Humanized anti-CD137 antibodies and uses thereof

Abstract

Disclosed herein are humanized anti-CD137 antibodies and methods of using them to induce CD137 signaling, thereby enhancing immune responses, such as T cell function. The antibodies disclosed herein can be used to treat diseases, such as cancer and immune disorders.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of International Patent Application No. PCT / CN2019 / 086364, filed May 10, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] CD137, also known as 4-1BB or tumor necrosis factor receptor subfamily 9 (TNFRSF9), is a member of the tumor necrosis factor (TNF) receptor family. It is involved in the regulation of activated T cells (CD4 + More than CD8 + More broadly, it is expressed by dendritic cells, B cells, follicular dendritic cells, natural killer cells, granulocytes, and in cells of the blood vessel wall at sites of inflammation.

[0003] CD137 is a costimulatory receptor on activated T cells, and crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137 can also induce peripheral monocyte proliferation and enhance T cell apoptosis induced by TCR / CD3-triggered activation and regulated CD28 costimulation, resulting in promotion of Th1 cell responses. Its expression is induced by lymphocyte activation, and in addition to CD137 ligand (CD137L), TNF receptor-associated factor (TRAF) adaptor proteins have been found to bind to the receptor and result in the transduction of signals that activate NF-κB.

[0004] Both antagonistic and agonistic antibodies specific for CD137 have been developed. US6,569,997, US8,137,667, Fisher et al., Cancer Immunol Immunother (2012) 61:1721-1733. Agonistic antibodies specific for CD137 have been reported to enhance T cell function and promote antitumor activity. Fisher et al., Cancer Immunol Immunother (2012) 61:1721-1733. On the other hand, agonistic antibodies specific for CD137 have also been reported to induce significant hepatotoxicity in patients. Segal et al., Clin Can Res., 2017, 23:1929-1936.

[0005] It is therefore of interest to develop effective and safe CD137 agonists for therapeutic use. Summary of the Invention

[0006] The present disclosure is based, at least in part, on the development of superior humanized anti-CD137 antibodies, which may be full-length antibodies, optionally including Fc variants with altered binding activity to one or more Fc receptors. Such humanized anti-CD137 antibodies have been demonstrated to have a variety of superior characteristics, as reported in the following examples.

[0007] Thus, one aspect of the disclosure provides a humanized antibody that binds to CD137. Such an antibody comprises: (i) a heavy chain variable domain (V H ), and (ii) a light chain variable domain (V L ). V H contains the same heavy chain complementarity determining regions (CDRs) 1 to 3 as reference antibody 371, with the heavy chain CDRs grafted into the human IGHV1-2*2 framework. L contains the same light chain CDRs 1-3 as reference antibody 371, with the light chain CDRs grafted into the human IGKV1-39*01 framework.

[0008] In some embodiments, a humanized anti-CD137 antibody may comprise a heavy chain CDR1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 12, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 16. Alternatively, or in addition, a humanized anti-CD137 antibody may comprise a light chain CDR1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 29, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 33.

[0009] In some embodiments, the humanized anti-CD137 antibodies disclosed herein may comprise a heavy chain framework region 1 (HC FR1) comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having three or fewer back mutations, a HC FR2 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof having three or fewer back mutations, a HC FR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof having three or fewer back mutations, and / or a HC FR4 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof having three or fewer back mutations.

[0010] Alternatively, or in addition, the humanized anti-CD137 antibodies disclosed herein may comprise a light chain framework region 1 (LC FR1) comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof having three or fewer back mutations, a LC FR2 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof having three or fewer back mutations, a LC FR3 comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having three or fewer back mutations, and / or a LC FR4 comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof having three or fewer back mutations.

[0011] In some examples, the humanized anti-CD137 antibody may be a humanized light chain variable region comprising one or more back mutations at positions K42 (e.g., K42G), P44 (e.g., P44V), F71 (e.g., F71Y), Y87 (e.g., Y87F), and V104 (e.g., V104L) in SEQ ID NO: 4. In one example, the humanized antibody comprises an LC FR1 comprising the amino acid sequence of SEQ ID NO: 35, an LC FR2 comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 39, an LC FR3 comprising a sequence selected from SEQ ID NO: 37 and SEQ ID NO: 40, and / or an LC FR4 comprising the sequence of SEQ ID NO: 38 or SEQ ID NO: 41. In a particular example, the humanized antibody of claim 4 comprises an LC FR1 comprising the sequence of SEQ ID NO: 35, an LC FR2 comprising the sequence of SEQ ID NO: 39, an LC FR3 comprising the sequence of SEQ ID NO: 40, and an LC FR4 comprising the sequence of SEQ ID NO: 38.

[0012] In some examples, a humanized anti-CD137 antibody disclosed herein may comprise a VH comprising the amino acid sequence of SEQ ID NO:3, 8, or 9, and / or a VL comprising the amino acid sequence of SEQ ID NO:4, 5, or 10. In a particular example, a humanized antibody may comprise a VH comprising the amino acid sequence of SEQ ID NO:3 and a VL comprising the amino acid sequence of SEQ ID NO:5.

[0013] In some embodiments, the humanized antibody may be a full-length antibody (e.g., an IgG molecule, such as an IgG1 molecule). Alternatively, the humanized antibody may be an antigen-binding fragment thereof. In some examples, the full-length antibody may comprise a wild-type Fc region. In other examples, the full-length antibody may comprise an Fc variant with altered effector activity. One example is an Fc region comprising the amino acid sequence of SEQ ID NO: 42. In certain examples, the humanized antibody disclosed herein may comprise a heavy chain having the amino acid sequence of SEQ ID NO: 6 and / or a light chain having the amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, the humanized anti-CD137 antibodies disclosed herein can be part of a multispecific antibody that further binds to FcγRIIB.

[0015] In another aspect, provided herein is an isolated nucleic acid or set of nucleic acids that collectively encodes any of the humanized anti-CD137 antibodies disclosed herein.In some embodiments, the isolated nucleic acid or set of nucleic acids is located on one vector.In other embodiments, the set of nucleic acids is located on two vectors.In some examples, the one or two vectors are one or two expression vectors.

[0016] In yet another aspect, a host cell is provided herein that comprises any of the isolated nucleic acids or sets of nucleic acids disclosed herein. For example, the host cell can comprise one or more expression vectors for producing a humanized anti-CD137 antibody.

[0017] In addition, the disclosure features pharmaceutical compositions including any of the humanized anti-CD137 antibodies disclosed herein, or nucleic acids encoding same.

[0018] In another aspect, the present disclosure provides a method of modulating an immune response in a subject, the method comprising administering to a subject in need of modulating an immune response an effective amount of any of the pharmaceutical compositions disclosed herein, comprising a humanized anti-CD137 antibody also disclosed herein. In some embodiments, the subject may be undergoing a therapy comprising an immune checkpoint inhibitor. In other embodiments, the method may further comprise administering to the subject an immune checkpoint inhibitor. Exemplary checkpoint inhibitors include anti-PD-1 or anti-PD-L1 antibodies. One example is pembrolizumab.

[0019] In some embodiments, the subject to be treated may be a human patient who has, is suspected of having, or is at risk of having cancer. Examples include prostate cancer, colon cancer, melanoma, etc. In some examples, the cancer is an advanced, metastatic, or unresectable malignant tumor. In some examples, the cancer is histologically or cytologically confirmed.

[0020] In some embodiments, the subject to be treated may be a human patient who has, is suspected of having, or is at risk of having an immune disorder.In some examples, the immune disorder may be an autoimmune disease.Examples include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes, multiple sclerosis, celiac disease, and graft-versus-host (GVH) disease.

[0021] In some embodiments, the subject (eg, a human patient) has undergone or is undergoing therapy for cancer or an immune disorder.

[0022] In some embodiments, the humanized anti-CD137 antibody (e.g., clone 3712-IgG1v) may be administered to a subject at a dose of about 0.3-10 mg / kg. In some embodiments, the humanized anti-CD137 antibody is administered to a subject once every 2-4 weeks, optionally once every 3 weeks.

[0023] Further provided herein is a method of producing a humanized anti-CD137 antibody, comprising (i) culturing a host cell as disclosed herein that comprises one or more expression vectors encoding any of the humanized anti-CD137 antibodies disclosed herein under conditions that allow expression of the anti-CD137 antibody, and (ii) harvesting the anti-CD137 antibody so produced from the cell culture. In some embodiments, the method may further comprise isolating the antibody from the host cell or the culture supernatant.

[0024] Also within the scope of the present disclosure are humanized anti-CD137 antibodies as disclosed herein for use in the treatment of cancer or immune disorders as also disclosed herein, and the use of such humanized anti-CD137 antibodies for the manufacture of a medicament for use in the treatment of cancer or immune disorders.

[0025] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0027] [Figure 1] Figure 1 is a graph showing FACS analysis of reference antibody 371 (mouse / human chimera) and its humanized versions, clones 3711 and 3712, for binding to CHO cells overexpressing human CD137. Serially diluted antibodies at final concentrations as indicated were incubated with CHO-human CD137 cells. The mean fluorescence intensity (MFI) shown on the y-axis indicates antibody binding. [Diagram 2] Figure 2 is a bar graph showing a comparison of reference antibody 371 and humanized antibodies 3711 and 3712 in a human CD8+ T cell co-stimulation assay. Serially diluted antibodies at final concentrations of 10, 20, and 40 ng / mL as indicated were added to plates where human CD8+ T cells were co-cultured with CHO-K1-huFcγRIIB cells. IFNγ concentration shown on the y-axis indicates activation of human CD8+ T cells. [Diagram 3] Figure 3 is a line graph showing the binding of huCD137-C6His to clone 3712 on a Biacore T200. Serial dilutions of clone 3712 (2.5, 5, 10, 20, 40, 80, 160, and 320 nM in duplicate) were injected sequentially over a flow cell with immobilized human CD137 protein with an association time of 180 seconds, and buffer flow was maintained for 180 seconds for dissociation. [Figure 4A]Figures 4A-4B are graphs showing the results of ELISA for the binding of CD137 protein and the humanized antibody 3712. Clone 3712 or Avastin (negative control) serially diluted at the final concentrations as indicated on the x-axis was added to plates coated with recombinant human (Figure 4A) or cyno (Figure 4B) CD137 protein. The absorbance shown on the y-axis indicates the binding of the antibody. [Figure 4B] Same as above. [Figure 5A] Figures 5A-5C are graphs showing FACS of the binding of clone 3712 and cell CD137. Clone 3712 or Avastin serially diluted at the final concentrations as indicated on the x-axis was incubated with CHO-human CD137 (Figure 5A), CHO-cynomolgus CD137 (Figure 5B), or parental CHO (Figure 5C) cells. The mean fluorescence intensity (MFI) shown on the y-axis indicates the binding of the antibody. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] Figures 6A-6B are graphs showing FACS of the binding of clone 3712 to endogenous CD137 on activated CD8 T cells. Clone 3712 or Avastin serially diluted at the final concentrations as indicated on the x-axis was incubated with human (Figure 6A) or cynomolgus (Figure 6B) PBMC pre-activated with anti-CD3 antibody. The mean fluorescence intensity (MFI) of CD8+ T cells shown on the y-axis indicates the binding of the antibody. [Figure 6B] Same as above. [Figure 7A]7A-7E are graphs showing FACS analysis of clone 3712 binding to CHO cells overexpressing human Fcγ receptors. Serial dilutions of clone 3712 or Avastin at final concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.078, 0.039, 0.0195, 0.0098, 0.0049, 0.0024, 0.0012 and 0.0006 μg / mL) as indicated on the x-axis were incubated with CHO-huFcγ receptor cells: CHO-huFcγRIA (Figure 7A), CHO-huFcγRIIA-R131 (Figure 7B), CHO-huFcγRIIA-H131 (Figure 7C), CHO-huFcγIIB (Figure 7D), and CHO-huFcγIIIA (Figure 7E). Mean fluorescence intensity (MFI) indicated on the y-axis indicates antibody binding. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 8A] 8A-8B are graphs showing the binding of clone 3712 to FcRn (FIG. 8A) and C1q (FIG. 8B) as measured by ELISA. In FIG. 8A, FcRn was added to plates coated with clone 3712 or Avastin at final concentrations of 10000, 5000, 2500, 1250, 625, 312.5, and 156.3 ng / mL, as indicated on the x-axis. FcRn bound to the coated antibody was detected by anti-His tag-HRP antibody. In FIG. 8B, clone 3712 or Avastin was coated on an ELISA plate at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, and 8 μg / mL, as indicated on the x-axis, and C1q was added to the plate at 2 mg / mL. C1q bound to the antibody was detected by anti-human C1q-HRP antibody. [Figure 8B] Same as above. [Figure 9]Figure 9 is a graph showing the results of CD137 reporter activity assay. Serial dilutions of clone 3712 or Avastin at final concentrations of 25000, 6250, 1562.5, 390.6, 97.7, 24.4, 6.10, 1.53, 0.381, 0.095, and 0.024 ng / mL, as shown on the x-axis, were added to plates where CD137 reporter cells were co-cultured with CHO-K1-huFcγRIIB, CHO-K1, or medium without cells. IL-8 concentration shown on the y-axis indicates activation of CD137 reporter cells. [Figure 10] Figure 10 is a graph showing the results of a human CD8+ T cell co-stimulation assay. Serial dilutions of clone 3712 or Avastin at final concentrations of 25000, 6250, 1562.5, 390.6, 97.7, 24.4, 6.10, 1.53, 0.381, and 0.095 ng / mL, as shown on the x-axis, were added to plates of human CD8+ T cells co-cultured with CHO-K1-huFcγRIIB, CHO-K1, or medium. IFNγ concentration, shown on the y-axis, indicates activation of human CD8+ T cells. [Figure 11] 11 is a graph showing the pharmacokinetics of clone 3712 in mice. Individual plasma concentration-time profiles of clone 3712 following IV dosing of 3 mg / kg in male C57BL / 6 mice are shown. [Figure 12A] Figures 12A-12B are graphs showing tumor growth curves of various groups in a mouse tumor model. Mouse colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice on day 0. Mice with established tumors were divided into control and treatment groups (n=6) on day 7, and treatments as indicated were administered by intraperitoneal injection. Tumor sizes were measured twice weekly by caliber and calculated as tumor volume using the formula 0.5 x length x width2. The mean ± SEM of tumor size is shown in Figure 12A, and Figure 12B shows individual mouse data. [Figure 12B] Same as above. [Figure 13A]Figures 13A-13B are graphs showing tumor growth curves in various groups. Mouse colon cancer MC38 cells were implanted subcutaneously into homozygous B-h4-1BB mice on day 0. Mice with established tumors were divided into control and treatment groups (n=6) on day 7, and the indicated treatments were administered by intraperitoneal injection. Figure 13A compares treatment with clone 3712 to two reference antibodies reported in the literature. Figure 13B shows the dosing effect of clone 3712. Tumor sizes were measured twice a week by Calibur and calculated as tumor volume using the formula 0.5 x length x width2. The mean ± SEM of tumor size is shown. Mean values ​​were compared using a multiple t-test in Prism. In both Figures 13A and 13B, statistically significant differences p<0.05 and p<0.01 when compared to the 10 mg / kg group of clone 3712 are indicated by * and **, respectively. [Figure 13B] Same as above. [Figure 14] Figure 14 is a graph showing the effect of combining clone 3712 with anti-PD-1 antibody in a mouse tumor model. Mouse melanoma B16-OVA cells were implanted subcutaneously on day 0 into WT B6 mice that had been transplanted with hPD-1 / 4-1BB mouse donor bone marrow cells after lethal irradiation. Tumor volumes were measured along three orthogonal axes (a, b, and c) and calculated as tumor volume = abc / 2. After tumors were established (day 14), mice were divided into control and treatment groups (n=5) treated with 100 μg of anti-PD-1 antibody on days 14, 21, and 28, or with 100 μg of anti-CD137 clone 3712 administered by intraperitoneal injection on day 14, or with combination treatments at the same dosing schedule as the single agents. Tumor growth was measured twice weekly. Relative tumor size was calculated by dividing tumor size by the initial tumor size on day 14. Mean tumor size ± SEM is shown. Mean values ​​were compared using multiple t-tests in Prism. Statistically significant differences p<0.05 compared with single agent groups are indicated with *. [Figure 15] FIG. 15 shows the T cell stimulatory activity for humanized antibodies presented at different concentrations. [Figure 16A]Figures 16A-16D include diagrams showing the binding of clone 3712 to various chimeric CD137 receptor proteins. Figures 16A and 16C show the binding of clone 3712 to CD137 receptor proteins Ly048, Ly049, and ly050 compared to human CD137. Figures 16B and 16D show the binding of clone 3712 to CD137 receptor proteins Ly051, Ly052, and ly110 compared to human CD137. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 17] FIG. 17 shows the binding of various anti-CD137 antibodies to the CD137 receptor. [Figure 18] FIG. 18 shows inhibition of tumor growth inhibition by clone 3712-IgG1v. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Provided herein is a humanized agonistic antibody that can bind to CD137 and FcγRIIB (e.g., via a suitable Fc moiety) and enhance CD137-mediated signal transduction in the presence of FcγRIIB. Such anti-CD137 antibodies can be derived from the reference anti-CD137 antibody clone 371 (mouse parent) disclosed herein. The humanized anti-CD137 antibodies disclosed herein have shown similar or superior biological activity compared to the parent clone, as shown in the following examples. For example, humanized anti-CD137 antibody clone 3712 (e.g., IgG1 format that may include IgG Fc variants) has CD137 binding affinity comparable to the parent antibody, selective binding to FcγRIIB, and superior T cell stimulating activity, and has shown superior anti-tumor activity than known anti-CD137 antibodies. When investigated in animal models, the humanized antibodies were safe. The humanized antibodies are expected to show a safety profile in humans. Additionally, the humanized anti-CD137 antibodies described herein may be synergistic in combination with anti-PD-1 and other immunotherapies, as well as cancer vaccines, cellular therapies, and / or other oncology therapeutics.

