions, and purification protocols all affect the heterogeneity of glycosylation patterns. The FDA and EMA recommend glycosylation pattern determination for glycoprotein drugs, indicating that the oligosaccharide content of glycoprotein products should be tested to ensure product consistency. Therefore, the glycosylation pattern and / or total glycosylation of the antibodies of this invention can vary, for example, + / - 1%, + / - 5%, + / - 10%, + / - 15%, + / - 20%, + / - 25%, + / - 30%, at least 30%, or at most 30%.

[0079] Those skilled in the art will understand that antibody glycosylation patterns can be determined by known and appropriate methods, such as capillary electrophoresis (Wacker et al., 2011), high-performance liquid chromatography with fluorescence detection (HPLC-FD), high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), and mass spectrometry. All of the above methods for determining glycosylation patterns require interpretation of the results, such as chromatogram interpretation. Interpretation of results may result in variations in glycosylation patterns, for example, ±5%. This can depend on the method used, and all methods have an error of 2-5%, for example, 2-4%.

[0080] This invention provides anti-OX40L antibodies and derivatives having the standard N-glycan (terminal glycosylation) pattern of human IgG1 expressed in CHO cells (e.g., CHO-K1 cells), as described, for example, as in Reusch & Tajeda, 2015 (“Fc glycansof therapeutic antibodies as critical quality attributes”, Glycobiology, Volume 25, Issue 12, December 2015, Pages 1325–1334) and / or Luo & Zhang 2023 (“Benchmark glycan profile of therapeutic monoclonal antibodies produced by Mammalian cell expression systems”, Pharma. Res.), but with reduced fucosylation. Luo & Zhang (2023) describe the most common N-glycan structures and terminal epitopes among FDA-approved monoclonal antibodies in Table 1. Examples of this application also characterize the normal glycosylation pattern of human IgG1 expressed in CHO-K1 cells. Therefore, the antibodies of this invention are themselves glycosylated. In one embodiment, the glycosylation pattern of the antibody of the present invention is determined by methods known in the art as described above. In one embodiment, the glycosylation pattern of the antibody of the present invention is determined relative to the expression system or synthesis or production method used for production, for example, the benchmark glycan analysis method disclosed in Luo & Zhang (2023). In one embodiment, the glycosylation pattern of the antibody of the present invention is determined relative to the monoclonal antibody reference standards established by the United States Pharmacopeia (USP), for example, the standards disclosed in Guo et al, 2022. In one embodiment, the glycosylation pattern of the antibody of the present invention has the standard N-glycan glycosylation pattern of human IgG1, but with reduced fucosylation. In a preferred embodiment, the antibody comprises a light chain (LC) and an IgG1 heavy chain (HC) as described herein, and is glycosylated with at least 2 kDa of total glycans. In a preferred embodiment, the antibody is glycosylated with up to 3 kDa of total glycans. In some embodiments, the antibody is glycosylated with at least 2 kDa of total glycans and up to 3 kDa of total glycans.

[0081] In some embodiments, the heavy chain constant region of the antibody is glycosylated with at least 2 kDa of total glycans. In some embodiments, the heavy chain constant region of the antibody is glycosylated with up to 3 kDa of total glycans. In some embodiments, the heavy chain constant region of the antibody is glycosylated with both at least 2 kDa and up to 3 kDa of total glycans.

[0082] In several embodiments of the invention, the provided antibody is engineered to contain a high proportion of glycan GO, specifically 50% to 80% of the total glycans, while reducing the core fucosylation level. In some embodiments, the antibody glycan contains 50% to 80% glycan GO. In some embodiments, the antibody glycan contains 50% to 80% glycan GO, for example 55% to 80%, for example 60% to 80%, for example 65% to 80%, for example 70% to 80%, for example 75% to 80%, for example 50% to 75%, for example 50% to 70%, for example 50% to 65%, for example 50% to 60%, for example 50% to 55% glycan GO.

[0083] In some embodiments, the total glycans of the antibody comprise 50% to 80% glycan GO. In other embodiments, the total glycans of the antibody comprise 50% to 80% glycan GO, for example 55% to 80%, 60% to 80%, 65% to 80%, 70% to 80%, 75% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55% glycan GO. Controlling the level of GO, combined with reduced core fucosylation, can enhance the functional properties of the antibody, including improving ADCC activity.

[0084] Fucosylation

[0085] The term "core fucosylation" used in this article refers to the addition of fucose ("fucosylation") to the reducing end of an N-linked glycan ("GicNAc"). In IgG1 antibodies, core fucosylation can occur at the N-glycosylation site of asparagine (Asn-297) in the Fc region. Reduction of core fucosylation in antibodies has been shown to alter Fc effector function, particularly Fcγ receptor binding and ADCC activity. This implies that antibodies lacking core fucosylation in the Fc glycan may exhibit higher ADCC activity at lower concentrations compared to their fucosylated counterparts (Yamane-Ohnuki and Satoh, 2009).

[0086] Generally, the terms "low fucosylation," "reduced fucosylation," or "decreased fucosylation" for glycoproteins do not refer to a smaller number of fucose residues linked to a single glycoprotein molecule. Rather, it refers to a glycoprotein preparation containing multiple individual glycoprotein molecules with different glycosylation characteristics. For example (and not limitingly), for the IgG1 antibody expressed in modified CHO cells according to this invention, "low fucosylation," "reduced fucosylation," or "decreased fucosylation" refers to a smaller number of individual glycoproteins with fucose residues on N-linked GICNAc residues. According to the present invention, such “low fucosylation” or “reduced fucosylation” can be characterized in a variety of ways (see other parts of this document), but in each case, it refers to a relatively low (or reduced) number of glycoproteins with fucosylate residues in the glycoprotein population compared to the same glycoprotein population produced in unmodified cell lines, or compared to the same glycoprotein population produced without exposure to inhibitors and / or analogs that reduce core fucosylation, or compared to the same glycoprotein population produced without exposure to culture conditions / additives that reduce fucosylation.

[0087] "Relative fucosylation" refers to the amount of fucosylated glycans compared to the total amount of glycans contained in the antibody molecule or antibody derivative.

[0088] On one hand, the present invention provides an anti-OX40L antibody comprising a variable heavy chain, wherein the core fucosylation of said heavy chain is less than 80%. In one embodiment, the fucosylation of said anti-OX40L antibody is only about 50%, for example, at most 40%, for example, at most 30%, for example, at most 20%, for example, at most 20%, for example, at most 10%, compared to the fucosylation level of the same glycoprotein produced in cells without this modification.

[0089] The fucosylation of the glycoprotein preparations of the present invention is only about 50%, for example, at most 40%, for example, at most 30%, for example, at most 20%, for example, at most 10%, of the same glycoprotein produced in unmodified cell lines, or produced without exposure to inhibitors and / or analogs that reduce core fucosylation, or produced without exposure to culture conditions / additives that reduce fucosylation.

[0090] The fucosylation of the glycoprotein preparations prepared in cells according to the present invention is only about 50%, for example, at most 40%, for example, at most 30%, for example, at most 20%, for example, at most 10%, of the same glycoprotein prepared in unmodified cell lines, or without exposure to inhibitors and / or analogs that reduce core fucosylation, or without exposure to culture conditions / additives that reduce fucosylation.

[0091] Another method for characterizing glycoproteins with reduced fucosylation is by measuring the ratio of fucosylated to non-fucosylated glycans in cell-prepared glycoprotein preparations, or glycoprotein preparations not exposed to inhibitors and / or analogs that reduce core fucosylation, or glycoprotein preparations not exposed to culture conditions / additives that reduce fucosylation. For example, the ratio of fucosylated to non-fucosylated glycans in glycoprotein preparations is approximately 1:10 to 1:15, 1:15 to 1:20, 1:20 to 1:40, 1:40 to 1:60, 1:60 to 1:80, 1:80 to 1:100, or 1:100 to 1:150.

[0092] Another method for characterizing glycoproteins with reduced fucosylation is by the relative weight percentage of non-fucosylated glycoproteins (compared to total glycoproteins, i.e., fucosylated and non-fucosylated glycoproteins). For example, compared to the same glycoprotein preparation prepared from unmodified cells, the percentage of non-fucosylated glycans in a glycoprotein preparation prepared from modified cells is at most 70%, for example, at most 60%, for example, at most 50%, for example, at most 40%, for example, at most 30%. For example, compared to the same glycoprotein preparation not exposed to cell culture conditions and / or additives that reduce glycoprotein fucosylation, the percentage of non-fucosylated glycans in a glycoprotein preparation prepared by exposure to cell culture conditions and / or additives that reduce glycoprotein fucosylation is at most 70%, for example, at most 60%, for example, at most 50%, for example, at most 40%, for example, at most 30%. Therefore, in some embodiments of the present invention, the polysaccharide having a core fucoidylation is less than 70% of the total polysaccharide, for example less than 60%, for example less than 50%, for example less than 40%, for example less than 30%, for example less than 20%.

[0093] In some embodiments, the antibodies prepared by the method of the present invention contain at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of non-core fucosylated glycans (e.g., lacking core fucosylation) compared to a control antibody. In some embodiments, the antibodies or antibody derivatives prepared by the method of the present invention contain at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of non-core fucosylated antibodies compared to a control antibody. In some embodiments, the compositions of antibodies or antibody derivatives prepared by the method of the present invention contain less than 100% non-core fucosylated antibodies and / or antibody derivatives.

[0094] Another method for characterizing glycoproteins with reduced fucosylation is by the relative content of fucose to glycans in the glycoprotein preparation, or the relative content of fucose to glycan components in the glycoprotein preparation. In one embodiment, the molar ratio of fucose to glycan fraction does not exceed about 1:10, 1:20, 1:25, 1:33, 1:50, 1:100, or 1:200.

[0095] In one embodiment, the amount of the prepared fucosylated antibody protein is determined by deglycosylation of the antibody protein with PNGase F followed by oligosaccharide analysis by HPLC, wherein the fucosylated oligosaccharides are quantified by integrating the glycan peak area, for example, the fucosylation of the protein is calculated based on the glycan peak area. The identity (and composition) of the glycan can be determined (and / or quantified) by any suitable method (e.g., mass spectrometry). Therefore, in some embodiments of the invention, core fucosylation is determined using capillary electrophoresis or HPLC, such as hydrophilic interaction liquid chromatography-HPLC (HILIC-HPLC), electrospray ionization mass spectrometry (ESI-MS), or hydrophilic interaction liquid chromatography-FLD-MS / MS equipped with fluorescence detection and tandem mass spectrometry.

[0096] Methods for determining core fucosylation include hydrazine hydrolysis or enzymatic hydrolysis (e.g., see Biochemical Experimentation Methods 23: Method for Studying Glycoprotein Sugar Chain (Japan Scientific Societies Press), edited by Reiko Takahashi (1989)), fluorescent labeling or radioisotope labeling of the released sugar chains, followed by chromatographic separation of the labeled sugar chains. Alternatively, the composition of the released sugar chains can be determined by analyzing the HPAEC-PAD method (e.g., see J Liq Chromatogr. 6:1557 (1983)). (See US 2004-0110282.)

[0097] In some embodiments of the present invention, core fucosylation is determined as (fucosylated glycan area) / (total glycan area). In some embodiments of the present invention, core fucosylation is determined as (fucosylated glycan concentration) / (total glycan concentration). The calculation of the fucosylation percentage takes into account the peak area of ​​all fucosylated substances within the total glycan area. The following formula (1) is used to calculate the fucosylation percentage: (1) Fucosylation percentage: Fucosylated glycan area / Total glycan area In antibodies with 100% core fucosylation, all glycans are core-fucosylated. In antibodies with 50% core fucosylation, every other glycan has one unfucosylated and every other glycan has one fucosylated. Defucosylation results in only a minor change in the overall molecular weight of the antibody because only the incorporation of the fucose moiety decreases / stops, while other sugars are largely unaffected.

[0098] In some embodiments of the present invention, the determination of core fucosylation is (amount of fucosylated polysaccharides) / (amount of total polysaccharides).

[0099] The results of fucosylation determination depend on the interpretation of results such as chromatograms. Interpretation of results can lead to variations in the degree of fucosylation, for example, ±5%. This variation varies depending on the method used; all fucosylation determination methods disclosed herein have an error of 2-5%, for example, 2-4%.

[0100] In some embodiments of the invention, the antibody is covalently linked to a ligand selected from: chromophores, fluorophores, radiotracers, drugs, peptides, proteins, enzymes, single- or double-stranded oligonucleotides and their analogues, biotin, and therapeutic portions such as cytotoxins, chemotherapeutic agents, cytokines, and radioisotopes.

