Bispecific anti-CD28X anti-CD22 antibody and its use

Abstract

To provide bispecific anti-CD28X-anti-CD22 antibodies and uses thereof.SOLUTION: The invention provides a bispecific antigen-binding molecule comprising a first antigen binding domain that specifically binds to human CD28 and a second antigen binding domain that specifically binds to human CD22. In certain embodiments, the bispecific antigen-binding molecule can inhibit the growth of tumors expressing CD22 such as B cell lymphoma. The antibodies and bispecific antigen-binding molecules are useful for treating a disease or disorder to which an upregulated or induced targeted immune response is desirable and / or therapeutically beneficial.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 781,689, filed on 19 December 2018, the entire contents of which are incorporated herein by reference. Sequence List

[0002] This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on 16 December 2019, is named 118003_49220_SL.txt and is 104,353 bytes in size.

[0003] The present invention relates to a bispecific antigen-binding molecule that binds to CD28 and a target molecule, such as CD22, and to a method of using the same. [Background technology]

[0004] CD28 is a type I transmembrane protein expressed on the surface of T cells, possessing a single extracellular Ig-V-like domain constructed as a homodimer. CD28 is a receptor for the CD80(B7.1) and CD86(B7.2) proteins and is activated by CD80 or CD86 expressed on antigen-presenting cells (APCs). Binding of CD28 to CD80 or CD86 provides a crucial stimulatory signal for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 provides a potent signal for the production of various interleukins. CD28 also enhances cellular signaling, such as pathways regulated by the NFκB transcription factor after TCR activation. CD28 co-signaling is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death.

[0005] Anti-CD28 antibodies have been proposed for therapeutic purposes related to T cell activation. One such anti-CD28 antibody, TGN1412 (an anti-CD28 superagonist), was used in a clinical trial in 2006. Six healthy volunteers were intravenously administered TGN1412 at a dose of 0.1 mg / kg. Within two hours, all six patients experienced a significant inflammatory response (cytokine storm), and all patients developed multiple organ failure within 16 hours. The subjects were treated with corticosteroids, and cytokine levels returned to normal within 2-3 days. The starting dose of 0.1 mg / kg in the Phase 1 study (related to CRS) was based on a 500-fold increase in the cynomolgus monkey "NOAEL" dose of 50 mg / kg (Suntharalingam, et al., Cytokine Storm). (in a Phase 1 Trial of the anti-CD28 Monoclonal Antibody TGN1412, NEJM 355:1018-1028 (2006)). Unfortunately, TGN1412 induced a cytokine storm, which was not anticipated by toxicity studies in cynomolgus monkeys or ex vivo human PBMC studies.

[0006] CD22 (also known as Siglec-2) is a member of the Siglec family and is a transmembrane protein that specifically recognizes α2,6-sialic acid and is selectively expressed on B lymphocytes (B cells).

[0007] CD22 has many intrinsic functions, including inhibiting B cell receptor (BCR) signaling through, for example, B cell homeostasis, B cell survival and migration, attenuation of TLR and CD40 signaling, and recruitment of SH2 domain-containing phosphatases by phosphorylation of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasm, as well as promoting adhesion between B cells and other cell types.

[0008] CD22 is not found on the surface of B cells in the early stages of development, nor is it expressed in stem cells. However, 60-70% of all B-cell lymphomas and leukemias express CD22.

[0009] Anti-CD22 antibodies have been investigated for the treatment of B-cell lymphoma and leukemia. However, the monoclonal antibody epratuzumab has only shown limited success. (Grant, et al. (2013) Cancer 119(21):10.1002 / cncr.28299) Therefore, improved anti-CD22 antibodies are needed in this field. Anti-CD28 antibodies that are safe for use in pharmaceutical compositions are also needed. Furthermore, bispecific antigen-binding molecules that bind to both CD28 and target antigens (e.g., CD22) are useful in therapeutic situations where specific targeting and T-cell-mediated killing of cells expressing the target antigen are desirable. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Suntharalingam, et al., Cytokine Storm in a Phase 1 Trial of the anti-CD28 Monoclonal Antibody TGN1412, NEJM 355:1018-1028(2006) [Non-Patent Document 2] Grant, et al. (2013) Cancer 119(21):10.1002 / cncr.28299 [Overview of the project] [Means for solving the problem]

[0011] In a first embodiment, the present invention provides a bispecific antigen-binding molecule that binds to CD28 and a target antigen. According to one exemplary embodiment, the bispecific antigen-binding molecule binds to CD28 and CD22; such a bispecific antigen-binding molecule is also referred to herein as an “anti-CD28 / anti-CD22 bispecific molecule”. The anti-CD22 portion of the anti-CD28 / anti-CD22 bispecific molecule is useful for targeting cancer cells expressing CD22 (e.g., cancerous B cells), and the anti-CD28 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of CD22 on cancer cells and CD28 on T cells promotes, for example, selective killing (cytolysis) of targeted cancer cells by activated T cells after TCR activation. Therefore, the anti-CD28 / anti-CD22 bispecific molecule of the present invention is particularly useful in the treatment of diseases and disorders caused by CD22-expressing tumors (e.g., B-cell proliferative disorders, e.g., B-cell lymphomas, e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma).

[0012] A bispecific antigen-binding molecule according to this aspect of the present invention comprises a first antigen-binding domain that specifically binds to human CD28 and a second antigen-binding domain that specifically binds to CD22. The present invention includes an anti-CD28 / anti-CD22 bispecific molecule (e.g., a bispecific antibody) in which each antigen-binding domain comprises a heavy chain variable region (HCVR) paired with a light chain variable region (LCVR). In certain exemplary embodiments of the present invention, the anti-CD28 antigen-binding domain and the anti-CD22 antigen-binding domain each comprise different distinct HCVRs paired with a common LCVR.

[0013] The present invention provides an anti-CD28 / anti-CD22 bispecific molecule in which the first antigen-binding domain that specifically binds to CD28 comprises any of the HCVR amino acid sequences listed in Table 6. The first antigen-binding domain that specifically binds to CD28 may also comprise any of the LCVR amino acid sequences listed in Table 6. According to a particular embodiment, the first antigen-binding domain that specifically binds to CD28 comprises any of the HCVR / LCVR amino acid sequence pairs listed in Table 6. The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which the first antigen-binding domain that specifically binds to CD28 comprises any of the heavy chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 6 and / or any of the light chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 6.

[0014] According to a particular embodiment, the present invention provides an anti-CD28 / anti-CD22 bispecific molecule in which a first antigen-binding domain that specifically binds to CD28 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 28 and 26, or a heavy chain variable region (HCVR) having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which the first antigen-binding domain that specifically binds to CD28 comprises a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which the first antigen binding that specifically binds to CD28 comprises an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs. 28 / 10 and 26 / 10.

[0017] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule comprising a first antigen-binding domain that specifically binds to CD28, comprising a heavy chain CDR3 (HCDR3) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 32; and a light chain CDR3 (LCDR3) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0018] In a particular embodiment, the first antigen-binding domain that specifically binds to CD28 includes the HCDR3 / LCDR3 amino acid sequence pair of SEQ ID NO: 32 / 16.

[0019] The present invention also provides an anti-CD28 / anti-CD22 bispecific antigen-binding molecule in which the first antigen-binding domain that specifically binds to CD28 comprises: a heavy chain CDR1 (HCDR1) domain having the amino acid sequence of SEQ ID NO: 28, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having the amino acid sequence of SEQ ID NO: 30, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] Some specific non-limiting examples of the anti-CD28 / anti-CD22 bispecific antigen-binding molecules of the present invention include a first antigen-binding domain that specifically binds to CD28, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, having the amino acid sequence of SEQ ID NOs: 28-30-32-12-14-16, respectively.

[0021] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which a second antigen-binding domain that specifically binds to CD22 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18, or a heavy chain variable region (HCVR) having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which a second antigen-binding domain that specifically binds to CD22 comprises an amino acid sequence selected from SEQ ID NO: 10, or a light chain variable region (LCVR) having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule in which a second antigen-binding domain that specifically binds to CD22 comprises an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 10.

[0024] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule comprising a second antigen-binding domain that specifically binds to CD22, comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 8 and 24, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from SEQ ID NO. 16, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0025] In a particular embodiment, the second antigen-binding domain that specifically binds to CD22 comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16 and 24 / 16.

[0026] The present invention also relates to a heavy chain CDR1 (HCDR1) domain having a second antigen-binding domain that specifically binds to CD22, wherein the second antigen-binding domain is an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 20, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The invention also provides an anti-CD28 / anti-CD22 bispecific antigen-binding molecule comprising an R2(HCDR2) domain; a light chain CDR1(LCDR1) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12; and a light chain CDR2(LCDR2) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0027] Certain non-limiting examples of the anti-CD28 / anti-CD22 bispecific antigen-binding molecules of the present invention include a second antigen-binding domain that specifically binds to CD22, comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24-12-14-16, respectively.

[0028] In related embodiments, the present invention includes an anti-CD28 / anti-CD22 bispecific antigen-binding molecule in which a second antigen-binding domain that specifically binds to CD22 comprises heavy and light chain CDR domains contained within a heavy and light chain variable region (HCVR / LCVR) sequence selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 10.

[0029] In another embodiment, the present invention provides nucleic acid molecules encoding any of the HCVR, LCVR, or CDR sequences of the anti-CD28 / anti-CD22 bispecific antigen-binding molecules disclosed herein, including nucleic acid molecules comprising polynucleotide sequences as described in Table 7 herein, and nucleic acid molecules comprising two or more polynucleotide sequences as described in Table 7 in any functional combination or arrangement thereof. Recombinant expression vectors having the nucleic acids of the present invention, and host cells into which such vectors have been introduced, are also included in the present invention, as are methods of producing antibodies by culturing host cells under conditions that enable antibody production and then recovering the produced antibodies.

[0030] The present invention includes an anti-CD28 / anti-CD22 bispecific antigen-binding molecule in which one of the antigen-binding domains that specifically binds to CD28 specifically binds to CD22, and is combined with, ligated to, or otherwise bound to one of the antigen-binding domains to form a bispecific antigen-binding molecule that binds to CD28 and CD22.

[0031] The present invention comprises an anti-CD28 / anti-CD22 bispecific antigen-binding molecule having a modified glycosylation pattern. In some applications, modifications to remove antibodies lacking undesirable glycosylation sites or fucose moieties present on oligosaccharide chains may be useful to increase, for example, antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0032] In another embodiment, the present invention provides a pharmaceutical composition comprising an anti-CD28 / anti-CD22 bispecific antigen-binding molecule disclosed herein and a pharmaceutically acceptable carrier. In a related embodiment, the present invention features a composition which is a combination of an anti-CD28 / anti-CD22 bispecific antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is advantageously any agent that can be combined with the anti-CD28 / anti-CD22 bispecific antigen-binding molecule. Examples of agents that can be advantageously combined with the anti-CD28 / anti-CD22 bispecific antigen-binding molecule are discussed in detail elsewhere herein.

[0033] In yet another embodiment, the present invention provides a therapeutic method for targeting / killing CD22-expressing cancer cells using the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention, comprising administering a therapeutically effective amount of a pharmaceutical composition containing the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention to a subject in need thereof.

[0034] The present invention also includes the use of the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention in the manufacture of therapeutic agents for diseases or disorders related to or caused by CD22 expression.

[0035] In yet another embodiment, the present invention provides a therapeutic method for targeting / killing CD22-expressing cancer cells using the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention, wherein the anti-CD28 / anti-CD22 bispecific antigen-binding molecule is combined with another antitumor bispecific antigen-binding molecule that binds to CD3 (e.g., anti-CD28 / anti-CD22 combined with an anti-CD3 / anti-CD20 antibody).

[0036] In yet another embodiment, the present invention provides a therapeutic approach for targeting / killing CD22-expressing cancer cells using the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention, wherein the anti-CD28 / anti-CD22 bispecific antigen-binding molecule is combined with a checkpoint inhibitor targeting PD-1, PD-L1, or CTLA-4 (e.g., anti-CD28 / anti-CD22 combined with an anti-PD-1 antibody). For example, in a particular embodiment, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a PD-1 targeting agent, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or semiprimab (Libtayo®). In certain embodiments, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a PD-L1 targeting agent, such as Atezolizumab (Tecentriq®), Avelumab (Bavencio®), or Durvalumab (Imfinzi®). In certain embodiments, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a CTLA-4 targeting agent, such as Ipilimumab (Yervoy®).

[0037] In yet another embodiment, the present invention provides a therapeutic method for targeting / killing cancer cells expressing CD22 using the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention, wherein the anti-CD28 / anti-CD22 bispecific antigen-binding molecule is combined with other antitumor bispecific antigen-binding molecules that bind to CD3 (e.g., anti-CD28 / anti-CD22 combined with an anti-CD3 / anti-CD20 bispecific antibody, e.g., REGN1979 (see U.S. Patent No. 9,657,102, where the anti-CD20 arm contains the HCVR / LCVR amino acid pair of SEQ ID NO: 1242 / 1258 and the anti-CD3 arm contains the amino acid pair of SEQ ID NO: 1250 / 1258)) and / or a checkpoint inhibitor that targets PD-1, PD-L1, or CTLA-4 (e.g., anti-CD28 / anti-CD22 combined with an anti-PD-1 antibody). For example, in certain embodiments, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a PD-1 targeting agent, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or cemiprimab (Libtayo®, e.g., cemiprimab containing the HCVR / LCVR amino acid pair, see U.S. Patent No. 9,987,500, SEQ ID NO: 162 / 170). In certain embodiments, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a PD-L1 targeting agent, such as atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®). In certain embodiments, the anti-CD28 / anti-CD22 antibody of the present invention can be combined with a CTLA targeting agent, such as ipilimumab (Yervoy®).

[0038] Other embodiments will become apparent from a further review of the detailed description below. [Brief explanation of the drawing]

[0039] [Figure 1]Figure 1 is a set of graphs showing the binding of anti-CD28 / anti-CD22 bispecific antibodies to human CD4+CD28-expressing T cells and target cells expressing human CD22 on their cell surface. [Figure 2] Figures 2A and 2B are a set of graphs showing that anti-CD28 / anti-CD22 bispecific antibodies increase luciferase production in the presence of primary T cell stimulation and CD22 target expression. Figure 2A is a set of graphs showing the activation of engineered reporter T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells in addition to a constant 200 pM REGN1945 (hIgG4 isotype negative control), as assessed by luciferase production. Figure 2B is a set of graphs showing the activation of engineered reporter T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells in addition to a constant 200 pM REGN2281 (anti-CD20 x anti-CD3), as assessed by luciferase production. [Figure 3] Figures 3A and 3B are a set of graphs illustrating that anti-CD28 / anti-CD22 bispecific antibodies increase IL-2 production in the presence of primary T cell stimulation and CD22 target expression. More specifically, Figure 3A is a set of graphs illustrating the activation of CD4+ T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86-negative cells in the presence of a constant 2nM REGN1945 (hIgG4 isotype control), as assessed by IL-2 production. Figure 3B is a set of graphs illustrating the activation of CD4+ T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86-negative cells in the presence of a constant 2nM REGN2281 (anti-CD20 x anti-CD3), as assessed by IL-2 production. [Figure 4]Figure 4 is a set of graphs showing that the combination of REGN5837 and semiprimab enhances IL-2 release more than REGN5837 treatment alone in cells engineered to express PD-L1. [Figure 5A] Figure 5A is a set of graphs showing that the combination of REGN5837 and semiprimab enhances IL-2 release in the presence of NALM6 cells engineered to express PD-L1. [Figure 5B] Figure 5B is a set of graphs showing that the combination of REGN5837 and semiprimab enhances IL-2 release more than REGN5837 treatment alone in RAJI cells engineered to express PD-L1. [Figure 6]Figure 6 is a graph showing that treatment of NSG mice with NALM-6-Luc tumors with REGN5837 in the presence of REGN1979 (anti-CD20xanti-CD3) is associated with significant tumor suppression. Briefly, human PBMCs were grafted into NSG mice (n=6-9 per group), and then NALM-6-luc B-cell leukemia cells were transplanted 12 days after grafting (day 0). Mice were administered 4 mg / kg of REGN5837 + 0.04 mg / kg of REGN1979 (hatched circle), 0.4 mg / kg of REGN5837 + 0.04 mg / kg of REGN1979 (closed upward triangle), 0.04 mg / kg of REGN5837 + 0.04 mg / kg of REGN1979 (diamond), 4 mg / kg of non-TAAxCD28 + 0.04 mg / kg of REGN1979 (square), 4 mg / kg of REGN5837 + 0.4 mg / kg of non-TAAxCD3 (white circle), or 4 mg / kg of non-TAAxCD28 + 0.4 mg / kg of non-TAAxCD3 (black inverted triangle) on days 8, 15, and 22 (arrows) after transplantation. Tumor growth was monitored by bio-imaging of tumor volume at 6, 10, 14, 17, 20, and 23 days post-transplant. Overall data are expressed as group mean ± SEM. Statistical significance was determined using two-way ANOVA and Tukey's post-hoc study. The following symbols were used to indicate statistical significance compared to non-TAAxCD28 + non-TAAxCD3 controls: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 7] Figures 7A-7C are graphs showing that REGN1979 activated human T cells and killed Nalm6 cells in a dose-dependent manner. More specifically, Figure 7A is a graph showing the percentage survival of Nalm6 cells in the presence of the indicated antibody. Figure 7B is a graph showing the percentage of CD25-expressing (CD25+)CD8+ cells in the presence of the indicated antibody. Figure 7C is a graph showing the proliferation of CD25+CD8+ cells as evaluated by CellTrace violet dilution in the presence of the indicated antibody. [Figure 8]Figures 8A, 8B, and 8C are graphs showing that REGN1979 activates human T cells and dose-dependently kills WSU-DLCL2 cells. More specifically, Figure 8A is a graph showing the percentage survival of WSU-DLCL2 cells in the presence of the indicated antibody. Figure 8B is a graph showing the percentage of CD25-expressing (CD25+) CD8+ cells in the presence of the indicated antibody. Figure 8C is a graph showing the proliferation of CD8+ cells in the presence of the indicated antibody, expressed as a percentage of cell division. [Figure 9] Figure 9 is a graph showing that REGN1979 induced the release of human cytokines IL-2, IL-4, IL-6, and IL-10 in assays using human PBMCs and WSU-DLCL2 cells. The cytokine release observed with REGN1979 was enhanced in the presence of a fixed concentration of CD22XCD28 compared to cytokine release induced by REGN1979 alone. [Figure 10]Figures 10A–10E are graphs showing that REGN1979 activated human T cells and dose-dependently depleted NHL. Addition of fixed-concentration CD22xCD28 bispecific antibody to REGN1979 enhanced its cytotoxic efficacy (EC50) by 2.3 and 3.5 times compared to REGN1979 with a one-arm CD28 control antibody or an unstimulated control, respectively. The observed target cell lysis mediated by REGN1979, measured by CD8+ and CD4+ cells or CellTrace violet dilution, was associated with CD25 upregulation of T cell activation and proliferation. More specifically, Figure 10A is a graph showing the percentage survival of NHL cells from patient bone marrow in the presence of the indicated antibody. Figure 10B is a graph showing the percentage of CD25-expressing (CD25+) CD8+ cells in the presence of the indicated antibody. Figure 10C is a graph showing the proliferation of CD8+ cells evaluated by CellTrace violet dilution in the presence of the indicated antibody. Figure 10D is a graph showing the percentage of CD25(CD25+)CD4+ cells in the presence of the indicated antibody. Figure 10E is a graph showing the proliferation of CD4+ cells evaluated by CellTrace violet dilution in the presence of the indicated antibody. [Figure 11]Figures 11A–11E are graphs showing that REGN5837 enhances the potential for REGN1979-mediated cytotoxicity, CD25 cell surface expression, and T cell proliferation in a concentration-dependent manner. Briefly, WSU-DLCL2 cells were incubated for 72 hours at 37°C with human PBMCs, which were predominantly lymphocytes with a target cell-to-PBMC ratio of 1:5, and with anti-CD20xCD3 (REGN1979) in concentrations ranging from 4.8 fM to 10 nM, either as monotherapy (i.e., without REGN5837) or in the presence of fixed concentrations of REGN5837 (ranging from 0.01 to 15 μg / mL). Conditions lacking REGN1979, containing only the indicated concentrations of REGN5837, are plotted as 0.04 pM. Viable cells were detected by flow cytometry using live / dead cell staining (11A). T cell activation (measured as CD25 expression; 11B, 11D) and CD4+ and CD8+ T cell proliferation (11C, 11E) were detected by flow cytometry using a phenotyping cocktail of Violet Cell Tracker dye and fluorophore-labeled antibodies against CD2, CD4, CD8, and CD25. More specifically, Figure 11A is a graph showing the percentage of dead cells and REGN5837 at the indicated concentration. Figure 11B is a graph showing the percentage of CD25+CD4+ cells and REGN5837 at the indicated concentration. Figure 11C is a graph showing the proliferation of CD4+ cells as evaluated by CellTrace Violet dilution using REGN5837 at the indicated concentration. Figure 11D is a graph showing the percentage of CD25+CD8+ cells. Figure 11E is a graph showing the proliferation of CD8+ cells as evaluated by CellTrace Violet dilution using REGN5837 at the indicated concentration. [Figure 12]Figures 12A–12G are graphs showing that REGN5837 enhances the potential and maximum level of REGN1979-mediated cytokine release from human T cells in a concentration-dependent manner in the presence of WSU-DLCL2 B-cell lymphoma cells. Briefly, WSU-DLCL2 cells were incubated for 72 hours at 37°C with human PBMCs, which were predominantly lymphocyte-rich, at a target cell-to-PBMC ratio of 1:5, either as monotherapy (i.e., without REGN5837) or in the presence of a fixed concentration of REGN5837 (ranging from 0.01 to 15 μg / mL) with anti-CD20xCD3 (REGN1979) in a concentration range (4.8 fM to 10 nM). The state lacking REGN1979 is plotted as 0.04 pM, containing only REGN5837 at the indicated concentration. The supernatant was evaluated for cytokine release of (12A)IL-2, (12B)IL-4, (12C)IL-6, (12D)IL-10, (12E)TNF-α, (12F)IFN-γ, and (12G)IL-17α using the BD Cytometric Bead Array Human Th1 / Th2 / Th17 Cytokine Kit. More specifically, Figure 12A is a graph showing the level of IL-2 released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells". Figure 12B is a graph showing the level of IL-4 released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells". Figure 12C is a graph showing the level of IL-6 released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells". Figure 12D is a graph showing the level of IL-10 released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells". Figure 12E is a graph showing the level of TNF-α released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells". Figure 12F is a graph showing the level of IFN-γ released from human T cells in the presence of REGN5837 at a concentration labeled "WSU-DLCL2 cells".Figure 12G is a graph showing the levels of IL-17α released from human T cells in the presence of REGN5837 at concentrations labeled as WSU-DLCL2 cells. [Figure 13] Figures 13A and 13B are graphs showing that treatment of NSG mice with WSU-DLCL2 tumors with REGN5837 in the presence of 0.4 or 4 mg / kg of REGN1979 is associated with significant tumor suppression. Briefly, female NSG mice (n=6-7 per group) were transplanted with a 1:1 mixture of WSU-DLCL2 B-cell lymphoma cells and human PBMCs (Day 0). On days 1, 8, and 15 post-transplant (arrows), mice were administered a combination of 1 mg / kg of REGN5837 and 0.4 mg / kg (13A) or 4 mg / kg (13B) of REGN1979 (or a non-crosslinked control). Tumor growth was monitored by caliper measurements on days 7, 10, 14, 16, 28, 31, 35, 38, 43, 46, 49, 53, 57, and 63 post-transplant. Overall data are expressed as group mean ± SEM. Statistical significance was determined using two-way ANOVA and Tukey's post-hoc study. The following symbols were used to indicate statistical significance between groups: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Asterisks indicate statistical significance between REGN1979 monotherapy and isotype controls, hash marks indicate significance between the REGN5837 and REGN1979 combination and isotype controls, and carets indicate significant differences between REGN1979 monotherapy and the REGN5837 and REGN1979 combination. More specifically, Figure 13A is a graph showing tumor growth in mice administered 1 mg / kg of REGN5837 and 0.4 mg / kg of REGN1979 (or non-crosslinked controls, non-TAAxCD3). Figure 13B is a graph showing tumor growth in mice administered 1 mg / kg REGN5837 and 4 mg / kg (or non-crosslinked control, non-TAAxCD3). [Figure 14]Figure 14 is a graph showing that treatment with REGN5837 in the presence of a sub-efficacious dose of REGN1979 was associated with significantly higher survival rates in NSG mice with WSU-DLCL2 tumors compared to REGN5837 or REGN1979 monotherapy. Briefly, female NSG mice (n=6-7 per group) were transplanted with a 1:1 mixture of WSU-DLCL2 B-cell lymphoma cells and human PBMCs (day 0). Mice were administered a combination of REGN5837 and REGN1979 or a control at days 1, 8, and 15 post-transplant (arrows). Statistical significance was determined using the Mantel-Cox test. The following symbols were used to indicate statistical significance between groups: *, p<0.05; ***, p<0.001. The caret symbol indicates statistical significance compared to isotype controls, the asterisk indicates significance compared to 0.4 mg / kg of REGN1979 monotherapy, and the hash mark indicates significance compared to 4 mg / kg of REGN1979 monotherapy. [Modes for carrying out the invention]

[0040] Before describing the present invention, it should be understood that the specific methods and experimental conditions described are subject to change and therefore the invention is not limited to such methods and conditions. It should also be understood that the scope of the present invention is limited only by the appended claims, and therefore the technical terms used herein are for the sole purpose of describing specific embodiments and are not limiting.