[0029] CD137 (also known as 4-1BB or TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor family. It is expressed on activated T cells (CD4 + More than CD8 +CD137 is expressed by DCs, follicular dendritic cells, natural killer cells, granulocytes, and more broadly by DCs (Gramaglia et al., Eur. J. Immunol., 30(2):392-402 (2000)), and by dendritic cells, B cells, follicular dendritic cells, natural killer cells, granulocytes, and in cells of the vascular wall at sites of inflammation. Expression of CD137 on dendritic cells has been shown to result in increased secretion of IL-6 and IL-12, as well as the ability of DCs to stimulate T cell responses to alloantigens and infiltrate tumors (Pan et al., J. Immunol., 172(8):4779-89 (2004)). Activated natural killer cells express CD137 after stimulation with cytokines, promoting natural killer cell proliferation and IFN-γ secretion without affecting cytolytic activity (Wilcox et al., J. Immunol., 169(8):4230-6 (2002)).

[0030] Accordingly, described herein are humanized anti-CD137 antibodies (e.g., agonistic anti-CD137 antibodies), nucleic acids encoding same, pharmaceutical compositions comprising the antibodies or the encoding nucleic acids, and the use of such antibodies in therapeutic applications.

[0031] Humanized antibody that binds to CD137 The present disclosure provides humanized antibodies that bind to CD137, particularly human and / or monkey CD137. Such antibodies may be agonistic antibodies, and upon binding to CD137, induce cell signaling mediated by CD137.

[0032] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact (i.e., full-length) polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), variants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified form of immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0033] A typical antibody molecule consists of a heavy chain variable region (V H ) and the light chain variable region (V L ), which are usually involved in antigen binding. H and V L The regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). H and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948, and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0034] Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. A humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have a modified Fc region as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

[0035] Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is H and V LThe variable regions of the parent V are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, the parent V H and V L Using the sequence as a search query, a human V antibody having an amino acid sequence that is homologous to the amino acid sequence of the parent non-human antibody is selected from any antibody gene database. H and V L Next, human V H and V L An acceptor gene is selected.

[0036] The CDR regions in the selected human acceptor gene can be replaced with the CDR regions from the parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important for interactions with the CDR regions can be used to replace the corresponding residues in the human acceptor gene.

[0037] In some embodiments, the anti-CD137 antibodies described herein have suitable binding affinity for a target antigen (e.g., CD137) or an epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A Refers to. A is the dissociation constant (K D The anti-CD137 antibodies described herein have a binding affinity of at least 10 to a target antigen or antigen epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (K D The increase in binding affinity can be expressed as K D The higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a decrease in the K A (or a number K DA higher K for binding the first antigen than A (or a smaller numerical value K D ) can be indicated. In such a case, the antibody has specificity for the first antigen (e.g., the first protein of the first conformation or its mimetic) compared to the second antigen (e.g., the same first protein of the second conformation or its mimetic, or the second protein). The difference in binding affinity (e.g., for specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 -fold. In some embodiments, any of the anti-CD137 antibodies can be further affinity matured to increase the binding affinity of the antibody for the target antigen or its antigen epitope.

[0038] Binding affinity (or binding specificity) can be determined by various methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of the bound binding protein as a function of the target protein concentration. The concentration of the bound binding protein ([Bound]) is generally related to the concentration of the free target protein ([Free]) by the following equation. [Bound]=[Free] / (Kd + [Free])

[0039] However, obtaining a quantitative measurement of affinity determined using a method such as ELISA or FACS analysis only gives K Aand can therefore be used for comparison, such as to determine whether a higher affinity, e.g., 2-fold higher, is to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., in vitro or in vivo assay, so that K A It is not necessary to make a precise determination of the

[0040] The humanized anti-CD137 antibodies described herein can be derived from the antibody clone 371, H and V L The sequences are provided below with the CDRs in bold (as determined by Kabat numbering). Further information on reference antibody 371 can be found in WO2019 / 113039, the relevant disclosure of which is incorporated herein by reference for the purposes or subject matter referred to herein. [ka] [ka]

[0041] A humanized anti-CD137 antibody derived from reference antibody 371 may contain substantially similar heavy and light chain complementarity regions (CDRs), each of which corresponds to a suitable human V H Framework and preferred V L An antibody having a "substantially similar" heavy or light chain CDR compared to the corresponding CDR of a reference antibody means that the heavy or light chain CDR in the antibody contains less than 10 amino acid residue changes (e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change) in the collection compared to the relevant corresponding CDR in the reference antibody. For example, a humanized antibody may contain only up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue changes in the entire heavy and / or light chain CDR region and have substantially similar affinity (e.g., K of the same order) as the reference antibody. Dvalues) bind to the same epitope on CD137.

[0042] In some cases, a humanized antibody disclosed herein may have the same heavy chain CDR3 as the reference antibody 371, and optionally the same light chain CDR3 as the reference antibody. Alternatively, or in addition, a humanized antibody may have the same heavy chain CDR1 and / or CDR2 as the reference antibody, and optionally the same light chain CDR1 and / or CDR2 as the reference antibody.

[0043] The change in amino acid residue can be a conservative amino acid residue substitution.As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.Variants can be prepared according to the method for modifying polypeptide sequences known to one of ordinary skill in the art, for example, the method found in the reference book that summarizes such methods, for example, Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include those made among amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0044] In some embodiments, the humanized anti-CD137 antibodies disclosed herein contain the same heavy and light chain CDRs as reference antibody 371. H and / or V LTwo antibodies that have CDRs mean that their CDRs are identical when determined by the same technique (e.g., the Kabat, Chothia, AbM, Contact, or IMGT techniques known in the art; see, e.g., bioinf.org.uk / abs / ).

[0045] In some embodiments, the humanized anti-CD137 antibodies disclosed herein comprise heavy and light chain CDRs derived from parent antibody 371 (substantially similar or identical), which are compatible with the appropriate recipient human V H Genes and V L In some instances, the recipient human V H The gene may be IGH1-2*02. Alternatively, or in addition, the recipient human V L The gene may be a Vκ gene, which may be IGKV1-39*01.

[0046] In some embodiments, the heavy and light chain CDRs from clone 371 are synthesized in a suitable recipient VHV domain without introducing additional mutations in the framework regions. H and V L In other embodiments, one or more back mutations may be introduced into the framework region to enhance binding activity, stability, and / or other favorable properties. As used herein, a "back mutation" refers to a mutation that occurs in a human recipient V gene. H or V L It refers to changing the amino acid residue at a particular position in the framework back to the amino acid residue at the corresponding position in the murine parent antibody framework.

[0047] Provided below are exemplary humanized VH and VL chains derived from reference antibody 371 (CDRs according to the Kabat numbering scheme are in bold, back mutations are in bold and underlined): [ka] [ka] [ka] [ka] [ka] [ka]

[0048] Tables 1 and 2 below provide the heavy and light chain framework region (FR) and CDR sequences of parent antibodies, as well as exemplary humanized VH sequences derived therefrom. H and V L Provide the chain. [Table 1] [Table 2]

[0049] In some embodiments, the humanized anti-CD137 antibodies disclosed herein comprise a heavy chain CDR1 (HC CDR1) comprising the sequence of SEQ ID NO: 12, a HC CDR2 comprising the sequence of SEQ ID NO: 14, a HC CDR3 comprising the sequence of SEQ ID NO: 16, and / or a light chain CDR1 (LC CDR1) comprising the sequence of SEQ ID NO: 29, a LC CDR2 comprising the sequence of SEQ ID NO: 31, and a LC CDR3 comprising the sequence of SEQ ID NO: 33.

[0050] In some embodiments, the humanized anti-CD137 antibody disclosed herein may further comprise the same heavy chain framework region 1 (HC FR1) as LYV371_VH-1 (e.g., SEQ ID NO: 18) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibody may further comprise the same HC FR2 as LYV371_VH-1 (e.g., SEQ ID NO: 19) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibody may further comprise the same HC FR3 as LYV371_VH-1 (e.g., SEQ ID NO: 20) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibody may further comprise the same HC FR4 as LYV371_VH-1 (e.g., SEQ ID NO: 21) or a variant thereof having no more than one, two or three back mutations. Exemplary back mutations may occur at one or more of positions K12 (e.g., K12V), V20 (e.g., V20L) in FR1, positions V37 (e.g., V37I), E46 (e.g., E46G), and W48 (e.g., W48I) in FR2, positions V68 (e.g., V68A), M70 (e.g., M70L), R72 (e.g., R72A), and A97 (e.g., A97T) in FR3.

[0051] In some embodiments, a humanized anti-CD137 antibody disclosed herein comprises (i) an HC FR1 of SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 25, (ii) an HC FR2 of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 26, (iii) an HC FR3 of SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 27, and / or (iv) an HC FR4 of SEQ ID NO: 21. In some examples, a humanized anti-CD137 antibody has heavy chain FR1, FR2, FR3, and FR4 that are the same as VH-1, VH-2, or VH-3 shown in Table 1.

[0052] In some embodiments, the humanized anti-CD137 antibodies disclosed herein may further comprise a light chain framework region 1 (LC FR1) as LYV371_VL-1 (e.g., SEQ ID NO: 35) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibodies disclosed herein may further comprise a LC FR2 as cLYV371_VL-1 (e.g., SEQ ID NO: 36) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibodies may further comprise a LC FR3 as LYV371_VL-1 (e.g., SEQ ID NO: 37) or a variant thereof having no more than one, two or three back mutations. Alternatively, or in addition, the humanized anti-CD137 antibody may further comprise the same LC FR4 as LYV371_VL-1 (e.g., SEQ ID NO: 338), or a variant thereof having one, two, or no more than three back mutations.

[0053] In some embodiments, the humanized anti-CD137 antibodies disclosed herein comprise a humanized light chain variable region comprising one or more (e.g., 1, 2, 3, or 4) backmutations at one or more positions: K42 (e.g., K42G) and P44 (e.g., P44V) in FR2, F71 (e.g., F71Y) and Y87 (e.g., Y87F) in FR3, and V104 (e.g., V104L) in FR4.

[0054] In some embodiments, the humanized anti-CD137 antibodies disclosed herein further comprise (i) an LC FR1 comprising the sequence of SEQ ID NO: 35, (ii) an LC FR2 comprising the sequence of SEQ ID NO: 36 or SEQ ID NO: 39, (iii) an LC FR3 comprising the sequence of SEQ ID NO: 37 or SEQ ID NO: 40, and / or (iv) an LC FR4 comprising the sequence of SEQ ID NO: 41. In some examples, the humanized anti-CD137 antibodies comprise the same LC FR1, LC FR2, LC FR3, and LC FR4 as VL-1, VL-2, or VL-3 shown in Table 2.

[0055] In certain examples, the humanized anti-CD137 antibodies disclosed herein comprise a VH chain comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:8, or SEQ ID NO:9. Alternatively, or in addition, the humanized anti-CD137 antibodies disclosed herein comprise a VL chain comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:10. Exemplary humanized anti-CD137 antibodies provided herein include clones 3711, 3712, 3713, 3714, 3715, 3716, 3717, 3718, and 3719. See Table 3 below for the VH and VL components of these exemplary humanized anti-CD137 antibodies. Such exemplary antibodies can be any antibody format disclosed herein, e.g., a single chain antibody, a Fab fragment, or a full-length antibody.

[0056] In some embodiments, the heavy chain of any of the anti-CD137 antibodies described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region of the antibodies described herein may comprise a single domain (e.g., CH1, CH2, or CH3) or any combination of single domains.

[0057] In one particular example, the heavy chain constant region is derived from human IgG (gamma heavy chain) of any of the IgG subfamilies described herein. In one example, the constant region is derived from a human Ig molecule, such as IgG1. The Fc region of any of the humanized anti-CD137 antibodies disclosed herein can be a wild-type Fc domain. Alternatively, the Fc domain can be an Fc variant that includes one or more mutations compared to the wild-type counterpart to regulate one or more effector activities. For example, it can include a modified constant region that is immunologically inert, e.g., does not cause complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be evaluated using the methods disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624, PCT Application PCT / GB99 / 01441, and / or UK Patent Application No. 9809951.8. In some embodiments, the anti-CD137 antibodies described herein may comprise a mutated Fc region compared to the wild-type counterpart such that the antibody has a higher binding affinity to an Fc receptor, e.g., FcγRIIB (CD32B). Such antibodies may efficiently engage FcγRIIB-expressing cells, thereby enhancing therapeutic efficacy. In other embodiments, the anti-CD137 antibodies described herein may comprise a mutated Fc region compared to the wild-type counterpart such that the antibody has a selective binding affinity to an Fc receptor, e.g., FcγRIIB (CD32B). Such antibodies may selectively and efficiently engage FcγRIIB-expressing cells, thereby enhancing therapeutic efficacy.

[0058] Fc variants for use in making humanized anti-CD137 antibodies can be found, for example, in WO2018 / 183520, the relevant disclosure of which is incorporated herein by reference for the purposes of the subject matter referenced herein. In some cases, the Fc variants can include an S / P substitution at position 228 (EU numbering). In one example, the humanized anti-CD137 antibodies disclosed herein can include an Fc domain that includes the following amino acid sequence provided below (SEQ ID NO: 42). ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] Any of the anti-CD137 antibodies described herein may comprise a light chain further comprising a light chain constant region, which may be any CL known in the art. In some examples, the CL is a kappa light chain.

[0060] Antibody heavy and light chain constant regions are well known in the art and are provided, for example, in the IMGT database (imgt.org) or at vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0061] Provided below is the amino acid sequence of one exemplary full-length humanized anti-CD137 antibody clone 3712 (IgG1v / kappa). [ka] [ka]

[0062] In some embodiments, the anti-CD137 antibody is a bispecific antibody capable of binding to both CD137 and FcγRIIB (without Fc-FcR interactions). Such a bispecific antibody may comprise a first antigen-binding region and a second antigen-binding region, each of which is a V H / V L The first antigen-binding region may bind to CD137 and the second antigen-binding region may bind to FcγRIIB.