[0101] In some embodiments of the present invention, the antibody is humanized. In some embodiments of the present invention, the antibody is a human IgG1 subclass antibody.

[0102] Method for preparing anti-OX40L antibodies with reduced fucosylation

[0103] Methods to reduce core fucosylation include chemical enzyme remodeling, culture medium supplementation, and engineered cell lines (see Li et al, 2017).

[0104] Inhibitors

[0105] Small molecule inhibitors of enzymes acting in the glycosylation pathway can be used to reduce core fucosylation. Inhibitors such as castanospermine, deoxymannojirimycin, and australine act early in the glycosylation pathway by inhibiting glucosidases I and II, producing antibodies with immature glycans (e.g., high levels of mannose) and low levels of fucosylation.

[0106] One aspect of the present invention is to provide a method for preparing an anti-OX40L antibody or antibody derivative, the method comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the culture medium contains inhibitors of glucosidase I and II, followed by purification of the antibody. In some embodiments of the invention, the cells are mammalian cells. In some embodiments of the invention, the inhibitor is selected from the following: spermine, deoxynojrimycin, and oslinine. In another embodiment of the invention, the inhibitor is α-L-fucosidase.

[0107] analog

[0108] Fucose analogs can reduce the incorporation of fucose into the complex N-glycosidic glycan chains of antibodies or antibody derivatives produced by host cells. Suitable fucose analogs are those that can be added to the host cell culture medium and inhibit the core fucylation of the complex N-glycosidic glycan chains of antibodies or antibody derivatives. Fucose analogs are typically taken up by host cells (e.g., via active transport or passive diffusion) (see Almahayni et al., (2022).

[0109] One aspect of the present invention is to provide an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding polypeptides encoding SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the culture medium contains a fucose analogue, and the antibody is subsequently purified. In some embodiments of the invention, the cells are mammalian cells.

[0110] In some embodiments, the fucose analogue is an inhibitor of fucokinase, GDP-fucosylationase, fucosyltransferase (FUT), GDP-mannose 4,6-dehydratase, GDP-fucosylationase, and / or fucosylate transporters (e.g., GDP-fucosylate transporters). In some embodiments, the fucose analogue (or its intracellular metabolites or products) inhibits one or more enzymes in the fucosylate rescue pathway. (As used herein, intracellular metabolites may be, for example, GDP-modified analogs, or fully or partially deesterified analogs. For example, fucose analogs (or intracellular metabolites or products of fucose analogs) may inhibit the activity of fucokinase or GDP-fucosylationase. In some embodiments, fucose analogs (or intracellular metabolites or products of fucose analogs) may inhibit fucosyltransferases (preferably 1,6-fucosyltransferases, such as FUT8 protein). In some embodiments, fucose analogs are inhibitors of fucosyltransferases (FUTs), such as...) Inhibitors of 1,6-fucosyltransferase (FUT8). In some embodiments, fucose analogs (or intracellular metabolites or products of fucose analogs) can inhibit the activity of enzymes in the de novo fucose synthesis pathway. For example, fucose analogs (or intracellular metabolites or products of fucose analogs) can inhibit the activity of GDP-mannose 4,6-dehydratase and / or GDP-fucose synthase. In some embodiments, fucose analogs (or intracellular metabolites or products of fucose analogs) can inhibit fucose transporters (e.g., GDP-fucose transporters).

[0111] In some embodiments, the fucose analogue is selected from the group comprising: L-fucose, 2-fluorofucose, 2-fluoro-peracetylated fucose (2FF), 5-alkynylfucose, alkynylfucose monoacetate, alkynylfucose triacetate, alkynylfucose diacetate, and 5-alkynylfucose peracetate. In some embodiments, the fucose analogue is 2-fluoro-peracetylated fucose (2FF). In some embodiments, the fucose analogue is alkynylfucose peracetate. In some embodiments, the fucose analogue is alkynylfucose triacetate. In some embodiments, the fucose analogue is alkynylfucose diacetate. In some embodiments, the fucose analogue is a mixture of alkynylfucose peracetate, alkynylfucose triacetate, and alkynylfucose diacetate. In some embodiments, the fucose analog is a mixture of alkynylfucose peracetate, alkynylfucose triacetate, alkynylfucose diacetate, and alkynylfucose monoacetate. In any of the embodiments, the fucose analog is not fucose. In some embodiments, the fucose analog is not alkynylfucose peracetate. In some embodiments, the fucose analog is not galactose or L-galactose.

[0112] In some embodiments of the present invention, the fucosyltransferase (FUT) inhibitor is a carbohydrate mimic. In some embodiments of the present invention, the carbohydrate mimic is selected from the group consisting of O-glycosides and C-glycosides, carbocyclic compounds, and nitrogen-containing heterocyclic compounds (e.g., piperidine and pyrrolidine). Those skilled in the art will understand that various carbohydrates and structural analogs containing GDP units in their structure can exhibit fucosyltransferase inhibitory activity.

[0113] In a preferred embodiment, the fucosyltransferase inhibitor is 2-fluoro-peracetylated fucose (2FF). 2FF is a fluorinated analog of fucose that can be taken up by cells and metabolized intracellularly into a fucosyltransferase inhibitor based on the desired donor substrate. It also prevents the de novo synthesis of the natural substrate, thereby leading to global, family-wide inhibition of fucosyltransferases and remodeling of cell surface glycans.

[0114] The effective amount of fucose analogue can be determined using standard cell culture methods. For example, cell culture assays can be used to help determine the optimal dosage range. Accurate dosage also depends on factors such as application time, host cell line, and cell density. The effective dosage can be deduced from dose-response curves obtained from in vitro model testing systems. Therefore, in one embodiment, fucose analogue is added to the culture medium to maintain its effective concentration.

[0115] In some embodiments, the concentration of fucose analogue in the culture medium is 10 nM to 50 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 10 nM to 10 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 100 nM to 5 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 100 nM to 3 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 100 nM to 2 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 100 μM to 1 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 1 μM to 1 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 10 μM to 1 mM. In some embodiments, the concentration of fucose analogue in the culture medium is 10 nM to 500 μM. In some embodiments, the concentration of fucose analogue in the culture medium is 1 μM to 500 μM. In some embodiments, the concentration of the fucose analog in the culture medium is from 1 μM to 250 μM. In some embodiments, the concentration of the fucose analog in the culture medium is from 10 μM to 100 μM. In a preferred embodiment, the concentration of the fucose analog in the culture medium is at least 150 µM, for example at least 200 µM, for example at least 250 µM. In some embodiments, the fucose analog is dissolved in the culture medium (at a temperature suitable for host cell maintenance / growth) at a concentration of at least 10 nM. In some embodiments, the fucose analog is dissolved in the culture medium (at a temperature suitable for host cell maintenance / growth) at a concentration of at least 100 nM.

[0116] Cell engineering modification

[0117] Cell engineering has successfully produced modified glycans by inserting or deleting key genes. Antibodies and their derivatives used in this method can be produced using recombinant expression techniques, hybridomas, myeloma, or other antibody-expressing cells. Recombinant expression of antibodies or their derivatives that bind to a target antigen typically involves constructing an expression vector containing a nucleic acid encoding said antibody or its derivative. Once the nucleic acid encoding such a protein is obtained, a vector for producing the protein molecule can be constructed using recombinant DNA techniques known in the art. (See Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3)). rd The standard techniques described in (ed., 2001) can be used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture, transgene incorporation, and recombinant protein expression.

[0118] Methods for engineering cell lines that cannot fucosylate proteins to reduce core fucosylation include gene knockout, gene knock-in, and RNA interference (RNAi), which are well-known and have been documented in Edwards. et al This is well illustrated in (2022). In gene knockout, the gene encoding FUT8 (α1,6-fucosyltransferase) is inactivated. FUT8 catalyzes the transfer of fucose residues from GDP-fucose to position 6 of the GlcNAc in the Asn link (N-link) of the N-glycan. FUT8 has been reported to be the only enzyme responsible for adding fucose to position 297 of the Asn link of the N-linked biantennary sugar. For example, deletion of the fucosyltransferase gene (FUT8) in Chinese hamster ovary (CHO) cells produces antibodies with complete defucosylation and enhanced antibody-dependent cytotoxicity (Yamane-Ohnuki et al., 2004). Furthermore, lectin-resistant CHO glycosylation mutants resistant to certain lectins have been screened. These cell lines were isolated by repeated screening for cells unable to bind specific lectins in the presence of a mutagen. It has been reported that other cell lines are also unable to fucosylate proteins (such as antibodies), for example, see U.S. Patent Nos. 7,425,466 and 7,214,775 (α1,6-fucosyltransferase, i.e., the FUT8 mutant).

[0119] RNAi often targets FUT8 gene expression, leading to reduced mRNA transcription levels or complete knockout of gene expression. Another approach is to disrupt the gene encoding GDP-mannose 4,6-dehydratase (GMD). GMD is involved in catalyzing the conversion of d-glucose to GDP-fucose, and is therefore crucial for the activity of α1,6-FucT downstream in this pathway for fucosylation (Kanda). et al (2007).

[0120] Gene knock-in technology can add genes encoding enzymes, such as β-1,4-N-acetylglucosamine transferase (GNTIII), GDP-6-deoxy-D-lythose-4-hexylulose reductase (RMD), or Golgi α-mannosidase II. Increased levels of these enzymes in cells deviate monoclonal antibodies from the fucosylation pathway (leading to reduced core fucosylation) and increase the amount of di-N-acetylglucosamine.

[0121] Another aspect of the present invention is to provide a method for preparing an anti-OX40L antibody, the method comprising expressing a polynucleotide encoding polypeptides encoding SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells (e.g., mammalian cells) to prevent core fucosylation. Cell lines that cannot fucosylate proteins are known in the art.

[0122] In some embodiments of the present invention, the cells do not encode functional fucosylation transferases. Those skilled in the art will understand that fucosylation-deficient cell lines can be generated in a variety of ways. Methods for generating fucosylation-deficient cell lines include, but are not limited to, gene knockout (e.g., homologous recombination, CRISPR-Cas9, and TALEN) and gene knockdown (e.g., via antisense oligonucleotides, ribozymes, and RNA interference).

[0123] In some implementations, the cell encodes functional β-1,4-N-acetylglucosamine transferase (GnTIII), GDP-6-deoxy-D-lythose-4-hexylose reductase (RMD), and / or Golgi α-mannosidase II (ManII).

[0124] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are recombinant cells. In some embodiments, the cells are selected from the following: CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), Syrian hamster cells, rat myeloma cells, mouse myeloma cells (e.g., SP2 / 0, NSO), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, Jurkat cells, Daudi cells, A431 cells (epidermal cells), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, MMT 060562 cells, Sertoli cells, BRL cells. 3A cells, HT1080 cells, human myeloma cells, tumor cells, human lymphoma cells (e.g., Namalwa cells), and cell lines derived from the above cells.

[0125] Using conventional hybridoma technology, monoclonal antibodies against the antigen can be obtained from immunized transgenic mice. In some embodiments, the host cells are derived from hybridomas.

[0126] Cell-free protein synthesis

[0127] Cell-free protein synthesis (CFPS) refers to a synthetic reaction system utilizing a reaction mixture containing biological macromolecules (e.g., biological extracts and / or specific reagents). The reaction mixture will contain a template (e.g., DNA, mRNA, etc.) for generating the macromolecule, monomers of the macromolecule to be synthesized (e.g., amino acids, etc.), and cofactors, enzymes, and other reagents required for synthesis (e.g., ribosomes, tRNA, polymerases, transcription factors, etc.). Such synthetic reaction systems are well known in the art and have been described in the literature.

[0128] In one embodiment, the antibody and antibody derivative are produced, synthesized, or obtained by a cell-free protein synthesis method (e.g., the method disclosed in Hunt et al., 2023 or Stech et al., 2017). In another embodiment, the method for producing an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO: 13 and SEQ ID NO: 14 is a cell-free protein synthesis method.

[0129] Regulation of the required level of fucosylation

[0130] Reduced fucosylation due to cell engineering / manipulation can be mitigated by adding fucose to the cell culture medium to promote its incorporation into cells, and modulated to the desired fucosylation level via the fucosylation salvage pathway. This facilitates control of the bioprocess within predetermined values, where a wide range of fucosylation levels can be achieved in the bioproduction of monoclonal antibodies through the strategic use of methods that control fucosylation levels. et al (2020).

[0131] Therefore, one embodiment of the present invention provides a method for producing an anti-OX40L antibody or antibody derivative, the method comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells to prevent core fucosylation, wherein the antibody or antibody derivative is restored or modulated to a desired level by exposure to fucose, wherein the cells are exposed to a culture medium containing fucose. In some embodiments, fucose is added to the culture medium at progressively increasing concentrations, for example up to 1 mM.