[0041] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. Where used herein, the term “about” means, when used in reference to a particular numerical value, that the value may vary by less than 1% from the stated value. For example, where used herein, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0042] Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but preferred methods and materials are described herein. All patents, applications and non-patent publications referenced herein are incorporated herein by reference in their entirety.

[0043] definition As used herein, the term "CD28" refers to an antigen expressed on T cells as a costimulatory receptor. Human CD28 contains the amino acid sequence described in Sequence ID No. 74 and / or has an amino acid sequence such as that described in NCBI acceptance number NP_006130.1. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of each protein, polypeptide or protein fragment unless expressly designated as being of non-human origin. Thus, the term "CD28" means human CD28 unless specifically designated as being of non-human origin, such as "mouse CD28" or "monkey CD28."

[0044] As used herein, “CD28-binding antibody” or “anti-CD28 antibody” includes antibodies that specifically recognize CD28 and their antigen-binding fragments, as well as antibodies that specifically recognize dimeric CD28 and their antigen-binding fragments. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD28 and / or cell surface-expressed CD28. Examples of soluble CD28 include the native CD28 protein and recombinant CD28 protein mutants, such as monomeric and dimeric CD28 constructs, which lack a transmembrane domain or are not bound to the cell membrane.

[0045] As used herein, the expression “cell surface expressed CD28” means one or more CD28 proteins expressed in vitro or in vivo on the surface of a cell such that at least a portion of the CD28 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. “Cell surface expressed CD28” includes CD28 proteins in the context of functional T cell costimulatory receptors on the cell membrane. The expression “cell surface expressed CD28” includes CD28 proteins expressed on the surface of a cell as part of a homodimer. “Cell surface expressed CD28” may include, or consist of, CD28 proteins expressed on the surface of a cell that normally expresses the CD28 protein. Alternatively, “cell surface expressed CD28” may include, or consist of, CD28 proteins expressed on the surface of a cell that does not normally express human CD28 but has been artificially engineered to express CD28 on that surface.

[0046] As used herein, the term "anti-CD28 antibody" includes both monovalent antibodies having single specificity and bispecific antibodies having a first arm that binds to CD28 and a second arm that binds to a second (target) antigen, wherein the anti-CD28 arm contains either an HCVR / LCVR or CDR sequence as listed in Table 1 herein. Examples of anti-CD28 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes, for example, antibodies and antigen-binding fragments of antibodies, including bispecific antibodies.

[0047] Where used herein, the term "CD22" refers to the human CD22 protein unless otherwise specified as being of non-human origin (e.g., "mouse CD22," "monkey CD22," etc.). The human CD22 protein has the amino acid sequence described by acceptance number CAA42006. The sequence of recombinant human CD22ecto (D20-R687) with the myc myc hexahistidine tag (disclosed as "hexahistidine" as SEQ ID NO: 60) is given by acceptance number NP_001762.2 and is also shown as SEQ ID NO: 50. The hCD22ecto domain (D20-R687).hFc can also be purchased from R&D Systems, Catalog#1968-SL-050.

[0048] As used herein, “CD22-binding antibody” or “anti-CD22 antibody” includes an antibody capable of binding to soluble CD22 and / or cell surface-expressed CD22, and its antigen-binding fragment. Soluble CD22 includes the native CD22 protein and recombinant CD22 protein variants, such as CD22 constructs lacking a transmembrane domain or not bound to the cell membrane.

[0049] As used herein, the term "anti-CD22 antibody" includes both monovalent antibodies having single specificity and bispecific antibodies comprising a first arm that binds to CD22 and a second arm that binds to a second (target) antigen, wherein the anti-CD22 arm comprises either an HCVR / LCVR or CDR sequence as listed in Table 1 herein. Examples of anti-CD22 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes, for example, antibodies and antigen-binding fragments of antibodies, including bispecific antibodies.

[0050] The term "antigen-binding molecule" includes, for example, antibodies and antigen-binding fragments of antibodies, including bispecific antibodies.

[0051] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD28). The term "antibody" also includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is C H 1. C H 2 and C H It contains three domains. Each light chain includes a variable light chain region (abbreviated as LCVR or VL in this specification) and a constant light chain region. The constant light chain region has one domain (C L Includes 1). V H and V L The region can be further subdivided into a hypervariability region called the Complementarity Determination Region (CDR), which incorporates a preserved region called the Framework Region (FR). H and V L It consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. In different embodiments of the present invention, the FRs of the anti-CD28 antibody and / or anti-CD22 antibody (or their antigen-binding moieties) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0052] The term “antibody,” as used herein, also includes the antigen-binding fragment of a complete antibody molecule. The terms “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived, for example, from a complete antibody molecule using any suitable standard technique, such as protein digestion or genetic engineering techniques, including the manipulation and expression of DNA encoding antibody-variable and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated, chemically or by molecular biological techniques, to, for example, position one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0053] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small module immunotherapies (SMIPs), and shark variable IgNAR domains, are also included in the expression “antigen-binding fragment” as used herein.

[0054] Antibody antigen binding typically involves at least one variable domain. The variable domain can have any size amino acid composition and generally contains at least one CDR adjacent to or within one or more framework sequences. V L domain bound to a V H domain in an antigen-binding fragment having a V H and a V L domain can be arranged relative to each other in any suitable arrangement. For example, the variable region can be a dimer, such as V H -V H , V H -V L or V L -V L dimers may be included. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0055] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary three-dimensional arrangements of variable and constant domains that can be found within the antigen-binding fragment of an antibody of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C[[ID=?]] H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H It seems there is an error in the original text where the tag in line 57 is incorrect as "?". Please check and correct it if possible for a more accurate translation.3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv)V L -C L Examples include: In any configuration of the variable domain and constant domain including any of the exemplary configurations described above, the variable domain and constant domain can be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments can be linked to each other and / or one or more monomers V H Or V L The material may include a homodimer or heterodimer (or other polymer) having any of the variable or constant domain configurations, non-covalently bonded to the domain (for example, by disulfide bonds).

[0056] Similar to fully antibody molecules, antigen-binding fragments may be monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any polyspecific antibody format, including the exemplary bispecific antibody format disclosed herein, can be adapted for use in relation to the antigen-binding fragment of the present invention using routine techniques available in the art.

[0057] The antibodies of the present invention may function by complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cell-mediated cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a bound antibody on target cells, thereby leading to the lysis of the target cells. CDC and ADCC are well known and can be measured using assays available in the art. (e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes) See et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in its ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, antibody isotypes can be selected based on whether or not it is desirable for the antibody to mediate cytotoxicity.

[0058] According to certain embodiments of the present invention, the anti-CD28 antibody and / or anti-CD22 antibody (monospecific or bispecific) of the present invention is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may, for example, include amino acid residues in the CDR and certain CDR3 that are not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by in vitro random or site-directed mutagenesis, or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence.

[0059] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression transfect vector in a host cell (as described further below), antibodies isolated from a recombinant connatorial human antibody library (as described further below), antibodies isolated from an animal transgenic for a human immunoglobulin gene (e.g., mouse) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means including splicing a human immunoglobulin gene sequence with another DNA sequence. Such recombinant human antibodies have a variable region and a constant region derived from a human germline immunoglobulin sequence. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if using animals that are transgenic for human Ig sequences, in vivo somatic mutagenesis), and therefore the recombinant antibody V H and V L The amino acid sequence of the region is human germline V H and V L While derived from and related to the sequence, this sequence may not be naturally present in the human antibody germline repertoire in vivo.

[0060] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable quadruple-chain construct of approximately 150–160 kDa, where the dimers are linked by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, and a molecule of approximately 75–80 kDa is formed, consisting of covalently bonded light and heavy chains (half-antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0061] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but is not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed using the human IgG1 hinge. The present invention comprises antibodies having one or more mutations in the hinge, CH2, or CH3 region, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0062] The antibodies of the present invention may be isolated antibodies. “Isolated antibodies,” as used herein, mean antibodies identified and isolated, and / or recovered, from at least one component of their natural environment. For example, antibodies isolated or removed from at least one component of an organism, or from tissues or cells in which antibodies naturally exist or are naturally produced, are “isolated antibodies” according to the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody subjected to at least one purification or isolation step. According to certain embodiments, isolated antibodies may substantially contain other cellular material and / or chemical substances.

[0063] The present invention also includes one-arm antibodies that bind to CD28 and / or CD22. As used herein, “one-arm antibody” means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies of the present invention may comprise either an HCVR / LCVR or CDR amino acid sequence as shown in Table 1.

[0064] The anti-CD28 antibody and / or anti-CD22 antibody, or its antigen-binding domain, as described herein, may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antigen-binding protein or antigen-binding domain is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention comprises an antibody and its antigen-binding domain derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more frameworks and / or CDR regions are mutated to replace the corresponding residue(s) of the germline sequence from which the antibody is derived, or the corresponding residue(s) of another human germline sequence, or a conserved amino acid substitution (such sequence changes are collectively referred to herein as “germline mutations”) of the corresponding germline residue(s). Those skilled in the art can readily produce many antibodies and antigen-binding fragments, including one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable domain sequences disclosed herein. In a particular embodiment, V H and / or V LIn other embodiments, all framework and / or CDR residues within the domain are mutated to residues found in the original germline sequence from which the antibody was induced. In other embodiments, only certain residues are mutated to the original germline sequence, for example, within the first eight amino acids of FR1, or within the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally induced). Furthermore, the antibody of the present invention, or its antigen-binding domain, may include any combination of two or more germline mutations within the framework and / or CDR region, for example, here, certain individual residues are mutated to corresponding residues of a particular germline sequence, while other certain residues different from the original germline sequence are mutated to corresponding residues of a different germline sequence. Once obtained, an antibody, or its antigen-binding fragment, containing one or more germline mutations, can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (in some cases), or reduced immunogenicity. Antibodies or antigen-binding fragments obtained by this general method are included in the present invention.

[0065] The present invention also includes an anti-CD28 antibody and / or an anti-CD22 antibody, as well as an antigen-binding molecule comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect of the present invention include a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conserved substitutions. For example, the present invention includes an anti-CD28 antibody and an antigen-binding molecule having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, conserved amino acid substitutions such as 10 or less, 8 or less, 6 or less, and 4 or less for any of the HCVR, LCVR, and / or CDR amino acid sequences listed in Table 6 herein.

[0066] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen may have multiple epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either structural or linear. Structural epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl groups on an antigen.

[0067] The terms "substantially identical" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, nucleotide sequence identity exists in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some cases, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0068] When applied to polypeptides, the terms “substantially similar” or “substantially identical” mean that two peptide sequences, when optimally aligned, such as by programmed GAP or BESTFIT using default gap weights, share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity. Preferably, the non-identical residue positions are distinguished by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the % sequence identity or similarity can be adjusted up to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. BioI.24:307-331. Examples of amino acid groups having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-alpartate, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed by Gonnet et al (1992) Science 256:1443-1445. A "somewhat conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0069] Polypeptide sequence similarity, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which, with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutaines. For example, GCG See Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlap region between the query sequence and the search sequence and % sequence identity (Pearson (2000) above). Another preferred algorithm when comparing the sequences of the present invention with a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J.Mol.BioI.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402.

[0070] The terms “proliferative disorder” and “proliferative disorder” refer to disorders associated with a degree of abnormal cell proliferation that would benefit from treatment with anti-CD28 / anti-CD22 bispecific antigen-binding molecules or the methods of the present invention. This includes chronic and acute disorders, including pathological conditions that make mammals susceptible to the disorder in question. In one embodiment, the proliferative disorder is cancer, which is a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation.

[0071] As used herein, “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as they appear herein.

[0072] "B-cell proliferative disorders" include Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), such as aggressive NHL, relapsed aggressive NHL, low-grade / follicular NHL, small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, giant lesion NHL, relapsed painless NHL, and rituximab-refractory painless NHL, among others. Painful NHL; refractory NHL, refractory painless NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Valdenström macroglobulinemia, lymphocyte-predominant Hodgkin lymphoma (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL); leukemias including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), pilocytic cell leukemia, and chronic myeloblastic leukemia; and other hematological malignancies.

[0073] The terms “non-Hodgkin lymphoma” or “NHL” as used herein refer to lymphatic cancers other than Hodgkin lymphoma. Hodgkin lymphoma can generally be distinguished from non-Hodgkin lymphoma by the presence of Reed-Sternberg cells in Hodgkin lymphoma, and the absence of such cells in non-Hodgkin lymphoma. Examples of non-Hodgkin lymphomas as used herein include any that are so as identified by a person skilled in the art (e.g., an oncologist or pathologist) according to a classification scheme known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See, in particular, the lists in Figures 11.57, 11.58 and 11.59.More specific examples, though not limited to, include: relapsed or refractory NHL, frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or pre-lymphocytic leukemia and / or small lymphocytic lymphoma, B-cell pre-lymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, pilocytic cell leukemia, plasmacytoma and / or plasmacytogenic myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular lymphoma. This includes cystic NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, invasive NHL (including invasive frontline NHL and invasive recurrent NHL), NHL that relapses after or is unresponsive to autologous stem cell transplantation, mediastinal primary B-cell large cell lymphoma, primary humoral lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small uncleaved cell NHL, giant lesion NHL, Burkitt lymphoma, progenitor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, and vascular central lymphoma. Bispecific antigen binding molecules

[0074] The antibodies of the present invention may be monospecific, bispecific, or polyspecific. Polyspecific antibodies may be specific to different epitopes of a single target polypeptide, or may contain antigen-binding domains specific to multiple target polypeptides. See, for example, Tutt et al., 1991, J.Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD28 antibody and / or anti-CD22 antibody of the present invention may be ligated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or its fragment may be functionally ligated to one or more other molecular entities, such as another antibody or antibody fragment, (e.g., by chemical coupling, gene fusion, non-covalent association, or other methods) to produce a bispecific or polyspecific antibody having secondary binding specificity.

[0075] The use of the terms “anti-CD28 antibody” and / or “anti-CD-22 antibody” herein is intended to include both monospecific anti-CD28 antibodies and / or monospecific anti-CD22 antibodies, as well as bispecific antibodies comprising a CD28-binding arm or a CD22-binding arm and an arm that binds to a target antigen. Accordingly, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds to human CD28 or CD22, and the other arm of the immunoglobulin is specific to a target antigen. The target antigen to which the other arm of the CD28 or CD22 bispecific antibody binds may be any antigen expressed on or near cells, tissues, organs, microorganisms, or viruses to which a targeted immune response is desired. The CD28-binding arm may comprise either of the HCVR / LCVR or CDR amino acid sequences listed in Table 1 herein. The CD22-binding arm may comprise either of the HCVR / LCVR or CDR amino acid sequences listed in Table 1 herein. In a particular embodiment, the CD28-binding arm binds to human CD28 and induces human T cell proliferation.

[0076] In relation to the bispecific antibody of the present invention, in which one arm of the antibody binds to CD28 and the other arm binds to a target antigen, the target antigen may be a tumor-associated antigen, such as CD22.

[0077] According to one exemplary embodiment, the present invention includes bispecific antigen-binding molecules that specifically bind to CD28 and CD22. Such molecules may be referred to herein, for example, as "anti-CD28 / anti-CD22," or "anti-CD28xCD22," or "CD28xCD22," or "anti-CD22 / anti-CD28," or "anti-CD22xCD28," or "CD22xCD28" bispecific molecule, or "αCD22xαCD28," or "αCD28xαCD22," or other similar names.

[0078] According to one exemplary embodiment, a bispecific antigen-binding molecule (e.g., a bispecific antibody) may have an effector arm and a targeting arm. The effector arm may be a first antigen-binding domain (e.g., an anti-CD28 antibody) that binds to an antigen on an effector cell (e.g., a T cell). The targeting arm may be a second antigen-binding domain (e.g., an anti-CD22 antibody) that binds to an antigen on a target cell (e.g., a tumor cell). According to one exemplary embodiment, the effector arm binds to CD28 and the targeting arm binds to CD22. Bispecific anti-CD28 / CD22 may provide a co-stimulatory signal to effector cells (e.g., T cells). The effector arm has no effect on stimulating T cells without clustering. The effector arm alone has little effect on stimulating T cells unless combined with the targeting arm. The tumor targeting arm may have incomplete tumor specificity. The antigen targeted by the targeting arm (e.g., CD22) may be expressed on a fraction of tumor cells. The specificity of the tumor targeting arm can be increased by combining it with an anti-CD3 bispecific antigen-binding molecule (e.g., an anti-CD3 / CD20 bispecific antibody) and overlapping it.

[0079] As used herein, the term “antigen-binding molecule” means a protein, polypeptide, or molecular complex comprising, or consisting of, at least one complementarity-determining region (CDR), which, alone or in combination with one or more further CDRs and / or framework regions (FRs), specifically binds to a particular antigen. In certain embodiments, the antigen-binding molecule is an antibody or an antibody fragment, as these terms are defined elsewhere herein.

[0080] As used herein, the expression “bispecific antigen-binding molecule” means a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within a bispecific antigen-binding molecule contains at least one CDR, either alone or in combination with one or more further CDRs and / or FRs, which specifically bind to a particular antigen. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., CD28), and the second antigen-binding domain specifically binds to a second antigen (e.g., CD22).

[0081] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody includes a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In relation to a bispecific antigen-binding molecule (e.g., a bispecific antibody) containing first and second antigen-binding domains, the CDR of the first antigen-binding domain may be designated with the prefix "D1", and the CDR of the second antigen-binding domain may be designated with the prefix "D2". Accordingly, the CDR of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and the CDR of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

[0082] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be bound to another multimerizing domain. The binding of one multimerizing domain to another promotes the binding between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second multimerizing domain of the same or similar structure or configuration. For example, the multimerizing domain may be immunoglobulin C H It may be a polypeptide containing 3 domains. An unrestricted example of a polymerized component is the Fc portion (C) of an immunoglobulin. H 2-C H This includes, for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0083] The bispecific antigen-binding molecule of the present invention typically comprises two multimerization domains, for example, two Fc domains, each being part of an independent antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0084] In certain embodiments, the multimerizing domain is an amino acid sequence of 1 to about 200 amino acids in length containing an Fc fragment or at least one cysteine ​​residue. In other embodiments, the multimerizing domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0085] The bispecific antigen-binding molecules of the present invention can be produced using any bispecific antibody format or technique. For example, an antibody or fragment thereof having primary antigen-binding specificity can be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment having secondary antigen-binding specificity (e.g., by chemical coupling, gene fusion, non-covalent association, or other methods) to produce a bispecific antigen-binding molecule. Specific examples of bispecific formats that can be used in connection with the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, bivariable domain (OVO)-Ig, Quadroma, knob-into-hole, common light chain (e.g., a common light chain with a knob-into-hole), CrossMab, CrossFab, (SEEO) body, leucine zipper, Ouobody, IgG1 / IgG2, biactive Fab(OAF)-IgG, and Mab 2 One example is the bispecific format (for a review of this format, see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the references mentioned therein).

[0086] In relation to the bispecific antigen-binding molecules of the present invention, the polymerized domain, for example, the Fc domain, may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring version of the Fc domain. For example, the present invention includes a bispecific antigen-binding molecule comprising one or more modifications in the Fc domain, resulting in a modified Fc domain having a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule includes a modification that increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes where the pH is in the range of about 5.5 to about 6.0) C H 2 or C HIt is included in 3 regions. Non-restrictive examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., LN / FIW or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / EID or T); or modifications at positions 428 and / or 433 (e.g., UR / S / P / Q or K) and / or 434 (e.g., H / F or V); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0087] The present invention also includes the first C H 3 domains and second Ig C H It contains a bispecific antigen-binding molecule comprising three domains, and the first and second IgC H The three domains differ from each other by at least one amino acid, and this difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking that amino acid difference. In one embodiment, first Ig C H The 3 domains bind to protein A, and the second IgC H The 3 domains include mutations that reduce or disable protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H3 may further include the Y96F modification (according to IMGT; Y436F according to the EU). Further modifications that can be found within the second CH3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V821 (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V4221); for IgG2 antibodies, N44S, K52N, and V821 (according to IMGT; according to EU, N384S, K392N, and V4221); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221).

[0088] In certain embodiments, the Fc domain may be a chimeric combination of Fc sequences derived from multiple immunoglobulin isotypes. For example, the chimeric Fc domain may be human IgG1, human IgG2, or human IgG4 C H C originating from 2 regions H Part or all of the 2 sequences and C derived from human IgG1, human IgG2, or human IgG4. H 3. It may include part or all of the sequence. The chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein is: [IgG4C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4C H 3] includes. Another example of a chimeric Fc domain that may be contained in any of the antigen-binding molecules described herein is: from the N-terminus to the C-terminus: [IgG1C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4C H 2]-[IgG1C HThis includes [3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the present invention are described in International Publication No. 2014 / 022540A1, the full contents of which are incorporated herein by reference. Chimeric Fc domains having these general structural configurations and their variants may alter Fc receptor binding, which in turn affects Fc effector function. Sequence variants

[0089] The antibodies and bispecific antigen-binding molecules of the present invention may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The antigen-binding molecules of the present invention may contain antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in the framework and / or CDR region are mutated to the corresponding residue(s) of the germline sequence from which the antibody is derived, or to the corresponding residue(s) of another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable domain sequences disclosed herein. In a particular embodiment, V H and / or V LAll framework and / or CDR residues within the domain are mutated to residues found in the original germline sequence from which the antigen-binding domain originally originated. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues found in the first eight amino acids of FR1, or only mutated residues found in the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally originated). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (in some cases), or reduced immunogenicity. A bispecific antigen-binding molecule containing one or more antigen-binding domains obtained by this general method is included in the present invention.