[0063] In some embodiments, the anti-CD137 antibody is a bispecific or multispecific (e.g., trispecific) antibody capable of binding both to CD137 and one or more other antigens of interest. Such a bispecific or multispecific antibody may comprise a first antigen-binding region, a second antigen-binding region, and optionally a third antigen-binding region, each of which is a V H / V L The first antigen-binding region can bind to CD137 and the second antigen-binding region can bind to another antigen of interest.

[0064] Preparation of humanized anti-CD137 antibody Methods for constructing humanized antibodies are well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is H and V L The variable regions of the parent V are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, the parent V H and V L Using the sequence as a search query, a human V antibody having an amino acid sequence that is homologous to the amino acid sequence of the parent non-human antibody is selected from any antibody gene database. H and V L Next, human V H and V L An acceptor gene is selected.

[0065] The CDR regions in the selected human acceptor gene can be replaced with the CDR regions from the parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important for interactions with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene.

[0066] In some examples, the anti-CD137 antibodies disclosed herein can be prepared by recombinant techniques, as exemplified below.

[0067] The nucleic acids encoding the heavy and light chains of the anti-CD137 antibody described herein can be cloned into one expression vector, with each nucleotide sequence being operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences of the heavy and light chains.

[0068] In some instances, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each of them can be isolated from the host cell that expresses it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow the formation of the antibody.

[0069] In general, the nucleic acid sequence encoding one or all chains of the antibody can be operably linked to a suitable promoter and cloned into a suitable expression vector using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and join together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0070] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0071] Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcription modulator to regulate transcription from a mammalian cell promoter carrying the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992), Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998), Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 that use estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0072] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli functions as a transcriptional modulator to regulate transcription from a lac operator-bearing mammalian cell promoter in combination with a tetracycline repressor (tetR) and a transcriptional activator (VP16) to create a tetR-mammalian cell transcriptional activator fusion protein, tTa (tetR-VP16), and a tetO-bearing minimal promoter from the human cytomegalovirus (hCMV) major immediate early promoter to create a tetR-tet operator system for controlling gene expression in mammalian cells [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than a tetR-mammalian cell transcription factor fusion derivative, can function as a potent trans-modulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16):1392-1399(2003)). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)).

[0073] In addition, the vectors can include, for example, some or all of the following: a selectable marker gene such as a neomycin gene for selecting stable or transient transfectants in mammalian cells, an enhancer / promoter sequence from the immediate early gene of human CMV for high levels of transcription, transcription termination and RNA processing signals from SV40 for mRNA stability, the SV40 polyoma origin of replication and ColE1 for proper episomal replication, an internal ribosome binding site (IRES), a versatile multiple cloning site, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0074] Examples of polyadenylation signals useful in practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0075] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cells can be cultured under suitable conditions for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, such as affinity purification. If desired, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time to allow for production of the antibody.

[0076] In some embodiments, the method for preparing the antibody described herein involves a recombinant expression vector encoding both the heavy and light chains of the anti-CD137 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cell) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cell can be incubated under suitable conditions to allow the formation of the antibody.

[0077] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-CD137 antibody and the other encoding the light chain of an anti-CD137 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the polypeptide chains of the antibody. If the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow the formation of the antibody. If the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0078] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A or Protein G-bound matrix.

[0079] Any of the nucleic acids encoding the heavy chain, light chain, or both, of the anti-CD137 antibodies described herein, vectors containing same (e.g., expression vectors), and host cells containing the vectors are within the scope of this disclosure.

[0080] The anti-CD137 antibodies thus prepared can be characterized using methods known in the art to detect and / or measure increased biological activity of CD137. For example, ELISA-type assays may be suitable for qualitative or quantitative measurement of CD137 promotion of T-cell proliferation.

[0081] treatment method The present disclosure provides a method of treating a disease, e.g., an immune disorder, such as cancer or an autoimmune disease, by administering a therapeutically effective amount of an anti-CD137 antibody. In some cases, any of the humanized anti-CD137 antibodies disclosed herein can be combined with an immune checkpoint inhibitor, such as an anti-PD-1 antibody. In one example, the anti-PD-1 antibody can be the anti-PD-1 antibody SSI-361 disclosed in WO2017 / 087599, the relevant disclosures of which are incorporated by reference for the subject matter and purposes referenced herein, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), avelumab (BAVENCIO®), durvalumab (IMFINZI®), or atezolizumab (TECENTRIQ®). In particular, the anti-PD-1 antibody can be pembrolizumab.

[0082] Pharmaceutical Compositions The antibodies described herein and the encoding nucleic acid or nucleic acid set, the vector containing it, or the host cell containing the vector can be mixed with a pharma- ceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. By "acceptable," it is meant that the carrier must be compatible with the active ingredient of the composition (and preferably be able to stabilize the active ingredient) and not be deleterious to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0083] The pharmaceutical composition used in the method of the present invention may contain a pharma- ceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, It may include proteins such as gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0084] In some instances, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids) that can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and a PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to obtain liposomes having the desired diameter.

[0085] The antibody or encoding nucleic acid may also be encapsulated, for example, in microcapsules prepared by droplet formation techniques or interfacial polymerization, such as hydroxyethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are known in the art; see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0086] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, and these matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), isobutyl sucrose acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0087] Pharmaceutical compositions used for in vivo administration must be sterile.This can be easily achieved, for example, by filtration through a sterile filtration membrane.Therapeutic antibody compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0088] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or for administration by inhalation or insufflation.

[0089] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical carriers, such as conventional tablet ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, as well as other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition, such that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may contain an inner dosage and an outer dosage component, the latter in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0090] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions containing a surfactant conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutical acceptable vehicles, may be added as required.

[0091] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition or in an emulsion formed upon mixing with oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20%, for example 5-20%, of oil. Fat emulsions contain fat droplets of 0.1-1.0 μm, especially 0.1-0.5 μm, and have a pH in the range of 5.5-8.0.

[0092] The emulsion composition may be prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0093] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic effect.

[0094] Compositions, preferably in sterile pharma- ceutically acceptable solvents, can be nebulized by the use of gases. Nebulized solutions can be breathed directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0095] therapeutic use To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

[0096] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats. The human subject in need of treatment may be a human patient who has, is at risk for, or is suspected of having a target disease / disorder, such as cancer, an autoimmune disease such as an immune disorder, or an infectious disease. In some embodiments, pharmaceutical compositions comprising humanized anti-CD137 antibodies are for use in enhancing immune responses in subjects, which are also within the scope of the present disclosure.

[0097] Examples of cancer include breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological malignancies, including acute lymphoblastic and myeloid leukemia, e.g., B-cell CLL; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia / lymphoma; intraepithelial neoplasia, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma. These include, but are not limited to, ovariectomy cancer; ovarian cancer, including those arising from epithelial, stromal, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including embryonic tumors such as seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, and germ cell tumors; thyroid cancer, including thyroid carcinoma and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms' tumor.

[0098] In one example, the cancer may be an advanced, metastatic, or unresectable malignant tumor. The malignant tumor may be histologically or cytologically confirmed. In particular, the malignant tumor may be advanced or metastatic.

[0099] The subject with target cancer can be identified by routine medical examination, for example, clinical examination, organ function examination, CT scan, or ultrasound.Target cancer can also be identified histologically and / or cytologically.In some embodiments, the subject treated by the methods described herein can be a human cancer patient who has undergone or is undergoing anti-cancer therapy, for example, chemotherapy, radiation therapy, immunotherapy, or surgery.

[0100] In some embodiments, subjects treated by the methods disclosed herein (e.g., involving clone 3712-IgG1v) may be human patients aged 18 years or older. Patients may (i) have histologically or cytologically confirmed metastatic or unresectable malignancies, (ii) have adequate bone marrow, liver, and renal function, and / or (iii) have recovered to baseline from all reversible AEs of prior anticancer therapy. Patients infected with HIV may be treated by the methods disclosed herein if the disease is under control of effective therapy.

[0101] Alternatively, or in addition, the subject may be a human patient who does not have one or more of the following: (1) receiving systemic anti-cancer therapy for 5 half-lives of the first dose of an anti-CD137 antibody disclosed herein, (2) prior radiation therapy within 14 days of the first dose of an anti-CD137 antibody disclosed herein, (3) active CNS metastases and / or carcinomatous meningitis, (4) receiving a live virus vaccine within 30 days, (5) having had a grade ≥ 3 allergic reaction to treatment with a monoclonal antibody, (6) QT interval or syndrome abnormalities, (7) having a grade ≥ 3 immune-related AE (irAE) or history of irAE, (8) receiving immunologically-based treatment for any reason, (9) receiving treatment with a systemic immune stimulant within 4 weeks prior to the first dose of an anti-CD137 antibody, (10) having received systemic treatment within the past 2 years. (11) have an active chronic autoimmune disease that has required or is receiving systemic therapy for an autoimmune or inflammatory disease; (12) have clinically significant cardiac disease such as unstable angina or acute myocardial infarction within 6 months; (13) have an active infection requiring intravenous (iv) anti-infective within 14 days prior to the first dose of anti-CD137 antibody; (14) show current evidence or history of interstitial lung disease or active non-infectious pneumonia requiring treatment such as oral or intravenous glucocorticoids to aid in management; (15) show evidence of severe or uncontrolled systemic disease; (16) have another disease or clinically significant abnormalities in laboratory parameters; and / or (17) have received a previous stem cell or bone marrow or solid organ transplant.

[0102] In some embodiments, the methods disclosed herein are for treating immune disorders. Immune disorders refer to dysfunction of the immune system. Examples include autoimmune diseases, immunodeficiencies, or allergies. In some embodiments, the target disease of treatment is an autoimmune disease. Examples include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), myasthenia gravis (MG), Graves' disease, idiopathic thrombocytopenic purpura (ITP), Guillain-Barre syndrome, autoimmune myocarditis, membranous glomerulonephritis, diabetes mellitus, type I or II diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, gastritis, celiac disease, vitiligo, hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encephalomyelitis, immune deficiency syndrome ... inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encephalomyelitis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encephalomyelitis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encephalomyelitis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encepha These include, but are not limited to, chronic neutropenia, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines, T lymphocytes typically seen in tuberculosis, sarcoidosis, and polymyositis, polyarteritis nodosa, cutaneous vasculitis, pemphigus, pemphigoid, Goodpasture's syndrome, Kawasaki disease, systemic sclerosis, antiphospholipid syndrome, Sjogren's syndrome, graft versus host disease (GVH), and immune thrombocytopenia.

[0103] Subjects with a target autoimmune disease may be identified by routine medical tests, such as the presence of antinuclear antibodies, antimitochondrial autoantibodies, antineutrophil cytoplasmic antibodies, antiphospholipid antibodies, anticitrullinated peptide (anti-CCP), antirheumatoid factor, immunoglobulin A, C-reactive protein tests, complement tests, erythrocyte sedimentation rate (ESR) tests, blood coagulation profiles, and protein electrophoresis / immunofixation electrophoresis, etc. In some embodiments, the subject treated by the methods described herein may be a human subject with an autoimmune disease who has undergone or is undergoing treatment for the autoimmune disease, such as immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory drug therapy, and / or analgesic drug therapy.

[0104] A subject suspected of having any of such target diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk of a disease / disorder can be a subject having one or more of the risk factors of that disease / disorder.

[0105] As used herein, "effective amount" refers to the amount of each active agent necessary to confer a therapeutic effect on a subject, alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is an increase in CD137 activity, an increase in T cell proliferation and survival, and / or an increase in the anti-tumor immune response. Determination of whether the amount of the composition described herein has achieved a therapeutic effect will be apparent to those of ordinary skill in the art. As recognized by those of ordinary skill in the art, the effective amount will vary depending on the particular condition being treated, the severity of the condition, age, general health, size, gender and weight of the subject, the duration of the treatment, the nature of any concurrent therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of the medical practitioner. These factors are well known to those of ordinary skill in the art and can be addressed by routine experimentation only. Generally, it is preferred to use the maximum dosage of the individual components or combinations thereof, i.e., the highest safe dosage based on sound medical judgment.

[0106] Empirical considerations such as half-life generally contribute to the determination of dosage. For example, an antibody that is compatible with the human immune system, such as a humanized antibody or a fully human antibody, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The dosing frequency can be determined and adjusted over the course of the therapy and generally, although not necessarily, is based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained release formulation of the antibody may be appropriate in some cases. A variety of formulations and devices for achieving sustained release are known in the art.

[0107] In one example, the dosage of the antibody described herein can be empirically determined in an individual who is given one or more doses of the antibody.The individual is given increasing doses of the agonist.The indicators of disease / disorder can be tracked to evaluate the effectiveness of the agonist.

[0108] In general, for administration of any of the antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. In some embodiments, the daily dosage may be 10 mg / kg. Depending on the condition, for repeated administration over several days or more, treatment is sustained until desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder, or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the antibody, or a maintenance dose of about 1 mg / kg every other week thereafter. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing from 1 to 4 times per week is contemplated. In some embodiments, a dosage range of about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) can be used. In some embodiments, the dosage frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, or every 3 months, or more. Each such period can be referred to as a cycle. In some embodiments, as many as 40 cycles, particularly 35 cycles, can be administered. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) can be changed over time.

[0109] In one example, the dosing frequency is once every three weeks. In particular, 0.3-10 mg / kg of a humanized anti-CD137 antibody (e.g., clone 3712-IgG1v) disclosed herein may be administered once every three weeks. As many as 35 cycles may be administered. The mode of administration in these embodiments may be intravenous.

[0110] In some embodiments, a dose ranging from about 0.3 to 5.00 mg / kg can be administered to a normal weight adult patient. In some examples, the dosage of the anti-CD137 antibodies described herein can be 10 mg / kg. The particular dosing regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as the characteristics of the individual agent, such as the half-life of the agent, and other considerations known in the art.

[0111] For purposes of this disclosure, the appropriate dosage of the antibodies described herein will depend on the particular antibody(ies), antibodies, and / or non-antibody peptides (or compositions thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is an increase in an anti-tumor immune response in the tumor microenvironment. Methods for determining whether a dosage has produced a desired result will be apparent to one of skill in the art. Administration of one or more antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled physician. Administration of the antibody may be essentially continuous over a preselected period of time or may be a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0112] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, for the purpose of curing, healing, mitigating, alleviating, altering, relieving, improving, ameliorating, or affecting the disorder, a symptom of the disease, or a predisposition to a disease or disorder.

[0113] Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease, or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means suspending, impeding, slowing, delaying, stabilizing, and / or postponing the progression of the disease. This delay can be for a variety of lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a particular time frame, when compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0114] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. Onset of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0115] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to enhance CD137 activity (and / or T cell proliferation) in vivo by at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0116] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the medical field. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, the subject can be administered via an injectable depot administration route, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0117] Injectable compositions may contain various carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0118] In one embodiment, antibody is administered via site-specific delivery technique or targeted local delivery technique.Examples of site-specific delivery technique or targeted local delivery technique include various implanted depot sources of antibody or local delivery catheters such as injection catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implanted devices, site-specific carriers, direct injection, or direct application.See, for example, PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0119] Targeted delivery of therapeutic compositions comprising antisense polynucleotide, expression vector or subgenomic polynucleotide can also be used.Receptor-mediated DNA delivery technology is described, for example, in Findeis et al., Trends Biotechnol.(1993)11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff,ed.)(1994); Wu et al., J.Biol.Chem.(1988)263:621; Wu et al., J.Biol.Chem.(1994)269:542; Zenke et al., Proc.Natl.Acad.Sci.USA(1990)87:3655; Wu et al., J.Biol.Chem.(1991)266:338.

[0120] Therapeutic compositions containing polynucleotides (e.g., those encoding an antibody described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can also be used during gene therapy protocols.