[0132] In some embodiments, the method includes an additional step of subsequent antibody isolation. In some embodiments, the method includes an additional step of subsequent antibody purification. In some embodiments, the method includes an additional step of optionally folding the antibody. The antibodies of this disclosure can be purified to homogeneity. Antibody isolation and purification can be performed using conventional protein isolation and purification methods known to those skilled in the art, such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g., protein A purification).

[0133] In some implementations, the cells are selected from mammalian cells, bacterial cells, yeast cells, and plant cells.

[0134] In the methods described above in this invention, in some embodiments, the antibody is IgG1. In some embodiments, the antibody is a complete antibody. In some embodiments, the antibody comprises a heavy chain variable region, a light chain variable region, and an Fc region. In some embodiments, the antibody derivative comprises an antibody Fc region and a ligand-binding domain for non-immunoglobulin proteins.

[0135] Detection methods for anti-OX40L antibodies

[0136] Blocking OX40L-OX40 interaction

[0137] This invention provides compositions and methods for producing antibodies and antibody derivatives that inhibit the binding of OX40 to OX40L, wherein the antibodies and antibody derivatives of this invention bind to human OX40L, thereby inhibiting the OX40 / OX40L interaction and consequently inhibiting OX40L-induced signal transduction. Preferably, the antibodies and antibody derivatives of this invention inhibit the hOX40L / OX40 interaction. In one embodiment, the antibody of this invention inhibits the interaction between OX40L and OX40. Quantification of the OX40L / OX40 interaction can be measured using techniques known to those skilled in the art, including, for example, ELISA using immobilized OX40L (e.g., biotinylated OX40L immobilized on the surface of streptavidin).

[0138] Improved ADCC effect

[0139] Core fucosylation of Fc N-linked glycans affects antibody effector function because the absence of fucose enhances antibody-dependent cytotoxicity (ADCC) responses and increases binding to Fc receptors. Cytotoxic activity against antigen-positive cultured cell lines can be assessed by measuring effector function (e.g., as described in Cancer Immunol. Immunother. 36:373 (1993)).

[0140] In some embodiments, the antibodies and antibody derivatives of this disclosure exhibit higher effector function (e.g., ADCC activity) compared to antibodies with normal fucosylation levels (i.e., antibodies produced without fucoside analogs or inhibitors, or antibodies produced from wild-type cell lines). Effector activity can be modulated by altering the concentration of fucoside analogs in the culture medium and / or the duration of exposure to fucoside analogs. ADCC activity can be measured using assays known in the art, and in exemplary embodiments, ADCC activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold compared to the core fucosylated parent antibody.

[0141] Fc receptor affinity assay

[0142] The antibodies of this invention are Fc engineered to enhance their affinity for various Fcγ receptors. In some embodiments, the antibodies and antibody derivatives of this disclosure exhibit higher affinity for Fc receptors (e.g., CD16A158V and CD16a158F) compared to antibodies with normal fucosylation levels (i.e., antibodies produced without fucosylate analogs or inhibitors, or antibodies produced from wild-type cell lines).

[0143] The affinity for the Fc receptor can be modulated by changing the concentration of the fucose analog in the culture medium and / or the duration of exposure to the fucose analog. The affinity for the Fc receptor can be measured using methods known in the art, and in exemplary embodiments, the affinity for the Fc receptor is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold compared to the core fucose-modified parent antibody.

[0144] T cell activation assay

[0145] On one hand, the present invention provides a method for detecting defucosylated anti-OX40L antibody or antibody derivative, the method comprising detecting the antibody in a T cell activation assay and verifying whether T cell activation is reduced compared to the fucosylated anti-OX40L antibody. In some embodiments, T cell activation is reduced by at least 5%, for example at least 10%, for example at least 15%, for example at least 25%, for example at least 30%.

[0146] Indications

[0147] This invention includes the use of antibodies or antibody derivatives for the prevention and / or treatment of at least one disease or condition associated with OX40L. In one embodiment, the invention relates to a method for the prevention and / or treatment of at least one disease or condition that can be treated by modulating OX40L, its biological or pharmacological activity, and / or biological pathways or signaling involved by OX40L, the method comprising administering to a subject in need a pharmaceutically active amount of the antibody or antibody derivative of the invention and / or a pharmaceutical composition comprising said antibody or antibody derivative. Specifically, the pharmaceutically active amount may be an amount sufficient to modulate OX40L, its biological or pharmacological activity, and / or biological pathways or signaling involved by OX40L.

[0148] In some embodiments, the present invention provides the use of antibodies or antibody derivatives for the prevention and / or treatment of inflammatory diseases and / or autoimmune diseases. In some embodiments, antibodies or antibody derivatives are used for the prevention and / or treatment of inflammatory diseases and / or autoimmune diseases. In one embodiment, antibodies or antibody derivatives are used for the prevention and / or treatment of inflammatory diseases. In one embodiment, antibodies or antibody derivatives are used to prepare a medicament for the prevention and treatment of inflammatory diseases. In one embodiment, antibodies or antibody derivatives are used for the prevention and / or treatment of autoimmune diseases. In one embodiment, antibodies or antibody derivatives are used to prepare a medicament for the prevention and treatment of autoimmune diseases. In one embodiment, the diseases are selected from graft-versus-host disease, allogeneic transplant rejection, asthma, systemic lupus erythematosus, arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, atopic dermatitis, psoriasis, hidradenitis suppurativa, immunoglobulin A nephropathy, Hashimoto's disease, Graves' disease, chronic sinusitis, and multiple sclerosis. In one aspect, the present invention provides a kit comprising the above-described antibodies or antibody derivatives.

[0149] In the context of this invention, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the occurrence of one or more symptoms associated with disease, reducing and / or alleviating one or more symptoms associated with disease, reducing the severity and / or duration of disease and / or any related symptoms, and / or preventing further increase in the severity of disease and / or any related symptoms, preventing, reducing or reversing any physiological damage caused by disease, and any pharmacological effects that are generally beneficial to patients treated with this method.

[0150] Subjects to be treated can be any warm-blooded animal, but especially mammals, and even more so humans. It will be apparent to those skilled in the art that subjects to be treated are, in particular, humans who have or are at risk of having the diseases or conditions described herein.

[0151] Pharmaceutical Composition

[0152] This invention includes antibodies and antibody derivatives, compositions, and methods for the prevention and / or treatment of inflammatory diseases and / or autoimmune diseases. The antibodies and antibody derivatives and / or compositions of this invention are typically administered via any commonly used route. This includes, but is not limited to, parenteral administration, orthotopic administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, or intravenous injection. Other formulations suitable for other routes of administration include oral formulations. Oral formulations contain commonly used excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions may be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, containing about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.

[0153] Generally, the method of administration of the compositions of the present invention corresponds to the dosage formulation, and the amount should be therapeutically effective and immunomodulatory. The amount administered depends on the subject to be treated. The precise amount of active ingredient to be administered depends on the physician's judgment.

[0154] The method of administration can vary considerably. Any conventional method of antibody administration is applicable. These methods are considered to include oral administration (in physiologically acceptable solid matrix or physiologically acceptable dispersion form), parenteral administration, injection, etc. The dosage of the drug composition will depend on the route of administration and will vary according to the subject's body size and health condition. Allogeneic reactive immune response and T-cell activity assays can be performed after administration.

[0155] The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce harmful, allergic, or other adverse reactions when administered to animals or humans. As used herein, "pharmaceuticalally acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption retardants. Such media and reagents are well known in the art for use with pharmaceutically active substances. Unless any conventional media or reagent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions should be considered.

[0156] The composition can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of proteins), which can be formed from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed from free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0157] The carrier can also be a solvent or dispersion medium, including water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Microbial activity can be inhibited by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0158] The effective amount of a therapeutic or prophylactic composition is determined based on the intended target. The term "unit dose" or "dose" refers to a physically independent unit applicable to a subject, each unit containing a predetermined amount of the composition calculated to produce the expected response associated with its administration (i.e., appropriate route and regimen). The amount to be administered (based on the number of treatments and unit dose) depends on the expected outcome and / or protection. The precise amount of the composition also depends on the physician's judgment and varies from person to person. Factors affecting the dosage include the subject's physiological and clinical condition, route of administration, intended therapeutic target (symptom relief or cure), and the potency, stability, and toxicity of the particular composition. Once formulated, it will be administered in a manner consistent with the dosage form and at an effective therapeutic or prophylactic amount. The formulation is readily available for administration in various dosage forms.

[0159] Example

[0160] Example 1: Affinity Maturation

[0161] In vitro affinity maturation (CAM) is based on the principles of mutation and selection. CAM has been successfully used to optimize antibodies, antibody fragments, or other peptide molecules, such as antibody mimics. Random mutations are introduced into the CDR using radiation, chemical mutagens, or error-prone PCR. Furthermore, genetic diversity can be increased through strand rearrangement. Typically, antibody fragments with low nanomolar affinities can be obtained through two or three rounds of mutation and selection using display methods such as phage display.

[0162] Purpose: To generate an anti-OX40L antibody with improved properties (e.g., potency, binding affinity to OX40L, and antibody viscosity) compared to existing anti-OX40L antibodies.

[0163] Materials and Methods: Affinity maturation: Affinity maturation was performed using scFv. Site-directed CDR mutagenesis was conducted to introduce mutations into the parental antibody (anti-OX40L, clone R4930) sequence. An antibody-phage library was constructed for in vitro screening under stringent conditions. Finally, the antibody was identified, characterized, and converted into its final antibody form. Antibody binding was validated, and the antibody was further characterized to identify lead candidates.

[0164] Antigen preparation: The R4930 gene was cloned into an scFv phage display vector. After transformation into E. coli, soluble scFv antibodies were expressed, and antibody-phage particles were packaged. Titration-ELISA was used to detect packaging and scFv production (data not shown).

[0165] Generation of CDR Mutant Libraries: Bioinformatics analysis of R4930 was performed to generate site-directed CDR mutant libraries. Homology modeling of the antibody Fv region was performed, and CDRs were transplanted into the template to identify CDR residues that might be involved in antigen binding. Eighteen sites were identified for the heavy chain and 12 sites for the light chain. Commonly used amino acids in specific strains were identified by analyzing the NGS database. Based on this, degenerate codons were designed to introduce mutations at the identified sites that might be involved in antigen binding, avoiding amino acids with unfavorable properties. The introduction of mutations can be described by a Gaussian distribution, with an average of four mutations per antibody chain. Primers based on degenerate codons were designed and used to introduce the mutations into the antibody sequence. The mutated antibody gene was cloned into the scFv phage display vector, generating three libraries, which were then packaged into antibody-phage particles. The total functional diversity generated was approximately 7 × 10⁻⁶. 8 CFU libraries were developed. Antibody clones with functional open reading frames were identified by DNA Sanger sequencing analysis. Antibody-phage particle packaging and purification resulted in each library exceeding 3 × 10⁻⁶ cells / mL. 12 CFU / mL.

[0166] Affinity maturation via in vitro screening: The generated antibody-phage libraries were used for affinity maturation via in vitro screening. For each individual library, the same total excess of antibody-phage particles relative to functional size was used. The specific amounts were combined into a single library for in vitro screening. Two rounds of in vitro screening were performed, with the second round of panning increasing the stringency through antigen restriction and competition. Negative screening against multiple negative antigens was performed in both rounds of panning. Four different strategies were employed for affinity maturation via in vitro screening. In the second round of panning, the antigen amount decreased sequentially by a factor of ten from strategy one to strategy four. The antibody-phage eluted after the first round of panning was amplified and used in the second round of panning, and the antibody-phage eluted after the second round of panning was used for antibody screening.

[0167] Antibody screening: Antibody-phage particles eluted after the second round of panning were used to infect *E. coli*. 384 clones were randomly selected from each strategy for antibody screening. A total of 1536 antibody clones were used to produce monoclonal scFv antibodies in the bacterial system. The binding activity of the generated antibody clones to OX40L directly immobilized on ELISA plates was tested. Two negative antigens (streptavidin and BSA) were directly immobilized on ELISA plates. Soluble scFv antibodies were detected using myc-tag with HRP-conjugated secondary antibodies. Antibody clones with the following defined parameters were identified as antigen-specific clones: ELISA binding signal to positive antigen ≥ 0.2 ELISA binding signal to negative antigen ≤ 0.2 The S / N ratio between positive and negative antigens is ≥ 3. Approximately one-third of all tested antibodies were identified as antigen-specific clones. Parental scFv antibodies were used as positive controls. Some antibodies showed higher binding signals than the parental scFv antibody (data not shown). Simultaneously, a negative control scFv (trastuzumab) was generated and tested, which showed no specific binding.