[0090] The present invention also includes antigen-binding molecules in which one or both of the antigen-binding domains comprise a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention comprises antigen-binding molecules comprising an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is one in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-alpartate, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "somewhat conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0091] The present invention also includes antigen-binding molecules comprising an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. The terms “substantially identical” or “substantially identical,” when referring to amino acid sequences, mean that two amino acid sequences share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity, when optimally aligned, for example, by a program such as GAP or BESTFIT using default gap weights. Preferably, the non-identical residue positions are distinguished by conservative amino acid substitutions. If two or more amino acid sequences differ from each other by conservative substitutions, the % sequence identity or similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art; see, for example, Pearson (1994) Methods Mol. BioI.24:307-331.

[0092] Polypeptide sequence similarity, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which, with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their mutaines. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and % sequence identity of the best overlap region between the query sequence and the search sequence (Pearson (2000) above). Another preferred algorithm when comparing the sequences of the present invention with a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, eg, Altschul et al. (1990) J.Mol.BioI.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402. pH dependent binding

[0093] The present invention includes an anti-CD28 / anti-CD22 bispecific antigen-binding molecule having pH-dependent binding characteristics. For example, the anti-CD28 antibody of the present invention may exhibit a decrease in binding to CD28 at acidic pH compared to neutral pH. Alternatively, the anti-CD22 antibody of the present invention may exhibit an enhancement in binding to CD22 at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0094] In some cases, "a decrease in the binding of... at acidic pH compared to neutral pH" is the K of the antibody binding to the antigen at acidic pH D value, relative to the K of the antibody binding to the antigen at neutral pH D value (or its inverse). For example, if an antibody or its antigen-binding fragment exhibits an acidic / neutral K D ratio of about 3.0 or greater, then, for the purposes of the present invention, the antibody or its antigen-binding fragment can be considered to exhibit "a decrease in binding to CD28 at acidic pH compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K D ratio of the antibody or antigen-binding fragment of the present invention is about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0095] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for decreased (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. Furthermore, modifications to the antigen-binding domain at the amino acid level may also yield antibodies with pH-dependent properties. For example, substituting one or more amino acids in the antigen-binding domain (e.g., within a CDR) with histidine residues may result in antibodies with reduced antigen binding at acidic pH compared to neutral pH. Antibodies containing Fc variants

[0096] According to a particular embodiment of the present invention, for example, an anti-CD28 / anti-CD22 bispecific antigen-binding molecule is provided, comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides a mutation in the Fc domain that increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes where the pH is in the range of about 5.5 to about 6.0), wherein the mutation is C H 2 or C HThe molecule contains antibodies and antigen-binding molecules in three regions. Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0097] For example, the present invention includes an anti-CD28 / anti-CD22 bispecific antigen-binding molecule comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Any possible combination of the Fc domain mutations and other mutations within the antibody variable domain disclosed herein is intended within the scope of the present invention.

[0098] Biological properties of antibody and antigen-binding molecules The present invention includes antibodies and antigen-binding fragments that bind to human CD28 and / or CD22 with high affinity. The present invention also includes antibodies and antigen-binding fragments that bind to human CD28 and / or CD22 with medium or low affinity, depending on the therapeutic context and desired specific targeting characteristics. For example, in the case of a bispecific antigen-binding molecule in which one arm binds to CD28 and the other arm binds to a target antigen (e.g., CD22), it may be desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-CD28 arm binds to CD28 with medium or low affinity. In this way, selective targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding overall / untargeted CD28 binding and the associated adverse side effects.

[0099] According to a particular embodiment, the present invention measures human CD22 (e.g., at 25°C) and Kn less than about 15 nM by surface plasmon resonance using an assay format such as that defined in Example 5 herein. D The present invention includes antibodies and antigen-binding fragments of antibodies that bind to human CD22. D They are joined together.

[0100] According to a particular embodiment, the present invention measures the K22 of monkeys (e.g., at 25°C) and the K22 of monkeys by surface plasmon resonance using an assay format such as that defined in Example 5 herein, to a K22 of less than about 60 μM. DIt includes antibodies and antigen-binding fragments of antibodies that bind with K. In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to simian CD22 with a K of less than about 60 μM, less than about 59 μM, less than about 58 μM, less than about 57 μM, less than about 56 μM, less than about 55 μM, less than about 54 μM, less than about 53 μM, less than about 52 μM, less than about 51 μM, less than about 50 μM, less than about 49 μM, less than about 48 μM, less than about 47 μM, less than about 46 μM, less than about 45 μM, less than about 44 μM, less than about 43 μM, less than about 42 μM, less than about 41 μM, less than about 40 μM, less than about 39 μM, less than about 38 μM, less than about 37 μM, less than about 36 μM, less than about 35 μM, less than about 34 μM, less than about 33 μM, less than about 32 μM, less than about 31 μM, less than about 30 μM, less than about 25 μM, less than about 20 μM, about 15 μM, or less than about 10 μM, measured by surface plasmon resonance using an assay format such as that defined in Example 5 herein or a substantially similar assay. D binds with K.

[0101] According to certain embodiments, the present invention includes antibodies and antigen-binding fragments of antibodies that have a K of less than about 45 μM when binding to human CD28 (e.g., at 25°C) measured by surface plasmon resonance using an assay format such as that defined in Example 5 herein. D It includes antibodies and antigen-binding fragments of antibodies that bind with K. In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to human CD28 with a K of less than about 45 μM, less than about 44 μM, less than about 43 μM, less than about 42 μM, less than about 41 μM, less than about 40 μM, less than about 39 μM, less than about 38 μM, less than about 37 μM, less than about 36 μM, less than about 35 μM, less than about 34 μM, less than about 33 μM, less than about 32 μM, less than about 31 μM, less than about 30 μM, less than about 25 μM, less than about 20 μM, less than about 15 μM, less than about 10 μM, measured by surface plasmon resonance using an assay format such as that defined in Example 5 herein or a substantially similar assay. D binds with K.

[0102] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD22 by surface plasmon resonance at 25°C, for example, using an assay format as defined in Example 5 herein, or by a substantially similar assay, with a dissociation half-life (t1 / 2) of more than about 7.5 minutes. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind to human CD22 by surface plasmon resonance at 25°C, for example, using an assay format as defined in Example 5 herein, or by a substantially similar assay, with a t1 / 2 of more than about 7 minutes, more than about 10 minutes, more than about 15 minutes, more than about 20 minutes, more than about 25 minutes, more than about 30 minutes, more than about 35 minutes, more than about 40 minutes, more than about 45 minutes, more than about 50 minutes, more than about 55 minutes, more than about 60 minutes, more than about 65 minutes, more than about 70 minutes, more than about 75 minutes, or more than about 100 minutes.

[0103] The present invention also includes antibodies and antigen-binding fragments thereof that bind to monkey CD22 by surface plasmon resonance at 37°C, for example, using an assay format as defined in the Examples herein, or by a substantially similar assay, with a dissociation half-life (t1 / 2) of more than 4.3 minutes. In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to CD28 by surface plasmon resonance at 25°C, for example, using an assay format as defined in Example 5 herein, or by a substantially similar assay, with a t1 / 2 of more than 4 minutes, more than 5 minutes, more than 6 minutes, more than 7 minutes, more than 8 minutes, more than 9 minutes, more than 10 minutes, more than 15 minutes, more than 20 minutes, more than 25 minutes, more than 30 minutes, more than 35 minutes, more than 40 minutes, more than 45 minutes, or more than 50 minutes.

[0104] The present invention also includes antibodies and antigen-binding fragments that bind to human CD28 with a dissociation half-life (t1 / 2) greater than approximately 2.3 minutes, as measured by surface plasmon resonance at 25°C, for example, using an assay format as defined in Example 5 herein, or by a substantially similar assay. In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to CD28 at a t1 / 2 of more than about 2 minutes, more than about 5 minutes, more than about 10 minutes, more than about 20 minutes, more than about 30 minutes, more than about 40 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes, more than about 200 minutes, more than about 300 minutes, more than about 40 minutes, more than about 500 minutes, more than about 600 minutes, more than about 700 minutes, more than about 800 minutes, more than about 900 minutes, more than about 1000 minutes, or more than about 1200 minutes, as measured by surface plasmon resonance at 25°C or 37°C, for example, using an assay format as defined in the examples herein, or by a substantially similar assay.

[0105] The present invention also includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that can bind to human CD28 and human and monkey CD22. According to certain embodiments, the bispecific antigen-binding molecules of the present invention specifically interact with cells expressing CD28 and / or CD22. The extent to which the bispecific antigen-binding molecules bind to cells expressing CD28 and / or CD22 can be assessed by fluorescence-activated cell classification (FACS), as shown in Example 6 herein. For example, the present invention includes bispecific antigen-binding molecules that specifically bind to human cell lines or cynomolgus monkey cells (e.g., T cells) that express CD28 but not CD22, and to human cell lines or cynomolgus monkey cells (e.g., B cells or Nalm6 cells) that express CD22 but not CD28. The present invention measures approximately 1.3 × 10⁻⁶ of the aforementioned cells and cell lines using a FACS assay or substantially similar assay as described in Example 6. -6 ~Approx. 2.3×10 -8 M, or smaller EC 50 It contains a bispecific antigen-binding molecule that binds based on its value.

[0106] The present invention comprises a bispecific antigen-binding molecule (e.g., a bispecific antibody) capable of binding to human CD28 and / or human CD22. According to certain embodiments, the bispecific antigen-binding molecule of the present invention specifically interacts with cells expressing CD28 and / or CD22. The extent to which the bispecific antigen-binding molecule binds to cells expressing CD28 and / or CD22 can be evaluated by flow cytometry, as shown in Example 7 herein. For example, the present invention comprises a bispecific antigen-binding molecule that specifically binds to human cells expressing CD28 but not CD22 (e.g., T cells), and to human cell lines expressing CD22 but not CD28 (e.g., HEK293 cells transduced with human CD22 genetically modified to lack CD80 and CD86, and Raji B cells). The present invention expresses a binding of approximately 1.14 × 10⁻¹⁴ to either of the aforementioned cells and cell lines, as measured by flow cytometry or a substantially similar assay as described in Example 7. -8 ~Approx. 9.76×10 -9 M, or smaller EC 50 It contains a bispecific antigen-binding molecule that binds based on its value.

[0107] The present invention also provides anti-CD28 / anti-CD22 bispecific antigen-binding molecules that induce or enhance the efficacy of CD20xCD3 T cell-mediated killing of tumor cells. For example, the present invention can be measured using an in vitro T cell-mediated tumor cell killing assay, for example, an assay format as defined in Example 8 herein (e.g., to evaluate the degree of Raji cell killing by human PBMCs in the presence of anti-CD20xCD3 and anti-CD28xCD22 antibodies), or a substantially similar assay, to obtain approximately 1.48 × 10⁻⁶ -10 M-EC 50The present invention comprises an anti-CD28xCD22 antibody that induces or enhances the efficacy of CD20xCD3 T cell-mediated toxicity of tumor cells. In certain embodiments, the antibody or antigen-binding fragment of the present invention measures T cell-mediated toxicity of tumor cells (e.g., PBMC-mediated toxicity of Raji cells) by an in vitro T cell-mediated toxicity assay, for example, using an assay format as defined in Example 8 herein, or a substantially similar assay, to an EC of less than about 150 pM, less than about 100 pM, less than about 75 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5.0 pM, less than about 4.0 pM, less than about 3.0 pM, less than about 2.5 pM, less than about 2.0 pM, or less than about 1.5 pM. 50 Use values ​​to guide the process.

[0108] The present invention also includes anti-CD28 / anti-CD22 bispecific antigen-binding molecules that exhibit one or more selected from the group consisting of T cell activation, induction of IL-2 release, induction of CD25+ upregulation in human PBMCs, and increased human T cell-mediated cytotoxicity in CD22-expressing cell lines (see, for example, Example 9 herein). The present invention also includes anti-CD28 / anti-CD22 bispecific antigen-binding molecules that, when combined with bispecific antibodies conjugating CD20 and CD3, for example, REGN1979, etc., enhance the killing of CD22-expressing tumor cells. The present invention also includes anti-CD28 / anti-CD22 bispecific antigen-binding molecules that, when combined with antibodies conjugating PD-1, for example, semiprimab, etc., enhance the killing of CD22-expressing tumor cells (see Examples 10-15).

[0109] Epitope mapping and related technologies The epitope on CD28 or CD22 to which the antigen-binding molecule of the present invention binds may consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD28 or CD22 protein. Alternatively, the epitope may consist of multiple non-adjacent amino acids (or amino acid sequences) of CD28 or CD22. The antibody of the present invention may interact with amino acids contained within a CD28 monomer or with amino acids on two different CD28 chains of a CD28 dimer. The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have multiple epitopes. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes may be either stereostructural or linear. Structural epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl groups on an antigen.

[0110] Using various techniques known to those skilled in the art, it is possible to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques that can be used to determine the epitope or binding domain of a particular antibody or antigen-binding domain include, for example, routine cross-blocking assays, e.g., AntibodiesThese include methods described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry. Generally, in hydrogen / deuterium exchange methods, the protein of interest is deuterium-labeled, and then the antibody is conjugated to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to reveal deuterium-labeled residues corresponding to specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography can also be used to identify the amino acids within the polypeptide with which the antibody interacts.

[0111] The present invention further includes anti-CD28 and anti-CD22 antibodies (for example, antibodies comprising any of the amino acid sequences listed in Table 6 herein) that bind to the same epitope as any of the specific exemplary antibodies described herein.

[0112] According to a particular embodiment, the present invention provides antibodies and antigen-binding fragments of antibodies that bind to an epitope on human CD22 containing one or more amino acids of SEQ ID NO: 34, SEQ ID NO: 35, and / or SEQ ID NO: 36, as determined by hydrogen / deuterium exchange detected by mass spectrometry as described in Examples 3 and 4.

[0113] Similarly, the present invention also includes anti-CD28 and / or anti-CD22 antibodies (for example, antibodies comprising the amino acid sequences listed in Table 6 of this specification) that compete with any of the specific exemplary antibodies described herein for binding to CD28 and / or CD22.

[0114] The present invention also includes a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human CD28 and a second antigen-binding fragment that specifically binds to human CD22, wherein the first antigen-binding domain binds to the same epitope on CD28 as any of the specific exemplary CD28-specific antigen-binding domains described herein, and / or the second antigen-binding domain binds to the same epitope on CD22 as any of the specific exemplary CD22-specific antigen-binding domains described herein.

[0115] Similarly, the present invention also includes a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human CD28 and a second antigen-binding fragment that specifically binds to human CD22, wherein the first antigen-binding domain competes for binding to CD28 with any of the specific exemplary CD28-specific antigen-binding domains described herein, and / or the second antigen-binding domain competes for binding to CD22 with any of the specific exemplary CD22-specific antigen-binding domains described herein.

[0116] It is possible to easily determine whether a specific antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule of the present invention, or whether it competes for binding, by using routine methods known in the art. For example, to determine whether a test antibody binds to the same CD28 (or CD22) epitope as the reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first bound to the CD28 protein (or CD22 protein). Next, the ability of the test antibody to bind to the CD28 (or CD22) molecule is evaluated. If the test antibody can bind to CD28 (or CD22) after saturated binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different CD28 (or CD22) epitope than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CD28 (or CD22) molecule after saturated binding with the reference bispecific antigen-binding molecule, then the test antibody can bind to the same CD28 (or CD22) epitope as the epitope bound by the reference bispecific antigen-binding molecule of the present invention. Additional routine experiments (e.g., peptide mutation and binding analysis) can then be performed to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric shielding (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to a particular embodiment of the present invention, for example, if, as measured by a competitive binding assay, one antigen-binding protein in a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess inhibits the binding of the other by at least 50%, preferably 75%, 90%, or even 99%, then the two antigen-binding proteins bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, then the two antigen-binding proteins are considered to have the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, then the two antigen-binding proteins are considered to have an "overlapping epitope."

[0117] To determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the binding method is performed in two directions: In the first direction, the reference antigen-binding molecule is bound to the CD28 protein (or CD22 protein) under saturated conditions, and then the binding of the test antibody to the CD28 (or CD22) molecule is evaluated. In the second direction, the test antibody is bound to the CD28 (or CD22) molecule under saturated conditions, and then the binding of the reference antigen-binding molecule to the CD28 (or CD22) molecule is evaluated. If, in both directions, only the first (saturated) antigen-binding molecule can bind to the CD28 (or CD22) molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CD28 (or CD22). As will be understood by those skilled in the art, an antibody competing for binding with a reference antigen-binding molecule does not necessarily need to bind to the same epitope as the reference antibody, but can sterically shield the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0118] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific to a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific to two different antigens (e.g., CD28 and CD22) can be appropriately positioned relative to each other to produce the bispecific antigen-binding molecule of the present invention using routine methods. (Exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecule of the present invention are discussed elsewhere in this specification.) In certain embodiments, one or more individual components (e.g., heavy and light chains) of the bispecific antigen-binding molecule of the present invention are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more heavy and / or light chains of the bispecific antigen-binding molecule of the present invention can be prepared using VELOCIMMUNE® technology. Using VELOCIMMUNE® technology (or any other human antibody production technique), a high-affinity chimeric antibody against a specific antigen (e.g., CD28 or CD22) having a human variable region and a mouse constant region is first isolated. Antibodies are characterized and selected based on desirable properties, including affinity, selectivity, and epitopes. The mouse constant region is replaced with a desired human constant region to generate a complete human heavy and / or light chain that can be incorporated into the bispecific antigen-binding molecule of the present invention.

[0119] Human bispecific antigen-binding molecules can be produced using genetically modified animals. For example, a genetically modified mouse can be used that is unable to rearrange and express an endogenous mouse immunoglobulin light chain variable sequence, and which expresses only one or two human light chain variable domains encoded by a human immunoglobulin sequence functionally linked to the mouse κ constant gene at the endogenous mouse κ locus. Using such a genetically modified mouse, a fully human bispecific antigen-binding molecule can be produced containing two different heavy chains that bind to the same light chain containing a variable domain derived from one of two different human light chain variable region gene segments. (For example, a detailed discussion of such recombinant mice and their use for producing bispecific antigen-binding molecules is provided in full in U.S. Patent Application No. 2011 / 0195454, which is incorporated herein by reference.)

[0120] Biologically equivalent The present invention encompasses antigen-binding molecules having amino acid sequences that, while different from those of the described antibodies, retain the ability to bind to CD28 and / or CD22. Such mutant molecules, when compared to the parent sequence, involve the addition, deletion, or substitution of one or more amino acids, but exhibit biological activity that is essentially equivalent to the biological activity of the described antigen-binding molecules. Similarly, the antigen-binding molecule-coding DNA sequences of the present invention encompass sequences that encode antigen-binding molecules that, when compared to the disclosed sequences, involve the addition, deletion, or substitution of one or more nucleotides, but are essentially biologically equivalent to the antigen-binding molecules described in the present invention. Examples of such mutant amino acids and DNA sequences are discussed above.

[0121] The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmacokinetic equivalents or pharmacokinetic substitutes and, under similar experimental conditions, at the same molar dose, whether administered in a single-dos or multiple-dos manner, their absorption rates and degree of absorption do not show significant differences. Some antibodies may be considered equivalent or pharmacokinetic substitutes if their degree of absorption is equivalent but their absorption rates are not. However, such differences in absorption rates may still be considered bioequivalent because they are intentional, reflected in labeling, and are not essential for achieving effective drug concentrations in the body, for example, in chronic use, and are not medically important to the particular drug being investigated.

[0122] In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference between them in terms of safety, purity, and potency.

[0123] In one embodiment, the two antigen-binding proteins are bioequivalent if the patient can switch between the reference product and the biological product one or more times, and there is no expected increase in the risk of side effects, including a clinically significant change in immunogenicity or a decrease in efficacy, compared to continuous treatment without such switching.

[0124] In one embodiment, two antigen-binding proteins are biologically equivalent if, to the extent that such mechanisms are known, they both act by a common mechanism or mechanism of action for the conditions of use.

[0125] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo studies in humans or other mammals measuring the concentration of an antibody or its metabolite as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals measuring the appropriate acute pharmacological effect of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, bioavailability, or bioequivalence of an antibody.

[0126] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by various substitutions of residues or sequences, or by removing terminal or internal residues or sequences that are not required for biological activity. For example, removing or substituting cysteine ​​residues that are not essential for biological activity with other amino acids can prevent the formation of unnecessary or incorrect intramolecular disulfide crosslinks during regeneration. In other contexts, bioequivalent antibodies may include the exemplary bispecific antigen-binding molecules described herein, including amino acid changes that modify the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0127] Species selectivity and species cross-response According to certain embodiments, the present invention provides an antigen-binding molecule that binds to human CD28 but not to CD28 from other species. The present invention also provides an antigen-binding molecule that binds to human CD22 but not to CD22 from other species. The present invention also includes an antigen-binding molecule that binds to human CD28 and CD28 from one or more non-human species; and / or an antigen-binding molecule that binds to human CD22 and CD22 from one or more non-human species.

[0128] According to certain exemplary embodiments of the present invention, an antigen-binding molecule is provided that may or may not bind to human CD28 and / or human CD22, and optionally to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee CD28 and / or CD22. For example, in certain exemplary embodiments of the present invention, a bispecific antigen-binding molecule is provided comprising a first antigen-binding domain that binds to human CD28 and cynomolgus monkey CD28, and a second antigen-binding domain that specifically binds to human CD22.

[0129] immune complex The present invention encompasses antigen-binding molecules ("immune complexes") that are complexed with a therapeutic moiety, such as a cytotoxin, chemotherapeutic agent, immunosuppressant, or radioisotope. Cytotoxic agents include drugs that are harmful to cells. Examples of cytotoxic agents and chemotherapeutic agents suitable for immune complex formation are known in the art (for example, see International Publication No. 05 / 103081, incorporated herein by reference in its entirety).

[0130] Therapeutic formulations and administration The present invention provides pharmaceutical compositions comprising the antigen-binding molecule of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and a variety of agents that provide improved mobility, delivery, tolerance, etc. Numerous suitable formulations can be found in the formulation collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., L.POFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0131] The dose of the antigen-binding molecule administered to a patient may vary depending on the patient's age and physique, the target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer it intravenously in single doses of approximately 0.01 to 20 mg / kg body weight, more preferably 0.02 to 7 mg / kg body weight, 0.03 to 5 mg / kg body weight, or 0.05 to 3 mg / kg body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective doses and schedules for administering the bispecific antigen-binding molecule can be determined empirically; for example, patient progression can be monitored by periodic assessments, and the dose can be adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0132] For example, various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-dependent endocytosis (see, for example, Wu et a I., 1987, J. Bio I. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption via the epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption from the epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0133] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard injection needle and syringe. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Pen-type delivery devices are reusable. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0134] Numerous reusable pens and auto-injector delivery devices are applicable to the delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples of disposable pen-type delivery devices applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, STARLET (trademark) and OPTICLIK (trademark) (Sanofi-Aventis, Frankfurt, Germany).

[0135] In certain circumstances, pharmaceutical compositions can be delivered by controlled-release systems. In one embodiment, a pump can be used (see Langer, cited above; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201). In another embodiment, a polymer material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be positioned near the target of the composition, and therefore only a fraction of the systemic dose is required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138). Other controlled-release systems are discussed in the review Langer, 1990, Science 249:1527-1533.

[0136] Injectable formulations may include intravenous, subcutaneous, intradermal, and intramuscular injections, as well as infusion formulations. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Therefore, examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable formulations thus prepared are preferably filled into suitable ampoules.