[0121] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (generally, see Jolly, Cancer Gene Therapy (1994) 1:51, Kimura, Human Gene Therapy (1994) 5:845, Connelly, Human Gene Therapy (1995) 1:185, and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or regulated.

[0122] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936, WO 94 / 03622, WO 93 / 25698, WO 93 / 25234, WO 93 / 11230, WO 93 / 10218, WO 91 / 02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, British Patent No. 2,200,651, and European Patent No. 0345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-1247). ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532) and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0123] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycation-condensed DNA (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147), ligand-conjugated DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985), eukaryotic cell delivery vehicle cells (see, for example, U.S. Patent No. 5,814,482, PCT Publication Nos. WO95 / 07994, WO96 / 17072, WO95 / 30763, and WO97 / 42338), and nuclear charge neutralization or fusion with cell membrane, with or without being linked to killed adenovirus alone. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publication Nos. WO 95 / 13796, WO 94 / 23697, WO 91 / 14445, and European Patent No. 0524968. Additional techniques are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0124] The particular dosage regimen employed in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history.

[0125] In some embodiments, a combination of two or more antibodies, or an antibody and another suitable therapeutic agent, can be administered to a subject in need of treatment. Antibodies can also be used in combination with other agents that function to enhance and / or complement the effectiveness of the agent.

[0126] The efficacy of treatment of a target disease / disorder can be assessed by methods well known in the art.

[0127] Combination therapy The anti-CD137 antibodies described herein may be utilized in combination with other types of therapies for the target disease, such as cancer, immune disorders, or infectious diseases.

[0128] When the anti-CD137 antibodies described herein are used to treat cancer, they can be combined with anti-cancer therapies, such as those known in the art. Additional anti-cancer therapies include chemotherapy, surgery, radiation, immunotherapy, gene therapy, etc. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure.

[0129] In some embodiments, anti-CD137 antibodies may be combined with other immunomodulatory therapies, such as, for example, inhibitors of checkpoint molecules (e.g., PD-1, PD-L1, PD-L2, CTLA-4, CD40, LAG3, TIM-3, or A2aR). As shown in Figure 14, combined treatment with anti-CD137 and anti-PD-1 antibodies produced synergistic effects. Tumor growth was significantly suppressed in mouse models compared to either of the two treatments alone.

[0130] Alternatively, or in addition, the treatment of the present disclosure may include chemotherapy agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, and the like. Microtubule disrupting agents such as cyclosporine, epothilones, and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethinylamine, oxaliplatin, ifosfamine, melphalan, merchlorthamine, mitomycin, mitoxantrone, nitrosporine, Antiproliferative / mitotic inhibitors, including leu, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes that metabolize L-asparagine systemically and lack the ability to synthesize their own asparagine cell-clearing, L-asparaginase); antiplatelet agents; antiproliferative / mitotic inhibitor alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines, and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazene-dacarbazine (DTIC); antiproliferative / mitotic inhibitor antimetabolites such as folic acid analogs (methotrexate);Platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (bleberdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab, cyclosporine ... mabs) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disrupting agents;

[0131] When the anti-CD137 antibody described herein is for the treatment of immune disorders, the antibody can be combined with other immunomodulatory treatments, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.). In some cases, the antibody can be combined with another therapy for autoimmune disease. Examples include but are not limited to intravenous Ig therapy; nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids; cyclosporine, rapamycin, ascomycin; cyclophosphamide; azathioprine; methotrexate; brequinar; FTY720; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin; immunosuppressants, or adhesion molecule inhibitors.

[0132] For examples of additional useful agents, see also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0133] When co-administered with an additional therapeutic agent, the preferred therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0134] Kits for use in treating disease The present disclosure also provides kits for use in treating or alleviating a target disease, such as cancer, immune disorders, and infectious diseases, as described herein. Such kits may include one or more containers containing an anti-CD137 antibody, such as any of those described herein, and optionally a second therapeutic agent, also as described herein, for use in combination with the anti-CD137 antibody.

[0135] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering the anti-CD137 antibody and, optionally, a second therapeutic agent to treat, delay the onset of, or alleviate a target disease, such as those described herein. The kit can further include instructions for selecting an individual suitable for treatment, for example, based on applying the diagnostic method described herein to identify whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0136] The instructions for use of anti-CD137 antibodies generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. The instructions provided with the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions transmitted on a magnetic or optical storage disk) are also acceptable.

[0137] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a disease, such as cancer, an immune disorder (e.g., an autoimmune disease), or an infectious disease. Instructions for performing any of the methods described herein can be provided.

[0138] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers) or infusion devices, such as mini-pumps, are also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-CD137 antibody, such as those described herein.

[0139] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0140] general technique The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully described, for example, in such publications as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (M. J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J. E. Elis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (D. M. Weir and CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994), Current Protocols in Immunology (JEColigan et al. al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995).

[0141] Without further elaboration, it is believed that a person skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the specific embodiments provided herein should be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0142] Example 1: Generation of humanized anti-CD137 antibodies Humanization and backmutation design Reference antibody 371 was humanized as described below. A sequence alignment was generated comparing the reference antibody 371 variable domain with human germline (Glanville J.et al.PNAS 2009;106(48)20216-21). Based on overall sequence identity, matching interface positions, and similarly classified CDR reference positions, germline families were identified as containing the most suitable acceptor frameworks for each of the light and heavy chains. IGKV1-39*01 for the light chain and IGHV1-2*02 for the heavy chain. The amino acid sequences of the identified human heavy and light chain variable acceptor frameworks are provided below. V H Amino acid sequence (AAP97932.1): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTTGTTYFDYWGQGTLVTVSS (SEQ ID NO: 43) V L Amino acid sequence (BAH04687.1): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIR (SEQ ID NO: 44)

[0143] Humanization of clone 371 was performed by CDR grafting into selected human frameworks (SEQ ID NOs: 3 and 4 for the heavy and light chains, respectively).

[0144] Homology modeling of the 371 antibody Fv fragment was performed. The clone 371 sequence was BLAST searched against the Protein Data Bank (PDB) antibody database to identify the template of the Fv fragment and to construct the domain interface. The structure template 1NMB (Malby et al., Structure, 1994 Aug 15;2(8):733-46) was selected because of 78% identity. The amino acid sequence alignment between the reference antibody 371 antibody (SEQ ID NO:2 before splitting and SEQ ID NO:1 after splitting) and the 1NMB template (SEQ ID NO:45) is shown below, where │ is a chain split and * indicates identical amino acid residues in both sequences. [ka]

[0145] The identity model was constructed using a customized build homology model protocol. Disulfide bridges were assigned and linked. Loops were optimized using the DOPE method. Based on the identity model of 1NMB, the sequence of reference antibody 371 was analyzed. Framework region (FR) residues that were considered important for binding activity, i.e., the reference FR residues and the V of the antibody, were analyzed. H -V L The interface residues were identified. The framework residues in the inner core were further analyzed. Reference antibody 371_V L -1 (ported reference antibody 371_V L Four residues in V41 (SEQ ID NO:2) were identified for back-mutation: K42 (a buried residue with a side chain charge), P44 (V H / V L (buried residue at the interface), F71 (buried canonical residue), and Y87 (V H / V L Residues buried in the interface). Humanized variant, reference antibody 371_V L -2 (SEQ ID NO:5) was designed to change the identified residues back to those of reference antibody 371 (K42G, P44V, F71Y, and Y87F) to test whether the identified residues are necessary to maintain optimal activity.

[0146] The humanized reference antibody 371 antibody, a recombinant fully human IgG4 / kappa, was constructed from human IgG4 containing the hinge S228P (EU numbering, Kabat numbering 241) stabilizing mutation (Angal et al., Mol. Immunol 30:105, 1993) and a human kappa light chain constant region. The humanized reference antibody 371, clone 3711, contains a V H -1 and V L Clone 3712 is a CDR-grafted version of V-1, which contains back mutations at four amino acid residues (K42G, P44V, F71Y, and Y87F). H -1 and V L -2.

[0147] Additional humanized V H Chain (V H -2 and V H -3) and V L Chain (V L -3) was also constructed. H and V L The amino acid sequence of V H and V L A chain is provided on top.

[0148] The above humanized V H and V L Humanized anti-CD137 antibodies containing random combinations of chains were constructed, see Table 3 below. [Table 3]

[0149] cDNA sequences encoding the variable domain sequences of anti-CD137 antibodies were synthesized as chimeras to the human IgG4 heavy chain constant region (Angal et al., Mol. Immunol 30:105, 1993) or the human kappa light chain constant region, containing the hinge S228P (EU numbering, Kabat numbering 241) stabilizing mutation. Transient expression in HEK293 and / or CHO was performed using plasmids containing the corresponding heavy and light chain sequences. These chimeric and humanized antibodies were purified by protein affinity chromatography. The purified antibodies were checked for endotoxin (<5EU / mg) and monomerization (>95%).

[0150] Example 2: Evaluation of anti-CD137 humanized antibodies K of CD137 antigen binding D measurement Chimeric and humanized antibodies were tested in an antigen-binding assay on Octet Red 96 to estimate binding kinetics. Antibodies were loaded onto an anti-human Fc (AHC) biosensor. The loaded sensor was bathed in serial dilutions of CD137 protein (300 nM, 1:3 drop, 7 points) in assay buffer (PBS with 0.1% BSA, 0.02% Tween-20, pH 7.2). The rate constants calculated using a monovalent (1:1) model are shown in Table 4 below. Humanized antibody 3711 showed similar binding kinetics as parental chimeric reference antibody 371. Antibody 3712 with a back mutation showed lower affinity due to a 3-fold higher off-rate. [Table 4]

[0151] CD137 binding FACS CHO cells overexpressing human CD137 were harvested using trypsin-EDTA partial digestion followed by centrifugation at 1000 g for 3 min. The cells were diluted to 5x10 in cold PBS-BSA (2%). 6The chimeric anti-CD137 antibody was diluted three times in PBS-BSA (final concentrations were 0.01, 0.1, 1, and 10 μg / mL) and 50 μL of each concentration was added to the CHO-CD137 cells. The cell solution was mixed and incubated for 2 hours at 4°C in the dark. The cells were then washed twice with PBS-BSA. The secondary antibody conjugate (goat F(ab') 2 Anti-human IgG-Fc (PE), pre-adsorbed (ab98596) was added at a concentration of 1 μg / mL 100 μL / well, the cells were mixed and incubated in the dark for 1 h at 4 °C. Afterwards, the cells were washed twice with PBS-BSA and subsequently fixed with 2% PFA before being subjected to FACS analysis. As shown in Figure 1, the reference antibody 371 and the humanized antibodies 3711 and 3712 showed similar binding affinity to CHO cells overexpressing human CD137.

[0152] T cell functional assays Freshly isolate PBMCs from four healthy volunteers and culture at 1x10 in RPMI-1640 containing 10% FBS. 6 / mL. EasySep™ Human CD8 + CD8+ T cells were isolated from the samples using a T Cell Isolation Kit (Stemcell, 17953). The resulting T cells were diluted at 5 × 10 5 / mL.

[0153] Costimulation assays of human CD8+ T cells were performed in co-culture with CHO cells expressing human FcγRIIB. To perform the co-culture assay, CHO cells engineered to express human FcγRIIB were cultured at 2.5 × 10 4 The cells were plated in a 96-well culture plate at a concentration of 1000 cells / well. The cells were incubated at 37°C and 5% CO 2 Cells were allowed to attach during overnight incubation in a cell culture incubator at 4 °C. Human CD8+ T cells from four donors were cultured at 1 × 10 5Added to cells / wells, added OKT3 at 0.1 μg / mL, and added CD137 antibody at final concentrations of 0, 0.01, 0.02, and 0.04 μg / mL. The culture plates were incubated for 3 days in a cell culture incubator at 37 °C and 5% CO 2 and incubated in an incubator for 3 days. The IFNγ content in the culture supernatant was determined by ELISA (eBioscience, 88-7316-88). As shown in Figure 2, chimeric and humanized antibodies showed the ability to costimulate human CD8-positive T lymphocytes in the presence of FcγRIIB-expressing CHO cells. Notably, it was observed that antibody 3711 showed weaker potency than the parental reference antibody 371 and clone 3712 (reverse mutation). Therefore, clone 3712, a humanized antibody containing a reverse mutation in the variable region of the light chain, was selected as the desired humanized sequence for Fc engineering.

[0154] Example 3: Fc engineering and characterization of anti-CD137 humanized antibody Preparation of antibody clone 3712 with Fc point mutations Using the humanized sequence of clone 3712 described above, a CD137 antibody clone 3712 containing the Fc region of human IgG1 / kappa (SEQ ID NOs: 6 and 7) including an Fc variant of human IgG1 was constructed (clone 3712-IgG1v / kappa). Clone 3712 was cloned into an expression vector for production in transiently expressing CHO cells and stable CHO cell lines.

[0155] K of CD137 antigen binding by Biacore D Measurement The flow cell of a CM5 sensor chip (GE Healthcare Life Sciences) was activated with freshly mixed 50 mmol / L NHS and 200 mmol / L EDC for 420 s (10 μL / min). Then, huCD137-C6His in 10 mmol / L NaAC (pH 5.0) was injected over the activated flow cell (10 μL / min) with HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20, pH 7.4) as the running buffer. The remaining active binding sites were blocked with a 420 s injection of 1 mol / L ethanolamine. Measurements were performed at 25° C. and HBS-EP was used as the running buffer. Injection of the tested analyte and surface regeneration of the CM5 sensor chip were included in each running cycle. Serially diluted clone 3712 (2.5, 5, 10, 20, 40, 80, 160, and 320 nM) was injected sequentially into both cells with a binding time of approximately 180 seconds. For dissociation, the buffer flow was maintained for approximately 180 seconds. To remove the tested antibody, 10 mM glycine-HCl was injected for approximately 30 seconds to regenerate. The above procedure was repeated for each concentration of serially diluted test antibody. The raw data of the surface plasmon resonance experiment was evaluated using Biacore T200 evaluation software 3.1 with a 1:1 binding model. Flow cell 1 was used as the reference flow cell. The affinity and kinetics data of the experiment are shown in Table 5 and the raw data are shown in Figure 3. The affinity (K D The activation energy (value) was 18.9 nM with fast on and fast off kinetics. [Table 5]

[0156] CD137 binding ELISA Binding of clone 3712-IgG1v to recombinant CD137 receptor protein was also analyzed in a standard ELISA format. Human or cynomolgus CD137 receptor protein was diluted to 1 μg / mL in DPBS and coated at 100 μL (0.1 μg) or 50 μL (0.05 μg) per well onto ELISA plates (Corning, Cat#: 9018, High Binding) and incubated overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST. Clone 3712 was diluted to 10 μg / mL in assay diluent and then serially diluted 3-fold in 11 points in assay diluent to final concentrations of 10000, 3333.3, 1111.1, 370.4, 123.5, 41.2, 13.7, 4.6, 1.52, 0.51 and 0.17 ng / mL. Diluted clone 3712-IgG1v was added to the assay plate in duplicate at 50 μL / well. The plate was incubated at room temperature for 1 hour and then washed three times with PBST. Goat anti-human IgG-H+L HRP conjugated at 1:100,000 dilution was added to the plate at 100 μL / well. The plate was then incubated at room temperature for 1 hour followed by washing four times with PBST. TMB substrate solution was added at 100 μl / well. Color was developed for 15 minutes and 100 μl / well of 2N H 2 SO 4 The absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0157] To determine whether clone 3712-IgG1v binds to mouse or rat CD137 receptors, as well as other human proteins such as human PD-1, LAG-3, VISTA, B7-H3, B7-H4, GITR, CD40, TIGIT, PD-L1, CD20, CD47, CD19, CD27, and LTBR, ​​protein samples were diluted to 1 μg / mL in DPBS and coated at 100 μL (0.1 μg) or 50 μL (0.05 μg) per well onto ELISA plates (Corning, Cat#: 9018, High Binding) and incubated overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST. Target-specific antibodies used as positive references / controls were diluted to 10 μg / mL in assay diluent, clone 3712-IgG1v was diluted to 10 μg / mL and 1 μg / mL in assay diluent, and the negative control Avastin was diluted to 10 μg / mL in assay diluent. These samples were added to the plate at 50 μL / well. The plate was incubated for 1 hour at room temperature and then washed three times with PBST. HRP-conjugated goat anti-human IgG-H+L at 1:100,000 dilution or goat anti-mouse IgG(H+L) secondary antibody, HRP at 1:50,000 dilution was added to the plate at 100 μL / well. The plate was incubated for 1 hour at room temperature followed by washing four times with PBST. 100 μl / well of TMB substrate solution was added. Color was developed for 15 minutes and 100 μL / well of 2N H 2 SO 4 The reaction was stopped by adding 0.5% CO. The absorbance at 450 nm and 620 nm was measured by a Tecan F200 Pro reader.