[0168] Antibody sequencing: All 444 antigen-specific antibodies were selected for DNA Sanger sequencing analysis. Sequence analysis revealed 271 unique mutant antibodies. These antibodies showed 1 to 7 mutations. Several hotspot mutations were identified, indicating preferred mutations at different locations.

[0169] Affinity ranking: To narrow down the number of antibodies used for conversion to the final form, affinity ranking was performed using the BLI octet system. First, an assay was established using parental scFv antibodies and biotinylated antigens produced in the bacterial system. The antigen was immobilized on a streptavidin sensor, which was incubated with bacterial scFv supernatant for binding. Dissociation was measured by incubation in buffer, and the dissociation rate was calculated after modeling binding and dissociation. We selected 271 clones for scFv production in the bacterial system. The above assay was performed on each antibody. The dissociation rate of the antibodies was calculated, and the antibodies were ranked accordingly. Of all the antibodies detected, 267 were detectable and fitted with high confidence. Among these 267 clones, 55 clones showed increased dissociation rates compared to the parental antibodies. Of these 55 antibodies, 13 showed at least a two-fold increase in dissociation rate compared to the parental antibodies.

[0170] Transformation to final form: 21 antibodies and parental antibody sequences were cloned into mammalian expression vectors containing constant domains of either the heavy or light chains. Transfection-grade plasmid DNA was generated and validated by DNA sequence analysis. This plasmid DNA was used for transient transfection of HEK cells. One week after antibody production in mammalian cell culture, the antibodies were purified using protein A affinity chromatography. The final buffer for antibody purification was phosphate-buffered saline (without additives). Quality control was performed at 280 nm using a UV / VIS spectrophotometer. The binding ability of the antibodies to the streptavidin-captured positive antigen human OX40L was detected in an ELISA. Streptavidin was used to control nonspecific binding. Each antibody was titrated at a starting concentration of 10 µg / ml (1 / square (10)) and incubated on coated and blocked ELISA plates. Detection was performed using HRP-conjugated secondary antibodies (anti-human IgG (Fc specific)-peroxidase antibody; Sigma Aldrich #A0170). All antibodies showed strong specific binding to the target antigen.

[0171] Antibody Affinity: To further characterize the antibodies, an affinity assay was established using the BLI octet system. Human IgG antibodies were immobilized on protein A sensor probes and titrated with human OX40L antigen. Binding and dissociation curves were modeled using concentrations of 50 nM or 15.8 nM, and antibody affinity was determined. The affinity between the parent antibody (R4930) and the screened anti-OX40L antibody candidate (BO2) was determined (see Table 1).

[0172] Allogeneic mixed lymphocyte reaction: Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat by density gradient centrifugation using Ficoll Paque Plus (Cytiva) (500g × 30 min). The buffy coat was obtained from anonymized healthy adult donors from the Akademiska University Hospital blood bank, and PBMC isolation was completed within 24 hours of blood collection. Under Swedish law, no ethical approval was required because the blood bank provided anonymous donor buffy coats for research purposes, and the biological material could not be traced back to a specific individual. The isolated PBMCs were mixed in PBS at a concentration of 2.0 × 10⁻⁶. 7Cells / mL were collected and stained using CFSE (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. A biphasic allogeneic mixed lymphocyte reaction was performed by mixing PBMCs from two different donors, meaning that the PBMCs from both donors were not inactivated. VPD450-stained PBMCs were washed and resuspended in AIM V medium (Gibco, Thermo Fisher Scientific Inc.) containing 10% heat-inactivated Ultralow IgG FBS (Gibco). The resuspended PBMCs were added to 96-well round-bottom cell culture plates, and pure medium or medium supplemented with the test sample was added to a final concentration of 2 × 10⁶ cells / mL. 6 Cells / mL, final volume 200 μL. The mixed lymphocyte reaction was incubated at 37°C and 5% CO2 for 7 days. The supernatant was retained after culture, and cytokine levels (detection of INF) were analyzed using a Legendplex (BioLegend) cytokine assay according to the manufacturer's instructions. γ (IL-6, TNFα levels). Add BO2 or R4930 at a concentration of 10 µg / mL at the start of culture.

[0173] Flow cytometry: Seven days after the mixed lymphocyte reaction, samples were washed and then stained with antibodies against cell surface antigens. For intracellular staining, the FoxP3 staining kit (BD Biosciences) was used according to the manufacturer's instructions. Samples were stained at 4°C in the dark and washed twice in staining buffer (BD Biosciences) before analysis using a BC Cytoflex flow cytometer (Beckman Coulter). T cells were defined as CD3+ cells. Proliferation was calculated using the proliferation index (FlowJo software). Tregs were defined as CD3+, CD4+, CD25hi, and FoxP3+. Naïve T cells were defined as CD3+, CD45RA+, and CCR7+. Memory T cells were defined as CD3+ and CD45RA-. The number of naïve cells was expressed as the memory cell / naïve cell ratio (M / N).

[0174] result: Table 1. Affinity of B02 and R4930 to OX40L.

[0175]

[0176] Statistical significance is defined as p<0.05。

[0177] The affinity of BO2 for OX40L was increased compared to that of R4930, as determined by the BLI octet system. This was reflected in the decrease in the Kd value of BO2 compared to R4930. The Kd value of BO2 was 1.9 nM, while that of R4930 was 2.9 nM.

[0178] Table 2. Proliferation, M / N, cytokines, activation, and T-Reg of OX118 and R4930. Assessed in a two-way MLR setting.

[0179]

[0180] Statistical significance is defined as p<0.05。

[0181] Compared to R4930, B02 significantly reduced cytokine abundance and had a higher T-Reg abundance. Furthermore, R4930 exhibited a favorable proliferation index, namely the ratio of memory cells to immature cells (M / N) (Table 2).

[0182] in conclusion: The inventors of this disclosure have demonstrated that, compared with the prior art anti-OX40L antibody R4930, BO2 exhibits higher antibody affinity for OX40L, reduced cytokine production and activation, and increased T-Reg abundance and proliferation. These data indicate that BO2 has an independent role in T cell activation.

[0183] Example 2: Optimization of the Fc region of anti-OX40L antibody OX118

[0184] Core fucosylation of Fc N-linked glycans affects the effector function of IgG antibodies because the absence of fucose increases antibody-dependent cytotoxicity (ADCC) responses and increases binding to Fcγ receptors.

[0185] One approach to reducing core fucosylation is to use the fucosyltransferase (FUT) inhibitor 2-fluoro-peracetylated fucose (2FF). 2FF is a competitive inhibitor of fucosyltransferase (FUT8), an enzyme essential for the transfer of fucose to the glycans forming in the Golgi apparatus (Rillahan Azim). et al. , 2012).

[0186] Purpose: By optimizing the Fc region of BO2 by reducing core fucosylation, a novel anti-OX40L antibody (OX118) was obtained. This antibody exhibits enhanced ADCC response compared to existing antibodies, thereby reducing T cell activation and improving clinical efficacy.

[0187] i) Through Treatment with 2-fluorofucose (2-FF) to reduce fucoidylation

[0188] First, the effect of 2-FF on reducing fucosylation levels was evaluated.

[0189] Materials and Methods: The composition of N-glycans was determined by enzymatic release of N-glycosidase F (PNGase F, Waters) after sample denaturation. The obtained N-glycans were fluorescently labeled using RapiFlour-MS (Waters) for detection. The fluorescently labeled N-glycans were flowed through a HILIC-FLD-MS, TripleTOF 4600 MS (AB SCIEX), and Nexera X2 HPLC system (SHIMADZU) onto a HILIC column (Waters). The mass-to-charge ratio (m / z) detected by mass spectrometry was then used to identify the N-glycans, and the relative abundance of each N-glycan was calculated based on the peak area of ​​the HILIC-FLD chromatogram. Data analysis was performed using PeakView software.

[0190] N-glycans can also be determined using the following method: The sample is first denatured with Rapidest-SF (Waters), then the N-glycans are released using PNGase F (Waters or equivalent). The resulting N-glycans are further labeled with the fluorescent label RapiFlour-MS (Waters) for detection and purified by solid-phase extraction (SPE, BEH Amide, Waters). These fluorescently labeled N-glycans are then analyzed using HILIC-FLD (e.g., 1290, Agilent). N-glycans are identified based on retention time, and the percentage of each N-glycan is calculated based on the peak area of ​​the HILIC-FLD chromatogram.

[0191] result: Tables 3 and 4 show the baseline glycosylation profiles of two human IgG1 B02 clones expressed in CHO K1 cells. Relative fucosylation (%) gradually decreased as the concentration of 2-FF used for treatment increased up to 400 µM. A fucosylation plateau of approximately 30% was observed at 2-FF concentrations above 200 µM. Figure 1The remaining portions of the glycosylation profile are normal for human IgG1 expressed in CHO K1 (man5 1.6–2.2%) and do not change dose-dependently with 2-FF concentration. As shown in Table 3, the glycosylation profile of IgG1 not exposed to 2-FF is normal for human IgG1 expressed in CHO K1. As shown in Table 3, the glycosylation profile of IgG1 with fucosylation reduced to 30% is normal for human IgG1 expressed in CHO K1.

[0192]

[0193] Table 4 shows the baseline glycosylation profile of human IgG1 (clone 8) expressed in CHO K1 cells. Treatment with 2-FF at concentrations increasing up to 400 μM resulted in a gradual decrease in fucosylation levels. Specifically, the fucosylation level decreased from 84.93% at 0 μM 2-FF to 2.29% at 400 μM. At 50 μM 2-FF, the fucosylation level decreased significantly to 12.98%, while the reduction in fucosylation saturated at approximately 150–200 μM, where the fucosylation level stabilized at approximately 3.20%–2.83%. Increasing 2-FF to 400 μM only resulted in a slight further decrease in fucosylation to 2.29%, indicating that fucosylation had plateaued at this concentration. Other components of the glycosylation profile, such as Man5 and the unassigned peak, remained constant throughout the increase in 2-FF concentration. The glycosylation profile of decreased fucosylation reflects an increase in G0%, which rose from 3.54% at baseline to 75.62% at 400 μM 2-FF, indicating the expected change in glycan structure with decreasing fucosylation. Table 3 shows similar results for another clone with a higher degree of fucosylation.

[0194] Table 4. N-Glycan detection results. Baseline glycosylation profile of human IgG1 (clone 8) expressed in CHO K1 cells and the effect of 2-FF on fucosylation pattern.

[0195]

[0196] b The following formula is used to calculate (fucosylation)% = (G0F-GN)% + (G0F)% + (G1Fa)% + (G1Fb)% + (G2F)%.

[0197] in conclusion: When the concentration of 2-FF was 200 µM, the effect of 2-FF on reducing fucosylation reached saturation, with fucosylation of approximately 30% (clone 125) and approximately 3% (clone 8).

[0198] ii) Fc receptor affinity assay

[0199] Purpose: This study investigated the relationship between the core fucosylation level of OX118 and the affinity of OX118 for different Fc receptors.

[0200] Materials and Methods: BLI experiments were performed on an Octet RED96e (ForteBio) instrument. Except for C1q, which was purchased from Sigma, all other recombinant proteins used for testing were purchased from Sino Biologicals (FcRn, CD16a 158V, CD16a 158F, CD32a 131Arg, CD32a 131His). The HIS1K sensor was used to capture his-tag-tagged recombinant proteins, such as FcRn, CD16a 158V, CD16a 158F, CD32a 131Arg, and CD32a 131His. The FAB2G sensor was used to capture the Fab region of the antibody in the C1q affinity assessment. For CD32a, CD16a, and C1q, the buffer used was 0.02% PBST containing 1% BSA, and the temperature was set to 25°C. The shaker speed was set to 1000 rpm. The pH of the FcRn binding buffer was set to pH 6. The binding and dissociation time for C1q was 300 seconds. The binding and dissociation time for FcRn was 60 seconds. The binding and dissociation time for CD32a and CD16a was 60 seconds and 150 seconds, respectively. Herceptin was used as an internal control antibody. The data acquisition rate was set to standard kinetics (5.0 Hz, 20 averages). Data analysis was performed using Octet® System Data Analysis (version 10.0). The binding of CD16a, CD32a, and C1q to the antibody Fc region is a weak affinity interaction (fast binding, fast dissociation) and is not suitable for complete kinetic analysis. Binding curves for all analyte concentrations have reached steady state, and their affinity can be assessed using the SSG KD values ​​from steady-state analysis. The affinity of CD64 and FcRn was assessed using the KD values ​​from kinetic analysis.