[0137] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses suitable for a single dose of the active ingredient. Examples of such dosage forms in unit doses include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in unit doses; in particular, it is preferable that the antibody is contained in about 5 to about 100 mg in the injection form and about 10 to about 250 mg in the other dosage forms.

[0138] Therapeutic use of antigen-binding molecules The present invention includes a method comprising administering to a subject in need of it a therapeutic composition comprising an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds CD28 to a target antigen (e.g., CD22). The therapeutic composition may comprise either the antibody or bispecific antigen-binding molecule disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression “subject in need of it” means a human or non-human animal exhibiting one or more symptoms or signs of cancer (e.g., a subject exhibiting a tumor or suffering from any of the cancers described below), or a human or non-human animal that would benefit from inhibition or reduction of CD22 activity or depletion of CD22+ cells.

[0139] The antibodies and bispecific antigen-binding molecules of the present invention (and therapeutic compositions comprising them) are particularly useful for treating any disease or disorder in which stimulation, activation, and / or targeting of the immune response is beneficial. In particular, the anti-CD28 / anti-CD22 bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention, and / or improvement of any disease or disorder associated with or mediated by CD22 expression or activity or the proliferation of CD22+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved includes the killing of effector cells, such as cells expressing CD22 in the presence of T cells. CD22-expressing cells that can be inhibited or killed using the bispecific antigen-binding molecules of the present invention include, for example, cancerous B cells.

[0140] The antigen-binding molecules of the present invention can be used, for example, to treat primary and / or metastatic tumors occurring in blood, bone marrow, lymph nodes (e.g., thymus, spleen), colon, liver, lung, breast, rectal cancer, central nervous system, and bladder cancer. According to one exemplary embodiment, the bispecific antigen-binding molecules of the present invention are used to treat B-cell proliferative disorders.

[0141] The present invention also encompasses methods for treating residual cancer in a subject. As used herein, the term “residual cancer” means the presence or persistence of one or more cancerous cells in a subject after treatment with anti-cancer therapy.

[0142] In a particular embodiment, the present invention provides a method for treating a disease or disorder associated with CD22 expression (e.g., B-cell proliferation disorder) comprising administering to a subject one or more bispecific antigen-binding molecules as described elsewhere herein after the subject has been shown to be unresponsive to other types of anticancer therapies. For example, the present invention includes a method for treating a B-cell proliferation disorder comprising administering an anti-CD28 / anti-CD22 bispecific antigen-binding molecule to a patient after the patient has received standard treatment for cancer, for example, a patient suffering from a B-cell proliferation disorder, at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or longer. In other embodiments, the bispecific antigen-binding molecule of the present invention containing the IgG4 Fc domain (anti-CD28 / anti-CD22 bispecific antigen-binding molecule) is first administered to a subject at one or more time points (for example, to provide robust initial depletion of prostate cancer cells), followed by the administration of an equivalent bispecific antigen-binding molecule containing a different IgG domain, e.g., the IgG1 Fc domain, at subsequent time points. It is envisioned that the anti-CD28 / anti-CD22 antibody of the present invention may be used in combination with other bispecific antigen-binding molecules, e.g., an anti-CD20 / anti-CD3 bispecific antibody. The bispecific antibody of the present invention may also be used in combination with checkpoint inhibitors, e.g., PD-1 and CTLA-4, and those targeting other targets. It may be advantageous to combine two bispecific antibodies that target the same tumor antigen (e.g., CD22), but one bispecific antibody targets CD3 on T cells and the other bispecific antibody targets a costimulatory molecule-like CD28. This combination may be used alone or in combination with a checkpoint inhibitor to enhance tumor cell killing.

[0143] Combination therapy and prescription The present invention encompasses compositions and therapeutic formulations comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic active ingredients, as well as therapeutic methods comprising administering such combinations to subjects in need.

[0144] Exemplary additional therapeutic agents that can be administered in combination with or in combination with the antigen-binding molecule of the present invention include, for example, chemotherapy, radiotherapy, PD-1-targeting checkpoint inhibitors (e.g., anti-PD-1 antibodies, e.g., pembrolizumab, nivolumab, or semiprimab, see U.S. Patent No. 9,987,500, SEQ ID NO: 162 / 170, HCVR / LCVR), CTLA-4, LAG3, TIM3, and other molecules targeting GITR, OX40, 4-1BB, etc. Examples include bivalent stimulating agonist antibodies, CD3x bispecific antibodies (see, for example, U.S. Patent No. 9,657,102 (REGN 1979), International Publication No. 2017 / 053856A1, International Publication No. 2014 / 047231A1, International Publication No. 2018 / 067331A1 and International Publication No. 2018 / 058001A1), other antibodies targeting CD22XCD3, CD22XCD28, or CD20XCD3, and other co-stimulating CD28x bispecific antibodies.

[0145] Other agents that can be beneficially administered in combination with the antibodies of the present invention include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors including small molecule cytokine inhibitors, as well as cytokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or antibodies that bind to their respective receptors. The pharmaceutical compositions of the present invention (for example, pharmaceutical compositions comprising anti-CD28 / anti-CD22 bispecific antigen-binding molecules disclosed herein) also include "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®) It may also be administered as part of a treatment regimen that includes one or more therapeutic combinations selected from "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, and cisplatin (e.g., Platinol®-AQ).

[0146] The present invention also encompasses therapeutic combinations comprising any of the antigen-binding molecules described herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvlll, cMet, IGF1 R, B-raf, PDGFR-o, PDGFR-I3, FOLH1, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptide body, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other manipulated molecules, e.g., diabody, triabody, tetrabody, minibody, and minimum recognition unit). The antigen-binding molecules of the present invention also encompass combinations The antigen-binding molecules of the present invention may also be administered in combination with and / or concurrently prescribed with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs.

[0147] The present invention encompasses compositions and therapeutic formulations comprising one or more antigen-binding molecules described herein in combination with chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (Cytoxan®); alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine. Methylamelamine and methylamelamine; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembitin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine N, ranimustine; antibiotics, for example, acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, Peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrectals, e.g., aminoglutethimide, mytotan, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edalaxate; defofamine; demecolcine; diazicone; erfornithine; eriptinium acetate; etoglu Side; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogwazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK(trademark); razoxane; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol(trademark), Bristol-Myers Squibb Oncology (Princeton, NJ) and docetaxel (Taxotere®; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeroda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine;This definition also includes any pharmaceutically acceptable salts, acids, or derivatives of the foregoing. This definition includes anti-hormonal agents that act to modulate or inhibit hormonal effects on tumors, such as anti-estrogens including tamoxifen and raloxifene, aromatases that inhibit 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as any pharmaceutically acceptable salts, acids, or derivatives of the foregoing.

[0148] Additional therapeutic active ingredients may be administered immediately before, simultaneously with, or immediately after the administration of the antigen-binding molecule of the present invention; (for the purposes of this disclosure, such an administration regimen is considered to be the administration of the antigen-binding molecule "in combination with" the additional therapeutic active ingredient).

[0149] The present invention encompasses pharmaceutical compositions formulated in conjunction with the antigen-binding molecule of the present invention and one or more additional therapeutic active ingredients as described elsewhere herein. Administration regimen

[0150] According to certain embodiments of the present invention, multiple doses of an antigen-binding molecule (e.g., an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds to CD22 and CD28) can be administered to a subject over a predetermined period of time. Methods according to this aspect of the present invention include administering multiple doses of the antigen-binding molecule to a subject in succession. As used herein, “succession” means administering each dose of the antigen-binding molecule to a subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present invention encompasses methods including the administration of a single initial dose of the antigen-binding molecule, followed by one or more second doses of the antigen-binding molecule, and then optionally one or more third doses of the antigen-binding molecule, in succession.

[0151] The terms “initial dose,” “second dose,” and “third dose” refer to the chronological order of administration of the antigen-binding molecule of the present invention. Therefore, the “initial dose” is the dose administered at the start of the treatment regimen (also called the “baseline dose”); the “second dose” is the dose administered after the initial dose; and the “third dose” is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of antigen-binding molecule, but generally may differ from one another in terms of the frequency of administration. However, in certain embodiments, the amounts of antigen-binding molecule contained in the initial, second, and / or third doses may change from one another during the course of treatment (e.g., adjusted upward or downward as needed). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as “loading doses” at the start of the treatment regimen, followed by subsequent doses administered at a lower frequency (e.g., “maintenance doses”).

[0152] In one exemplary embodiment of the present invention, each of the second and / or third doses is administered 1 to 26 weeks after the immediately preceding dose (for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or more). When used herein, the expression "immediately preceding dose" means the dose of the antigen-binding molecule administered to the patient in a series of multiple doses, without any intervening doses, before the administration of the next dose in the sequence.

[0153] Methods according to this aspect of the present invention may involve administering to a patient any number of second and / or third doses of an antigen-binding molecule (e.g., an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds to CD22 and CD28). For example, in one particular embodiment, only a single second dose is administered to the patient. In another embodiment, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in one particular embodiment, only a single third dose is administered to the patient. In another embodiment, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0154] In embodiments including multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks after the most recent dose. Similarly, in embodiments including multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 4 weeks after the most recent dose. Alternatively, the frequency of administering the second and / or third doses to the patient may vary throughout the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

[0155] In one embodiment, an antigen-binding molecule (e.g., a bispecific antigen-binding molecule that specifically binds to CD22 and CD28) is administered to the subject as a weight-based dose. The "weight-based dose" (e.g., a dose expressed in mg / kg) is the dose of the antibody or its antigen-binding fragment or the bispecific antigen-binding molecule, which will vary depending on the subject's body weight.

[0156] In another embodiment, the antibody or its antigen-binding fragment or bispecific antigen-binding molecule is administered to the subject as a fixed dose. “Fixed dose” (e.g., dose in mg units) means that a single dose of the antibody or its antigen-binding fragment or bispecific antigen-binding molecule is used for all subjects regardless of any specific subject-related factor, such as weight. In a particular embodiment, the fixed dose of the antibody or its antigen-binding fragment or bispecific antigen-binding molecule of the present invention is based on a predetermined body weight or age.

[0157] Generally, preferred doses of the antigen-binding molecules of the present invention are in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient, and generally may be in the range of about 1 to 50 mg per kilogram of body weight. For example, antibodies or their antigen-binding fragments or bispecific antigen-binding molecules can be administered in single doses of about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, and about 50 mg / kg. Intermediate values ​​and ranges of the values ​​listed are also intended to be part of the present invention.

[0158] In some embodiments, the antigen-binding molecule of the present invention is administered in a fixed dose of about 25 mg to about 2500 mg. In some embodiments, the antigen-binding molecule of the present invention is administered in doses of about 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, It is administered as a fixed dose of approximately 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1500 mg, 2000 mg, or 2500 mg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0159] Diagnostic applications of antibodies The bispecific antibodies of the present invention can also be used, for example, to detect and / or measure CD28 or CD22, or CD28-expressing cells or CD22-expressing cells, in a sample for diagnostic purposes. For example, an anti-anti-CD28xanti-CD22 antibody, or a fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression of CD28 or CD22 (e.g., overexpression, underexpression, insufficient expression, etc.). An exemplary diagnostic assay for CD28 or CD22 may, for example, involve contacting a sample obtained from a patient with the antibody of the present invention, where the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope, for example, 3 H, 14 C, 32 P, 35 S, or 125I; a fluorescent or chemiluminescent moiety, e.g., fluorescein isothiocyanate or rhodamine; or an enzyme, e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CD28 or CD22 in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence-activated cell sorting (FACS). Samples that can be used in the CD28 or CD22 diagnostic assays according to the present invention include any tissue or fluid sample that can be obtained from a patient and contains a detectable amount of CD28 or CD22 protein or fragment thereof in a normal or pathological state. Generally, baseline or standard levels of CD28 or CD22 are first established by measuring the levels of CD28 or CD22 in specific samples obtained from healthy patients (e.g., patients without diseases or conditions associated with abnormal CD28 or CD22 levels or activity). This baseline level of CD28 or CD22 can then be compared to the levels of CD28 or CD22 measured in samples obtained from individuals suspected of having a CD28 or CD22-related disease or condition. [Examples]

[0160] The following examples are provided to give a complete disclosure and explanation of how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of the inventors' invention. Although efforts have been made to ensure accuracy regarding the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0161] Example 1: Construction of an anti-CD22xCD28 antibody Generation of anti-CD28 antibodies Anti-CD28 antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., genetically modified mice containing DNA encoding human immunoglobulin weight and κ light chain variable regions) with human CD28 protein fused to the Fc portion of mouse IgG2a, or with cells expressing CD28 or DNA encoding CD28. The antibody immune response was monitored by a CD28-specific immunoassay. Once the desired immune response was achieved, spleen cells were collected and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce CD28-specific antibodies. Using this technique, several anti-CD28 chimeric antibodies (i.e., antibodies having a human variable domain and a mouse constant domain) were obtained. In addition, several fully human anti-CD28 antibodies were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. Patent Application No. 2007 / 0280945A1.

[0162] Certain biological properties of exemplary anti-CD28 antibodies produced according to the method of this embodiment are described in detail in the following examples.

[0163] Anti-CD22 antibody generation Genetically modified mice (VELOCIMMUNE® mice, see above) are modified to use human CD22 antigen (e.g., hCD22 ecto(D20-R687) see hFc, R&D Systems, Catalog#1968-SL-050; Accession# Anti-CD22 antibodies were obtained by immunization with CAA42006 (see also Figure 3) or by immunizing genetically modified mice containing DNA encoding human immunoglobulin weight and κ light chain variable regions with human CD22 antigen.

[0164] After immunization, spleen cells were collected from each mouse, and B cells were sorted by (1) fusing them with mouse myeloma cells to preserve their viability and form hybridoma cells, and then screening them for CD22 specificity, or (2) using human CD22 fragments as a sorting reagent that binds to and identifies reactive antibodies (antigen-positive B cells) (as described in U.S. Patent Application No. 2007 / 0280945A1).

[0165] Chimeric antibodies against CD22, possessing both a human variable region and a mouse constant region, were first isolated. The antibodies were characterized, and desired properties, including affinity and selectivity, were selected. If necessary, the mouse constant region was replaced with a desired human constant region, such as a wild-type or modified IgG1 or IgG4 constant region, to generate fully human anti-CD22 antibodies. While the selected constant region may vary depending on the specific application, high affinity antigen binding and target specificity reside in the variable region.

[0166] Generation of bispecific antibodies that bind to CD28 and CD22 Bispecific antibodies containing an anti-CD22 specific binding domain and an anti-CD28 specific binding domain were constructed using standard methods, where the anti-CD22 antigen-binding domain and the anti-CD28 antigen-binding domain each contain distinct HCVRs paired with a common LCVR. In some cases, bispecific antibodies were constructed using heavy chains from anti-CD28 antibodies, heavy chains from anti-CD22 antibodies, and a common light chain (see Table 1).

[0167] The bispecific antibody created according to this embodiment contains two independent antigen-binding domains (i.e., binding arms). The first antigen-binding domain contains a heavy chain variable region derived from the anti-CD28 antibody ("CD28-VH"), and the second antigen-binding domain contains a heavy chain variable region derived from the anti-CD22 antibody ("CD22-VH"). Both anti-CD22 and anti-CD28 share a common light chain. CD28-VH / CD22-VH pairing creates antigen-binding domains that specifically recognize CD28 on T cells and CD22 on tumor cells.

[0168] Example 2: Heavy and light chain variable region amino acids and nucleic acid sequences Table 1 lists the heavy and light chain variable regions and CDR amino acid sequence identifiers of the selected anti-CD22 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. Table 1: Amino acid sequence identifiers for CD22 antibodies [Table 1] Table 2: Nucleic acid sequence identifiers for CD22 antibodies [Table 2]

[0169] Table 3 lists the amino acid sequence identifiers for the heavy and light chain variable regions (HCVR and LCVR) and CDR of the selected anti-CD28 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 4. Table 3: Amino acid sequence identifiers of CD28 antibodies [Table 3] Table 4: Nucleic acid sequence identifiers of CD28 antibodies [Table 4]

[0170] Table 5 summarizes the component parts of the various anti-CD22x anti-CD28 bispecific antibodies constructed. Tables 6 and 7 list the HCVR, LCVR, CDR, and heavy and light chain sequence identifiers of the bispecific antibodies. Table 5: Summary of component parts of anti-CD22x anti-CD28 bispecific antibodies [Table 5]

[0171] Table 6 shows the amino acid sequence identifiers of the bispecific anti-CD22x anti-CD28 antibodies excited in this specification. The corresponding nucleic acid sequence identifiers are listed in Table 7. Table 6: Amino Acid Sequences of Anti-CD22 x Anti-CD28 Bispecific Antibodies [Table 6] Table 7: Nucleic Acid Sequences of Anti-CD22 x Anti-CD28 Bispecific Antibodies [Table 7] Example 3: Epitope Mapping of the Binding of REGN5837 to CD22 by Hydrogen-Deuterium Exchange

[0172] H / D exchange epitope mapping (HDX-MS) with mass spectrometry was performed to determine the amino acid residues of CD22 (recombinant human CD22, SEQ ID NO: 50) that interact with H4sH33037P2 (see Table 1, HCVR / LCVR pair of SEQ ID NOs: 2 / 10) (anti-hCD22 monoclonal antibody; parental anti-hCD22 antibody of REGN5837). An overview of the H / D exchange method is described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal.Chem. 73:256A-265A.

[0173] The HDX-MS experiment was carried out on an integrated HDX / MS platform composed of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical gradient, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0174] Labeling solutions were prepared as PBS buffer in D2O with a pD of 7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, corresponding to pH 7.4 at 25°C). For deuterium labeling, 11 μl of CD22.mmH (REGN5140 (SEQ ID NO: 50), 56.7 μM) or CD22.mmH (Ag-Ab complex) premixed with H4sH33037P2 (see above) in a 1:0.6 molar ratio was incubated in double incubation with 44 μl of D2O labeling solution at various time points at 20°C (e.g., non-deuterated control = 0 seconds; deuterium labeling for 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 55 μl of pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample, and incubated at 20°C for 5 minutes. Next, the quenched sample is injected into the Waters HDX Manager, and online pepsin / protease is performed. Digestion XIII was performed. The digested peptides were separated using a C8 column (1.0 mm × 50 mm, Nova Bioassays) with a 13-minute gradient of 10%-32%B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0175] Using the Byonic search engine (Protein Metrics), LC-MS / MS data of non-deuterated CD22 samples were searched for in databases containing CD22 and its randomized sequences. Search parameters (in ELN units) were set with non-specific enzyme digestion as the default and human glycosylation as a common variable modification. Next, the list of identified peptides was imported into HDX Workbench software (version 3.3) to calculate deuterium uptake for each peptide detected by LC-MS from all deuterated samples. For a given peptide, deuterium uptake (D) and the percentage of deuterium uptake (%D) were calculated using the center of gravity mass (intensity-weighted average mass) at each time point (see below).

number

[0176] A total of 427 peptides from hCD22.mmH (SEQ ID NO: 50) were identified from both hCD22.mmH alone and hCD22.mmH (HCVR / LCVR pairs of SEQ ID NO: 2 / 10) samples in complex with H4sH33037P2, representing 92.0% sequence coverage of hCD22. Peptides showing a D incorporation percentage difference greater than 5% were defined as significantly protected (Table 8). For hCD22.mmH, peptides corresponding to amino acids 481-505 (NVQYAPRDVRVRKIKPLSEIHSGNS; SEQ ID NO: 57) and 523-537 (FWEKNGRLLGKESQLNF; SEQ ID NO: 58) were significantly protected by H4sH33037P2. Table 8: Selected CD22.mmH peptides significantly protected by binding to H4sH33037P2. [Table 8-1] [Table 8-2] Example 4: Epitope mapping of H4sH33041P2 bound to CD22 by hydrogen-deuterium exchange.

[0177] H / D exchange epitope mapping with mass spectrometry (HDX-MS) was performed to determine the amino acid residue of CD22 (recombinant human CD22, SEQ ID NO: 50) that interacts with H4sH33041P2 (using the anti-hCD22 monoclonal antibody with HCVR / LCVR pairs, SEQ ID NO: 18 / 10, and the parental anti-hCD22 of REGN5838). An overview of the H / D exchange method is described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0178] The HDX-MS experiments were performed on an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0179] Labeling solutions were prepared as PBS buffer in D2O with a pD of 7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, corresponding to pH 7.4 at 25°C). For deuterium labeling, 11 μl of CD22.mmH (REGN5140 (SEQ ID NO: 50), 56.7 μM) or CD22.mmH (Ag-Ab complex) premixed with H4sH33041P2 in a 1:0.6 molar ratio was incubated in double incubation with 44 μl of D2O labeling solution at various time points at 20°C (e.g., non-deuterated control = 0 seconds; deuterium labeling for 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 55 μl of pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample, and incubated at 20°C for 5 minutes. Next, the quenched sample was injected into a Waters HDX Manager and subjected to online pepsin / protease XIII digestion. The digested peptides were separated using a C8 column (1.0 mm × 50 mm, Nova Bioassays) with a 13-minute gradient of 10%-32%B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0180] Using the Byonic search engine (Protein Metrics), LC-MS / MS data of the non-deuterated CD22 sample was searched against a database containing CD22 and its randomized sequences. The search parameters (units are in ELN) were set with non-specific enzymatic digestion as the default and human glycosylation as a common variable modification. Next, the list of identified peptides was imported into the HDX Workbench software (version 3.3) to calculate the deuterium incorporation of each peptide detected by LC-MS from all deuterated samples. For a given peptide, the deuterium incorporation (D) and percentage of deuterium incorporation (%D) were calculated as described below using the centroid mass (intensity-weighted average mass) at each time point. [Number]

[0181] A total of 454 peptides were identified from both hCD22.mmH alone and hCD22.mmH in the complex with the H4sH33041P2 sample from hCD22.mmH (SEQ ID NO: 50), representing 90.5% sequence coverage of hCD22. Peptides showing a D incorporation value percentage difference of more than 5% were defined as being significantly protected. For hCD22.mmH, the peptide corresponding to amino acids 246 - 277 (CEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNL; SEQ ID NO: 59) was significantly protected by H4sH33041P2. Table 9 presents the results from selected peptides that were significantly protected by binding to H4sH33041P2. Table 9: Selected CD22.mmH Peptides with Significant Protection upon Binding to H4sH33041P2 [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] Example 5: Binding affinity and rate constant of CD22xCD28 bispecific antibody induced by surface plasmon resonance

[0182] Equilibrium dissociation constant (K) of hCD22.mmH (SEQ ID NO: 50) and mfCD22.mmH (SEQ ID NO: 51) that bind to purified anti-CD22xCD28 bispecific mAbs or anti-CD22 divalent parental mAbs. D The values ​​(see Table 1, mAB33037P2;HCVR / LCVR:SEQ ID NO: 2 / 10) and mAb33041P2;HCVR / LCVR:SEQ ID NO: 18 / 10) were determined using a real-time surface plasmon resonance biosensor with a Biacore T-200 or Biacore 4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567:HCVR / LCVR:SEQ ID NO: 33 / 34) to capture purified anti-CD22xCD28 bispecific or anti-CD22 parental mAbs (see Tables 1 and 2 for mAb33037P2 and mAb33041P2). This Biacore binding test was performed in a buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v Surfactant P20 (HBS-EP running buffer). Different concentrations of hCD22 (SEQ ID NO: 50) and mfCD22 (SEQ ID NO: 51) (ranging from 90 nM to 3.33 or 0.37 nM, 3-fold dilution) with a C-terminal myc.myc hexahistidine tag (disclosed as SEQ ID NO: 60, "hexahistidine") prepared in HBS-EP running buffer were injected onto the mAb capture surface at a flow rate of 30 μl / min. The binding of CD22.mmH (SEQ ID NO: 50) to the captured monoclonal antibody was monitored for 5 minutes, and the dissociation of CD22.mmH in HBS-EP running buffer was monitored for 10 minutes. All binding reaction rate experiments were performed at 25°C. Kinetic association (k a ) and dissociation (k dThe rate constant was determined by fitting the real-time sensorgram to a 1:1 coupling model using Scrubber 2.0c curve fitting software. The coupling dissociation equilibrium constant (K) D The half-life (t1 / 2) and dissociation half-life were calculated from the velocity constant as follows: K D (M=k) d / k a , and t1 / 2(min) = 0.693 / k d / 60

[0183] The binding reaction rate parameters of human and cynoCD22 to purified mAbs at 25°C are described later in Tables 10-12.