[0158] Binding data were plotted using Graphpad 7.0, “[agonist] vs. response--variable slope (four parameters)” to obtain binding EC 50Values ​​for binding EC50 were calculated. Representative data are shown in Figures 4A-4B. A summary of binding EC50 values ​​is shown in Table 6. For these assays, Avastin was used as the control antibody. Results of these studies show that the mean binding EC50 values ​​of clone 3712-IgG1v to human and cynomolgus CD137 were 50 The values ​​shown are 0.37 nM (i.e. 52.9 ng / mL) and 0.33 nM (i.e. 47.4 ng / mL), respectively. Clone 3712-IgG1v showed no binding affinity to mouse or rat CD137 protein.

[0159] Additionally, other human proteins, including PD-1, LAG-3, VISTA, B7-H3, B7-H4, GITR, CD40, TIGIT, PD-L1, CD20, CD47, CD19, CD27, and LTβR, were tested using ELISA assays to determine the binding of clone 3712-IgG1v. No binding affinity was observed. This study demonstrated the specific binding of clone 3712 to the human and cyno CD137 receptors. [Table 6]

[0160] CD137 binding FACS CHO cells overexpressing human or cynomolgus CD137 and CHO parental cells were harvested by centrifugation at 1000g for 3 min. Cells were resuspended in ice-cold assay buffer at 2*10^6 / mL and transferred to 96-well V-bottom plates at 100μL / well. Clone 3712-IgG1v or Avastin antibody was diluted to 300μg / mL in assay buffer and then serially diluted 3-fold in assay buffer to prepare 15 points at 3× final concentration, with concentrations of 300000, 100000, 33333.3, 11111.1, 3703.7, 1234.6, 411.5, 137.2, 45.7, 15.2, 5.08, 1.69, 0.56, 0.19 and 0.063ng / mL. Diluted clone 3712-IgG1v or Avastin was added to the assay plate at 50 μL / well. Plates were mixed and incubated in the dark at 4° C. for 2 hours. Cells were washed three times by centrifugation at 1000 g for 3 minutes and goat F(ab') 2 The cells were resuspended in ice-cold assay buffer containing anti-human IgG-Fc(PE), 100 μL / well. The plates were incubated for an additional hour at 4° C. in the dark. The cells were washed twice by centrifugation at 1000 g for 3 minutes and resuspended in ice-cold assay buffer containing 2% PFA. The cells were then immediately subjected to a flow cytometer.

[0161] To determine whether clone 3712-IgG1v binds to mouse CD137 receptor as well as other human cell targets such as human LAG-3, VISTA, B7-H3, B7-H4, GITR, CD40, TIGIT, PD-L1, CD20, CD47, CD19, CD27, LTBR, ​​BTLA, CD160 and CD200R1, CHO cells overexpressing these targets were harvested by centrifugation at 1000g for 3 minutes. Cells were resuspended in ice-cold assay buffer at 2*10^6 / mL and transferred to 96-well V-bottom plates at 100μL / well. Target-specific antibodies were diluted to 30μg / mL or 6μg / mL in assay buffer, clone 3712-IgG1v was diluted to 30μg / mL or 3μg / mL in assay buffer, and Avastin was diluted to 30μg / mL in assay buffer. Diluted target-specific antibodies, clone 3712-IgG1v, or Avastin were added to the assay plate at 50 μL / well. Plates were incubated in the dark at 4° C. for 2 hours. Cells were washed three times by centrifugation at 1000 g for 3 minutes and then purified with goat F(ab') 2 Cells were resuspended in 100 μL of ice-cold assay buffer containing anti-human IgG-Fc (PE) or PE goat anti-mouse IgG antibodies. Plates were incubated for an additional hour at 4° C. in the dark. Cells were washed twice by centrifugation at 1000 g for 3 minutes, resuspended in ice-cold assay buffer containing 2% PFA, and immediately analyzed on a flow cytometer.

[0162] Mean fluorescence intensity (MFI) was calculated from the total cell population in FCM experiments. Binding MFI in dose response was plotted using Graphpad 7.0, “[agonist] vs. response—variable slope (four parameters)” to obtain the FCM binding EC 50 The values ​​of were calculated. Clone 3712-IgG1v showed strong binding to both human and cynomolgus CD137 overexpressed in CHO by FCM. No binding was observed to parental CHO cells. The reference antibody Avastin was used as a control to show the specificity of CD137 binding. Representative data are shown in Figures 5A-5C and the results are summarized in Table 7. [Table 7]

[0163] To demonstrate the specificity of clone 3712-IgG1v for CD137, CHO cells overexpressing other human receptor proteins, such as LAG-3, VISTA, B7-H3, B7-H4, GITR, CD40, TIGIT, PD-L1, CD20, CD47, CD19, CD27, LTBR, ​​BTLA, CD160, CD200R1, and PD-1, were tested by FCM. Consistent with the specificity demonstrated in the protein ELISA experiments above, no binding signal was observed for clone 3712. As expected from the ELISA data, clone 3712-IgG1v did not bind to mouse cell CD137.

[0164] Binding to endogenous CD137 on activated CD8+ T cells To confirm that clone 3712-IgG1v can bind to endogenous human and cynomolgus CD137, PBMCs activated with immobilized anti-CD3 antibodies were examined for binding. To obtain activated T cells in human PBMCs, 24-well plates were coated overnight at 4°C with 300 μL of DPBS containing 2 μg / mL AffiniPure goat anti-mouse IgG, Fcγ fragment specific. Plates were washed three times with DPBS. Antibody OKT3 was added to the plates at 1 μg / mL, 100 μL / well. Plates were incubated for 1 h at 37°C. A vial of freshly thawed human PBMCs was transferred to a 50 mL tube containing 25 mL of pre-warmed medium (RPMI1640 with 10% FBS) and spun at 250 g for 10 min. The cell pellet was resuspended in 2 mL of complete medium (containing 50 uM 2-Me) and the cell density was adjusted to 3.3*10^6 cells / mL with complete medium, after which 900 μL / well of the cell suspension was transferred to the plate. The culture medium was incubated at 37 °C for 24 h at 5% CO 2The plates were incubated at 37°C for approximately 48 hours. After incubation, the cells were harvested and washed once with DPBS. The cell pellet was resuspended in cold assay buffer and the cell density was adjusted to 2*10^6 cells / mL. 100μL of the cell suspension was transferred to a 96-well V-bottom plate. Clone 3712-IgG1v or Avastin antibody was diluted to 300μg / mL in assay buffer and then serially diluted 10-fold in assay buffer to prepare concentrations of 300000, 30000, 300, 30, 3, 0.3 and 0.03ng / mL, 3x final concentration. 50μL / well of diluted Clone 3712-IgG1v or Avastin was added to the assay plate. The plate was incubated in the dark at 4°C for 30 minutes. The cells were washed three times by centrifugation at 1000g for 3 min and resuspended in 50 μL of ice-cold assay buffer containing FITC anti-human CD3, PE anti-human CD8a and APC anti-human IgG Fc antibodies. The plates were incubated for another 30 min at 4°C in the dark. The cells were then washed twice by centrifugation at 1000g for 3 min and resuspended in 100 μL of ice-cold assay buffer containing 7-AAD. After incubation for 5 min at 4°C in the dark, the cells were immediately analyzed on a flow cytometer.

[0165] To obtain activated T cells in cynomolgus monkey PBMCs, 24-well plates were pre-treated with 2 μg / mL AffiniPure F(ab') 2 Plates were coated overnight at 4°C with 300μL DPBS containing fragment goat anti-human IgG, Fcγ fragment specific. Plates were washed 3 times with DPBS. Anti-CD3 antibody, Ly305, capable of binding and activating cynomolgus CD3, was added to the plates at 3μg / mL, 100μL / well. Plates were incubated at 37°C for 1 hour. A vial of freshly thawed cynomolgus PBMCs was transferred to a 50mL tube containing 15mL pre-warmed medium (RPMI1640 with 10% FBS) and spun at 250g for 10 minutes. The cell pellet was resuspended in 2mL medium, adjusted to a cell density of 3.3*10^6 cells / mL with complete medium, and 900μL / well of the cell suspension was transferred to the plates. Culture medium was incubated at 4°C for 10 min at 37°C for 1 h at 37°C for 1 h at 37°C.2 The plates were incubated at 37°C for approximately 48 hours at 5°C. After incubation, the cells were harvested and washed once with DPBS. The cell pellet was resuspended in cold assay buffer and the cell density was adjusted to 2*10^6 cells / mL. 100μL of the cell suspension was transferred to a 96-well V-bottom plate. Clone 3712-IgG1v-Biotin or Avastin-Biotin antibody was diluted to 300μg / mL in assay buffer and then serially diluted 10-fold in assay buffer to prepare concentrations of 300000, 30000, 300, 30, 3, 0.3 and 0.03ng / mL, 3× final concentration. 50μL / well of the diluted Clone 3712-IgG1v-Biotin or Avastin-Biotin was added to the assay plate. The plate was incubated in the dark at 4°C for 60 minutes. The cells were washed three times by centrifugation at 1000g for 3 min and resuspended in 50 μl of ice-cold assay buffer containing FITC anti-human CD8 antibody (clone: ​​SK1) and APC streptavidin. The plate was incubated for another 60 min at 4°C in the dark. The cells were then washed twice by centrifugation at 1000g for 3 min and resuspended in 100 μL of ice-cold assay buffer containing 7-AAD. The plate was incubated for 5 min at 4°C in the dark and immediately analyzed on a flow cytometer.

[0166] As shown in Figures 6A-6B, clone 3712-IgG1v showed dose-dependent binding to activated human or cynomolgus monkey CD8+ T cells. Clone 3712-IgG1v showed similar EC 50 Clone 3712-IgG1v was found to bind to human and cynomolgus monkey cellular CD137 receptors at 100-fold lower concentrations (37-50 ng / mL or approximately 0.3 nM). Clone 3712-IgG1v was also found to bind to endogenous CD137 on activated human or cynomolgus monkey CD8+ T cells in a dose-dependent manner. Thus, clone 3712 shows high specificity for CD137 by FCM, as expected from the ELISA studies described herein.

[0167] Fc receptor and complement C1q binding FcγRI binding assay CHO-K1-huFcγRI cells were harvested by centrifugation at 1000g for 3 min. Cells were resuspended in ice-cold assay buffer at 2*10^6 / mL and transferred to 96-well V-bottom plates at 100μL / well. Clone 3712-IgG1v or Avastin antibody was diluted to 30μg / mL in assay buffer, then serial 2-fold dilutions were made in assay buffer to prepare 15 points at concentrations of 30000, 15000, 7500, 3750, 1875, 937.5, 468.8, 234.4, 117.2, 58.6, 29.3, 14.6, 7.32, 3.66 and 1.83ng / mL, 3× final concentration. Diluted clone 3712-IgG1v or Avastin was added to the assay plate at 50μL / well. The plates were mixed and incubated in the dark for 2 hours at 4° C. Cells were washed three times by centrifugation at 1000 g for 3 minutes and the goat F(ab') 2 The cells were resuspended in ice-cold assay buffer containing 100 μL / well of anti-human IgG-Fc (PE) secondary antibody. The plates were incubated for an additional hour at 4° C. in the dark. The cells were washed twice by centrifugation at 1000 g for 3 minutes and resuspended in ice-cold assay buffer containing 2% PFA. The cells were immediately subjected to flow cytometry analysis.

[0168] CHO-K1-huFcγRI cells express high levels of human FcγRI with high affinity for IgG1 (Ravetch and Bolland 2001, Roopenian and Akilesh 2007) and were used in the FcγRI binding assay of clone 3712-IgG1v. Binding was examined through multiple concentrations of antibody including 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.078, 0.039, 0.0195, 0.0098, 0.0049, 0.0024, 0.0012, and 0.0006 μg / mL (15 dilution points, 2-fold serial dilutions from a top of 10 μg / mL). Binding was measured using PE-conjugated, polyclonal, anti-human FcF(ab') 2The fragments were detected by flow cytometry. Three independent experiments were performed. Figure 7A shows representative data for clone 3712-IgG1v, which shows no binding to FcγRI, whereas the reference human IgG1 antibody, Avastin, has an EC 50 The results showed high binding affinity with values.

[0169] FcγRIIA binding assay CHO-K1-huFcγRIIA-Arg131 and CHO-K1-huFcγRIIA-His131 cells were harvested by centrifugation at 1000g for 3 min. Cells were resuspended in ice-cold assay buffer at 2*10^6 / mL and transferred to 96-well V-bottom plates at 100μL / well. Clone 3712-IgG1v or Avastin antibody was diluted to 300μg / mL in assay buffer and then serial 2-fold dilutions were made in assay buffer to prepare the following concentrations: 300000, 150000, 75000, 37500, 18750, 9375, 4688, 2344, 1172, 586, 293, 146, 73.2, 36.6 and 18.3ng / mL, 3x final concentration. Diluted clone 3712-IgG1v or Avastin was added to the assay plate at 50 μL / well. Plates were mixed and incubated in the dark at 4° C. for 2 hours. Cells were washed three times by centrifugation at 1000 g for 3 minutes and goat F(ab') 2 The cells were resuspended in ice-cold assay buffer containing 100 μL / well of anti-human IgG-Fc (PE) secondary antibody. The plates were incubated for an additional hour at 4° C. in the dark. The cells were washed twice by centrifugation at 1000 g for 3 minutes and resuspended in ice-cold assay buffer containing 2% PFA. The cells were immediately subjected to flow cytometry.

[0170] CHO-K1-huFcγRIIA-Arg131 and CHO-K1-huFcγRIIA-His131 cells express the activating FcγRIIA receptor with low binding affinity for IgG1. Both were investigated for the FcγRIIA binding assay. Binding was examined in a concentration range containing 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, 0.049, 0.024, 0.012, and 0.006 μg / mL (15 dilution points, two-fold serial dilution starting from 100 μg / mL at the top). Binding was redetected by flow cytometry using a PE conjugate, polyclonal, anti-human Fc F(ab’) 2 fragment. At least three independent experiments were performed. Figures 7B and 7C show representative data of clone 3712-IgG1v that showed weak binding to huFcγRIIA-Arg131 and no binding to huFcγRIIA-His131. The reference control, abatacept, showed moderate binding in both assays, as expected for normal human IgG1.

[0171] FcγRIIB binding assay CHO-K1-huFcγRIIB-Ile232 cells were harvested by centrifugation at 1000 g for 3 minutes. The cells were resuspended in ice-cold assay buffer at 2*10^6 / mL and transferred to a 96-well V-bottom plate at 100 μL / well. Clone 3712-IgG1v or abatacept antibody was diluted to 300 μg / mL in assay buffer and then 3× final concentrations of 300000, 150000, 75000, 37500, 18750, 9375, 4688, 2344, 1172, 586, 293, 146, 73.2, 36.6, and 18.3 ng / mL were prepared using two-fold serial dilution in assay buffer. Diluted clone 3712 or abatacept was added to the assay plate at 50 μL / well. The plate was mixed and incubated at 4 °C for 2 hours in the dark. The cells were washed three times by centrifugation at 1000 g for 3 minutes and 100 μL / well of goat F(ab’) 2The cells were resuspended in ice-cold assay buffer containing anti-human IgG-Fc (PE) secondary antibody. The plates were incubated for an additional hour at 4°C in the dark. The cells were washed twice by centrifugation at 1000g for 3 minutes and resuspended in ice-cold assay buffer containing 2% PFA. The cells were immediately subjected to flow cytometry.