[0201] result: The affinity of OX118 for CD16A, 158V, and CD16a 158F increases with decreasing fucosylation percentage. However, the affinity of OX118 for CD32a, C1q, and FcRn does not change with increasing fucosylation. Figure 2A reduced saturation effect of fucosylation was observed at approximately 50% fucosylation level; below this level, Fc receptor affinity was not further enhanced.

[0202] in conclusion: Decreased OX118 fucosylation leads to increased affinity for CD16a, but has no effect on binding to FcRn, C1q, or CD32a. This effect saturates at approximately 50% OX118 fucosylation.

[0203] iii) Reduced fucosylation and T cell activation

[0204] Purpose: This study investigated the relationship between the core fucosylation level of OX118 and the ability of OX118 to induce T cell activation.

[0205] Materials and Methods: Treatment with the fucosyltransferase inhibitor 2-fluoro-peracetylated fucose (2FF) yielded two OX118 variants with complete (96%) and reduced (34%) core fucosylation levels, respectively.

[0206] result: Compared to the control, the addition of both OX118 variants induced a concentration-dependent decrease in T cell activation. Figure 3 Decreased OX118 core fucosylation levels (34%) led to a more pronounced concentration-dependent decrease in T cell activation, with increased doses resulting in a greater reduction in T cell activation compared to fully fucosylated OX118 variants (96%). Figure 3 When the concentration of the OX118 variant is 10... 3 and 10 4 These data were statistically significant at ng / mL.

[0207] in conclusion: OX118 induces a concentration-dependent decrease in T cell activation, regardless of whether it is fully fucosylated (97%) or reduced fucosylated (34%). The decrease in T cell activation is stronger when the core fucosylation of OX118 is reduced (34%) compared to when OX118 is fully fucosylated (97%).

[0208] Overall, Fc optimization of BO2 was achieved through exposure to 2-FF, resulting in a novel anti-OX40L antibody (OX118). Exposure to 2-FF resulted in decreased fucosylation levels and enhanced affinity for the Fc receptor (CD16a). This effect saturated at approximately 50% fucosylation, below which no further enhancement of Fc receptor affinity was observed. Furthermore, compared to the control and fully fucosylated OX118, OX118 with reduced fucosylation significantly decreased T cell activation.

[0209] Example 3: Allogeneic mixed lymphocyte reaction

[0210] Purpose: Study on the in vitro effects of OX118 on different cell types 。

[0211] Materials and Methods: CTLA-4 Ig (clone Belatacept, Bristol Myers Squibb, SEQ ID NO: 15) was purchased from Appoteket Hjärtat AB. Anti-OX40L IgG1 reference antibody (clone R4930 / Oxelumab; IgG1, Fc activity) and anti-OX40L IgG4 reference antibody (clone Amlitelimab; IgG4, Fc-silenced, SEQ ID NO: 16, SEQ ID NO: 17) were prepared by Proteogenix via transient expression in CHO K1 cells. OX118 with reduced core fucosylation (34%) was used in experiments (the rest of the glycosylation profile was normal for human IgG1 expressed in CHO K1: man5 5.2%, G06 5.2%, G0-GN 1%, G0F 22%, G1Fa 1.43%, G1Fb 0.54%, G2F 0.29%).

[0212] Allogeneic mixed lymphocyte reaction: Peripheral blood mononuclear cells (PBMCs) were separated from the white membrane layer by density gradient centrifugation using Ficoll Paque Plus (Cytiva) (500g x 30 min). The white membrane layer was obtained from anonymized healthy adult donors from the blood bank of Akademiska University Hospital, and PBMC separation was completed within 24 hours after blood collection. No ethical approval was required under Swedish law because the blood bank provided anonymous donor white membrane layers for research purposes, and the biological material could not be traced back to a specific individual. The separated PBMCs were centrifuged in PBS at 2.0 × 10⁻⁶. 7PBMCs were mixed at a concentration of 100 cells / mL and stained with CFSE (Thermo Fisher Scientific Inc) according to the manufacturer's instructions. PBMCs from two different donors were mixed in a two-way allogeneic mixed lymphocyte reaction, i.e., PBMCs from either donor were not inactivated. VPD450-stained PBMCs were washed and resuspended in AIM V medium (Gibco, Thermo Fisher Scientific Inc) containing 10% heat-inactivated Ultralow IgG FBS (Gibco). The resuspended PBMCs were aliquoted into 96-well round-bottom cell culture plates, and pure medium or medium supplemented with the test sample was added to a final concentration of 2 × 10⁻⁶ cells / mL. 6 Cells / mL, final volume 200 μL. The mixed lymphocyte reaction was incubated at 37°C and 5% CO2 for 7 days. The supernatant was collected after culture and cytokine levels (IL-10, IL-4, and TNFα) were analyzed using the Legendplex (BioLegend) cytokine assay according to the manufacturer's instructions.

[0213] Flow cytometry: Seven days after the mixed lymphocyte reaction, samples were washed and then stained with antibodies against cell surface antigens. For intracellular staining, a FoxP3 staining kit (BD Biosciences) was used according to the manufacturer's instructions. Samples were stained at 4°C in the dark and washed twice with staining buffer (BD Biosciences), then analyzed using a BC Cytoflex flow cytometer (Beckman Coulter). T cells were defined as CD3+ cells. Proliferating T cells were defined as those with low CFSE. Treg cells were defined as CD3+, CD4+, CD25hi, and FoxP3+. Naïve T cells were defined as CD3+, CD45RA+, and CCR7+.

[0214] Charts and Statistical Analysis: GraphPad Prism 10 software was used for results visualization and statistical analysis of the underlying data. Data analysis employed one-way ANOVA and Dunnett's multiple comparison test.

[0215] result: Compared with controls and other OX40L-targeting antibodies (IgG1 Ref and IgG4; 10 µg / mL) and CTLA-4 Ig (15 µg / mL), OX118 (10 µg / mL) reduced the proliferation of CD4+ and CD8+ cells. Figure 4 ).

[0216] Compared with controls and other OX40L-targeting antibodies (IgG1 Ref and IgG4; 10 µg / mL) and CTLA-4 Ig (15 µg / mL), OX118 (10 µg / mL) increased the percentage of Treg cells in all proliferating cells. Figure 5 A). This effect was observed to be concentration-dependent, at a concentration of 10 OX118. 2 -10 5 At ng / mL, an increase in the percentage of Treg cells was observed in all proliferating cells. Figure 5 B).

[0217] Compared with controls and other OX40L-targeting antibodies (IgG1 Ref and IgG4; 10 µg / mL) and CTLA-4 Ig (15 µg / mL), the addition of OX118 (10 µg / mL) to the biphasic mixed lymphocyte assay resulted in an increase in the naive T cell population in proliferating CD4+ cells. Figure 6 ).

[0218] Compared to the control, the addition of OX118 (10 µg / mL) to the biphasic mixed lymphocyte reaction resulted in a decrease in IL-4. Compared to the control and other OX40L-targeting antibodies IgG1 Ref (10 µg / mL) and CTLA-4 Ig (15 µg / mL), OX118 (10 µg / mL) resulted in a decrease in TNFα levels. IL-10 levels were comparable to those of the control and other OX40L-targeting antibodies IgG1 Ref (10 µg / mL) and CTLA-4 Ig (15 µg / mL). Figure 7 ).

[0219] in conclusion: OX118 reduced the proliferation of CD4+ and CD8+ cells in vitro, while increasing the number of proliferating Treg cells. Furthermore, OX118 reduced the levels of pro-inflammatory cytokines IL-4 and TNFα in vitro.

[0220] Example 4: Xenograft-host disease model

[0221] Purpose: To evaluate the potential role of OX118 (a fucosylated anti-OX40L antibody) in preventing the development of graft-versus-host disease in a xenograft setting, in which NSG mice were challenged by human PBMCs.

[0222] Materials and Methods: This study used 6-9 week old female NSG (NOD.Cg- Prkdc scid Il2rg tm1Wjl / SzJ) mice. Mice were randomly assigned to different treatment groups (control, OX118 10 mg / kg, and OX118 50 mg / kg) based on body weight. PBMCs were obtained from a healthy, randomized, and unidentified donor. PBMCs were used immediately after separation from the leukocyte removal system (LRS) separation chamber.

[0223] On day 0, 10 million freshly isolated human PBMCs were administered via the tail vein.

[0224] Twenty-four hours after PBMC injection, the first dose of OX118 was administered intraperitoneally (ip), followed by weekly administration for three weeks (four ip administrations on days 1, 8, 15, and 22). Five mice were used in each group. Human IgG1 isotype was used as a control (BioXCell, catalog number #BE0297). OX118 was administered at doses of 10 mg / kg (low dose) or 50 mg / kg (high dose), weekly until day 22. Activity scores were assessed daily (see Table 5). Mice with a score of 3 for more than two days or a score below 3 for one day were euthanized.

[0225] Table 5. Activity scoring criteria.

[0226]

[0227] The GvHD score was assessed daily using an integer scoring method (see Table 6) to monitor the development of GvHD. The score ranged from 0 (asymptomatic) to 2 (severe symptoms) and was divided into five different aspects.

[0228] Table 6. Evaluation criteria for GvHD scores.

[0229]

[0230] Flow cytometry: Blood samples were collected on days 8, 15, and 22 for flow cytometry. Cells were centrifuged (400 xg, 5 min) and the supernatant was removed. Fc blocking antibody (10 µg / well, diluted 1:100 with FC buffer) was used. The specific antibody was then added to Zombie Aqua Fixable Viability staining solution (diluted 1:100 with PBS buffer) according to the manufacturer's instructions, and the plates were incubated at 4°C in the dark for 30 min. The antibodies used included: hCD3 (clone UCHT1, Biolegend), hCD45 (clone H130, Biolegend), hCD4 (clone RPA-T4, Biolegend), hCD8 (clone RPA-T8, Biolegend), hCD25 (clone M-A251, Biolegend), HLA-DR (clone L243, Biolegend), hFoxp3 (clone PCH101, Thermo Scientific; this antibody was used with an intracellular staining protocol), and HCD62L (clone DREG-56, Biolegend). All antibodies were used according to the manufacturer's instructions. FC buffer: PBS containing 2% FBS; FBS (#A21-102 – PAA), PBS (#14190169 – Gibco / Fisher Scientific). Fc blocking antibody: CD16 / 32 purified (2.4G2), 0.5 mg / ml (#553142 – BD Biosciences). Zombie Aqua Fixable Viability Kit (#423102 – BioLegend).

[0231] After washing the cells with 200 µl of FC buffer, the plates were centrifuged at 400 × g for 5 min, and the supernatant was removed. The samples were resuspended in 400 µl of FC buffer and analyzed using an Attune NXT flow cytometer (Thermo Fisher) (violet (405 nm) / blue (488 nm) / yellow (561 nm) / red (638 nm) laser configuration). Flow cytometry was used to characterize the phenotype of blood immune cells. The cell populations studied are detailed in Table 7.

[0232] Table 7. Gating strategies for assessing blood immune cell phenotypes.

[0233]

[0234] Analysis: Double-cell exclusion is performed based on forward scattering height and forward scattering area to retain only single cells. Cell debris is then excluded based on forward / lateral scattering. Finally, live / dead cells (catalog number 423102, Biolegend) are distinguished. Gating is implemented according to Table 5.

[0235] result: OX118 treatment can prevent GvHD in mice transplanted with human peripheral blood mononuclear cells (PBMCs): GvHD was induced in mice, defined as an increase in GvHD score (mean GvHD score on day 30: control: 6.8; 10 mg / kg OX118 group: 0.8; 50 mg / kg OX118 group: 1.2). Compared with the control, OX118 treatment inhibited the formation of hCD45+ leukocyte population in peripheral blood. This effect was observed in both low and high doses of OX118 (10 mg / kg and 50 mg / kg, respectively). Figure 8 On day 21, peripheral chimerism accounted for 1-3% of all peripheral blood cells, compared to 63% in the control group. This is consistent with previously published literature (Tripathi). et al. Compared to (2019), this indicates that the control of chimeric / graft proliferation has been improved by 3-4 times.

[0236] OX118 can reduce the activation of T cells in peripheral blood of mice after GvHD induction: In mice 21 days after GvHD induction, OX118 treatment reduced the relative populations of activated CD4+, CD8+, and CD14+ T cells compared to control. This effect was observed in both low and high doses of OX118 (10 mg / kg and 50 mg / kg, respectively). Figure 9 ).

[0237] OX118 can increase the proportion of regulatory T cells (T-Reg) in proliferating CD4+ T cells after GvHD induction in mice: Twenty-one days after induction of GvHD in mice, treatment with OX118 (pooled data from all OX118-treated mice) increased the number of T-regs compared to control. Figure 10 ).