[0184] The equilibrium dissociation constant (K) of the purified anti-CD22xCD28 bispecific mAb or anti-CD28 divalent parental mAb (see Tables 3 and 4 for mAb14226P2) is 14226P2. DThe values ​​were determined using a real-time surface plasmon resonance biosensor with a Biacore T-200 instrument. The CM4 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567; HCVR / LCVR SEQ ID NO: 33 / 34) to capture purified anti-CD22xCD28 bispecific or anti-CD28 parent mAb (see above). This Biacore binding test was performed in a buffer consisting of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, and 0.05% v / v Surfactant P20 (HBS-EP running buffer). Different concentrations of hCD28 (ranging from 600 nM to 2.47 nM, 3-fold dilution) with a C-terminal myc.myc hexahistidine tag (disclosed as "hexahistidine" as SEQ ID NO: 60) prepared in HBS-EP running buffer were injected onto the mAb capture surface at a flow rate of 50 μl / . Binding of CD28.mmH (SEQ ID NO: 54) to the captured monoclonal antibody was monitored for 5 minutes, and dissociation of CD28.mmH in HBS-EP running buffer was monitored for 10 minutes. All binding reaction rate experiments were performed at 25°C. Kinetic binding (ka) and dissociation (kd) rate constants were determined by fitting a real-time sensorgram to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding-dissociation equilibrium constant (K) D The dissociation half-life (t1 / 2) was calculated from the velocity constant as follows: K D (M=k) d / k a , and t1 / 2(min) = 0.693 / k d / 60

[0185] The binding reaction rate parameters of human CD28 to purified mAbs at 25°C are described later in Table 13. [Table 10] Table 10: Human CD22 mmH binding reaction rate to anti-CD22 x CD28 bispecific mAbs at 25°C Table 11: Reaction rates of monkey CD22.mmH(XP_005588899.1) binding to anti-CD22xCD28 bispecific mAb at 25°C [Table 11] Table 12: Reaction rates of monkey CD22.mmH(EHH59463.1) binding to anti-CD22xCD28 bispecific mAbs at 25°C [Table 12-1] [Table 12-2] Table 13: Human CD28-mmH binding reaction rate to anti-CD22xCD28 bispecific mAbs at 25°C [Table 13] Example 6: Binding specificity of anti-CD28 and anti-CD22xCD28 bispecific antibodies against target cell line (Nalm6), effector cell line (Jurkat), and cynomolgus monkey T and B cells, as measured by flow cytometry.

[0186] Using flow cytometry analysis, the binding of CD22xCD28 bispecific antibodies to human CD22-expressing Nalm6 cells, human CD28-expressing Jurkha T cells, and cynomolgus monkey T(CD28+) and B(CD22+) cells was determined. In short, 1 × 10⁻⁶ 5Cells / wells were incubated for 30 minutes at 4°C with serial dilutions ranging from 133 nM to 61 pM of CD22xCD28 bispecific antibody or H4sH15260P (an isotype-controlled human IgG4 antibody that binds to human antigens without cross-reactivity to human or cynomolgus monkey CD28 or CD22) for Jurkat and Nalm6 cells. Cynomolgus monkey PBMCs were incubated with a single 67 nM antibody. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS, and PE conjugate anti-human secondary antibody was added to the cells for a further 30 minutes of incubation. A further phenotyping antibody cocktail (anti-CD2, anti-CD20, anti-CD16, anti-CD14) was added to the wells containing cynomolgus monkey PBMCs. Wells containing no antibodies or only secondary antibodies were used as controls.

[0187] After incubation with secondary antibody, cells were washed and resuspended in 200 μl of cold PBS containing 1% filtered FBS, and analyzed by flow cytometry using BD LSR_Fortessa. Cynomolgus monkey T cells were identified as CD2+ / CD16-, and B cells were identified as CD20+. FACS-bound EC 50 The values ​​were calculated using a four-parameter nonlinear regression analysis in Prism software.

[0188] Table 14 presents binding data of CD22xCD28 bispecific antibodies to the surface of CD22-expressing cell lines, as determined by flow cytometry. Table 14 also presents binding data of CD22xCD28 bispecific antibodies to the surface of human CD28-expressing cell lines, as determined by flow cytometry.

[0189] REGN5837 bound to Nalm6 cells EC 50 The EC50 value was 1.3E-08M. REGN5838 bound to Nalm6 cells, with an EC50 value of 1.8E-08M. The isotype control antibody did not show binding to CD22-expressing cell lines.

[0190] REGN5837 bound to Jurkha T cells EC50 Value 2.1E-08M. REGN5838 bound to Jurkha T cells. 50 The value was 2.3E-08M. The isotype control antibody did not show binding to cell lines expressing CD28.

[0191] Table 15 presents data on the binding of CD22xCD28 bispecific antibodies to the surface of cynomolgus monkey (Cambodian origin) T and B cells, as determined by flow cytometry.

[0192] REGN5837 bound to B cells in all 12 of the 12 cynomolgus monkeys tested, and to T cells in 11 of the 12 monkeys tested. Binding to CD20+ B cells ranged from 12.6 to 30.3 times that of secondary cells, with a median of 15.7 times. Binding to CD2+ / CD16- T cells ranged from 1.2 to 5.2 times that of secondary cells, with a median of 3.5 times. Positive binding was defined as greater than 1.2 times that of secondary cells. REGN5838 bound to B cells in all 12 of the 12 cynomolgus monkeys tested, and to T cells in 11 of the 12 monkeys tested. Binding to CD20+ B cells ranged from 6.5 to 13.5 times that of secondary cells, with a median of 9.3 times. Binding to CD2+ / CD16- T cells ranged from 1.2 to 4.7 times that of secondary cells, with a median of 3.8 times. Positive binding was defined as greater than 1.2 times binding to secondary cells. Isotype control antibodies did not show binding to cynomolgus monkey T or B cells. Table 14: Binding and binding multiplier results of flow cytometry experiments on recombinant target and effector cells [Table 14] Table 15: Binding multiplier results from flow cytometry experiments on cynomolgus monkey (Cambodian origin) T and B cells. [Table 15-1] [Table 15-2]

[0193] Example 7: Binding specificity of anti-CD28 and anti-CD22xCD28 bispecific antibodies to human CD4+ T cells and recombinant target cells using flow cytometry. Using flow cytometry analysis, effector cells expressing CD22xCD28 bispecific (REGN5837;REGN5838) and human CD28 of the control antibody (human CD4) were identified. + Binding to T cells and target cells expressing human CD22 (HEK293 / hCD20 / hCD22 and Raji / CD80 and CD86-negative B- cells) was investigated. HEK293 / hCD20 cells were included as a CD28 and CD22-negative cell line.

[0194] Human CD4 + T cells were isolated from human peripheral blood mononuclear cells (PBMCs) obtained from healthy donor leukocyte packs. PBMC isolation was performed by density gradient centrifugation using 50 mL SepMate® tubes according to the manufacturer's recommended protocol. Briefly, 15 mL of Ficoll-Paque PLUS was layered into a 50 mL SepMate tube, followed by the addition of 30 mL of leukocytes diluted 1:2 with D-PBS + 2% FBS. Subsequent steps were performed according to the SepMate manufacturer's protocol. CD4 + Subsequently, T cells were isolated from PBMCs using Miltenyi Biotec's Human CD4 Microbead Kit, following the manufacturer's instructions. + T cells, 5 x 10⁶ per vial 6 The cells were frozen in FBS containing 10% DMSO at the cell concentration.

[0195] Target cells, including the HEK293 cell line and human Raji B cells, were prepared as follows.

[0196] Stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (amino acids M1-P297 of acceptance number NP_068769.2) was transduced with human CD22 (amino acids M1-A847 of acceptance number NP_001762.2). Human CD22-positive cells were isolated by fluorescence-activated cell sorting (FACS) and converted into single clones. The resulting clonal cell line (HEK293 / hCD20 / hCD22 clone E4) was maintained in DMEM + 10% + P / S / G + NEAA with 500 g / mL of G418 added.

[0197] Human Raji B cells (ATCC#CCL-86) endogenously expressing CD20, CD22, Fcγ receptor (FcγR), CD80, and CD86 on their cell surface were genetically modified by deleting CD80 and CD86 using CRIPSR technology. CD80 and CD86 are known ligands for CD28. The engineered Raji / CD80 and CD86-negative cells were maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine supplemented with HEPES and sodium pyruvate.

[0198] The cells were stained as follows:

[0199] In short, human CD4+ T cells, HEK293 / hCD20, HEK293 / hCD20 / hCD22, and Raji / CD80 and CD86-negative cells were resuspended in a staining buffer containing D-PBS + 2% FBS. Raji cells were incubated with mouse IgG (final 625 mg / mL) to block the endogenous FcΓ receptor. In short, 2 × 10⁶ cells were collected in a 96-well plate. 5Cells / wells were incubated at 4°C for 30–60 minutes with serial dilutions of antibodies ranging from 6.1 pM to 100 nM. Negative control samples without antibody were included. Cells were washed once with cold staining buffer and incubated with allophycocyanin (APC)-labeled anti-human secondary antibody for 30–45 minutes. After incubation, cells were washed once with cold D-PBS buffer without FBS and incubated with LIVE / DEAD Fixable Green Dead Cell Stain (Invitrogen) according to manufacturer's instructions to distinguish live and dead cells. Cells were then fixed in BD Cytofix Buffer according to manufacturer's instructions, washed, resuspended in staining buffer, and analyzed by flow cytometry using an iQue Screener flow cytometer. EC 50 For determination, geometric mean fluorescence intensity (MFI) values ​​were analyzed using a 4-parameter logistic equation on a 9-point response curve using GraphPad Prism. The binding multiplier was calculated using the following equation: Binding ratio = maximum geometric value within the tested dose range MFI value Background geometric MFI value [0nM]

[0200] The CD22xCD28 bispecific antibody has the ability to bind to human CD22 and CD28 in primary human CD4 overexpressing CD22 (HEK293 / hCD20 / hCD22) or endogenously (Raji / CD80 and CD86 negative). + T cells and manipulated cells were evaluated by flow cytometry. A negative cell line (HEK293 / hCD20) was included as a control.

[0201] EC 50 The multiple-combined values ​​are summarized in Figure 1 and Table 16. Table 16: Human CD4 + Flow cytometry experiments on T cells and manipulated target cells 50 And the result of multiple concatenation: [Table 16] Abbreviations: NC = Not calculable (displayed for curves where the bond did not reach saturation); ND = Not measured Table 16 Human CD4 + EC of antibodies against T cells and manipulated cell lines, e.g., HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative B cells 50 And a table of multiple bond values.

[0202] As expected, neither the CD28 antibody nor the parent (REGN5705;HCVR / LCVR SEQ ID NO: 35 / 36) or its bispecific format (REGN5837, REGN5838, and 1-arm CD28 control (SEQ ID NO: 48)) bound to negative HEK293 / hCD20 cells. Due to nonspecific binding, weak binding of approximately 1.8x was detected at the highest concentration with the isotype control antibody (Figure 1 and Table 16).

[0203] The binding of anti-CD22x anti-CD28 antibodies is HEK293 / hCD20 / hCD22, (16.44x for REGN5837, 34.9x for REGN5838, EC 50 (approximately 11.4 nM) and Raji / CD80 and CD86 negative cells (38.35x for REGN5837, EC2) 50 Approximately 9.76 nM, 81.74x for REGN5838, EC 50 Binding was observed at approximately 14.9 nM. No significant binding was detected in 1-arm CD28 or isotype controls (Figure 1 and Table 16).

[0204] The binding of antibodies targeting human CD28 to primary human CD4 + It was detected in T cells. The parent CD28 antibody, REGN5705, showed an EC of approximately 4.13 nM above the background. 50 The antibody bound to cells at 37.48x, while the bispecific antibodies REGN5837, REGN5838, and the one-arm control showed binding at 9.25x, 10.63x, and 10.97x, respectively. As expected, the isotype control did not bind to cells (Figure 1 and Table 16). Example 8: Co-stimulation with anti-CD22xCD28 bispecific antibodies enhances targeted cytotoxicity, T cell activation, and cytokine release induced by anti-CD20xCD3 bispecific antibodies.

[0205] The enhancement of CD22xCD28 to CD20xCD3 targeted killing was evaluated using a 96-hour cytotoxic assay targeting Raji cells (Raji-80 / 86DKO) engineered to lack CD80 and CD86 expression. Briefly, human PBMCs were added to RPMI medium in a 1 × 10⁶ solution. 6 Lymphocytes were enriched by seeding cells / mL and incubating overnight at 37°C, removing attached macrophages, dendritic cells, and some monocytes. The following day, Raji-80 / 86DKO cells were labeled with 1 μM of the fluorescent tracking dye CFDA-SE, and attached cell-depleted naive PBMCs were labeled with 1 μM of the fluorescent tracking dye CellTrace. The cells were labeled with Violet. Labeled target cells and PBMCs (effector / target cell ratio 10:1) were co-incubated for 96 hours at 37°C with serial dilutions (concentration range: 5 nM to 0.64 pM) of CD20xCD3 bispecific antibody REGN1979, which has one heavy chain arm composed of SEQ ID NO: 42, the other heavy chain arm composed of SEQ ID NO: 43, and the light chain of SEQ ID NO: 44, and fixed concentrations of CD22xCD28 costimulatory molecules REGN5837 or REGN5838, a one-arm CD28 bispecific control (REGN5678), or 2.5 ug / ml (16.7 nM) of IgG4s isotype control (H4sH10154P3, isotype control with HCVR / LCVR pair of SEQ ID NO: 37 / 38). Cells were collected from plates and analyzed by FACS on a FACS BD LSRFortessa-X20. For FACS analysis, cells were stained with a Fixable Live / Dead Far Red-reactive (Invitrogen) dye. 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected per sample. To assess the specificity of cell elimination, cells were gated to a live CFDA-SE-labeled population. The percentage of the live population was recorded and used to calculate viability.

[0206] T cell activation was assessed by incubating cells with antibodies directly conjugated to CD2, CD4, CD8, and CD25. The percentage of CD8+ cells expressing CD25 was reported as a measure of T cell activation. Furthermore, as T cells proliferated, CellTraceViolet was diluted, and the MFI, as measured by FACS, decreased. Therefore, T cell proliferation was reported as a decrease in the MFI of CellTraceViolet on CD8+ T cells. EC of target Raji cells lacking CD80 and CD86 expression and binding. 50 The values ​​were calculated using a four-parameter nonlinear regression analysis in Prism software.

[0207] The supernatant from this assay was collected for cytokine level analysis. The concentrations of IL-17a, IFNγ, TNFα, IL-10, IL-6, IL-4, and IL-2 were analyzed using a Cytometric Bead Array (CBA) kit according to the manufacturer's instructions. Cytokine levels were interpolated from curves created using kit standards and reported as pg / mL. Maximum cytokine levels were calculated using four-parameter nonlinear regression analysis in Prism software.

[0208] The results of assays were tested to evaluate the ability of the anti-CD20xCD3 bispecific antibody REGN1979 (see above) to kill target cells expressing human CD20 and CD22 in unstimulated human T cells in combination with a co-stimulated CD22xCD28 antibody or a one-arm CD28 or isotype control antibody.

[0209] REGN1979 activated human T cells and dose-dependently depleted Raji cells lacking CD80 and CD86 expression. Adding a fixed concentration CD22xCD28 bispecific antibody to REGN1979 compared it to REGN1979 with a single-arm CD28 or isotype control antibody, demonstrating the superior cytotoxicity (EC) of REGN1979. 50 ) was increased by 3.5 to 6.4 times (Table 17).

[0210] The observed REGN1979-mediated target cell lysis was associated with T cell activation and proliferation, as measured by CD25 upregulation or CellTrace violet dilution for CD8+ cells, respectively. Addition of fixed-concentration CD22xCD28 bispecific antibody to REGN1979 enhanced the potential for REGN1979-induced T cell activation and proliferation by 2.1–2.6-fold and 7.4–8.4-fold, respectively, compared to REGN1979 with one-arm CD28 or isotype control antibodies (Table 17).

[0211] REGN1979 induced the release of human cytokines. The released cytokines observed with CD22xCD28 bispecific antibody and REGN1979 were enhanced in the presence of fixed-concentration CD22xCD28 costimulatory molecules with fixed-concentration one-arm CD28 or isotype control antibodies (Table 18).

[0212] In summary, co-stimulation increased the potential for targeted cytotoxicity, T cell activation, and cytokine release compared to CD20xCD3 combined with a control antibody. Table 17: EC of cytotoxicity and T cell activation 50 Value (average of 3 experiments) [Table 17-1] [Table 17-2] Table 18: Cytokine release (pg / mL) [Table 18]

[0213] Example 9: Bioassay of CD22 bispecific antibody T cell activation is achieved by stimulating T cell receptors (TCRs) that recognize specific peptides presented by tumor histocompatibility complex class I or II (MHCI or MHCII) proteins on antigen-presenting cells (APCs) (Goldrath et al. 1999). Activated TCRs then initiate a cascade of signaling events, which can be monitored by various transcription factors, e.g., activator protein 1 (AP-1), nuclear factor 1 (NFAT) of activated T cells, or nuclear factor κ-light chain enhancer (NFκB) of activated B cells. The T cell response is then further refined by the involvement of co-receptors constitutively or inductively expressed on T cells, e.g., CD28, CTLA-4 (cytotoxic T lymphocyte protein 4), PD-1 (programmed cell death protein 1), LAG-3 (lymphocyte-activating gene 3), or other molecules (Sharpe et al.). (al. 2002). The co-stimulatory molecule CD28 is activated by its endogenous ligand, CD80 or CD86, expressed on APCs. CD28 enhances cellular signaling pathways, such as those regulated by NFκB transcription factors after TCR activation. CD28 co-signaling is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death (Smeets et al. 2012).

[0214] Anti-CD22xCD28 bispecific antibody used in luciferase-based reporter bioassay and primary human CD4 + This was characterized by an IL-2 functional assay using T cells.

[0215] Luciferase-based reporter assay : A T-cell / APC (antigen-presenting cell) luciferase-based reporter assay was developed to evaluate the effect of CD28 activation on NFκB activity, mediated by the involvement of anti-CD28 x anti-CD22 bispecific antibodies.

[0216] Manipulation of reporter T cells: Clonal reporter T cell lines were created by transducing immortal human Jurkat T cells (ATCC#TIB-152) with NFκB-luciferase (NFκB-Luc) lentiviral reporter (Qiagen) according to the manufacturer's instructions. The clonal reporter line (Jurkat / NFκB-Luc clone 1C11) was maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine with 1 μg / mL of puromycin added.

[0217] APC operation: Stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (amino acids M1-P297 of acceptance number NP_068769.2) was transduced with human CD22 (amino acids M1-A847 of acceptance number NP_001762.2). Human CD22-positive cells were isolated by fluorescence-activated cell sorting (FACS) and converted into single clones. The resulting clonal cell line (HEK293 / hCD20 / hCD22 clone E4) was maintained in DMEM+10%+P / S / G+NEAA with 500 μg / mL of G418 added.

[0218] Human Raji B cells (ATCC#CCL-86) endogenously expressing CD20, CD22, Fcγ receptor (FcγR), CD80, and CD86 on their cell surface were genetically modified by deleting CD80 and CD86 using CRIPSR technology. CD80 and CD86 are known ligands for CD28. The engineered Raji / CD80 and CD86-negative cells were maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine supplemented with HEPES and sodium pyruvate.

[0219] T cell / APC stimulation: In this experiment, engineered reporter T cells were stimulated with two bispecific antibodies. The first stimulus was delivered by T cells activating the bispecific antibody REGN2281 (an anti-CD20x anti-CD3 antibody having one heavy chain arm composed of SEQ ID NO: 39, one heavy chain arm composed of SEQ ID NO: 40, and one light chain arm of SEQ ID NO: 41), targeting the CD3 molecule on the engineered reporter T cells and CD20 on HEK293 or on Raji / CD80 and CD86-negative B cells. The first stimulus avoids the need for TCR activation by their ligands, which are specific peptides presented on MHC molecules. The second stimulus is driven by a CD28 bispecific antibody (i.e., an anti-CD28x anti-CD22 bispecific antibody). This antibody mimics T cell CD28 activation by its ligand CD80 / CD86, which is expressed on APCs. This interacts with CD28 on T cells and CD22 on HEK293 cells or Raji / CD80 and CD86-negative B- cells, driving the activation of CD28 on manipulated reporter T cells. Simultaneous activation of the TCR and CD28 leads to enhanced NFκB transcriptional activity, thereby increasing the production of reporter genes and luciferase.

[0220] Luciferase assay setup: Cell suspensions and antibody dilutions for screening anti-CD22x anti-CD28 bispecific antibodies were prepared using RPMI1640 with 10% FBS and P / S / G added as the assay medium.

[0221] One day before screening, manipulated reporter T cells were placed in cell culture medium in a quantity of 0.5 × 10⁶ cells. 6 Cells were cultured at a concentration of cells / mL. Anti-CD28xanti-CD22 bispecific antibodies and controls, serially diluted 1:3, were tested in the presence of a constant 200 pM of REGN2281 (anti-CD20xanti-CD3, see above) or REGN1945 (hIgG4 isotype control with HCVR / LCVR pair, SEQ ID NO: 45 / 46). Ten-point dilutions ranged from 15 pM to 100 nM, and the final dilution did not contain CD28 antibody.

[0222] The reagents were added in the following order: 1) A fixed concentration of REGN2281 (anti-CD20x anti-CD3, see above) or REGN1945 (hIgG4 isotype control, see above) at a final concentration of 200 pM was added to each well of a 96-well white flat-bottom plate; 2) 4 × 10 5 cells / mL (final cell concentration 1 x 10 4 HEK293 cells or Raji / CD80 and 2×10⁶ cells resuspended in cells / well. 6 cells / mL (final cell concentration 5 x 10 4 CD86-negative B cells resuspended in cells / well were added to the corresponding plate; 3) serially diluted antibodies were added to the corresponding wells; 4) reporter T cells cultured overnight were added to 2 × 10⁶ wells. 6 Resuspend in / mL, final concentration 5 × 10 4 Cells were added to the plate in wells. After incubating the plate at 37°C / 5% CO2 for 4-6 hours, 100 μL of ONE-Glo® was added. Cells were lysed by adding the (Promega) reagent, and luciferase activity was detected. The emitted light was captured in relative light units (RLU) using a multi-label plate reader, Envision (PerkinElmer). All serial dilutions were tested in double-row. Tested.

[0223] Antibody EC 50 The values ​​were determined by fitting the data to a four-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism® software. The lead factor was calculated using the following formula:

number

[0224] Primary human CD4 + IL-2 functional assay using T cells : Primary CD4 + The T cell / APC functional assay was developed to evaluate the effect of CD28 activation on IL-2 production in conjunction with an anti-CD22x anti-CD28 bispecific antibody.