[0172] A transfected CHO cell line, CHO-K1-huFcγRIIB-Ile-232, containing cells expressing high levels of FcγRIIB, was used for the FcγRIIB binding assay. Test concentrations included the following: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, 0.049, 0.024, 0.012, and 0.006 μg / mL (15 dilution points, 2-fold serial dilutions from a top of 100 μg / mL). Binding by the test antibodies was measured using polyclonal, anti-human Fc F(ab') 2 At least three independent experiments were performed, and representative data are shown in Figure 7D. Clone 3712-IgG1v and Avastin showed moderate binding affinity to human FcγRIIB, but in contrast to Avastin, clone 3712-IgG1v did not show saturation at high concentrations (50 and 100 μg / mL).

[0173] FcγRIIIA binding assay The CHO-K1-huFcγRIIIA-Phe158 cells were harvested by centrifugation at 1000 g for 3 minutes. The cells were resuspended in ice-cold assay buffer at 2×10^6 / mL and transferred to a 96-well V-bottom plate at 100 μL / well. Clone 3712-IgG1v or the avastin antibody was diluted to 300 μg / mL in assay buffer and then serially diluted 2-fold in assay buffer to prepare final concentrations of 300000, 150000, 75000, 37500, 18750, 9375, 4688, 2344, 1172, 586, 293, 146, 73.2, 36.6 and 18.3 ng / mL, 3× final concentration. The diluted clone 3712-IgG1v or avastin was added to the assay plate at 50 μL / well. The plate was mixed and incubated at 4 °C for 2 hours in the dark. The cells were washed 3 times by centrifugation at 1000 g for 3 minutes and resuspended in ice-cold assay buffer containing goat F(ab’) 2 The anti-human IgG-Fc(PE) secondary antibody was resuspended in ice-cold assay buffer containing 100 μL / well. The plate was incubated for an additional 1 hour at 4 °C in the dark. The cells were washed 2 times by centrifugation at 1000 g for 3 minutes and resuspended in ice-cold assay buffer containing 2% PFA. The cells were immediately subjected to flow cytometry.

[0174] CHO-K1-huFcγRIIIA-Phe158 cells express high levels of human FcγRIIIA, which has a relatively high affinity for IgG1. Therefore, these cells were used in the FcγRIIIA binding assay. Binding was examined through a series of antibody concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, 0.049, 0.024, 0.012 and 0.006 μg / mL (from an initial 100 μg / mL, serially diluted 2-fold, 15 dilution points). Binding was detected by flow cytometry using a PE-conjugated, polyclonal, anti-human Fc F(ab’)2 fragment. Three independent experiments were performed. Figure 7E shows representative data indicating that clone 3712-IgG1v has no detectable binding to FcγRIIIA, while the reference human IgG1 antibody, avastin, shows the expected moderate binding.

[0175] FcRn binding assay Clone 3712-IgG1v and control IgG1 monoclonal antibody (Avastin) were diluted to 1 μg / mL in DPBS and coated on ELISA plates (Corning, Cat#: 9018, high binding) at 100 μL / well overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST (pH 6.0). Human FcRn protein was diluted to 10 μg / mL in assay diluent (pH 6.0) and then serial 2-fold dilutions were made in assay diluent (pH 6.0) to prepare final concentrations of 10000, 5000, 2500, 1250, 625, 312.5, and 156.25 ng / mL. Diluted FcRn protein was added to assay plates in duplicate at 50 μL / well. Plates were incubated at room temperature for 1 hour and then washed 3 times with PBST (pH 6.0). HRP-conjugated mouse anti-His tag antibody at 1:20,000 dilution (pH 6.0) was added to the plate at 100 μL / well. Plates were incubated at room temperature for 1 hour and then washed 4 times with PBST (pH 6.0). TMB substrate solution was added at 100 μl / well. Color was developed for 12 minutes and then 100 μL / well of 2N H 2 SO 4 The reaction was stopped by adding 0.5% CO. The absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0176] Binding of FcRn to clone 3712-IgG1v and human IgG1 Avastin was determined by ELISA. Clone 3712-IgG1v and Avastin were separately coated on plastic ELISA plates and then incubated with human FcRn protein with His tag at concentrations of 10000, 5000, 2500, 1250, 625, 312.5, and 156.3ng / mL (2-fold serial dilution, 7 dilution points). FcRn bound to the coated antibodies was detected by anti-His tag HRP antibody. Three independent experiments were performed and representative data are shown in Figure 8A. Clone 3712-IgG1v showed the expected binding to FcRn, although the signal intensity was weaker compared to the binding of the reference antibody Avastin. Biacore binding of 3712 to human FcRn was also performed and the results confirmed positive binding with similar kinetics to human IgG1 and IgG4.

[0177] C1q ELISA Clone 3712 and control IgG1 monoclonal antibody (Avastin) were diluted to 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL in DPBS and coated on ELISA plates (Corning, Cat#: 9018, High Binding) in duplicate at 100 μL / well overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST. Human C1q protein was diluted to 2 μg / mL in assay diluent. Diluted C1q protein was added to assay plates at 50 μL / well. Plates were incubated at room temperature for 1 hour, and then washed three times with PBST. HRP-conjugated sheep anti-human C1q antibody at 1:800 dilution was added to plates at 100 μL / well. Plates were incubated for 1 hour at room temperature and then washed 4 times with PBST. TMB substrate solution was added at 100 μl / well. Color was developed for 7 minutes and 100 μL / well of 2N H 2 SO 4The reaction was stopped by adding 0.5% CO. The absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0178] Complement activation and lysis of cells via the classical pathway is initiated via the binding of C1q to the Fc portion of IgG molecules. Binding of C1q to clone 3712-IgG1v and human IgG1 Avastin was determined by ELISA. Clone 3712-IgG1v and Avastin were separately coated on plastic ELISA plates at multiple concentrations, 0.125, 0.25, 0.5, 1, 2, 4, and 8 μg / mL, and then incubated with recombinant human C1q with a His tag at a concentration of 2 μg / mL. C1q bound to the antibodies was detected by anti-human C1q-HRP antibody. Three independent experiments were performed and representative data are shown in Figure 8B. Clone 3712-IgG1v showed reduced binding to C1q compared to the reference antibody Avastin.

[0179] Clone 3712-IgG1v is a humanized recombinant IgG1 / κ with point mutations in the Fc region engineered to reduce binding to C1q and activating Fcγ receptors, minimizing Fc effector activity. The engineered Fc was designed to retain binding to FcRγIIB and FcRn. The experiments described here demonstrate that clone 3712 exhibits no or minimal binding to human FcγRI, FcγRIIA-Arg131, FcγRIIA-His131, FcγRIIIa, and complement C1q, whereas the reference human IgG1 antibody Avastin exhibited the binding profile expected from the literature. Clone 3712 showed binding to human FcγRIIB and FcRn. The results are consistent with the expected profile of the engineered Fc of clone 3712-IgG1v, where binding to human FcγRIIB and FcRn is retained, but binding to other Fc receptors and C1q is lost.

[0180] Activation of CD137 signaling in reporter assays CHO-K1 or CHO-K1-huFcγRIIB-Ile232 cells were harvested by centrifugation at 250g for 5 min. Cells were resuspended in pre-warmed F12K medium containing 1% FBS at 2.5*10^6 cells / mL and transferred to a 96-well cell culture plate at 100μL / well. Meanwhile, 100μL / well of F12K medium containing 1% FBS was added to the third cell culture plate. DPBS was added to the edge of the cell culture plate. Plates were incubated at 4°C for 5 min at 5% CO 2 The cells were incubated overnight at 37°C in 100% CO. The next day, GS-H2-huCD137 cells were harvested after centrifugation at 250g for 5 min. The GS-H2-huCD137 cells were resuspended at 6*10^4 cells / mL in pre-warmed MEM medium containing 1% FBS, and 50μL / well of the cell suspension was added to the cell culture plate. Clone 3712-IgG1v or Avastin antibody was diluted to 25μg / mL in MEM medium containing 1% FBS, then serially diluted 4-fold in assay buffer to prepare final concentrations of 25000, 6250, 1562.5, 390.625, 97.65625, 24.414, 6.104, 1.526, 0.381, 0.095, and 0.024ng / mL. Diluted clone 3712-IgG1v or Avastin was added to the cell culture plate at 50 μL / well. The plate was mixed and incubated at 5% CO 2 The cells were incubated in 5% CO for approximately 18 hours at 37° C. After incubation, the cell culture supernatants were subjected to IL-8 detection using a human IL-8 assay kit from Cisbio.

[0181] Clone 3712-IgG1v showed dose-dependent agonist activity under crosslinking conditions. When CD137 reporter cells were cocultured with CHO-K1-huFcγRIIB cells, clone 3712 induced CD137 activation, resulting in IL8 secretion from reporter cells. Clone 3712-IgG1v did not activate CD137 without crosslinking (coculture with or without control CHO cells). Avastin was used as a control to show the specificity of CD137 activation. IL-8 concentrations in the culture supernatants were quantified and used to evaluate the activation of CD137 reporter cells. Representative data are shown in Figure 9. At least three independent experiments were performed and the results were comparable. Mean EC calculated from the CD137 reporter dose-response curve of clone 3712-IgG1v 50 The value was 44.4 ng / mL (approximately 0.31 nM, n=6).

[0182] Human CD8+ T cell costimulation assay CHO-K1 or CHO-K1-huFcγRIIB-Ile232 cells were harvested by centrifugation at 250g for 5 min. Cells were resuspended at 2.5*10^6 cells / mL in pre-warmed F12K medium containing 10% FBS and transferred to a 96-well cell culture plate at 100μL / well. Meanwhile, 100μL / well of F12K medium containing 10% FBS was added to the third cell culture plate. DPBS was added to the edge of the cell culture plate. Plates were incubated at 4°C for 5 min at 5% CO 2The cells were incubated overnight at 37°C in RPMI medium containing 10% FBS. The following day, a vial of freshly thawed human CD8+ T cells was transferred to a 50mL tube containing 25mL of pre-warmed medium (RPMI1640 with 10% FBS) and spun at 250g for 10 minutes. The cell pellet was resuspended in 2mL medium at 0.5-1*10^6 cells / mL and 50μL / well of the cell suspension was added to the plate. Clone 3712-IgG1v or Avastin antibody was diluted to 100μg / mL in RPMI medium containing 10% FBS and 0.4μg / mL OKT3, then 4-fold serial dilutions were prepared in assay buffer to obtain final concentrations of 100000, 25000, 6250, 1562.5, 390.6, 97.7, 24.4, 6.1, 1.53 and 0.38ng / mL, 4x final concentration. Diluted clone 3712-IgG1v or Avastin was added to the cell culture plate at 50 μL / well. The plate was mixed and incubated at 5% CO 2 and incubated in for about 72 hours at 37° C. After incubation, the cell culture supernatant was subjected to IFN-γ detection using a human IFN-γ assay kit from Cisbio.

[0183] Clone 3712-IgG1v showed dose-dependent agonist activity in activating human CD8+ T cells when co-cultured with CHO-K1-huFcγRIIB cells. No activity was observed for clone 3712 when human CD8+ T cells were co-cultured with CHO-K1 cells or medium. Avastin was used as a control to demonstrate the specificity of CD137 activation. Representative data from at least three independent experiments are shown in Figure 10. CD8+ T cells from six donors were tested and the results were comparable. Mean EC calculated from CD8+ T cell costimulation assay dose-response curve of clone 3712 50 The value was 30.6 ng / mL (approximately 0.21 nM, n=12).

[0184] Example 4: Pharmacokinetic study of humanized antibodies C57BL / 6 mice (6-7 weeks old, 19-20 g, male, purchased from SLAC Laboratory Animal Co.LTD) were used in this study. Antibodies were formulated in PBS and administered via tail vein injection at 3 mg / kg to groups of 4 mice. No abnormal clinical symptoms were observed throughout the in-life study.

[0185] Blood was collected by continuous bleeding at pre-dose, 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 5 days, 8 days, 11 days, 15 days, and 21 days. 10 μL of blood per time point was added to 40 uL of PBS-BSA solution. Samples were then mixed thoroughly and centrifuged at 2000 g for 5 minutes at 4° C. Supernatants were placed on dry ice immediately after collection and stored at approximately −70° C. until analysis. Blood antibody concentrations were determined by ELISA. Figure 11 shows blood antibody concentrations of clone 3712-IgG1v after a single intravenous injection of 3 mg / kg. Results and pharmacokinetic parameters are summarized in Table 8 below. [Table 8]

[0186] Example 5: In vivo evaluation of the efficacy of humanized antibodies material and method mouse Human CD137 knock-in mice (C57BL / 6, B-h4-1BB) and CD137 / PD-1 double knock-in mice (C57BL / 6, B-hPD-1 / h4-1BB) were purchased from Biocytogen, Inc. (Beijing, China). C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were maintained under specific pathogen-free conditions. Animal care and use were in accordance with institutional and NIH protocols and guidelines, and all studies were approved by the Animal Care and Use Committees of the relevant institutions.

[0187] cell line Mouse colon cancer MC38 cells were purchased from ShunRan Biotech (Shanghai, China) and incubated at 4 °C for 1 h at 5% CO 2 B16-OVA was cultured in 5% CO and maintained in vitro in DMEM supplemented with 10% heat-inactivated fetal bovine serum. 2 and were maintained in vitro in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mmol / L L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0188] Efficacy Testing Mouse colon cancer MC38 cells (0.5x10^6 cells in 0.1mL PBS) were subcutaneously implanted into homozygous B-h4-1BB mice. The tumor size was approximately 150±50mm 3 Mice were divided into control and treatment groups (n=5-6) if tumor size was 0.5×length×width. Treatment was by intraperitoneal injection twice a week. Tumor size was measured twice a week by caliber and was calculated as 0.5×length×width. 2 Tumor volumes were calculated using the formula: The mean tumor sizes ± SEM are shown in Figures 12A and 12B.

[0189] Tumor growth inhibition (TGI) was determined during the dosing period by the formula TGI=(Ct-Tt) / (Ct-C0)X100, where Ct=mean tumor volume of control at time t, Tt=mean tumor volume of treated at time t, and C0=mean tumor volume of control at time 0, which is the same for all randomized groups.

[0190] The antitumor efficacy of anti-CD137 antibodies in syngeneic mouse tumor models has been well documented (Melero et al. 1997; Murillo et al. 2008). Because clone 3712 does not cross-react with mouse CD137, we investigated the antitumor activity of clone 3712 using mice with the human CD137 extracellular domain knocked in to replace the mouse sequence.

[0191] Dosage regimen In the first study, dosing schedules of clone 3712-IgG1v were compared in a mouse MC38 syngeneic tumor model, as shown in Figures 12A and 12B. When administered intraperitoneally at 3 mg / kg either once only or multiple times on a twice weekly schedule, clone 3712 showed substantial antitumor efficacy against established tumors, with 100% and 103% tumor growth inhibition for the single and multiple dose groups, respectively, at the end of the experiment 21 days after dosing (Table 9). Excel's T.TEST P values ​​were performed to compare treated and vehicle control groups.