[0238] in conclusion

[0239] OX118 treatment, regardless of dosage, can prevent the induction and progression of GvHD in vivo by inhibiting peripheral chimerism, activating peripheral T cell populations, and enriching peripheral T-reg cell populations.

[0240] Example 5: Xenogeneic GvHD model – baseline OX118 (treatment A) vs. anti-human OX40L IgG4 isotype (treatment B).

[0241] Objective: To evaluate and compare the efficacy of different allotype anti-OX40L antibodies in a xenograft-versus-host disease (xeno-GvHD) model, with a focus on tissue infiltration of chimeric and human CD45+ (hCD45+) cells.

[0242] Materials and Methods: NOG mice received a total body irradiation dose of 1.0 Gy. After a 24-hour rest period, 3 million human PBMCs (peripheral blood mononuclear cells) were injected via the tail vein, marked as day 0. Treatment began on day 1 with treatment A (OX118), treatment B (anti-hOX40L IgG4), and control (human IgG1 isotype) administered at a dose of 10 mg / kg, respectively. GvHD scores (see Table 6) and body weight were assessed every other day.

[0243] Flow cytometry: Blood samples were collected, and whole blood was centrifuged at 3000 rpm for 5 minutes at 4°C to separate plasma (plasma was frozen at -80°C for subsequent cytokine analysis). Cells were resuspended in lysis buffer and incubated at room temperature for 2 minutes. The remaining leukocytes were resuspended in DPBS, and mouse Fc blocker (rat anti-mouse CD16 / CD32), human Fc blocker (Human TruStain FcX), and Live / Dead (Zombie NIR, BioLegend, 426106) were added. The mixture was incubated at room temperature in the dark for 15 minutes. Antibody was then added, and the mixture was incubated at 4°C in the dark for 30 minutes. For chimerism detection, mCD45 (PerCP, BioLegend, 103130) and hCD45 (AF700, BioLegend, 368514) were added. All samples were analyzed using Cytek (Aurora-5L). If possible, record more than 5000 hCD45+ events.

[0244] IHC: At the end of the study, organs (skin, colon, and lung) were harvested for immunohistochemical staining of human CD45+ cells in the tissues. Paraffin sections: Paraffin blocks were placed on a Leica HistoCore Arcadia C for 15–30 minutes before sectioning. The paraffin blocks were sectioned to a thickness of 4 µm using a Leica HistoCore BIOCUT microtome. The sections were dried at 60°C for 60 minutes. Automated IHC was performed using a BOND RX-equipped instrument. The hCD45 antibody used was CST, catalog number #13917S, diluted 1:200.

[0245] result: By day 21, the clinical scores of the control group rapidly increased, while the scores of treatments A and B remained at similar levels until around day 40. At this point, the clinical scores of treatment A decreased, while the clinical severity of the mice treated with B gradually increased. Figure 11 A). Similarly, mice treated with A gained weight after day 40, while mice treated with B lost weight. Figure 11 B).

[0246] Chimerism (indicated by circulating hCD45+ cells) was significantly lower in treatment A than in treatment B and the control group. Figure 11 C).

[0247] Furthermore, compared to treatment B, treatment A resulted in reduced hCD45+ cell infiltration in the colon, skin, and lungs. Figure 11 D).

[0248] in conclusion: Compared with anti-OX40L IgG4 (treatment B) and IgG1 isotype control, OX118 (treatment A) demonstrated superior control in the xenogeneic GvHD model. OX118 effectively reduced tissue infiltration of hCD45+ cells, supporting its potential application in the treatment of GvHD.

[0249] Example 6: Antibody-dependent cell-mediated cytotoxicity (ADCC) assay

[0250] Objective: To evaluate and compare the ADCC activity of OX118 with that of Herceptin® (trastuzumab; Roche, SH0346) as a positive control.

[0251] Materials and Methods: OX118 is a humanized IgG1 antibody targeting OX40L. ADCC bioassay effector cell variant V (high affinity) (BPS Bioscience, 60541) was used as the effector cells, while SKBR-3 cells (ATCC, HTB-30) were used as the target cells for systemic control. CHO-K1 / OX40L cells (GenScript, M00563) were used as the test target cells. Herceptin® (Roche, SH0346) was used as the systemic positive control, and human IgG1 (Sino Biological, MA17JU1250) was used as the negative control. This assay was performed using three different batches of OX118 drug substance (DS).

[0252] Target cells were seeded at a density of 10,000 cells per well in 96-well plates. Control and test samples were added to the wells, and the plates were incubated at 22–28°C for 30 minutes. Then, effector cells were added to the wells at a density of 60,000 cells per well, and the plates were incubated in a cell culture incubator at 37°C and 5% CO2 for another 6 hours.

[0253] After incubation, add 75 μL of Bio-Lite to each well.TM The Luciferase Assay System (Vazyme, DD1201) was used, and the luminescence signal was measured using a microplate reader. The signal was plotted against the logarithm of the antibody concentration (ng / mL), and the dose-response curve was fitted using GraphPad software with a four-parameter fit (4P-Fit) to calculate the half-maximal effective concentration (EC50). 50 )value.

[0254] result: Figure 13 shows the dose-response curves of OX118 ADCC activity compared to Herceptin®. Table 8 below summarizes the EC50 values ​​of different batches of OX118 active pharmaceutical ingredient: Table 8. ADCC effect of OX118 active pharmaceutical ingredient

[0255] in conclusion: Compared to the systemic positive control Herceptin®, OX118 exhibited potent ADCC activity with a significantly lower EC50 value. The consistent EC50 values ​​across three different batches of OX118 DS indicate robust and reproducible ADCC activity. In contrast, the negative control (human IgG1) showed no ADCC activity, confirming the specificity of the reaction.

[0256] Example 7: Combination of OX118 and FcyR

[0257] Objective: To evaluate the binding affinity of the Fc region of OX118 for various Fcγ receptors (including FcγRIa (CD64), FcγRIIa (CD32a 131H, CD32a 131R), FcγRIIIa (CD16a 158V, CD16a 158F) and neonatal Fc receptor (FcRn)), with Herceptin® as a positive control.

[0258] Materials and Methods: Biolayer interferometry (BLI) was performed using a Sartorius RED96e system to assess the binding affinity of OX118 active pharmaceutical ingredient (DS) to the Fcγ receptor. Three different batches of OX118 DS were used in this assay: batch #1 (toxicology batch), batch #2, and batch #3 (GMP DS batch). Herceptin® (Roche, SH0346) was used as a positive control, and human IgG1 was used as a negative control.

[0259] FcγR protein was immobilized, and after baseline equilibration, binding analysis was performed by adding serially diluted antibody samples followed by dissociation in buffer. Assays were performed at 25°C using 0.02% PBSTB as the run buffer. Data acquisition employed standard kinetics (5.0 Hz, mean 20), and data analysis used Octet® system data analysis software to calculate binding kinetic parameters (KD, kon, koff).

[0260] result: Tables 9, 10, 11, and 12 show the binding affinity of OX118 and Herceptin® for various Fcγ receptors.

[0261] Table 9. Binding affinity of OX118 active pharmaceutical ingredient to FcγRIa (CD64)

[0262] Table 10. Binding affinity of OX118 active pharmaceutical ingredient to FcγRIIa

[0263] Table 11. Binding affinity of OX118 active pharmaceutical ingredient to FcγRIIIa

[0264] Table 12. Binding affinity of OX118 active pharmaceutical ingredient to FcRn

[0265] Conclusion: OX118 exhibits enhanced binding affinity for FcγRIIIa compared to Herceptin®. Consistency among three different batches of OX118 demonstrates robust manufacturing and reproducibility. Binding affinities for other Fcγ receptors (including FcγRIa, FcγRIIa, and FcRn) are comparable to Herceptin®. OX118 demonstrates enhanced affinity for FcγRIIIa, the major mediator of ADCC, while its affinity for other FcRs is similar to that of the reference antibody (Herceptin®).

[0266] Example 8: Binding affinity of OX118 to C1q

[0267] Objective: To evaluate the binding affinity of OX118 to C1q, a key component of the complement system, which is involved in initiating the classical complement pathway. Binding to C1q is an important functional property of antibodies because it can trigger an immune response, thereby enhancing pathogen clearance. This study compared the binding of OX118 to C1q with Herceptin®, an IgG1 isotype antibody known to have C1q binding activity (as a positive control), to determine whether OX118 retains this important functional property.

[0268] Materials and Methods: Biolayer interferometry (BLI) was performed using a Sartorius Octet RED96e system to determine the binding affinity of OX118 active pharmaceutical ingredient (DS) to the C1q protein. Herceptin® (Roche, SH0346) was used as a positive control. Three different batches of OX118 DS were tested: batch #1 (toxicology batch), batch #2, and batch #3 (GMP DS batch).

[0269] The assay was performed at 25°C using 0.02% PBSTB as the run buffer. C1q protein was immobilized on the sensor, and serially diluted antibody was added. Binding analysis was performed after baseline equilibration. Dissociation was performed using buffer. Data were acquired at a standard kinetic rate (5.0 Hz, average 20), and analysis was performed using Octet® system data analysis software to calculate binding parameters (KD, Rmax).

[0270] result: Table 13 below summarizes the comparison of the binding activity of OX118 to C1q with that of Herceptin®: Table 13. Binding activity of OX118 active pharmaceutical ingredient with C1q

[0271] in conclusion: The binding activity of OX118 to C1q is comparable to that of Herceptin®, and there is no significant difference in binding affinity between different batches of OX118. This indicates that OX118 can interact with the complement system. The consistency of C1q binding between different production batches also confirms the robustness of the OX118 manufacturing process.

[0272] Sequence Overview

[0273] The novel anti-OX40L monoclonal antibody (mAB; OX118) was developed based on affinity maturation and Fc region optimization of the previously clinically tested mAb R4930 (US 7,501,496 B1). The CDR sequence was determined using the Chothia numbering scheme. Underlined bold amino acids differ from their corresponding amino acids in R4930.

[0274] OX118 VL sequence (SEQ ID NO: 1)

[0275] CRASQGISS L LAWYQQKPEKAPKSLI FDR S R LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTF

[0276] OX118 LC CDR1 (SEQ ID NO: 2)

[0277] RASQGISS L LA

[0278] OX118 LC CDR2 (SEQ ID NO: 3)

[0279] DR S R LQS

[0280] OX118 LC CDR3 (SEQ ID NO: 4)

[0281] QQYNSYPYT

[0282] OX118 / R4930 VH sequence (SEQ ID NO: 5)

[0283] EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSIISGSGGFTYYADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYCAKDRLVAPGTFDYWGQGALVTVSS

[0284] OX118 / R4930 HC CDR1 (SEQ ID NO: 6)

[0285] GFTFNSY

[0286] OX118 / R4930 HC CDR2 (SEQ ID NO: 7)

[0287] SGSGGF

[0288] OX118 / R4930 HC CDR3 (SEQ ID NO: 8)

[0289] DRLVAPGTFDY

[0290] R4930 VL sequence (SEQ ID NO: 9)

[0291] CRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTF

[0292] R4930 LC CDR1 (SEQ ID NO: 10)

[0293] RASQGISSWLA

[0294] R4930 LC CDR2 (SEQ ID NO: 11)

[0295] AASSLQS

[0296] R4930 LC CDR3 (SEQ ID NO: 12)

[0297] QQYNSYPYT

[0298] OX118 light chain (SEQ ID NO: 13)

[0299] DIQMTQSPSSLSASVGDRVTITCRASQGISS L LAWYQQKPEKAPKSLI FDR SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0300] OX118 heavy chain (SEQ ID NO: 14)

[0301] EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSIISGSGGFTYYADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYCAKDRLVAPGTFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0302] Belatacept (CTLA-4 Ig fusion protein; SEQ ID NO: 15)

[0303] MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPPCPDSDQEPKSSDKTHTSPPSPAPELLGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0304] Amlitelimab heavy chain (anti-OX40L IgG4; SEQ ID NO: 16)

[0305] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0306] Amlitelimab light chain (anti-OX40L IgG4; SEQ ID NO: 17)

[0307] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSHSVSFTFGPGTKVDIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0308] References

[0309] Almahayni K, Spiekermann M, Fiore A, Yu G, Pedram K, Möckl L. Smallmolecule inhibitors of mammalian glycosylation. Matrix Biol Plus. 2022 Mar16;16:100108. doi: 10.1016 / j.mbplus.2022.100108. Erratum in: Matrix BiolPlus. 2022 Dec 13;16:100126. PMID: 36467541; PMCID: PMC9713294.