[0225] Human primary CD4+ T cell isolation: Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs. PBMC isolation was achieved by density gradient centrifugation using 50 mL SepMate® tubes according to the manufacturer's recommended protocol. Briefly, 15 mL of FicollPaque PLUS was stacked in a 50 mL SepMate tube, followed by the addition of 30 mL of leukocytes diluted 1:2 with D-PBS + 2% FBS. Subsequent steps were carried out according to the SepMate manufacturer's protocol. CD4 + Subsequently, T cells were isolated from PBMCs using Miltenyi Biotec's Human CD4 Microbead Kit, following the manufacturer's instructions. + T cells, 5 x 10⁶ per vial 6 The cells were frozen in FBS containing 10% DMSO at the cell concentration.

[0226] Primary CD4 treated with CD28 antibody + IL-2 release from T cells: In this assay, primary CD4 +T cells are activated by crosslinking CD3 on their surface using an anti-CD20 x anti-CD3 bispecific antibody (REGN2281, see above) combined with either HEK293 cells engineered to express human CD20 or Raji cells expressing endogenous CD20, where CD80 and CD86 are silenced using CRISPR technology (Raji / CD80 and CD86 negative cells). Binding the CD20 arm of REGN2281 to CD20-expressing cells drives CD3 receptor clustering, providing the primary signal necessary for T cell stimulation. Importantly, in some cases, co-culturing primary leukocytes with genetically different cells can lead to allogeneic determinant incompatibility, resulting in T cell activation. This allows for sufficient primary stimulation without the exogenous addition of the anti-CD20 x anti-CD3 bispecific antibody. Regardless of primary stimulation, a co-stimulation, which can be provided by crosslinking CD28 molecules, is needed to detect quantifiable IL-2 release. Here, the bispecific CD28 antibody (i.e., the anti-CD28 x anti-CD22 bispecific antibody) is CD4 + CD28 on T cells and CD22 on HEK293 / hCD20 or RAJI / CD80 and CD86-negative cells interact, driving CD28 clustering and activation. The combined involvement of TCR and CD28 enhances IL-2 production, which is then released into the cell culture medium. IL-2 is detected and quantified from the cell supernatant using a homogeneous, no-wash AlphaLisa kit from PerkinElmer.

[0227] Human CD4 that was isolated and frozen in advance + T cells were thawed on the day of the assay in stimulation medium (X-VIVO15 cell culture medium supplemented with 0.01 mM BME containing 10% FBS, HEPES, NaPyr, NEAA, and 50 U / ml benzonase nuclease). The cells were centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium, and 1 × 10⁶ cells were placed in a 96-well round-bottom plate. 5 The cells were seeded at a concentration of cells / well. HEK293 cells engineered to express human CD20 alone or in combination with human CD22 were placed in primary stimulation medium at a rate of 10 × 106 Raji / CD80 and CD86-negative cells were treated with 15 μg / mL mitomycin C at a concentration of cells / mL. 10 × 10⁶ cells were placed in primary stimulation medium. 6 The cells were treated with 20 μg / mL mitomycin C at a concentration of cells / mL. After incubation at 37°C in 5% CO2 for 1 hour, HEK293 and Raji cells were washed three times with D-PBS containing 2% FBS and CD4. + 1 × 10⁶ wells containing T cells 4 For HEK293 cells or Raji / CD80 and CD86-negative cells per well, 5 × 10 4 The final cell / well concentration was added. Subsequently, anti-CD28 x anti-CD22 bispecific or control antibodies, serially diluted 1:3 in the range of 15 pM to 100 nM, were added to the wells in the presence of 2 nM REGN2281 (anti-CD20 x anti-CD3) or REGN1945 (negative hIgG4 isotype control, see above). The final 10-point dilution did not contain CD28 antibody. After incubating the plates for 72 hours at 37°C in 5% CO2, the cells were pelleted by centrifugation, and 40 μL of the supernatant was collected. From this, 5 μL was tested in the human IL-2 AlphaLISA assay according to the manufacturer's protocol. Measurements were acquired with a multi-label plate reader Envision, and the raw RFU (relative fluorescence units) values ​​were plotted. All serial dilutions were tested in double series.

[0228] Antibody EC 50 The values ​​were determined by fitting the data to a four-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism® software. The lead factor was calculated using the following formula:

number

[0229] The ability of anti-CD22xanti-CD28 bispecific antibodies to provide co-stimulation via CD28 on T cells in the absence or presence of CD22 target expression was evaluated using reporter cell-based bioassays that utilize luciferase activity as readout.

[0230] Activation curves for engineered reporter T cells co-incubated with HEK293 / hCD20 or HEK293 / hCD20 / hCD22 cells in addition to a constant 200 pM REGN1945 (hIgG4 isotype control) or REGN2281 (anti-CD20 x anti-CD3) are shown in Figure 2 (A and B). 50 The derived factor values ​​are summarized in Tables 19 and 20.

[0231] When reporter T cells and HEK293-derived APCs were treated with 200 pM REGN1945, none of the CD28 bispecific antibodies showed an increase in luciferase activity in the absence of TCR stimulation, regardless of the HEK293 strain used in the assay. Increased luciferase activation was observed only with the parental CD28 antibody (REGN5705) and HEK293 / hCD20 cells (2.18x) and HEK293 / hCD20 / hCD22 cells (2.05x). Single-arm CD28 and isotype control antibodies did not elicit a luciferase response in this setting (Table 19 and Figure 2).

[0232] When reporter T cells and HEK293-derived APCs were treated with 200 pM REGN2281, both anti-CD22x anti-CD28 bispecific antibodies (REGN5837 and REGN5838) were found to be EC 50 This induced strong luciferase activity in CD22-expressing HEK293 cells, as indicated by an increase in the derived factor. One-arm CD28 control antibody and parental CD28 antibody (REGN5705; see HCVR / LCVR SEQ ID NOs. 35 / 36) showed similar activity in both HEK293 strains. Isotype control antibodies did not elicit a luciferase response in this setting (Table 20 and Figure 2).

[0233] When reporter T cells and Raji / CD80 and CD86-negative cells were treated with 200 pM REGN1945, both anti-CD22x anti-CD28 bispecific antibodies (REGN5837 and REGN5838) and the parental CD28 antibody induced luciferase activity, while the one-arm CD28 control antibody and isotype control did not (Table 19 and Figure 2).

[0234] When reporter T cells and Raji / CD80 and CD86-negative cells were treated with 200 pM REGN2281, all CD28 bispecific antibodies (REGN5837 and REGN5838), including the parental CD28 antibody, as well as the one-arm CD28 control antibody, induced luciferase activity. EC only for anti-CD22x anti-CD28 and parental CD28 antibodies. 50 The value could not be determined for the 1-arm CD28 control because it did not reach a saturation level. No activation was detected in the isotype control (Table 20 and Figure 2). Primary human CD4 + IL-2 functional assay using T cells:

[0235] The ability of anti-CD22xanti-CD28 bispecific antibodies to provide costimulation via CD28 on T cells in the absence or presence of CD22 target expression, as measured by functional primary CD4 cytokine production. + Evaluation was performed using a T-cell assay.

[0236] The activation curves are shown in Figure 3 (A and B), and EC 50 The induction factor values ​​are HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 CD86-negative cells were co-incubated with CD4 in the presence of either a constant 2nM REGN1945 (hIgG4 isotype control) or REGN2281 (anti-CD20x anti-CD3). + Table 21 summarizes information about T cells.

[0237] In wells containing HEK293 / hCD20 or HEK293 / hCD20 / CD22 cells and a certain amount of REGN1945, no measurable IL-2 release was observed due to the lack of sufficient allogeneic primary T cell stimulation (Figure 3). However, in wells containing Raji / CD80 and CD86-negative cells and a certain amount of REGN1945, IL-2 release was detected due to a significant allogeneic response providing sufficient primary stimulation, even in the absence of antibody-mediated CD3 clustering (Figure 3 and Table 21).

[0238] When a constant 2nM concentration of REGN2281 and parent CD28 mAb (REGN5705, see above) were added, measurable IL-2 levels were detected in samples containing HEK293 / hCD20 or HEK293 / hCD20 / CD22 cells. In contrast to the bivalent CD28 mAb, when an anti-CD22xanti-CD28 bispecific mAb was added to wells containing HEK293 / hCD20 cells and REGN2281, IL-2 release was not dramatically enhanced. Only in the presence of HEK293 / hCD20 / CD22 cells and REGN2281 did the anti-CD22xanti-CD28 bispecific mAb significantly enhance IL-2 release (Figure 3 and Table 22).

[0239] Tables 19-22 are shown below.

[0240] Table 19 shows the EC levels of luciferase activity in engineered T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or RAJI / CD80 and CD86-negative cells and a constant 200 pM REGN1945 (isotype control). 50 A table showing the values, maximum values, and derived multiples is provided.

[0241] Table 20 shows the EC levels of luciferase activity in engineered T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86-negative cells and a constant 200 pM REGN2281 (anti-CD20 x anti-CD3). 50 A table showing the values, maximum values, and derived multiples is provided.

[0242] Table 21 shows the results of co-incubating HEK293 / hCD20, HEK293 / hCD20 / hCD22, or RAJI / CD80 and CD86-negative cells with a certain amount of 2nM REGN1945 (isotype control) and CD4 + IL-2 release from T cells 50 A table showing the values, maximum values, and derived multiples is provided.

[0243] Table 22 HEK293 / hCD20, HEK293 / hCD20 / hCD22, Raji / CD80, and CD86-negative cells, as well as CD4 cells co-incubated with a certain amount of 2nM REGN2281 (anti-CD20 x anti-CD3). + IL-2 release from T cells 50 A table showing the values, maximum values, and derived multiples is provided. Table 19: Luciferase activity in manipulated reporter T cells in the absence of TCR stimulation in 200 pM REGN1945 (isotype control) EC 50 Value, maximum value, and derived multiple value: [Table 19] Abbreviation: ND = Not measured Table 20: Luciferase activity in manipulated reporter T cells in the presence of TCR stimulation with 200 pM REGN2281 (αCD20xαCD3) 50 Value, maximum value, and derived multiple value: [Table 20] Abbreviations: NC = Not calculable (displays curves where the response did not reach saturation); ND = Not measured. Table 21: Primary human CD4 in the presence of 2nM REGN1945 (isotype control) + EC of IL-2 release from cells 50 Value, maximum value, and derived multiple value [Table 21] Abbreviations: NC = Not calculable (displays curves where the response did not reach saturation); ND = Not measured. Table 22: Primary human CD4 in the presence of 2nM REGN2281 (αCD20xαCD3) + IL-2 release from T cells 50 Value, maximum value, and derived multiple value [Table 22] Abbreviations: NC = Not calculable (displays curves where the response did not reach saturation); ND = Not measured. Example 10: Effect of anti-CD22X anti-CD28 antibody + semiprimab combination on IL-2 release from cells engineered to express PD-L1.

[0244] Materials and methods APC operation: RAJI cells RAJI is a B lymphocyte cell line isolated from an 11-year-old male of the ATCC(registered trademark) CCL-86(trademark) class. RAJI is maintained in RPMI + 10% FBS + P / S / G + NaPyr + HEPES.

[0245] RAJI CD80 and CD86 negative CD80 and CD86 expression in RAJI cells was eliminated using the CRISPR / Cas9 system.

[0246] NALM6 clone G5 The NALM6 clone is an acute lymphoblastic leukemia (ALL) cell line isolated from a 19-year-old male [NALM6 clone G5 (ATCC, #CRL-3273)]. NALM6 cells are maintained in RPMI + 10% FBS + P / S / G.

[0247] WSU-DLCL2 WSU-DLCL2 is a human DLBCL cell line isolated from the pleural fluid of a 41-year-old Caucasian male (Leibnitz Institute-DSMZ, Cat.#ACC575).

[0248] PD-L1 engineered cell line NALM-6, RAJI CD80 and CD86-negative (RAJI / CD80-CD86-), and WSU-DLCL2 cell lines were genetically engineered to stably express human PD-L1 (amino acids M1-T290 of acceptance number NP_054862.1). The resulting cell lines, NALM6 / PD-L1, RAJI / CD80-CD86- / PD-L1, and WSU-DLCL2 / PD-L1, were maintained in their respective culture media with 0.5 μg / mL of puromycin added for RAJI / CD80-CD86- cells, and 1 μg / mL of puromycin added for NALM-6 / PD-L1 and WSU-DLCL2 / PD-L1 cells.

[0249] T cell activation assay for T cell proliferation and IL-2 release The effect of REGN5837 on IL-2 release was evaluated in the presence of fixed concentrations of semiprimab using human primary T cells and allogeneic human B-cell lymphoma cell lines [NALM-6, NALM-6 / PD-L1, RAJI / CD80-CD86-, RAJI / CD80-CD86- / PD-L1, WSU-DLCL2, WSU-DLCL2 / PD-L1]. Co-culturing primary leukocytes with genetically different cells may lead to incompatibility of allogeneic determinants, potentially resulting in T cell activation. For assays using NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1) cells, the T cell activation assay was performed using enriched human primary T cells from three donors, while the assay using WSU-DLCL2(+ / -PD-L1) cells used T cells from one donor.

[0250] Isolation of T cells for use in a T cell activation assay to test combination therapy with REGN5837 + REGN2810. For experiments using NALM-6 and RAJI / CD80-CD86- cells, CD3+ T cells were isolated from PBMCs (555109, 555130, and 555131) of three donors, and for assays using WSU-DLCL2 cells, PBMCs from one donor (555175) were used. For donor 555109, PBMCs were isolated from peripheral blood using density gradient centrifugation. Briefly, 15 ml of Ficoll-Paque PLUS was added to a 50 ml conical tube, followed by 30 ml of blood diluted 1:1 with PBS containing 2% FBS. After centrifugation at 400xg for 30 minutes without braking, the mononuclear cell layer was transferred to a new tube, diluted 5x with PBS containing 2% FBS, and centrifuged at 300xg for 8 minutes. For donors 555130, 555131, and 555175, PBMCs were isolated from the peripheral blood of healthy donors using the Stem Cell Technologies EasySep Direct Human PBMC Isolation Kit according to the manufacturer's protocol. The isolated PBMCs were frozen in FBS containing 10% DMSO. For CD3+ T cell isolation, the frozen PBMC vials were thawed in a 37°C water bath and diluted in a stimulating medium containing 50 U / ml benzonase nuclease (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME). The cells were centrifuged at 1200 rpm for 10 minutes, resuspended in EasySep buffer, and isolated using the Stem Cell Technologies EasySep T Cell Isolation Kit according to the manufacturer's protocol.

[0251] Primary CD3 treated with CD28 antibody + IL-2 release from T cells: T cell activation assay using human OVCAR-3, PEO1, NALM-6, RAJI / CD80-CD86-, and WSU-DLCL2 cells (+ / -PD-L1) CD3+ T cells resuspended in stimulating medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) were placed in 1 × 10⁶ wells of a 96-well round-bottom plate. 5 Cells were seeded at a concentration of cells / well. Cells containing or not containing PD-L1 (+ / -PD-L1), NALM-6, RAJI / CD80-CD86-, and WSU-DLCL2 cells were treated with mitomycin C at 20 μg / mL (RAJI) or 15 μg / mL (NALM-6 and WSU-DLCL2) to stop their growth. After incubation at 37°C and 5% CO2 for 1 hour, the cells treated with mitomycin C were washed three times with D-PBS containing 2% FBS and then resuspended in stimulating medium. NALM-6, RAJI / CD80-CD86-, and WSU-DLCL2 (+ / -PD-L1) cells were seeded at 2.5 × 10⁶ for RAJI and WSU-DLCL2 cells. 4 Cells / well; for NALM-6 cells, 5 × 10 4 The final concentration of semiprimab or unbound IgG4 was added to the wells containing CD3+ T cells. P A control (20 nM) was added to the wells. In assays using WSU-DLCL2 (+ / - PD-L1) cells, a constant concentration of beratacept (hCTLA4.hIgG1) or unbound IgG1 control (50 nM) was added to the wells. Subsequently, REGN5837 or non-TAAxCD28 control antibody was titrated to 3.1 pM to 200 nM in a 1:4 dilution series for NALM-6 (+ / - PD-L1) cells, and to 0.6 pM to 1000 nM in a 1:6 dilution series for WSU-DLCL2 and RAJI / CD80-CD86-(+ / - PD-L1) cells, and added to the wells. The final point of the 10-point concentration curve did not include REGN5837 or the non-TAAxCD28 control antibody. After incubating the plates for 72 hours (WSU-DLCL2(+ / -PD-L1)) or 96 hours (NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1)) at 37°C in 5% CO2, 50 μL of the supernatant was collected and IL-2 release was measured.

[0252] For IL-2 release, 5 μL of supernatant was tested using the Human IL-2 AlphaLISA Kit according to the manufacturer's protocol. IL-2 measurements were obtained from Perkin Data was acquired using Elmer's Envision multi-label plate reader. A standard curve of known IL-2 concentrations was included and used to derive pg / ml values.

[0253] All serial dilutions were tested in triplicate for IL-2 release. The EC50 values ​​of the antibodies were determined from a four-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism™ software. The maximum level of IL-2 release is given as the mean maximum response detected within the tested dose range. Furthermore, the data reported for assays using WSU-DLCL2 cells include IL-2 generated in the absence or presence of 1000 nM titrating antibody to capture the decrease in IL-2 observed as the concentration of non-TAAxCD28 antibody increases.

[0254] Summary of results and conclusions IL-2 functional assay using primary human CD3+ T cells: The ability of anti-CD22xanti-CD28 bispecific antibodies to provide costimulation via CD28 on T cells in the presence of B-cell lymphocyte cell lines endogenously expressing CD22 was evaluated using a functional primary CD3+ T-cell assay that measures IL-2 cytokine production.

[0255] Activation curves of T cells incubated with NALM-6 (+ / -PD-L1) (Figure 5A) or RAJI / CD80-CD86-(+ / -PD-L1) (Figure 5B) are shown in Figures 5A and 5B. EC50 and Max IL-2 values ​​are constant at 20 nM hIgG4 PTable 23A summarizes the results for CD3+ T cells incubated with NALM-6(+ / -PD-L1) cells in the presence of either an isotype control or semiprimab, and Table 23B summarizes the results for CD3+ T cells incubated with RAJI / CD80-CD86-(+ / -PD-L1). Figure 4 shows the activation curves for CD3+ T cells incubated with WSU-DLCL2(+ / -PD-L1) cells. Constant 20nM hIgG4 P Table 24 summarizes the EC50 and IL-2 values ​​(reported for REGN5837 or non-TAAxCD28 at 0 nM or 1000 nM) of T cells incubated with WSU-DLCL2 (+ / -PD-L1) cells in the presence of either an isotype control or semiprimab, and in the presence of a certain 50 nM hIgG1 isotype control or either CTLA-4 receptor or bera-tacept.

[0256] In the presence of human primary T cells and allogeneic B-cell lymphocyte cell lines RAJI / CD80-CD86- and NALM-6, REGN5837 increased IL-2 release in a concentration-dependent manner. A non-TAAxCD28 control antibody slightly increased IL-2 at high antibody concentrations. In the absence of PD-L1 on RAJI / CD80-CD86- or NALM-6 cells, the addition of 20 nM semiprimab had no effect on IL-2 release. In the presence of PD-L1-expressing RAJI / CD80-CD86- or NALM-6 cells, the maximum IL-2 released in response to treatment with REGN5837 alone was reduced compared to T cells incubated with non-PD-L1-expressing cells. The addition of semiprimab slightly enhances REGN5837-mediated IL-2 release under conditions involving NALM-6 / PD-L1 cells, but in the presence of RAJI / CD80-CD86- / PD-L1 cells, it significantly enhances IL-2 release to levels observed under conditions involving PD-L1-deficient RAJI / CD80-CD86- cells.

[0257] In the presence of human primary T cells and the allogeneic B-cell lymphocyte cell line WSU-DLCL2, no concentration-dependent increase in IL-2 release was observed. Conversely, the non-TAAxCD28 control antibody was observed to decrease IL-2 release in a concentration-dependent manner. Unlike NALM-6 and RAJI / CD80-CD86- cells, which express little to no CD28 ligand, the WSU-DLCL2 cell line is known to express CD28 ligand. Since the CD28-binding arm of REGN5837, and therefore the CD28 arm of the non-TAAxCD28 antibody, is known to compete with the CD28 ligand for binding to CD28, the non-TAAxCD28 control antibody blocks CD28 activation by the CD28 ligand expressed on WSU-DLCL2 cells, thereby reducing IL-2 release. Unlike non-TAAxCD28 controls, IL-2 is not reduced by REGN5837 due to its ability to immobilize WSU-DLCL2 cells via its CD22 binding arm, causing it to behave similarly to and essentially replace CD28 ligands. In the presence of WSU-DLCL2 / PD-L1 cells, basal IL-2 release is reduced compared to WSU-DLCL2 cells that do not express PD-L1. Adding REGN5837 in the absence of semiprimab slightly enhances IL-2 release. Adding 20nM semiprimab enhances basal activity, which can be further slightly enhanced with dose escalation of REGN5837, resulting in higher max IL-2 release for the REGN5837 and semiprimab combination than for either treatment alone. Incubation of WSU-DLCL2 / PD-L1 cells in the presence of non-TAAxCD28, as observed in WSU-DLCL2 cells, is reduced by semiprimab or hIgG4. PIL-2 levels decrease regardless of the presence or absence of an isotype control. To further investigate the effect of CD28 ligand expression on masking the effects of REGN5837, soluble CTLA-4 receptor beratacept or hIgG1-compatible isotype control was added at 50 nM. Beratacept binds to CD28 ligand, CD80, and CD86 with high affinity, blocking their interaction and therefore CD28 activation. In the presence of 50 nM beratacept, basal IL-2 release is dramatically reduced because the CD28 ligand cannot bind to CD28 to provide co-stimulatory signaling. Even under these conditions, REGN5837 can bind to CD28 and provide co-stimulation, as evidenced by dose-dependent enhancement of IL-2 release. Addition of 20nM semiprimab alone does not enhance IL-2 release in the presence of beratacept, but when semiprimab is combined with an increased dose of REGN5837, max IL-2 release is increased compared to REGN5837 alone in the presence of cells engineered to overexpress PD-L1. Table 23A: The combination of REGN5837 with semiprimab enhances IL-2 release more than REGN5837 treatment alone in NALM-6 cells engineered to express PD-L1. [Table 23A-1] [Table 23A-2] ND: No concentration-dependent response was observed, so it was not measured. Table 23B: The combination of REGN5837 with semiprimab is used with RAJI / CD80 engineered to express PD-L1. - CD86 - In cells, it enhances IL-2 release more effectively than REGN5837 treatment alone. [Table 23B-1] [Table 23B-2] NC: The data did not fit the 4-parameter logistic equation, so the calculation was not performed. Table 24: The combination of REGN5837 with semiprimab enhances IL-2 release more than REGN5837 treatment alone in WSU-DLCL2 cells engineered to express PD-L1. [Table 24] ND: No concentration-dependent response was observed, so it was not measured. NC: The data did not fit the 4-parameter logistic equation, so the calculation was not performed.