[0192] Individual mouse data are shown in Figure 12B. A single injection of clone 3712-IgG1v at 3 mg / kg was able to control the growth of pre-established MC38 carcinomas in 3 weeks, demonstrating the potent antitumor activity of clone 3712-IgG1v in a preclinical tumor model. [Table 9]

[0193] Efficacy of clone 3712 compared with urelumab and utomilumab analogues In the next experiment, we compared the efficacy of clone 3712-IgG1v and the reference antibodies urelumab and utomilumab analogues prepared in-house according to the published sequence. When administered once at 10 mg / kg on day 7, clone 3712 and urelumab showed considerable tumor growth inhibition, whereas utomilumab was inferior compared to clone 3712 (Figure 13A). As shown in Table 6 below, utomilumab analogue, urelumab analogue, and clone 3712 administered at 10 mg / kg resulted in 61%, 80%, and 91% tumor growth inhibition, respectively, at the end of the experiment (day 21 after injection of a single dose).

[0194] In this experiment, the dose escalation of clone 3712-IgG1v was also carried out. As shown in Figure 13B, clone 3712-IgG1v administered once at 3, 5, and 10 mg / kg showed a significant inhibition of tumor growth, but the TGI was less than 50% at all time points, so the dose of 1 mg / kg was insufficient. The detailed tumor volume and analysis data of TGI are shown in Table 10. The P value of Excel's T.TEST was performed to compare the treatment group and the vehicle control group.

Table 10

[0195] Antitumor effect of the combination of clone 3712 and anti-PD-1 antibody The potential for synergistic antitumor activity between CD137 agonist antibody and PD-1 blocking antibody has been reported (Tolcher et al. 2017, Azpilikueta et al. 2016). Using the B16 melanoma syngeneic tumor model, the combination of clone 3712-IgG1v and anti-PD-1 was tested. WT B6 mice at 7-8 weeks of age were lethally irradiated with a single dose of 950 rad. The next day, 2-3×10 6 hPD-1 / 4-1BB mouse donor bone marrow cells were adoptively transferred. Before starting the tumor efficacy experiment, the mice were maintained with sulfamethoxazole and trimethoprim (Bactrim) antibiotics diluted in the drinking water for 4 weeks after reconstitution. Approximately 1x10^6 B16-OVA cells were subcutaneously injected into the right flank of the bone marrow chimeric mice. Tumor volume was measured along three orthogonal axes (a, b, and c), and tumor volume was calculated as tumor volume = abc / 2. After the tumors were established (about 9-12 days, about 100 mm 3), mice were treated with 100 μg (approximately 5 mg / kg) of anti-PD-1 antibody on days 14, 21, and 28, or with 100 μg (approximately 5 mg / kg) of anti-4-1BB antibody by intraperitoneal injection on day 14, or with combination treatments as indicated. Tumor growth was measured twice weekly. Relative tumor size was calculated by dividing tumor size by the initial tumor size on day 14. As shown in FIG. 14, the combination of clone 3712-IgG1v and anti-PD-1 resulted in more significant inhibition of tumor growth compared to either of the single agent treatment groups. The TGI numbers at the end of the study on day 52 for the single agent anti-PD-1, clone 3712-IgG1v, and combination groups were 42%, 66%, and 91%, respectively.

[0196] Example 6: Epitope mapping of CD137 antibodies The extracellular part of the CD137 receptor protein consists of four domains. Genes for human / mouse chimeras of the CD137 receptor were synthesized using standard laboratory techniques. Various chimeras were designed by exchanging domains or modules of the human CD137 receptor with the corresponding mouse CD137 receptor. The chimeras were designed based on evaluation of the human and mouse sequences as well as 3D investigation of the human CD137 receptor. The synthesized genes were assigned project-specific ID numbers (see Table 11). [Table 11]

[0197] Binding of clone 3712-IgG1v to the chimeric CD137 receptor protein was analyzed in a standard ELISA format. For the data shown in Figures 16A-B, clone 3712-IgG1v was diluted to 1 μg / mL in DPBS and coated onto ELISA plates (Corning, Cat#: 9018, High Binding) at a volume of 50 μL (0.05 μg) per well and incubated overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST. CD137 receptor human / mouse chimera was diluted to 100 or 10 μg / mL in assay diluent, then serially diluted 4-fold in 11 points in assay diluent to final concentrations of 100000, 25000, 6250, 1562.5, 390.63, 97.66, 24.41, 6.10, 1.53, 0.38 and 0.095 ng / mL or 10000, 2500, 625, 156.25, 39.06, 9.77, 2.44, 0.61, 0.15, 0.038 and 0.0095 ng / mL. Diluted CD137 receptor was added to the assay plate at 50 μL / well in duplicate. Plates were incubated at room temperature for 1 hour and then washed 3 times with PBST. Goat anti-human IgG-H+L HRP conjugated at 1:100,000 20 dilution was added to the plate at 100 μL / well. The plate was then incubated at room temperature for 1 hour followed by washing 4 times with PBST. TMB substrate solution was added at 100 μl / well. Color was developed for 15 minutes and stopped with 100 μL / well 2N H2SO4. Absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0198] For the data shown in Figure 16C-D, chimeric CD137 receptor protein samples were diluted to 5 μg / mL in DPBS and coated onto ELISA plates (Corning, Cat#: 9018, High Binding) at 50 μL (0.25 μg) per well and incubated overnight at 4°C. Plates were decanted, washed once, and assay diluent was added at 200 μL / well. After 1 hour incubation at room temperature, plates were washed once with PBST. Clone 3712 was diluted to 100 or 10 μg / mL in assay diluent and then serially diluted 4-fold in 11 points in assay diluent to final concentrations of 100000, 25000, 6250, 1562.5, 390.63, 97.66, 24.41, 6.10, 1.53, 0.38 and 0.095 ng / mL or 10000, 2500, 625, 156.25, 39.06, 9.77, 2.44, 0.61, 0.15, 0.038 and 0.0095 ng / mL. 50 μL / well of diluted clone 3712 was added to the assay plate in duplicate. Plates were incubated at room temperature for 1 hour and then washed 3 times with PBST. Goat anti-human IgG-H+L conjugated with HRP at 1:100,000 dilution or goat anti-mouse IgG(H+L) secondary antibody HRP, 10 at 1:50,000 dilution was added to the plate at 100 μL / well. The plate was incubated at room temperature for 1 hour, followed by washing 4 times with PBST. TMB substrate solution was added at 100 μl / well. Color was allowed to develop for 15 minutes, and then the reaction was stopped by adding 100 μL / well of 2N H2SO4. Absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0199] None of the human CD137 antibodies tested bound to mouse CD137. Thus, if a given antibody does not bind to a particular chimera, this indicates that the antibody is specific for one of the domains that has been replaced with a mouse domain in that chimera.

[0200] The binding pattern for clone 3712 indicates that the amino acid residues important for binding are likely located in domains 2 and 3 (CRD2–3), in contrast to utomirumab and urelumab, which bind to CRD3–4 and CRD1–2, respectively ( Chin et al., 2018 , Li et al., 2018 ).

[0201] Example 7: Evaluation of additional anti-CD137 humanized antibodies (i) Binding affinity KD measurements of CD137 antigen binding were performed according to the protocol described in Example 2 above. Chimeric and humanized antibodies were tested in an Octet Red 96 antigen binding assay to estimate binding kinetics. Antibodies were loaded onto an anti-human Fc (AHC) biosensor. The loaded sensor was immersed in serial dilutions of CD137 protein (300 nM, 1:3 drop, 7 points) in assay buffer (PBS with 0.1% BSA, 0.02% Tween-20, pH 7.2). The calculated kinetic constants using a monovalent (1:1) model are shown in Table 12 below. Except for clone 3714, all humanized antibodies showed similar or slightly lower but acceptable affinities compared to the parental chimeric reference antibody 371. [Table 12]

[0202] Evaluation of antibodies by T cell functional assay was performed according to the protocol described in Example 2. Clones 3711, 3714, and 3717 showed low T cell stimulatory activity (Figure 15).

[0203] (ii) Competitive binding of clone 3712 and CD137 ligand to the CD137 receptor The competition between CD137L and clone 3712 for binding to CD137 receptor was determined in a standard ELISA format. The competitive binding profile of clone 3712 to CD137 receptor was compared to that of reference anti-CD137 antibodies 371, 370, 372, 375, 390, and 402 (see WO2019 / 113039, the relevant disclosures of which are incorporated by reference for the subject matter and purposes referred to herein). CD137 receptor was diluted to 1 μg / mL in DPBS and then coated on ELISA plates (Corning, Catalog No.: 9018, High Binding) at a volume of 50 μL (0.05 μg) per well and incubated overnight at 4°C. The plate was decanted, washed once, and assay diluent was added at 200 μL / well. After incubation at room temperature for 1 hour, the plate was washed once with PBST. Clones 3712, 371, 370, 372, 375, 390, 402 and SSI-361 (negative control antibody) were diluted to 9 μg / mL in assay diluent and serially diluted 11 points in assay diluent to final concentrations of 9000, 3000, 900, 300, 90, 30, 9, 3, 0.9, 0.3 and 0.09 ng / mL. Diluted CD137 receptor was added to the assay plate in duplicate at 50 μL / well. Plates were incubated at room temperature for 1 hour. CD137L was diluted to 6 μg / mL in assay diluent and then serially diluted 11 points in assay diluent to final concentrations of 6000, 2000, 600, 200, 60, 20, 6, 2, 0.6, 0.2, 0.06 ng / ml or diluted to a final concentration of 40 ng / ml. Diluted CD137L was added to the assay plate at 50 μL / well in duplicate. Plates were incubated at room temperature for 1 hour and then washed 3 times with PBST. Goat anti-human IgG-H+L HRP conjugated at 1:100,000 20 dilution was added to the plate at 100 μL / well. Plates were then incubated at room temperature for 1 hour followed by washing 4 times with PBST. TMB substrate solution was added at 100 μl / well. Color was developed for 15 minutes and stopped with 100 μL / well 2N H2SO4.The absorbance at 450 nm and 620 nm was determined by a Tecan F200 Pro reader.

[0204] The results shown in FIG. 17 suggest that the binding of clone 3712 to the CD137 receptor partially blocked the binding of the CD137 receptor to CD137L.

[0205] Example 8: Efficacy evaluation of clone 3712 in an in vivo melanoma model in A375 NPG mice Humanized V of clone 3712 above H and V L The sequence was used to construct an anti-CD137 antibody (clone 3712-IgG1v, disclosed in Example 3 above) that contains the Fc region of human IgG1 / kappa (SEQ ID NOs: 6 and 7), including the Fc variant of human IgG1. 3712-IgG1v was cloned into an expression vector for production in transient expressing CHO cells and stable CHO cell lines according to the methods described in Example 3 above.

[0206] cell culture The A375 cell line was obtained from iCell Bioscience Inc. and maintained in vitro as monolayer cultures in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged every 3–5 days by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0207] animal The study included NPG (NOD-Prkdc) provided by Joinn-lab. scid Il2rg null ) mice were used, all of which were male, 4 weeks old and weighing approximately 19-31 g. A total of 20 mice were randomized into three groups based on body weight.

[0208] Human immune cells and tumor transplantation G-CSF-mobilized peripheral blood CD34(+) hematopoietic stem cells were isolated from peripheral blood stem cells (PBSCs) by magnetic activated cell sorting (MACS) and then transplanted into sublethally X-irradiated NPG mice by bone marrow cavity transplantation. Twelve weeks after transplantation, each mouse received 2 × 10 6 A375 cells are implanted subcutaneously into the right flank.

[0209] Immunomodulatory Treatment Mice in group 1 were treated with PBS and mice in group 2 were treated with clone 3712-IgG1v at 5 mg / kg via intravenous injection twice weekly for a total of 9 doses on days 1, 4, 8, 11, 15, 18, 22, 25 and 29.

[0210] Endpoints Tumors were measured in two dimensions using calipers twice a week starting on day 4, and the volume was calculated according to the formula V = 0.5 a × b 2 The tumor growth inhibition (TGI) was expressed as follows: TGI(%)=100×(1−T / C), where T and C are the mean tumor volumes of the treatment and control groups on day 29, respectively.

[0211] As shown in FIG. 18, treatment with 3712-IgG1v (Group 2) showed significant tumor growth inhibition (TGI=41%, p=0.09) compared to the PBS-treated control group (Group 1).

[0212] Example 9: Evaluation of cytokine releasing activity In vitro cytokine release assays using human whole blood and PBMCs suggested a lower risk of inducing a cytokine release storm for 3712-IgG1v, either in soluble or solid form, compared to CD28 agonist antibody (TGN1412) and CD3 agonist antibody (OKT3).

[0213] (i) Soluble 3712-IgG1v-induced cytokine release from high-density precultured PBMCs PBMCs from four healthy donors were tested in high-density culture experiments to evaluate the potential risk of cytokine induction by soluble 3712-IgG1v. The CD28 agonist antibody TGN1412 and the CD3 antibody OKT3 were used as positive controls, and Avastin was used as a negative control. Culture supernatants 2 and 24 hours after antibody treatment were analyzed for levels of IFN-γ, TNF-α, IL-6, and IL-2. At the 2-hour time point, none of the tested antibodies induced any changes in these cytokines. After 24 hours, TGN1412 and / or OKT3 caused an elevation of IFN-γ, TNF-α, IL-6, and IL-2 in all donors, whereas 3712-IgG1v and Avastin did not induce any significant changes over a wide range of concentrations (3 ng / mL to 300 μg / mL). The data suggest that the risk of inducing a cytokine storm for 3712-IgG1v may be low.

[0214] (ii) Cytokine release from PBMCs induced by plate-bound 3712-IgG1v Plate-bound 3712-IgG1v was tested in culture with PBMC from four healthy donors to assess its ability to induce cytokine production. CD28 agonist antibody TGN1412 and CD3 antibody OKT3 were used as positive controls, and Avastin was used as a negative control. Culture supernatants after 2 and 24 hours of incubation were analyzed for levels of IFN-γ, TNF-α, IL-6, and IL-2. At the 2 hour time point, none of the tested antibodies induced any changes in these cytokines. After 24 hours, TGN1412 and / or OKT3 caused an increase in IFN-γ, TNF-α, IL-6, and IL-2 in all donors except TNF-α and IL-6 in donor 1, whereas 3712-IgG1v and Avastin did not induce any significant changes over a wide range of concentrations (0.1 μg / well to 100 μg / well). The data suggest that 3712-IgG1v may have a low risk of inducing a cytokine storm.

[0215] (iii) Soluble 3712-IgG1v-induced cytokine release in whole blood The immunomodulatory activity of 3712-IgG1v(LYV1) was also examined using whole blood samples from a cohort of 15 healthy donors. Whole blood within 4 hours of collection was incubated with 3712-IgG1v or control. Cytokine levels in plasma were assessed using the Luminex platform. Whole blood incubated with PWM was used as a positive control, and whole blood incubated with PBS was used to determine background responses. Plasma samples were examined for IL-6, IL-8, IL-10, IFN-γ and TNF-α. As expected, incubation of whole blood with the positive control PWM significantly increased the secretion of all cytokines compared to whole blood incubated with PBS.

[0216] To benchmark the data generated on the samples against the data reported from the clinic, two clinical antibodies, Remtrada and Erbitux, with known cytokine release rates were run in parallel. Erbitux is recognized to have a low level of reaction associated with injection and was used to set the baseline for positive cytokine release. In contrast, Remtrada is associated with a high rate of injection reactions in the clinic (Bugelski, Achuthanandam, Capocasale, Treacy, & Bouman-Thio, 2009) and was used to establish the clinical management of high responders.

[0217] The mean levels of IL-6, IL-8, IL-10, IFN-γ, and TNF-α after treating whole blood with Remtrada were significantly higher than those of the cultures treated with 3712-IgG1v or Erbitux®. Statistical analysis showed no significant difference in the levels of IL-6, IL-10, IFN-γ, and TNF-α produced in response to 3712-IgG1v compared to Erbitux® regardless of concentration.