[0310] Chatenoud L, Thervet E, Primo J, Bach JF. Anti-CD3 antibody induceslong-term remission of overt autoimmunity in nonobese diabetic mice. ProcNatl Acad Sci U S A. 1994 Jan 4;91(1):123-7. doi: 10.1073 / pnas.91.1.123.PMID: 8278351; PMCID: PMC42898.

[0311] Compaan DM, Hymowitz SG. The crystal structure of the costimulatoryOX40-OX40L complex. Structure. 2006 Aug;14(8):1321-30. doi: 10.1016 / j.str.2006.06.015. PMID: 16905106.

[0312] Edwards E, Livanos M, Krueger A, Dell A, Haslam SM, Mark Smales C,Bracewell DG. Strategies to control therapeutic antibody glycosylation duringbioprocessing: Synthesis and separation. Biotechnol Bioeng. 2022 Jun;119(6):1343-1358. doi: 10.1002 / bit.28066. Epub 2022 Feb 28. PMID: 35182428; PMCID:PMC9310845.

[0313] Guo J, Tu H, Jing L, McCarthy D, Atouf F. USP Reference StandardMonoclonal Antibodies: Tools to Verify Glycan Structure. Pharmaceuticals(Basel). 2022 Mar 5;15(3):315. doi: 10.3390 / ph15030315. PMID: 35337113;PMCID: PMC8951495.

[0314] Haile Y, Adegoke A, Laribi B, Lin J, Anderson CC. Anti-CD52 blocksEAE independent of PD-1 signals and promotes repopulation dominated bydouble-negative T cells and newly generated T and B cells. Eur J Immunol.2020 Sep;50(9):1362-1373. doi: 10.1002 / eji.201948288. Epub 2020 May 27. PMID:32388861.

[0315] Kanda Y, Imai-Nishiya H, Kuni-Kamochi R, Mori K, Inoue M, Kitajima-Miyama K, Okazaki A, Iida S, Shitara K, Satoh M. Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy forgenerating completely non-fucosylated recombinant therapeutics. J Biotechnol.2007 Jun 30;130(3):300-10. doi: 10.1016 / j.jbiotec.2007.04.025. Epub 2007 May6. PMID: 17559959.

[0316] Li W, Zhu Z, Chen W, Feng Y, Dimitrov DS. Crystallizable FragmentGlycoengineering for Therapeutic Antibodies Development. Front Immunol. 2017Nov 13;8:1554. doi: 10.3389 / fimmu.2017.01554. PMID: 29181010; PMCID:PMC5693878.

[0317] Luo, S., Zhang, B. Benchmark Glycan Profile of Therapeutic MonoclonalAntibodies Produced by Mammalian Cell Expression Systems. Pharm Res (2023).https: / / doi.org / 10.1007 / s11095-023-03628-4

[0318] Mishra N, Spearman M, Donald L, Perreault H, Butler M. Comparison oftwo glycoengineering strategies to control the fucosylation of a monoclonalantibody. J Biotechnol. 2020;324S:100015. doi: 10.1016 / j.btecx.2020.100015.Epub 2020 Feb 20. PMID: 34154738.

[0319] Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as criticalquality attributes. Glycobiology. 2015 Dec;25(12):1325-34. doi: 10.1093 / glycob / cwv065. Epub 2015 Aug 11. PMID: 26263923; PMCID: PMC4634315.

[0320] Rillahan, C., Antonopoulos, A., Lefort, C. et al. Global metabolicinhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat ChemBiol 8, 661–668 (2012). https: / / doi.org / 10.1038 / nchembio.999

[0321] Tripathi T, Yin W, Xue Y, Zurawski S, Fujita H, Hanabuchi S, Liu YJ,Oh S, Joo H. Central Roles of OX40L-OX40 Interaction in the Induction andProgression of Human T Cell-Driven Acute Graft-versus-Host Disease.Immunohorizons. 2019 Mar;3(3):110-120. doi: 10.4049 / immunohorizons.1900001.PMID: 31240276; PMCID: PMC6592051.

[0322] Wacker C, Berger CN, Girard P, Meier R. Glycosylation profiles oftherapeutic antibody pharmaceuticals. Eur J Pharm Biopharm. 2011 Nov;79(3):503-7. doi: 10.1016 / j.ejpb.2011.06.010. Epub 2011 Jul 2. PMID: 21745568.

[0323] Yamane-Ohnuki N, Satoh M. Production of therapeutic antibodies withcontrolled fucosylation. MAbs. 2009 May-Jun;1(3):230-6. doi: 10.4161 / mabs.1.3.8328. Epub 2009 May 28. PMID: 20065644; PMCID: PMC2726589.

[0324] Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Iida S, Nakano R, Wakitani M, Niwa R, Sakurada M, Uchida K, Shitara K, Satoh M. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal hostcell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng. 2004 Sep 5;87(5):614-22. doi: 10.1002 / bit.20151. PMID: 15352059.

[0325] Yoshioka T, Nakajima A, Akiba H, Ishiwata T, Asano G, Yoshino S,Yagita H, Okumura K. Contribution of OX40 / OX40 ligand interaction to thepathogenesis of rheumatoid arthritis. Eur J Immunol. 2000 Oct;30(10):2815-23.doi: 10.1002 / 1521-4141(200010)30:10<2815::AID-IMMU2815>3.0.CO;2-#. PMID:11069062.

[0326] Item A

[0327] A1. An antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing a VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0328] A2. The antibody of item 1, wherein the light chain has the amino acid sequence of SEQ ID NO: 13.

[0329] A3. The antibody of item 1, wherein the core fucosylation is less than 70% of the total polysaccharide, for example less than 60%, for example less than 50%, for example less than 40%, for example less than 30%, for example less than 20%.

[0330] A4. An antibody of any one of items 1 to 3, wherein the core fucosylation is determined by quantitative analysis of the glycan, i.e. (area of ​​fucosylated glycan) / (area of ​​total glycan).

[0331] A5. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing inhibitors of glucosidase I and II, and subsequently purifying the antibody.

[0332] A6. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing a fucose analogue, and subsequently the antibody is purified.

[0333] A7. The method of item 6, wherein the fucose analogue is an inhibitor of fucokinase, GDP-fucosylationase, fucosyltransferase (FUT), GDP-mannose 4,6-dehydratase, GDP-fucosylationase and / or fucose transporter such as GDP-fucosylation transporter.

[0334] A8. The method of item 6, wherein the fucose analogue is an inhibitor of fucosyltransferase (FUT), such as 1,6-fucosyltransferase (FUT8).

[0335] A9. A method for producing an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells to prevent core fucosylation.

[0336] A10. The method of item 9, wherein the cells are selected from the following cells: CHO cells (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS cells (e.g., COS-7), Syrian hamster cells, rat myeloma cells, mouse myeloma cells (e.g., SP2 / 0, NSO), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, Jurkat cells, Daudi cells, A431 cells (epidermal cells), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, MMT 060562 cells, Sertoli cells, BRL cells. 3A cells, HT1080 cells, human myeloma cells, tumor cells, human lymphoma cells (e.g., Namalwa cells), and cell lines derived from the above cells.

[0337] A11. The method of any one of items 9 or 10, wherein the fucosylation of the antibody or antibody derivative is restored and / or titrated to the desired level by exposure to fucose, wherein the cells are exposed to a culture medium containing fucose.

[0338] A12. A method for testing a defucosylated anti-OX40L antibody or an antibody derivative thereof, comprising testing the antibody in a T-cell activation assay and verifying whether T-cell activation is reduced compared to the fucosylated anti-OX40L antibody.

[0339] A13. An antibody or antibody derivative according to any of the preceding claims, used for the prevention and / or treatment of inflammatory diseases.

[0340] A14. An antibody or antibody derivative according to any of the preceding claims, used for the prevention and / or treatment of autoimmune diseases.

[0341] A15. The antibody or antibody derivative according to items 13 to 15, wherein the disease is selected from graft-versus-host disease, allogeneic transplant rejection, asthma, systemic lupus erythematosus, arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, atopic dermatitis, psoriasis, hidradenitis suppurativa, immunoglobulin A nephropathy, Hashimoto's disease, Graves' disease, chronic sinusitis, and multiple sclerosis.

[0342] item

[0343] 1. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (VL).

[0344] 2. An anti-OX40L antibody having a variable light chain and a variable heavy chain, wherein the variable light chain comprises the following CDRs: a. SEQ ID NO: 2; b. SEQ ID NO: 3; and c. SEQ ID NO: 4; And the variable heavy chain mentioned therein includes the following CDRs: d. SEQ ID NO: 6; e. SEQ ID NO: 7; and f. SEQ ID NO: 8.

[0345] 3. An anti-OX40L antibody comprising a heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: g. SEQ ID NO: 2; h. SEQ ID NO: 3; and i. SEQ ID NO: 4; Furthermore, the heavy chain described herein comprises a constant region and a VH, wherein the VH comprises the following CDRs: j. SEQ ID NO: 6; k. SEQ ID NO: 7; and l. SEQ ID NO: 8, and, The core fucosylation of the heavy chain constant region is less than 80%.

[0346] 4. An anti-OX40L antibody comprising a heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: m. SEQ ID NO: 2; n. SEQ ID NO: 3; and o. SEQ ID NO: 4; And the heavy chain mentioned therein contains the following CDRs: p. SEQ ID NO: 6; q. SEQ ID NO: 7; and r. SEQ ID NO: 8, and, The core fucosylation of the heavy chain is less than 80%.

[0347] 5. An anti-OX40L antibody comprising an IgG1 heavy chain and a light chain, wherein the light chain comprises a VL, and the VL comprises the following CDRs: s. SEQ ID NO: 2; t. SEQ ID NO: 3; and u. SEQ ID NO: 4; And the IgG1 heavy chain contains the following CDRs: v. SEQ ID NO: 6; w. SEQ ID NO: 7; and x. SEQ ID NO: 8, and, The core fucosylation of the IgG1 heavy chain is less than 80%.

[0348] 6. An antibody comprising a light chain, the light chain comprising the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the antibody is less than 80%.

[0349] 7. An anti-OX40L antibody comprising a heavy chain and a light chain, the light chain comprising the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0350] 8. The antibody according to any one of the preceding claims, wherein the antibody comprises an IgG1 heavy chain.

[0351] 9. The antibody according to any one of the preceding claims, wherein the antibody comprises the IgG1 heavy chain constant region.

[0352] 10. The antibody according to any one of the preceding claims, wherein the antibody comprises an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region is glycosylated.

[0353] 11. An antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing a VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0354] 12. An anti-OX40L antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing the VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

[0355] 13. The antibody according to any of the preceding claims, wherein the core fucosylation of the heavy chain constant region is less than 80%, as determined by capillary electrophoresis, LC-MS, high-performance liquid chromatography with fluorescence detection (HPLC-FD), high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), mass spectrometry, hydrazine hydrolysis and / or enzymatic hydrolysis.

[0356] 14. The antibody according to any one of the preceding claims, comprising the variable heavy chain of SEQ ID NO: 5 and the variable light chain of SEQ ID NO: 1.

[0357] 15. The antibody according to any of the preceding claims, comprising the heavy chain of SEQ ID NO: 14 and the light chain of SEQ ID NO: 13.

[0358] 16. The antibody according to any one of the preceding claims, wherein the antibody is glycosylated with at least 2 kDa of total glycans.

[0359] 17. The antibody according to any of the preceding claims, wherein the antibody is glycosylated with up to 3 kDa of total glycans.

[0360] 18. The antibody according to any of the preceding claims, wherein the antibody is glycosylated with at least 2 kDa of total polysaccharides and at most 3 kDa of total polysaccharides.

[0361] 19. The antibody according to any of the preceding claims, wherein the heavy chain constant region is glycosylated with at least 2 kDa of total glycans.

[0362] 20. The antibody according to any of the preceding claims, wherein the heavy chain constant region is glycosylated with up to 3 kDa of total glycans.

[0363] 21. The antibody according to any of the preceding claims, wherein the heavy chain constant region is glycosylated by at least 2 kDa of total glycans and at most 3 kDa of total glycans.

[0364] 22. The antibody according to any of the preceding claims, wherein the antibody glycan comprises 50% to 80% glycan GO.

[0365] 23. The antibody according to any one of the preceding claims, wherein the antibody glycan comprises 50% to 80% glycan GO, for example 55% to 80%, for example 60% to 80%, for example 65% to 80%, for example 70% to 80%, for example 75% to 80%, for example 50% to 75%, for example 50% to 70%, for example 50% to 65%, for example 50% to 60%, for example 50% to 55% glycan GO.

[0366] 24. The antibody according to any of the preceding claims, wherein the total polysaccharides of the antibody comprise 50% to 80% polysaccharide GO.