[0258] Example 11: Antitumor efficacy of REGN5837 administration in the presence and absence of REGN1979 introduction REGN5837 is CD22 + B cells and CD28 + This is a human IgG4-based bispecific antibody (bsAb) designed to target B-cell NHL (e.g., DLBCL) by cross-linking T cells. The "signal 2" provided by REGN5837 in combination with other agents that provide "signal 1" (e.g., delivering the signal via primary T cell stimulation through TCR or CD3 clustering), such as the CD20xCD3 bispecific antibody (bsAb) REGN1979, can enhance the response to CD20xCD3 by providing amplified T cell activation and T-cell-mediated killing of B-cell NHL. Furthermore, REGN5837 may increase efficacy in patients who do not respond to CD20xCD3 monotherapy.

[0259] In the study described later, the antitumor efficacy of CD22xCD28bsAb REGN5837 was evaluated in immunodeficient NSG mice with established B-cell leukemia tumors on day 8, in the presence or absence of sub-effective doses of CD20xCD3bsAb (REGN1979).

[0260] In short, human peripheral blood mononuclear cells (PBMCs) were grafted intraperitoneally (IP) into mice (n=6-9 per group), and human NALM-6 B-cell leukemia cells, engineered to express luciferase to enable bioluminescence imaging (NALM-6-luc), were intravenously (IV) transplanted 12 days later. The antitumor efficacy of REGN5837 at doses of 0.04, 0.4, and 4 mg / kg, combined with a fixed dose of REGN1979 at 0.04 mg / kg, was evaluated against REGN5837 and REGN1979 monotherapy and non-crosslinked IgG4. P-PVA The antibody was compared to the control bsAb. Mice were administered the antibody by intraperitoneal (IP) injection 8, 15, and 22 days after transplantation of NALM-6-luc cells. Tumor burden was assessed twice a week during the experimental period.

[0261] Materials and methods Human-derived cell lines NALM-6-luc: The NALM-6 cell line is an acute lymphoblastic leukemia cell line isolated from a 19-year-old male patient (DSMZ, cat#ACC128); this line was modified with EF1a-luciferase-2A-GFP-Puro lentivirus (GenTarget) to facilitate in vivo imaging of tumor cell growth.

[0262] PBMCs: Human PBMCs were obtained from ReachBio, Cat.#0500-401, donor #0180905 (tumor growth experiment) and 0180621 (serum antibody experiment).

[0263] Experimental Design Testing system Female NSG mice (8-9 weeks old) were used in all experiments. Human PBMCs were implanted as IP in all mice, followed by IV transplantation of NALM-6-luc B-cell leukemia cells 12 days after implantation. The experimental design is detailed in Table 25. Tumor growth was monitored twice a week using bioluminescence imaging throughout the study period. For all experiments, mice were housed in the Regeneron animal facility under standard conditions. All experiments were conducted in accordance with the guidelines of the Regeneron Animal Care and Use Committee.

[0264] NSG mouse engraftment Female immunodeficient NSG mice: 4 x 10 7 Human PBMCs were engrafted in IP. Human CD45 was detected in all viable cells in postorbital blood collection and flow cytometry. + T cell levels were checked 11 days after the evaluation of the cell percentage; engraftment levels were 0.16-16% hCD45 + The study involved cells. Following the transplantation of PBMC-engrafted NSG mice, NALM-6-luc cells were applied.

[0265] NALM-6-Luc culture conditions and tumor transplantation NALM-6 cell lines were modified with EF1a-luciferase-2A-GFP-Puro lentivirus (GenTarget) to enhance in vivo imaging of tumor cell growth. The cell lines were maintained during RPMI under puromycin selectivity in 10% FBS supplemented with PSG (penicillin, streptomycin, and glutamine).

[0266] NALM-6-luc cells were collected by centrifugation and placed in PBS in a 2.5 × 10⁶ solution. 7 The cells were resuspended at a concentration of 5 × 10⁶ cells / mL. 200 μl (5 × 10⁶) was administered to NSG mice 12 days after engraftment of PBMCs. 6 NALM-6-luc cells were injected intravenously.

[0267] Antibody administration for tumor measurement Before administering the test substance or control, mice were assigned to stratified groups according to tumor volume and T cell engraftment level. Before administering the test substance or control, mice were assigned to stratified groups according to tumor size and T cell engraftment level. Antibody (REGN5837) REGN1979, REGN5671 [non-TAAxCD28 non-crosslinked control bsAb], or H4sH17664D [non-TAAxCD3 non-crosslinked control bsAb]) were administered as monotherapy or in combination by IP injection on days 8, 15, and 22 post-transplant (for in vivo efficacy) at the doses listed in the table. Table 25: Experimental design for evaluating tumor growth [Table 25] a One mouse in this group died early in the experiment and was excluded. This death was not due to tumor burdon, and since one mouse died in the control group, it was unlikely to be related to the administration of the test substance.

[0268] Tumor measurement and specified endpoints Mice transplanted with NALM-6-luc tumors were imaged twice a week using an IVIS Spectrum instrument, and the data were analyzed using Living Image software. Prior to imaging, the mice were intravenously injected with luciferin substrate. Ten minutes later, the mice were anesthetized with isoflurane, and bioluminescence (total luminous flux, expressed as photons / second [p / s]) was quantified. The experiment was terminated when the mice began to show signs of graft-versus-host disease (GVHD) (evaluated as a weight loss of 20% or more) according to IACUC criteria.

[0269] Statistical analysis of tumor growth Tumor volume over time was analyzed using two-way analysis of variance (ANOVA), followed by Tukey's post-hoc trial for multiple comparisons. A difference was considered statistically significant if p < 0.05. Statistical analysis was performed using GraphPad Prism software (version 8). result Antitumor efficacy of REGN5837 administration in the presence and absence of REGN1979 Immunodeficient NSG mice with NALM-6-luc tumors were injected via IP with the antibodies described above or a non-crosslinked control.

[0270] In tumor-bearing mice, treatment with 0.04, 0.4, and 4 mg / kg of REGN5837 in the presence of 0.04 mg / kg of REGN1979 resulted in a statistically significant suppression of tumor growth at 23 days post-transplant compared to non-crosslinked control bsAbs (non-TAAxCD28 and non-TAAxCD3 bsAbs) (p<0.05, p<0.01, and p<0.001, respectively) (Figure 6). Significant suppression of tumor growth was observed in the 0.4 and 4 mg / kg groups at 20 days post-transplant (p<0.05 for both groups). Neither REGN5837 (4 mg / kg) monotherapy nor REGN1979 (0.04 mg / kg) monotherapy significantly reduced tumor growth compared to non-crosslinked control bsAbs. There was no difference between the REGN5837 + REGN1979 combination therapy and any of the bsAb monotherapy, and statistical significance was reached. Rapid tumor growth was observed when the non-crosslinked control bsAb was administered throughout the entire treatment period, and all mice were euthanized on day 23. In all groups, GVHD was observed in at least one mouse at the end of the experiment (assessed as a weight loss of 20% or more).

[0271] In independent experiments using different sets of mice, blood was collected and serum antibody concentrations were measured at the following time points: 1 and 4 hours after administration on day 7, 1 hour before and 4 hours after administration on days 14 and 21, and once on day 29. During the administration period, trough concentrations of antibodies in serum were measured 1 hour before administration on days 14 and 21. Trough concentrations of antibodies in serum during the administration period were measured 1 hour before administration on days 14 and 21. 0.04 mg / kg REGN Administration of REGN5837 doses of 0.04, 0.4, and 4 mg / kg in the presence of 1979 was associated with trough concentrations of REGN5837 ranging from below the limit of quantification (BLQ) to 0.1, 1.6–2.3, and 16.5–21.1 μg / mL, respectively. The trough concentration of REGN1979 in serum was BLQ in all cases (data not included).

[0272] conclusion In the presence of 0.04 mg / kg of REGN5837, doses of 0.04, 0.4, and 4 mg / kg of REGN5837 were effective in suppressing NALM-6 B-cell leukemia tumor growth in mice. No significant tumor suppression was observed compared to the control group with either 4 mg / kg of REGN5837 or 0.04 mg / kg of REGN1979 monotherapy.

[0273] Example 12: FACS-based cytotoxicity against CD22 cells + human PBMCs + / - CD22xCD28 costimulatory bispecific antibodies (fixed CD22xCD28, titrated CD20xCD3) Materials and methods CD22xCD28 enhancement of CD20xCD3 targeted killing was evaluated in 96-hour cytotoxic assay-targeted Nalm6 or WSU-DLCL2 cells. Briefly, human PBMCs were added to RPMI medium in a 1x10⁶ mixture. 6Lymphocytes were enriched by seeding cells / mL and incubating overnight at 37°C, removing adherent macrophages, dendritic cells, and some monocytes. The following day, Nalm6 or WSU-DLCL2 cells were labeled with 1 μM of the fluorescent tracking dye CFDA-SE, and adherent cell-depleted naive PBMCs were labeled with 1 μM of the fluorescent tracking dye CellTrace Violet. Labeled target cells and PBMCs (effector / target cell ratio 4:1 for Nalm6, 5:1 for WSU-DLCL2) were co-incubated with or without serial dilutions of the CD20xCD3 bispecific antibody REGN1979 (concentration range: 5 nM to 0.64 pM) with a fixed concentration of 2.5 μg / ml (16.7 nM) of CD22xCD28 REGN5837. In assays targeting Nalm6 cells, a fixed amount of CD22xCD28REGN5838, 2.5 ug / ml (16.7 nM) of a single-arm control, was added along with either a CD28 bispecific control (REGN5678) or an IgG4s isotype control (H4sH10154P3). After incubation at 37°C for 96 hours, cells were collected from plates and analyzed by FACS on a FACS BD LSRFortessa-X20. For FACS analysis, cells were stained with a Fixable Live / Dead Far Red reactive (Invitrogen) dye. 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected per sample. To assess the specificity of cell elimination, cells were gated to a live CFDA-SE labeled population. The percentage or number of live target cells was recorded and used to calculate viability.

[0274] T cell activation was assessed by incubating cells with antibodies directly conjugated to CD2, CD4, CD8, and CD25. The percentage of CD8+ cells expressing CD25 was reported as a measure of T cell activation. Furthermore, as T cells proliferate, CellTraceViolet is diluted, and the MFI, as measured by FACS, decreases. T cell proliferation was therefore reported as a decrease in CellTraceViolet MFI in CD8+ T cells, or as the percentage of CD8+ cells with reduced CellTraceViolet MFI.

[0275] The supernatant from this assay was collected for cytokine level analysis. The concentrations of IL-17a, IFNγ, TNFα, IL-10, IL-6, IL-4, and IL-2 were analyzed using a Cytometric Bead Array (CBA) kit according to the manufacturer's instructions. Cytokine levels were interpolated from curves created using kit standards and reported as pg / mL. EC50 values ​​for target cell killing, T cell activation, proliferation, and cytokine levels, as well as maximum cytokine levels, were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0276] Results, summary, and conclusion: The anti-CD20xCD3 bispecific antibody REGN1979 was tested in combination with a costimulatory CD22xCD28 antibody or a one-arm CD28 or isotype control antibody to determine its ability to kill target cells expressing human CD20 and CD22 in naive human T cells.

[0277] REGN1979 was activated to dose-dependently kill Nalm6 (Figure 7) or WSU-DLCL2 (Figure 8) cells in human T cells. Adding a fixed concentration CD22xCD28 bispecific antibody to REGN1979 enhanced the cytotoxic efficacy (EC50) of REGN1979 by 4.7 to 5.2 times against Nalm6 cells (Table 26) or by 17.5 times against WSU-DLCL2 cells (Table 27) compared to REGN1979 alone, when compared with 1-arm CD28 or isotype control antibodies.

[0278] The observed REGN1979-mediated target cell lysis was associated with T cell activation and proliferation, as measured by CD25 upregulation or CellTrace violet dilution for CD8+ cells, respectively (Figures 7 and 8). Addition of fixed-concentration CD22xCD28 bispecific antibody to REGN1979 enhanced the potential for REGN1979-induced T cell activation and proliferation by 2.3–2.6-fold and 5.4–7.1-fold in the presence of Nalm6 cells compared to REGN1979 with 1-arm CD28 or isotype control antibodies (Table 26), or by 8.2-fold and 16.1-fold in the presence of WSU-DLCL2 cells compared to REGN1979 alone (Table 27).

[0279] In assays using human PBMCs and WSU-DLCL2 cells, REGN1979 induced the release of human cytokines. The cytokine release observed with REGN1979 was enhanced in the presence of fixed concentrations of CD22xCD28 compared to cytokine release induced by REGN1979 alone (Table 28, Figure 9).

[0280] In summary, co-stimulation increased the potential for targeted cytotoxicity, T cell activation, and cytokine release compared to CD20xCD3 combined with a control antibody or observed alone. Summary table of data: Table 26: EC50 values ​​for cytotoxicity and T cell activation at Nalm6 targets (single experiment) [Table 26] Table 27: EC50 values ​​for cytotoxicity and T cell activation at the WSU-DLCL2 target (average of two experiments) [Table 27-1] [Table 27-2] Table 28: Cytokine release from WSU-DLCL2 cytotoxic assay (average of two experiments) [Table 28]

[0281] Example 13: FACS-based cytotoxicity of NHL+ human PBMC+ / - CD22xCD28 stimulation (fixed CD22xCD28, CD20xCD3) Experimental Procedure The CD22xCD28 enhancement of CD20xCD3 targeted killing was evaluated using a 96-hour cytotoxicity assay targeting NHL cells isolated from primary NHL patient biopsies, including autologous PBMCs, in the presence of human stromal cells (HS-5). Briefly, HS-5 cells were seeded at 5000 cells / well in flat-bottomed 96-well plates and incubated overnight. The following day, PBMCs from NHL patients were labeled with 1 μM of the fluorescent tracking dye CellTrace Violet. Bone marrow and labeled PBMCs (effector / target cell ratio 10:1) were seeded in wells containing stromal cells and co-incubated at 37°C for 96 hours at 2.5 ug / ml (16.7 nM) with serial dilutions of CD20xCD3 bispecific antibody REGN1979 (concentration range: 6.7 nM to 10.7 pM) and fixed-concentration CD22xCD28 costimulatory molecule REGN5837 or one-arm control CD28 bispecificity (REGN5678). Cells were collected from plates and analyzed by FACS on a FACS BD LSR Fortessa-X20. For FACS analysis, cells were stained with an antibody cocktail (CD45, CD19, CD4, CD8, CD25) and a fixable live / dead near-IR reactive dye (Invitrogen). 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected per sample. To evaluate the specificity of cell elimination, target cells were gated to a live CD45+ violet-negative CD19+ population. Survival rates were calculated based on the number of target cells in the treated wells, normalized to the number of target cells in the untreated wells.

[0282] T cells were gated as CD4+ or CD8+ populations labeled with live CD45+ violet. The percentage of CD8+ and CD4+ cells expressing CD25 was reported as a measure of T cell activation. Furthermore, as T cells proliferated, CellTraceViolet was diluted, and the MFI, as measured by FACS, decreased. T cell proliferation was therefore reported as a decrease in CellTraceViolet's MFI on CD8+ and CD4+ T cells.

[0283] EC50 values ​​for targeted cell killing, T cell activation, and proliferation were calculated using a four-parameter nonlinear regression analysis in Prism software.

[0284] Results, summary, and conclusion: The anti-CD20xCD3 bispecific antibody REGN1979 was tested for its ability to kill NHL cells from patient bone marrow in combination with a costimulatory CD22xCD28 antibody or a one-arm CD28 control antibody in naive autologous T cells.

[0285] REGN1979 activated human T cells and dose-dependently depleted NHL. Addition of a fixed concentration CD22xCD28 bispecific antibody to REGN1979 enhanced its cytotoxic efficacy (EC50) by 2.3 and 3.5 times compared to REGN1979 with a one-arm CD28 control antibody or a control without co-stimulation (Table 29).

[0286] The observed REGN1979-mediated target cell lysis was associated with T cell activation and proliferation, as measured by CD25 upregulation or CellTrace violet dilution for CD8+ and CD4+ cells, respectively. Addition of fixed-concentration CD22xCD28 bispecific antibody to REGN1979 enhanced the potential for REGN1979-induced T cell activation and proliferation by 2.8–4.8-4.2-fold and 2.8–4.2-fold, respectively, compared to REGN1979 with one-arm CD28 or isotype control antibodies (Table 29 and Figure 10).

[0287] In summary, co-stimulation increased the potential for targeted cytotoxicity and T cell activation compared to CD20xCD3 combined with a control antibody. Summary table of data: Table 29: EC50 values ​​for cytotoxicity and T cell activation [Table 29-1] [Table 29-2] Example 14: In vitro and in vivo characterization of the antitumor efficacy of REGN5837 alone or in combination with REGN1979 in a model of diffuse large B-cell lymphoma (DLBCL). Materials and Methods - Introduction to the study and summary of results In vitro and in vivo studies were conducted for evaluation:

[0288] (1) CD28 + T cells CD22 + The ability of REGN5837 to enhance primary T cell activation by crosslinking with target cells. T cell activation was assessed using the levels of cytotoxicity against target cells, expression of cell-surface markers of T cell activation, T cell proliferation, and cytokine release as readouts. REGN1979, CD3 molecules and CD20 on T cells + Experiments were conducted in the presence or absence of CD20xCD3bsAb, which cross-links target cells and induces T cell activation.

[0289] (2) Antitumor efficacy of CD22xCD28bsAbREGN5837 in immunodeficient NSG mice with DLBCL tumors in the presence of 0.4 or 4 mg / kg of REGN1979.

[0290] REGN5837 and REGN1979 were tested in combination at various concentrations to evaluate the effects of REGN5837 on REGN1979-mediated T cell toxicity (WSU-DLCL2), upregulation of the late T cell activation marker (CD25), T cell proliferation, and cytokine release from primary human T cells in human DLBCL cell lines. REGN5837 was tested for T cell toxicity, CD4 + and CD8 + CD25 cell-surface expression on T cells, and CD4 + and CD8 +REGN5837 enhanced the potential of REGN1979 to mediate T cell proliferation in a concentration-dependent manner. Similarly, REGN5837 enhanced the potential of REGN1979 to mediate cytokine release in a concentration-dependent manner. At concentrations in the range of 77.2 pM to 100 nM, REGN5837 increased the potential of REGN1979-mediated T cell cytotoxicity to target cells; at concentrations in the range of 77.2 pM to 2.78 nM, REGN5837 increased the potential of REGN1979-mediated T cell activation and proliferation, but higher concentrations of REGN5837 did not further increase the potential of REGN1979 (Table 30). While the addition of REGN5837 did not substantially increase the maximum levels of REGN1979-mediated target cell killing and T cell proliferation, REGN5837 concentration-dependently enhanced the maximum levels of REGN1979-mediated release of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17α.

[0291] Immunodeficient NSG mice (n=6-7 per group) were subcutaneously transplanted (SC) with WSU-DLCL2 cells and human PBMCs in a 1:1 ratio. The antitumor efficacy of REGN5837 at 1 mg / kg in combination with REGN1979 at 0.4 or 4 mg / kg doses was compared with REGN5837 and REGN1979 monotherapy, as well as with non-crosslinked IgG4. P-PVA The treatment was compared with a control bsAb. Mice were administered antibodies by intraperitoneal (IP) injection on days 1, 8, and 15 after transplantation of WSU-DLCL2 cells. Treatment with 1 mg / kg of REGN5837 in the presence of 0.4 or 4 mg / kg of REGN1979 resulted in statistically significant suppression of WSU-DLCL2 tumor growth by day 28 post-transplant compared to REGN5837 or REGN1979 monotherapy and non-crosslinked control bsAb. REGN1979 monotherapy resulted in slight suppression of tumor growth, but REGN5837 monotherapy was ineffective compared to the non-crosslinked control.

[0292] In summary, when REGN5837 and REGN1979 were tested in vitro at various concentrations, REGN5837 was found to be CD22 +The presence of REGN5837 enhanced the potential of REGN1979 to mediate human T cell activation in the presence of WSU-DLCL2 cells. The maximum level of REGN1979-mediated cytokine release, rather than cytotoxicity, T cell activation, or proliferation, was increased in the presence of REGN5837. In vivo, 1 mg / kg of REGN5837 in the presence of either 0.4 or 4 mg / kg of REGN1979 was more effective in suppressing WSU-DLCL2 B-cell lymphoma tumor growth in mice compared to REGN5837 or REGN1979 monotherapy alone. Table 30: Summary of the effects of REGN5837 on REGN1979-mediated T cell activation using human PBMCs (measured by cytotoxicity to target cells, CD25 expression, and T cell proliferation). [Table 30-1] [Table 30-2] a REGN1979 was tested in the concentration range of 4.8 fM to 10 nM. b EC 50 The multiple change is EC 50 (REGN5837 not included) / EC 50 It was calculated as ([M]REGN5837).

[0293] Example 15: Evaluation of the effect of REGN5837 on REGN1979-mediated human T cell activation and testing of REGN5837 and REGN1979 in combination at various concentrations in the presence of WSU-DLCL2 cells. In NSG mice transplanted with human PBMCs and WSU-DLCL2 cells, the antitumor efficacy of human CD22xCD28bsAb REGN5837 was evaluated in both in vitro and in vivo studies by measuring the following parameters, in the presence or absence of substantive doses of human CD20xCD3bsAb (REGN1979). a) CD22 + T cell toxicity against target cells; b) CD4 +and CD8 + Upregulation of CD25 on the surface of T cells, a marker of T cell activation; c) T cell proliferation; d) Cytokine release (IL-4, IL-2, IL-6, IL-10, TNF-α, IFN-γ, and IL-17α).

[0294] Materials and methods cell line WSU-DLCL2: WSU-DLCL2 is a human DLBCL cell line isolated from the pleural fluid of a 41-year-old Caucasian male (Leibnitz Institute-DSMZ, Cat.#ACC575).

[0295] Human PBMC Leukopak was obtained from a human donor (donor #1500A) from the New York Blood Center for cytotoxicity, T cell activation, T cell proliferation, and cytokine release assays.

[0296] Human PBMCs were obtained from ReachBio for in vivo mouse experiments (Cat.#0500-401).

[0297] Experimental Design REGN5837 and REGN1979 were tested in combination at various concentrations to evaluate the effects of REGN5837 on REGN1979-mediated T cell toxicity, T cell proliferation, cell-surface expression of the late T cell activation marker (CD25), and cytokine release from human T cells in WSU-DLCL2 cells. The percentage of target cell killing, T cell activation, and T cell proliferation were measured as described herein.

[0298] The antitumor efficacy of REGN5837 alone and in combination with REGN1979 in DLBCL models using WSU-DLCL2 cells and PBMCs was evaluated as described herein. See Table 31.

[0299] In vitro evaluation of the effect of REGN5837 on the potential mediation of T cell activation by REGN1979. Human primary T cell isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs by density gradient centrifugation using 50 mL SepMate® tubes according to the manufacturer's recommended protocol. Briefly, 15 mL of FicollPaque PLUS was layered into a 50 mL SepMate tube, followed by the addition of 30 mL of whole blood diluted 1:2 with D-PBS. The tube was centrifuged at 1200 x g at room temperature for 10 minutes with the brake released. The top layer containing plasma and PBMCs was decanted into a new tube. Subsequent steps were performed according to the SepMate manufacturer's protocol. The isolated PBMCs were placed in 5 mL cryovials at a density of 250 × 10⁶. 6 The cells were frozen in FBS containing 10% DMSO at a concentration of cells / mL. The PBMCs were thawed in a 37°C water bath and resuspended in a stimulating medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / mL of benzonase nuclease at a rate of 10 mL / 100 million PBMCs. The mixture was then centrifuged at 300xg for 10 minutes. CD3 + T cells are EasySep® Human CD3 cells from StemCell Technologies. + Follow the manufacturer's recommended instructions to isolate the T cell. It was isolated from reddened PBMCs. PBMC-based flow cytometry-based T cell activation assay using PBMCs

[0300] The ability of REGN5837 to enhance T cell activation mediated by allogeneic primary stimulation or "Signal 1" provided by REGN1979 was evaluated using WSU-DLCL2 target cells and human PBMCs as effector cells. PBMCs were enriched for lymphocytes as described herein. Target and effector cells were incubated with the test substance and control antibody as described herein. Flow cytometry was performed to evaluate the upregulation of markers of T cell cytotoxicity, proliferation, and T cell activation as described herein. Furthermore, the effect of REGN5837 on REGN1979-mediated cytokine release was evaluated as described herein. Non-TAAxCD28 bsAb (containing the same CD28-binding and non-binding arms as REGN5837) was tested as a non-crosslinked control of REGN5837.