[0218] In summary, the effect of 3712-IgG1v on cytokine release was investigated in a comprehensive series of in vitro assays including high-density pre-culture of human PBMCs prior to the cytokine release assay and 24-hour whole human blood from healthy donors. 3712-IgG1v was tested after coating the culture plates and cultured with human PBMCs for 2 hours and 24 hours prior to cytokine detection. Not only 3712-IgG1v but also the clinical reference antibody Avastin® or Erbitux® showed no significant activity in inducing cytokine release in these assays, suggesting that 3712-IgG1v would have a low risk of inducing cytokine storm in humans.

[0219] Example 10: Pharmacokinetics and Toxicology Tests in Cynomolgus Monkeys Based on target sequence homology, conserved binding affinity, and FcγRIIB-dependent CD137 agonism, cynomolgus monkeys were identified as the only pharmacologically relevant nonclinical species to evaluate the PK and safety profile of 3712-IgG1v.

[0220] Pharmacokinetic studies To understand the PK profile of 3712-IgG1v following intravenous infusion, the intended route of administration in humans, two single-dose (3, 10, or 30 mg / kg, or 0, 10, 30, or 100 mg / kg) studies and one repeat-dose study (0, 10, 30, or 100 mg / kg) were conducted in naive cynomolgus monkeys.

[0221] Single-dose pharmacokinetics Following a single intravenous dose of 3712-IgG1v, systemic exposure was achieved in all animals, with no significant differences between sexes. Dose-normalized systemic exposure (AUC and C max The data suggested a linear pattern of kinetics over the dose range of 3 to 100 mg / kg. In the dose range of 3 to 30 mg / kg, mean T 1 / 2 The IVD ranged from 64.6 to 133 h, the CL was 0.355 to 0.488 mL / hr / kg, and the mean Vdss was 54.9 to 69.7 mL / kg, indicating that 3712-IgG1v was probably distributed in the blood and slowly cleared from the circulation. Formation of anti-3712-IgG1v antibodies (ADA) was detected in most animals, with a higher incidence in animals that received lower doses, suggesting that 3712-IgG1v is immunogenic in monkeys.

[0222] Repeated Dose Pharmacokinetics Repeated dose PK was evaluated in conjunction with a repeat dose toxicity study in cynomolgus monkeys. After repeated intravenous doses of 3712-IgG1v (0, 10, 30, or 100 mg / kg QW × 5), a significant decrease in systemic exposure was observed in animals in the 10 and 30 mg / kg groups, which may be the result of increased clearance and the influence of ADAs. ADA formation was detected primarily in the low dose groups, with 10 / 10, 8 / 10, and 2 / 10 animals in the 10, 30, and 100 mg / kg groups, respectively, found to be positive on day 29. The high prevalence of ADAs in the 10 and 30 mg / kg groups impaired the ability to accurately assess PK. In the 100 mg / kg dose group, where most animals were ADA negative, mild accumulation was noted, with an accumulation ratio (AR) of 1.6 in males and 1.7 in females.

[0223] Toxicology Testing Evaluation of potential hazards to humans receiving 3712-IgG1v was performed in two US-GLP-compliant toxicity studies in naive cynomolgus monkeys. The first was a single-dose study to evaluate potential acute toxicity of intravenous infusion of 3712-IgG1v (0, 10, 30, or 100 mg / kg) and to determine the maximum tolerated dose (MTD). The second was a pivotal 29-day repeat-dose toxicity study, conducted by administering five intravenous infusions of 0, 10, 30, or 100 mg / kg, QW for 29 days with a 6-week recovery period. Potential target organ and subchronic toxicity, as well as the reversibility, persistence, or delayed onset of any toxicity, were evaluated. Safety pharmacology and injection site reactions were also evaluated as part of the repeat-dose general toxicity study.

[0224] Single-dose toxicity A US-GLP-compliant single-dose toxicity study was conducted to evaluate the potential acute toxicity of 3712-IgG1v in naive cynomolgus monkeys. A total of eight monkeys were assigned to four groups (1 / sex / group) and received 0 (vehicle), 10, 30, or 100 mg / kg of 3712-IgG1v via a 60-minute intravenous infusion. Animals were observed for survival, clinical observations, body weight, food consumption, clinical pathology (hematology, serum chemistry, coagulation, and urinalysis), toxicokinetics (TK), and immunogenicity assessments for 14 days after dosing. In addition, non-GLP-compliant analyses of immunotoxicity (cytokines), lymphocyte phenotype, and soluble target antigens were performed. On day 15, all animals were necropsied and underwent gross (macroscopic) and histopathological (microscopic) examination.

[0225] There were no unscheduled deaths during the course of the study. No treatment-related effects were observed on clinical observations, body weight, food consumption, clinical pathology, immunotoxicity, or lymphocyte phenotype. Additionally, necropsy examination revealed no macroscopic or microscopic findings related to the test article.

[0226] Systemic exposure to 3712-IgG1v (C max and AUC 0-168 Both ADA and CD137 (both IgG1v and IgG2a) appeared to increase proportionally with increasing dose, and no significant gender differences in systemic exposure were observed at any dose level. ADA was positive in 1 / 2, 1 / 2, 1 / 2, and 1 / 2 of the animals receiving 0, 10, 30, and 100 mg / kg 3712-IgG1v, respectively. However, titers were very low in all but one female animal in the 10 mg / kg group (day 14 values ​​were 36, 1120, 70, and 12 for the 0, 10, 30, and 100 mg / kg groups, respectively). After intravenous infusion of clone 3712-IgG1v, serum CD137 protein increased dose-dependently from a baseline level of 0.1 ng / mL to 1.3 ng / mL, suggesting that 3712-IgG1v can bind and stabilize CD137 protein in the circulation of cynomolgus monkeys.

[0227] In conclusion, single intravenous infusions of 3712-IgG1v at 10, 30, and 100 mg / kg were well tolerated. Therefore, the MTD of a single intravenous dose of 3712-IgG1v in cynomolgus monkeys was considered to be ≥100 mg / kg.

[0228] Repeated dose toxicity Potential subchronic toxic effects of 3712-IgG1v were investigated in a US-GLP-compliant 29-day repeat-dose toxicity study in cynomolgus monkeys. The study design included a 42-day recovery phase to assess reversibility, persistence, or delayed onset of any adverse effects. A total of 40 monkeys were assigned to four groups (5 monkeys / sex / group) and received 0 (vehicle), 10, 30, and 100 mg / kg of 3712-IgG1v intravenously infused once weekly for 29 days (QW × 5) over 60 min. At the start of dosing, monkeys were approximately 2.5-3.5 years old and weighed in the range of 2.0-2.7 kg. On day 30, animals in the main group (3 / sex / group) were euthanized and necropsied, while animals in the recovery group (2 / sex / group) were observed for an additional 42 days until euthanasia on day 71.

[0229] Animals were evaluated for mortality, clinical observations (including injection site observations), body weight, food consumption, body temperature, safety pharmacology (ECG, heart rate, blood pressure, respiratory parameters and neurological examination), ophthalmology, clinical pathology (hematology, coagulation, serum chemistry and urinalysis), TK, immunogenicity (ADA analysis), organ weights and gross and histopathological examinations. In addition, non-GLP compliant analyses of immunotoxicity (cytokines), lymphocyte phenotype and soluble target antigens were performed.

[0230] Systemic exposure was achieved in all animals. There was no gender difference in systemic exposure after the first intravenous infusion, and systemic exposure (AUC0-168h and Cmax) increased proportionally with dose. ADA formation was detected in 10 / 10, 8 / 10, and 2 / 10 animals in the 10, 30, and 100 mg / kg groups, respectively, on day 29. A decrease in systemic exposure after the fourth dose was observed in the 10 mg / kg group, indicating a significant effect of ADA formation on drug exposure. However, mild accumulation in the 100 mg / kg group was observed, and most animals were ADA negative.

[0231] No unscheduled deaths occurred during the course of the study. There were no test article-related changes with regard to clinical observations, local irritation at the injection site, body weight, food consumption, temperature, safety pharmacology, ophthalmology, clinical pathology (hematology, coagulation, clotting, and urinalysis), safety pharmacology (electrocardiography, blood pressure, heart rate, respiratory and neurological examinations), immunology (B and T lymphocyte phenotyping, cytokine analysis), or pathology changes (organ weights, macroscopic and microscopic observations). All differences observed in clinical pathology and immunology parameters were small in magnitude, not dose-related, and / or within the historical reference ranges for this laboratory and therefore were not considered to be test article-related.

[0232] In conclusion, repeated intravenous infusions of 3712-IgG1v at 10, 30, or 100 mg / kg for 29 days (QW × 5) in male and female cynomolgus monkeys were well tolerated. No test article-related toxicity or toxic organs were identified. The no observed adverse effect level (NOAEL) was considered to be 100 mg / kg in this study. At this dose level, the mean Cmax and AUC 0-168 were 4930 μg / mL and 355,000 μg·h / mL in males and 4010 μg / mL and 300,000 μg·h / mL in females, respectively.

[0233] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0234] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to adapt the disclosure to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0235] Equivalent While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications in which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and equivalents thereof, embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0236] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0237] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, in some cases including the entire document.

[0238] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0239] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the specifically identified element or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", in one embodiment refers only to A (optionally including elements other than B), in another embodiment refers only to B (optionally including elements other than A), in yet another embodiment refers to both A and B (optionally including other elements), etc.

[0240] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one of, but also including more than one of, some element or list of elements, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one" or "exactly one," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term, such as, for example, "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0241] As used herein in the specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") may refer in one embodiment to at least one A (optionally including more than one A) and no B (and optionally including elements other than B), in another embodiment to at least one B (optionally including more than one B) and no A (and optionally including elements other than A), in yet another embodiment to at least one A (optionally including more than one A) and at least one B (optionally including more than one B) (and optionally including other elements), etc.

[0242] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. 1. A humanized antibody that binds to CD137, The antibody comprises: (i) a heavy chain variable domain (V Ｈ (ii) a heavy chain comprising a light chain variable domain (V Ｌ and a light chain comprising The V Ｈ comprising heavy chain complementarity determining regions (CDRs) 1-3 set forth as SEQ ID NOs: 12, 14 and 16, respectively, said heavy chain CDRs being grafted into a human IGHV1-2*2 framework; The V Ｌ comprises light chain CDRs 1-3 set forth as SEQ ID NOs: 29, 31 and 33, respectively, said light chain CDRs being grafted into a human IGKV1-39*01 framework; The V Ｌ comprises one or more back mutations to the IGKV1-39*01 framework, The one or more back mutations are selected from the group consisting of K42G, P44V, F71Y, Y87F, and V104L relative to SEQ ID NO:4; Humanized antibodies.

2. the antibody comprises a heavy chain framework region 1 (HC FR1) comprising the amino acid sequence of SEQ ID NO:18, SEQ ID NO:22 or SEQ ID NO:25, a HC FR2 comprising the amino acid sequence of SEQ ID NO:19, SEQ ID NO:23 or SEQ ID NO:26, a HC FR3 comprising the amino acid sequence of SEQ ID NO:20, SEQ ID NO:24 or SEQ ID NO:27, and a HC FR4 comprising the amino acid sequence of SEQ ID NO:21; and / or said antibody comprises a light chain framework region 1 (LC FR1) comprising the amino acid sequence of SEQ ID NO: 35, an LC FR2 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof having said back mutations of K42G and / or P44V, an LC FR3 comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having said back mutations of F71Y and / or Y87F, and an LC FR4 comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof having said back mutations of V104L; The humanized antibody of claim 1.

3. The humanized antibody of claim 1 or 2, wherein the antibody comprises an LC FR1 having the amino acid sequence of SEQ ID NO:35, an LC FR2 having the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:39, an LC FR3 having the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:40, and an LC FR4 having the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:

41.

4. The humanized antibody of claim 3, wherein the antibody comprises an LC FR1 comprising the amino acid sequence of SEQ ID NO: 35, an LC FR2 comprising the amino acid sequence of SEQ ID NO: 39, an LC FR3 comprising the amino acid sequence of SEQ ID NO: 40, and an LC FR4 comprising the amino acid sequence of SEQ ID NO:

38.

5. (i) The V Ｈ comprises the amino acid sequence of SEQ ID NO:3, Ｌ comprises the amino acid sequence of SEQ ID NO:5; (ii) the V Ｈ comprises the amino acid sequence of SEQ ID NO:3, Ｌ comprises the amino acid sequence of SEQ ID NO:10; (iii) the V Ｈ comprises the amino acid sequence of SEQ ID NO:8, Ｌ comprises the amino acid sequence of SEQ ID NO:5; (iv) Said V Ｈ comprises the amino acid sequence of SEQ ID NO:8, Ｌ comprises the amino acid sequence of SEQ ID NO:10; (v) the V Ｈ comprises the amino acid sequence of SEQ ID NO:9, Ｌ comprises the amino acid sequence of SEQ ID NO:5; or (vi) the V Ｈ comprises the amino acid sequence of SEQ ID NO:9, Ｌ comprises the amino acid sequence of SEQ ID NO:10; The humanized antibody of claim 1.

6. The V Ｈ comprises the amino acid sequence of SEQ ID NO:3, Ｌ The humanized antibody of claim 1, comprising the amino acid sequence of SEQ ID NO:

5.

7. The humanized antibody of any one of claims 1 to 6, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof that is an IgG molecule.

8. The humanized antibody of claim 7 , wherein the antibody is an IgG1 molecule.

9. The humanized antibody of claim 7 or 8, wherein the antibody comprises a wild-type Fc region.

10. The humanized antibody of claim 7 or 8, wherein the antibody comprises an Fc region variant comprising the amino acid sequence of SEQ ID NO:

42.

11. The humanized antibody of claim 1 , wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:6 and the light chain comprises the amino acid sequence of SEQ ID NO:

7.

12. The humanized antibody of any one of claims 1 to 11, wherein the humanized antibody is part of a multispecific antibody that further binds to FcγRIIB.

13. An isolated nucleic acid or set of nucleic acids that collectively encodes the humanized antibody of any one of claims 1 to 12.

14. 14. The isolated nucleic acid or set of nucleic acids of claim 13, wherein the nucleic acid or set of nucleic acids is located on one vector or on two vectors.

15. 15. The isolated nucleic acid or set of nucleic acids of claim 14, wherein said one or two vectors are one or two expression vectors.

16. A host cell comprising the isolated nucleic acid or set of nucleic acids according to any one of claims 13 to 15.

17. A pharmaceutical composition comprising a humanized antibody according to any one of claims 1 to 12, or a nucleic acid or set of nucleic acids according to any one of claims 13 to 15, and a pharma- ceutically acceptable carrier.

18. 20. The pharmaceutical composition of claim 17 for use in modulating an immune response in a human patient having, suspected of having, or at risk of having cancer, an immune disorder or an infectious disease.

19. The method of claim 1, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, and melanoma; The immune disorder is an autoimmune disease selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes, multiple sclerosis, celiac disease, and graft-versus-host (GVH) disease; 19. The pharmaceutical composition of claim 18.

20. 20. The pharmaceutical composition of claim 18 or 19, for use in combination with an immune checkpoint inhibitor.

21. The pharmaceutical composition of claim 20, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

22. 22. The pharmaceutical composition of claim 21, wherein the immune checkpoint inhibitor is pembrolizumab.

23. (i) the human patient has an advanced, metastatic, or unresectable malignant tumor; (ii) the human patient has histologically or cytologically confirmed cancer; or (iii) the human patient is undergoing therapy for said cancer or said immune disorder; A pharmaceutical composition according to any one of claims 18 to 22.

24. The humanized antibody is administered to the human patient at a dose of 0.3 to 10 mg / kg; and / or The humanized antibody is administered to the human patient once every 2-4 weeks; A pharmaceutical composition according to any one of claims 18 to 23.

25. 1. A method for producing a humanized anti-CD137 antibody, comprising: (i) culturing the host cell of claim 16 under conditions allowing expression of the anti-CD137 antibody; (ii) harvesting the anti-CD137 antibody so produced from the cell culture; and A method comprising:

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