[0367] 25. The antibody according to any one of the preceding claims, wherein the total polysaccharides of the antibody comprise 50% to 80% of polysaccharide GO, for example 55% to 80%, for example 60% to 80%, for example 65% to 80%, for example 70% to 80%, for example 75% to 80%, for example 50% to 75%, for example 50% to 70%, for example 50% to 65%, for example 50% to 60%, for example 50% to 55% of polysaccharide GO.

[0368] 26. The antibody according to any of the preceding claims, wherein the core fucosylation is less than 70% of the total antibody polysaccharide, for example less than 60%, for example less than 50%, for example less than 40%, for example less than 30%, for example less than 20%.

[0369] 27. The antibody according to any of the preceding claims, wherein the core fucosylation is determined by quantitative analysis of the glycan, i.e. (area of ​​fucosylated glycan) / (area of ​​total glycan).

[0370] 28. The antibody according to any one of items 1 to 26, wherein the core fucosylation is determined as (amount of fucosylated glycan) / (amount of total glycan).

[0371] 29. The antibody according to any of the preceding claims, wherein the core fucosylation is determined by capillary electrophoresis or HPLC, such as hydrophilic interaction liquid chromatography-HPLC (HILIC-HPLC), electrospray ionization mass spectrometry (ESI-MS), or hydrophilic interaction liquid chromatography-FLD-MS / MS equipped with fluorescence detection and tandem mass spectrometry.

[0372] 30. The antibody according to any one of the preceding claims, wherein the antibody is covalently linked to a ligand selected from: chromophores, fluorophores, radiotracers, drugs, peptides, proteins, enzymes, single- or double-stranded oligonucleotides and their analogs, biotin, and therapeutic portions such as cytotoxins, chemotherapeutic agents, cytokines, and radioisotopes.

[0373] 31. The antibody according to any of the preceding claims, wherein the antibody is humanized.

[0374] 32. The antibody according to any of the preceding claims, wherein the antibody is a human subclass IgG1, preferably wherein the IgG1 has the standard N-glycan glycosylation pattern of human IgG1, but with reduced fucosylation.

[0375] 33. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing inhibitors of glucosidase I and II, and subsequently purifying the antibody.

[0376] 34. The method according to item 33, wherein the cell is a mammalian cell.

[0377] 35. The method according to any one of claims 33 to 34, wherein the inhibitor is selected from the group consisting of castanospermine, deoxymannojirimycin, and australine.

[0378] 36. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding polypeptides SEQ ID NO: 13 and SEQ ID NO: 14 in cells, wherein the cells are exposed to a culture medium containing a fucose analogue, and subsequently the antibody is purified.

[0379] 37. The method according to item 36, wherein the cell is a mammalian cell.

[0380] 38. The method according to any one of claims 36 to 37, wherein the fucose analogue is an inhibitor of fucokinase, GDP-fucosylationase, fucosyltransferase (FUT), GDP-mannose 4,6-dehydratase, GDP-fucosylationase and / or fucose transporter such as GDP-fucosylation transporter.

[0381] 39. The method according to items 36 to 37, wherein the fucose analogue is an inhibitor of fucosyltransferase (FUT), such as 1,6-fucosyltransferase (FUT8).

[0382] 40. The method according to any one of claims 36 to 37, wherein the fucose analogue is selected from the group comprising: L-fucose, 2-fluorofucose, 2-fluoroperacetylated fucose (2FF), 5-alkynylfucose, alkynylfucose monoacetate, alkynylfucose triacetate, alkynylfucose diacetate, and 5-alkynylfucose peracetate.

[0383] 41. The method according to any one of claims 36 to 37, wherein the fucose analogue is 2-fluoro-peracetylated fucose (2FF).

[0384] 42. The method according to any one of claims 36 to 41, wherein a fucose analogue is added to the culture medium to maintain its effective concentration.

[0385] 43. A method for producing an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding polypeptides SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells to prevent core fucosylation.

[0386] 44. The method according to item 43, wherein the cell does not encode a functional fucosyltransferase.

[0387] 45. The method according to claim 43, wherein the cell encodes a functional β-1,4-N-acetylglucosamine transferase (GnTIII), GDP-6-deoxy-D-lythose-4-hexylose reductase (RMD), and / or Golgi α-mannosidase II (ManII).

[0388] 46. ​​The method according to any one of items 43 to 45, wherein the cell is a mammalian cell.

[0389] 47. The method according to any one of items 43 to 46, wherein the cell is a recombinant cell.

[0390] 48. The method according to any one of claims 43 to 47, wherein the cells are selected from the following cells: CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), Syrian hamster cells, rat myeloma cells, mouse myeloma cells (e.g., SP2 / 0, NSO), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, Jurkat cells, Daudi cells, A431 cells (epidermal cells), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, MMT 060562 cells, Sertoli cells, BRL cells. 3A cells, HT1080 cells, human myeloma cells, tumor cells, human lymphoma cells (e.g., Namalwa cells), and cell lines derived from the above cells.

[0391] 49. The method according to any one of items 43 to 48, wherein the fucosylation of the antibody or antibody derivative is restored and / or titrated to the desired level by exposure to fucose, wherein the cells are exposed to a culture medium containing fucose.

[0392] 50. The method according to item 49, wherein fucose is added to the culture medium in increasing concentrations, for example up to 1 mM.

[0393] 51. A method for producing an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in cells, subsequently purifying the antibody, and optionally folding the antibody.

[0394] 52. The method according to claim 51, wherein the cell is selected from mammalian cells, bacterial cells, yeast cells, and plant cells.

[0395] 53. The method according to any one of items 33 to 52, wherein the antibody is IgG1.

[0396] 54. The method according to any one of items 33 to 53, wherein the antibody is a complete antibody.

[0397] 55. The method according to any one of claims 33 to 54, wherein the antibody comprises a heavy chain variable region, a light chain variable region, and an Fc region.

[0398] 56. The method according to any one of claims 33 to 55, wherein the antibody derivative comprises an antibody Fc region and a ligand-binding domain of a non-immunoglobulin protein.

[0399] 57. The method according to any one of claims 33 to 56, wherein the method includes the additional step of isolating the antibody.

[0400] 58. The method according to any one of claims 33 to 57, wherein the method includes an additional step of purifying the antibody.

[0401] 59. A method for testing the potency of a defucosylated anti-OX40L antibody or an antibody derivative thereof, comprising testing the antibody in a T-cell activation assay and verifying whether T-cell activation is reduced compared to the fucosylated anti-OX40L antibody.

[0402] 60. A method for testing the potency of the defucosylated anti-OX40L antibody or antibody derivative described in any of the preceding claims, comprising testing the antibody in a T-cell activation assay and verifying whether T-cell activation is reduced compared to the fucosylated anti-OX40L antibody.

[0403] 61. The method according to any one of claims 59 to 60, wherein the T cell activation is reduced by at least 5%, for example at least 10%, for example at least 15%, for example at least 25%, for example at least 30%.

[0404] 62. A pharmaceutical composition comprising the antibody described in any one of claims 1 to 32 and at least one pharmaceutically acceptable excipient.

[0405] 63. The antibody or antibody derivative according to any one of the preceding claims for the prevention and / or treatment of at least one disease or condition associated with OX40L.

[0406] 64. The antibody or antibody derivative according to any of the preceding claims for the prevention and / or treatment of inflammatory diseases.

[0407] 65. Use of an antibody or antibody derivative as defined in any one of items 1 to 32 in the preparation of a medicament for the prevention and treatment of inflammatory diseases.

[0408] 66. The antibody or antibody derivative described in any of the preceding claims for the prevention and / or treatment of autoimmune diseases.

[0409] 67. Use of an antibody or antibody derivative as defined in any one of items 1 to 32 in the preparation of a medicament for the prevention and treatment of autoimmune diseases.

[0410] 68. An antibody or antibody derivative according to any one of items 63 to 67, wherein the disease is selected from graft-versus-host disease, allogeneic transplant rejection, asthma, systemic lupus erythematosus, arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, atopic dermatitis, psoriasis, hidradenitis suppurativa, immunoglobulin A nephropathy, Hashimoto's disease, Graves' disease, chronic sinusitis, and multiple sclerosis.

[0411] 69. A kit comprising the antibody or antibody derivative described in any one of items 1 to 32.

Claims

1. An antibody comprising a heavy chain of SEQ ID NO: 14 and a light chain containing a VL sequence of SEQ ID NO: 1, wherein the core fucosylation of the heavy chain is less than 80%.

2. The antibody of claim 1, wherein the light chain has the amino acid sequence of SEQ ID NO:

13.

3. The antibody of claim 1, wherein the core fucosylation is less than 70% of the total polysaccharide, for example less than 60%, for example less than 50%, for example less than 40%, for example less than 30%, for example less than 20%.

4. The antibody as claimed in any of the preceding claims, wherein the antibody is glycosylated with at least 2 kDa of total glycans.

5. The antibody as claimed in any of the preceding claims, wherein the antibody is glycosylated with up to 3 kDa of total glycans.

6. The antibody as claimed in any of the preceding claims, wherein the antibody is glycosylated with at least 2 kDa of total polysaccharides and at most 3 kDa of total polysaccharides.

7. The antibody as claimed in any of the preceding claims, wherein the antibody polysaccharide comprises 50% to 80% polysaccharide GO.

8. The antibody as claimed in any of the preceding claims, wherein the antibody glycan comprises 50% to 80% glycan GO, for example 55% to 80%, for example 60% to 80%, for example 65% to 80%, for example 70% to 80%, for example 75% to 80%, for example 50% to 75%, for example 50% to 70%, for example 50% to 65%, for example 50% to 60%, for example 50% to 55% glycan GO.

9. The antibody as claimed in any of the preceding claims, wherein the core fucosylation is determined by quantitative analysis of the glycans, i.e., (area of ​​fucosylated glycans) / (area of ​​total glycans).

10. The antibody as claimed in any of the preceding claims, wherein the core fucosylation is defined as (amount of fucosylated glycans) / (amount of total glycans).

11. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO:13 and SEQ ID NO:14 in cells, wherein the cells are exposed to a culture medium containing inhibitors of glucosidase I and II, and subsequently purifying the antibody.

12. A method for producing an anti-OX40L antibody or antibody derivative, comprising expressing a polynucleotide encoding a polypeptide encoding SEQ ID NO:13 and SEQ ID NO:14 in cells, wherein the cells are exposed to a culture medium containing a fucose analogue, and subsequently the antibody is purified.

13. The method of claim 12, wherein the fucose analogue is an inhibitor of fucokinase, GDP-fucosylationase, fucosyltransferase (FUT), GDP-mannose 4,6-dehydratase, GDP-fucosylationase, and / or a fucose transporter such as GDP-fucosylation transporter.

14. The method of claim 12, wherein the fucose analogue is an inhibitor of fucosyltransferase (FUT), such as 1,6-fucosyltransferase (FUT8).

15. A method for producing an anti-OX40L antibody or antibody derivative comprising expressing a polynucleotide encoding a polypeptide of SEQ ID NO: 13 and SEQ ID NO: 14 in genetically modified cells to prevent core fucosylation.

16. The method of claim 15, wherein the cells are selected from the following cells: CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), Syrian hamster cells, rat myeloma cells, mouse myeloma cells (e.g., SP2 / 0, NSO), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, Jurkat cells, Daudi cells, A431 cells (epidermal cells), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, MMT 060562 cells, Sertoli cells, BRL cells. 3A cells, HT1080 cells, human myeloma cells, tumor cells, human lymphoma cells (e.g., Namalwa cells), and cell lines derived from the above cells.

17. The method according to any one of claims 15 or 16, wherein the fucosylation of the antibody or antibody derivative is restored and / or titrated to the desired level by exposure to fucose, wherein the cells are exposed to a culture medium containing fucose.

18. A method for testing the potency of a defucosylated anti-OX40L antibody or an antibody derivative thereof, comprising testing the antibody in a T-cell activation assay and verifying whether T-cell activation is reduced compared to the defucosylated anti-OX40L antibody.

19. An antibody or antibody derivative according to any one of the preceding claims, for the prevention and / or treatment of inflammatory diseases.

20. An antibody or antibody derivative according to any one of the preceding claims, for the prevention and / or treatment of autoimmune diseases.

21. The antibody or antibody derivative according to claims 19 to 20, wherein the disease is selected from graft-versus-host disease, allogeneic transplant rejection, asthma, systemic lupus erythematosus, arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, atopic dermatitis, psoriasis, hidradenitis suppurativa, immunoglobulin A nephropathy, Hashimoto's disease, Graves' disease, chronic sinusitis, and multiple sclerosis.

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