[0301] Lymphocyte concentration in PBMCs Human PBMCs were placed in complete culture medium (RPMI cell culture medium supplemented with 10% FBS, penicillin-streptomycin-glutamine) at a rate of 1 × 10⁶ 6 Lymphocytes were enriched by seeding at cell / mL and incubating overnight at 37°C to remove adherent cells, such as macrophages, dendritic cells, and some monocytes.

[0302] Incubation of PBMCs and target cells with test substances PBMCs containing a high concentration of lymphocytes were collected and labeled with 1 μM Violet Cell Tracker fluorescent tracking dye. WSU-DLCL2 target cells were labeled with 1 μM Vybrant CFDA-SE fluorescent dye.

[0303] Subsequently, the dye-labeled PBMCs were mixed with dye-labeled target cells (5 × 10) in a 5:1 ratio. 3 Target cells (WSU-DLCL2) were seeded in 96-well round-bottom plates.

[0304] Seeded PBMCs and target cells were incubated for 72 hours at 37°C with the test substance or their respective controls at final concentrations ranging from 12.9 pM to 100 nM (REGN5837 or non-TAAxCD28) and 4.8 fM to 10 nM (REGN1979 or non-TAAxCD3).

[0305] Flow cytometry analysis After incubation with the test substance and control, dye-labeled cells were stained with LIVE / DEAD staining and a cocktail of antibodies against fluorophore-labeled CD2, CD4, CD8, and CD25. Counting beads (20 μl / well) were added immediately before sample analysis using a BD Celesta flow cytometer. Flow cytometry data were used to measure the upregulation of markers for target cell survival, T cell proliferation, and T cell activation. EC 50 The values ​​were determined from a four-parameter logistic equation on a 9-point dose-response curve using GraphPad Prism software. Maximum responses for cytotoxicity, T cell activation (CD25 upregulation), and proliferation were determined as the Prism curve fitting n maximum response plateau obtained by fitting the Prism curve. E compared to control C 50 The relative change wa is EC50 No REGN5837 / EC50 [M] REGN5837 Calculated as an, and the relative change in the maximum cytokine release is Max [M] REGN5837 / Max No REGN5837 It was calculated as follows.

[0306] target cell survival The percentage of surviving target cells under each experimental condition was normalized to the number of beads collected per well, and then calculated as the number of CFDA-SE-labeled live target cells per well. The percentage of target cell viability was determined as the ratio of the number of surviving target cells under any experimental condition to the number of surviving target cells under the antibody-free control condition (target cells in the presence of PBMCs only).

[0307] The percentage of cytotoxicity to target cells under each experimental condition reported in this manner was determined by subtracting the survival rate percentage (calculated as described above) from 100.

[0308] CD4 + and CD8 + CD25 expression on T cells Upregulation of CD25 (marker of late-stage activated T cells) is achieved by using raw, CD2 + , and CD4 + or CD8 + Evaluation was performed by gating on the cells. We reported the percentage of activated T cells expressing CD25 out of all T cells expressing either CD4 or CD8.

[0309] CD4 + and CD8 + T cell proliferation Primary CD4 + and CD8 + T cell proliferation is all CD4 + and CD8 + The percentage of T cells that underwent division was calculated and evaluated using flow cytometry. Since the fluorescence intensity of each cell decreases twofold with each division, the fluorescence intensity of cells stained with Violet Cell Tracker was used as a readout for cell division.

[0310] Cytokine release analysis The levels of cytokines (IL-4, IL-2, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A) in cell culture supernatant were quantified using the BD Cytometric Bead Array Human Th1 / Th2 / Th17 Cytokine Kit according to the manufacturer's instructions.

[0311] In vivo model of DLBCL using WSU-DLCL2 cell xenografts Female NSG mice were used in all experiments. All mice were SC-transplanted with WSU-DLCL2 B-cell lymphoma cells and administered antibodies via IP. Tumor growth was measured several times a week using calipers during the study period. For all experiments, mice were housed in an animal facility under Regeneron standard conditions. All experiments were performed in Regeneron in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

[0312] WSU-DLCL2 cell culture conditions and tumor transplantation The WSU-DLCL2 cell line was obtained from Leibnitz Institute-DSMZ and maintained in RPMI-1640 medium containing 10% FBS supplemented with penicillin, streptomycin, glutamine, and 1 mM HEPES.

[0313] WSU-DLCL2 cells (3 × 10 6 Collect cells (s), 5 × 10 5 The cells were mixed with PBMCs and resuspended in a 1:1 mixture of PBS and GFR Matrigel. 100 μl of the cell mixture was SC-injected into the right flank of female NSG mice.

[0314] Antibody administration for tumor measurement Before administering the test substance or control, mice were assigned to stratified groups according to tumor load.

[0315] Antibodies (REGN5837, REGN1979, REGN5671 [non-TAAxCD28 non-crosslinked control bsAb], or H4sH17664D [non-TAAxCD3 non-crosslinked control bsAb]) were administered as monotherapy or in combination by IP injection on days 1, 8, and 15 post-transplant at the doses listed in Table 31. Table 31: Experimental design for evaluating tumor growth and survival [Table 31-1] [Table 31-2] a One mouse died during the experiment and was removed.

[0316] Tumor measurement and specified endpoints Tumor growth was monitored over time using tumor caliper measurements X and diameter Y (length measured vertically and width). Tumor volume was calculated (X*Y*[X / 2], where X is the minor axis). Mice were euthanized when the tumor reached a specified tumor endpoint (tumor diameter > 20 mm or tumor ulceration). This endpoint followed the IACUC criteria.

[0317] Statistical analysis of tumor growth and survival Tumor volume over time was analyzed using two-way analysis of variance (ANOVA), followed by a Tukey post-hoc trial for multiple comparisons. Survival rates over time were analyzed for all groups using the Mantel-Cox trial, and further comparisons were made by conducting the Mantel-Cox trial for each group. A difference was considered statistically significant if p < 0.05. Statistical analysis was performed using GraphPad Prism 8 software.

[0318] result Effect of REGN5837 on REGN1979's ability to mediate T cell toxicity against WSU-DLCL2 target cells and cytokine release from human PBMCs. REGN5837 and REGN1979 were tested in combination at various concentrations to evaluate the effects of REGN5837 on REGN1979-mediated cytotoxicity, T cell activation, T cell proliferation, and cytokine release from human PBMC-derived T cells in WSU-DLCL2 cells, as previously described. Table 30 shows the effects of REGN5837 on REGN1979-mediated cytotoxicity, T cell activation, and T cell proliferation in WSU-DLCL2 cells. Table 32 summarizes the numerical results from two human donors showing the effect of REGN5837 on cytokine release.

[0319] Effects of REGN5837 on REGN1979-mediated cytotoxicity and human T cell proliferation REGN1979-mediated cytotoxicity, REGN1979-mediated T cell activation, and human CD4 derived from human PBMCs in WSU-DLCL2 target cells. + and CD8 + By evaluating the REGN1979-mediated proliferation of T cells, the efficacy of REGN1979 (EC) can be assessed. 50 The effect of increasing the concentration of REGN5837 on cytotoxicity and efficacy (maximum response) against WSU-DLCL2 cells was evaluated. REGN5837 has cytotoxic effects on WSU-DLCL2 cells and T cell activation (CD4 + and CD8 + (Measured as CD25 expression on T cells), and CD4 + and CD8 + The potential of REGN1979 to mediate T cell proliferation was enhanced in a concentration-dependent manner. At concentrations in the range of 77.2 pM to 100 nM, REGN5837 increased the potential of REGN1979-mediated T cell toxicity to target cells; at concentrations in the range of 77.2 pM to 2.78 nM, REGN5837 increased the potential of REGN1979-mediated T cell activation and proliferation, but higher concentrations of REGN5837 did not further increase the potential of REGN1979. These data are presented in the graph in Figure 11 and in Table 30.

[0320] REGN1979-mediated cytokine release from human PBMCs in the presence of REGN5837 The potential for REGN1979-mediated cytokine release from human PBMCs and the effect of increasing REGN5837 concentration on the maximal response were evaluated. In the presence of human PBMCs and WSU-DLCL2 cells, increasing the concentration of REGN5837 enhanced the maximal levels of REGN1979-mediated release of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A in a concentration-dependent manner (Figure 12). Furthermore, increasing the concentration of REGN5837 tended to enhance the potential for REGN1979 to mediate cytokine release. REGN1979-mediated EC 50The values, maximum cytokine levels, and relative increases exceeding background cytokine levels (without REGN5837) are summarized in Figure 12 and Table 32. Table 32 [Table 32-1] [Table 32-2] Antitumor efficacy of REGN5837 administration in the presence and absence of REGN1979 in amounts below the effective dose.

[0321] Immunodeficient NSG mice with WSU-DLCL2 tumors were administered via IP injection with the antibodies or non-crosslinked controls already described herein.

[0322] In tumor-bearing mice, treatment with 1 mg / kg REGN5837 in the presence of either 0.4 or 4 mg / kg REGN1979 resulted in a statistically significant suppression of tumor growth by day 28 (6 days after the final antibody administration) compared to non-crosslinked control bsAbs (non-TAAxCD28 and non-TAAxCD3 bsAbs) (Figures 13A and 13B). The combination of 1 mg / kg REGN5837 and 0.4 mg / kg REGN1979 resulted in a significant reduction in tumor volume by day 46 compared to REGN1979 monotherapy.

[0323] Both 0.4 and 4 mg / kg of REGN1979 monotherapy resulted in low tumor suppression compared to the non-crosslinked control by day 28, but REGN5837 monotherapy was ineffective. Rapid tumor growth was observed when the non-crosslinked control bsAb was administered throughout the treatment period, and all mice were euthanized on day 125.

[0324] The Mantel-Cox test detected statistically significant differences in survival across all groups (p=0.0001), and additional Mantel-Cox tests were conducted for group-by-group comparisons. Compared to mice administered with a non-crosslinked control antibody (no survival), mice administered 1 mg / kg of REGN5837 in combination with either 0.4 or 4 mg / kg of REGN1979 showed a significant increase in survival (85% and 70% survival, respectively) (Figure 14).

[0325] Furthermore, compared to mice treated with monotherapy using either REGN5837 or REGN1979, mice treated with 1 mg / kg of REGN5837 in combination with either 0.4 mg / kg or 4 mg / kg of REGN1979 showed a significant increase in survival.

[0326] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. The present invention provides, for example, the following items: (Item 1) a) The first antigen-binding domain (D1) that binds to human CD28, b) The second antigen-binding domain (D2) that specifically binds to human CD22 on target tumor cells and Isolated bispecific antigen-binding molecules containing these molecules. (Item 2) The isolated bispecific antigen-binding molecule according to item 1, wherein the second antigen-binding domain (D2) binds to an epitope on human CD22 containing one or more amino acids of SEQ ID NO: 57, SEQ ID NO: 58, and / or SEQ ID NO: 59. (Item 3) The bispecific antigen-binding molecule was measured by surface plasmon resonance at 25°C to have a K content of less than approximately 15 nM. D An isolated bispecific antigen-binding molecule as described in item 1 or 2, which binds to human CD22. (Item 4) The bispecific antigen-binding molecule was measured by surface plasmon resonance at 25°C and had a K content of less than approximately 60 μM. D An isolated bispecific antigen-binding molecule described in any one of items 1-3, which binds to Macaca fascicularis CD22. (Item 5) The bispecific antigen-binding molecule was measured by surface plasmon resonance at 25°C and had a K content of less than approximately 45 μM. D An isolated bispecific antigen-binding molecule described in any one of items 1 to 4, which binds to human CD28. (Item 6) The aforementioned bispecific antigen-binding molecule was measured by an in vitro FACS binding assay and yielded approximately 1 × 10⁻⁶ values. -8 M-EC 50 An isolated bispecific antigen-binding molecule according to any one of items 1 to 5, which binds to the surface of a cell expressing human CD28. (Item 7) The aforementioned bispecific antigen-binding molecule was measured by an in vitro FACS binding assay and yielded approximately 1 × 10⁻⁶ values. -8 M-EC 50 An isolated bispecific antigen-binding molecule according to any one of items 1 to 6, which binds to the surface of a cell expressing human CD22. (Item 8) An isolated bispecific antigen-binding molecule according to any one of items 1 to 7, which exhibits a co-stimulatory effect when used in combination with an anti-CD20xCD3 bispecific antibody. (Item 9) The aforementioned co-stimulatory effect is one or more of the following: (activation of T cells, induction of IL-2 release, induction of CD25+ upregulation in human PBMCs; and induction of human T cell-mediated cytotoxicity against CD22-expressing cell lines), as described in item 8. (Item 10) The aforementioned first antigen-binding domain (D1) is: a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 26, b) Three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 10, An isolated bispecific antigen-binding molecule, including one of the items 1-9. (Item 11) a) HCDR1 containing the amino acid sequence of SEQ ID NO: 28, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 32 Isolated bispecific antigen-binding molecules as described in item 10, including those listed above. (Item 12) a) LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 Isolated bispecific antigen-binding molecules as described in item 12, including those listed above. (Item 13) The aforementioned first antigen-binding domain: a) A set of HCVR CDRs (HCDR1, HCDR2, HCDR3) comprising the amino acid sequences of SEQ ID NOs. 28, 30, and 32, and a set of LCVR CDRs (LCDR1, LCDR2, LCDR3) comprising the amino acid sequences of SEQ ID NOs. 12, 14, and 16, Isolated bispecific antigen-binding molecules as described in item 10, including those listed above. (Item 14) The isolated bispecific antigen-binding molecule according to item 10, wherein the first antigen-binding domain comprises an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 26 / 10. (Item 15) The aforementioned second antigen-binding domain: a) Three HCDRs contained within an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18, b) Three LCDRs contained within LCVR containing the amino acid sequence of SEQ ID NO: 10, An isolated bispecific antigen-binding molecule, including one of the items 1 to 14. (Item 16) The aforementioned second antigen-binding domain: a) HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 4 and 20, b) HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 6 and 22, c) An isolated bispecific antigen-binding molecule as described in item 15, comprising HCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 24. (Item 17) The aforementioned second antigen-binding domain: a) LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 Isolated bispecific antigen-binding molecules as described in item 16, including the following. (Item 18) The aforementioned second antigen-binding domain: a) A set of HCVR CDRs (HCDR1, HCDR2, HCDR3) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 4, 6, 8; and 20, 22, 24; and a set of LCVR CDRs (LCDR1, LCDR2, LCDR3) containing amino acid sequences of SEQ ID NOs: 12, 14, 16; Isolated bispecific antigen-binding molecules, including those described in item 17. (Item 19) a) A first antigen-binding domain comprising an HCVR CDR containing the amino acid sequences of SEQ ID NOs. 28, 30, and 32, and an LCVR CDR containing the amino acid sequences of SEQ ID NOs. 12, 14, and 16, b) A second antigen-binding domain comprising an HCVR CDR containing the amino acid sequences of SEQ ID NOs: 4, 6, and 8, and an LCVR CDR containing the amino acid sequences of SEQ ID NOs: 12, 14, and 16, An isolated bispecific antigen-binding molecule, including one of the items 1 to 18. (Item 20) a) A first antigen-binding domain comprising an HCDR containing the amino acid sequences of SEQ ID NOs. 28, 30, and 32, and an LCDR containing the amino acid sequences of SEQ ID NOs. 12, 14, and 16, b) A second antigen-binding domain comprising an HCDR containing the amino acid sequences of SEQ ID NOs. 20, 22, and 24, and an LCDR containing the amino acid sequences of SEQ ID NOs. 12, 14, and 16, An isolated bispecific antigen-binding molecule, including one of the items 1 to 18. (Item 21) a) A first antigen-binding domain containing an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 26 / 10, b) A second antigen-binding domain containing an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 2 / 10, An isolated bispecific antigen-binding molecule, including one of the items 1 to 18. (Item 22) a) The first antigen-binding domain comprises an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 26 / 10; b) An isolated bispecific antigen-binding molecule according to any one of items 1 to 18, wherein the second antigen-binding domain contains an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 18 / 10. (Item 23) An isolated bispecific antigen-binding molecule that competes for binding to CD22 or binds to the same epitope on CD22 as the reference antibody, wherein the reference antibody comprises a first antigen-binding domain having an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 26 / 10, and a second antigen-binding domain having an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 2 / 10 or 18 / 10. (Item 24) An isolated bispecific antigen-binding molecule that competes for binding to human CD28 or binds to the same epitope on human CD28 as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain having an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 26 / 10, and a second antigen-binding domain having an HCVR / LCVR pair containing the amino acid sequence of SEQ ID NO: 2 / 10 or 18 / 10. (Item 25) A pharmaceutical composition comprising a bispecific antigen-binding molecule described in any one of items 1 to 24, and a pharmaceutically acceptable carrier or diluent. (Item 26) A nucleic acid molecule containing a nucleotide sequence that encodes a bispecific antibody as described in any one of items 1 to 24. (Item 27) An expression vector containing the nucleic acid described in item 26. (Item 28) Host cells containing the expression vector described in item 27. (Item 29) A method for inhibiting B-cell proliferation impairment in a subject, comprising administering to the subject an isolated bispecific antibody described in any one of items 1 to 24 or a pharmaceutical composition described in item 25, thereby inhibiting the growth of the B-cell lymphoma in the subject. (Item 30) The method according to item 29, further comprising administering a second therapeutic agent to the subject. (Item 31) The method according to item 30, wherein the second therapeutic agent includes an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated with an antitumor agent, a checkpoint inhibitor, or a combination thereof. (Item 32) A method for treating a patient suffering from B-cell proliferative disorder or another CD22-expressing cell malignancy, comprising administering to the subject an isolated bispecific antibody described in any one of items 1 to 24 or a pharmaceutical composition described in item 25, thereby treating the patient suffering from B-cell lymphoma or another CD22-expressing cell malignancy. (Item 33) The method according to item 32, further comprising administering a second therapeutic agent to the subject. (Item 34) The method according to item 33, wherein the second therapeutic agent includes an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated with an antitumor agent, a checkpoint inhibitor, or a combination thereof. (Item 35) The method according to either item 31 or 34, wherein the second therapeutic agent is a different bispecific antibody comprising a first antigen-binding domain that binds to the same tumor target antigen and a second antigen-binding domain that binds to CD3 on T cells.

Claims

1. An isolated bispecific antibody or its antigen-binding fragment, (a) A first antigen-binding domain that specifically binds to human CD28, comprising: (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 26 and three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprising the respective amino acid sequences of SEQ ID NOs: 28, 30, and 32; and a light chain variable region (LCVR) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprising the respective amino acid sequences of SEQ ID NOs: 12, 14, and 16; and (b) A second antigen-binding domain that specifically binds to human CD22 on target tumor cells, comprising: an HCVR containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2 and three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) containing the respective amino acid sequences of SEQ ID NOs: 4, 6, and 8; and a light chain variable region (LCVR) containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) containing the respective amino acid sequences of SEQ ID NOs: 12, 14, and 16. An isolated bispecific antibody or its antigen-binding fragment, containing the above.

2. (a) The first antigen-binding domain comprises an HCVR containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 26 and an LCVR containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10; (b) The second antigen-binding domain comprises an HCVR containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 2 and an LCVR containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

10. An isolated bispecific antibody or its antigen-binding fragment according to claim 1.

3. The isolated bispecific antibody or antigen-binding fragment according to claim 1 or 2, wherein the second antigen-binding domain (D2) binds to an epitope on human CD22 containing the amino acid sequence shown in SEQ ID NO: 57 and / or SEQ ID NO:

58.

4. The bispecific antibody or its antigen-binding fragment has a K content of less than 15 nM, as measured by surface plasmon resonance at 25°C. Ｄ An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which binds to human CD22.

5. The bispecific antibody or its antigen-binding fragment has a K content of less than 60 μM, as measured by surface plasmon resonance at 25°C. Ｄ An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to Macaca fascicularius CD22.

6. The bispecific antibody or its antigen-binding fragment has a K content of less than 45 μM, as measured by surface plasmon resonance at 25°C. Ｄ An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to human CD28.

7. The bispecific antibody or its antigen-binding fragment was measured by an in vitro FACS binding assay and yielded 2.1 × 10⁻⁶. －８ EC less than M ５０ An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to the surface of a cell expressing human CD28.

8. The bispecific antibody or its antigen-binding fragment was measured by an in vitro FACS binding assay and yielded 1.3 × 10⁻⁶. －８ EC less than M ５０ An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which binds to the surface of a cell expressing human CD22.

9. The isolated bispecific antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the bispecific antibody or its antigen-binding fragment exhibits a co-stimulatory effect when used in combination with an anti-CD20xCD3 bispecific antibody.

10. The isolated bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the aforementioned co-stimulatory effect is one or more of the following: activation of T cells, induction of IL-2 release, induction of CD25+ upregulation in human PBMCs, and increased human T cell-mediated cytotoxicity against CD22-expressing cell lines.

11. An isolated bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 10, which is a bispecific antibody.

12. An isolated bispecific antibody or its antigen-binding fragment according to claim 11, (a) The bispecific antibody comprises a human IgG heavy chain constant region; optionally, the human IgG heavy chain constant region is isotype IgG1 or IgG4; and / or (b) The bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype. Isolated bispecific antibodies or their antigen-binding fragments.

13. A pharmaceutical composition comprising an isolated bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier or diluent.

14. A bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 12 (i) The nucleic acid sequence encoding the HCVR of the first antigen-binding domain, (ii) The nucleic acid sequence encoding the HCVR of the second antigen-binding domain, (iii) Nucleic acid sequences encoding the LCVR of the first and second antigen-binding domains and A group of nucleic acid molecules that include [the specified element].

15. A group of expression vectors comprising the nucleic acid molecule described in claim 14.

16. A host cell comprising a bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 12, the group of nucleic acid molecules according to claim 14, or the group of expression vectors according to claim 15.

17. The pharmaceutical composition according to claim 13, for use in inhibiting B cell proliferation disorders in a target.

18. The pharmaceutical composition according to claim 13, for use in combination with a second therapeutic agent in inhibiting B cell proliferation disorder in a target.

19. The pharmaceutical composition according to claim 18, wherein the second therapeutic agent comprises an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated with an antitumor agent, a checkpoint inhibitor, or a combination thereof.

20. The pharmaceutical composition according to claim 13, for use in the treatment of a subject suffering from B-cell proliferative disorder or another CD22-expressing cell malignancy.

21. The pharmaceutical composition according to claim 13, for use in combination with a second therapeutic agent in the treatment of a subject suffering from B-cell proliferative disorder or another CD22-expressing cell malignancy.

22. The pharmaceutical composition according to claim 21, wherein the second therapeutic agent comprises an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated with an antitumor agent, a checkpoint inhibitor, or a combination thereof.

23. The pharmaceutical composition according to claim 18 or 21, wherein the second therapeutic agent is a different bispecific antibody comprising a first antigen-binding domain that binds to an antigen on the same target tumor cell and a second antigen-binding domain that binds to CD3 on a T cell.

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