Bispecific anti-CD28X anti-CD22 antibodies and uses thereof

Abstract

The present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to human CD28 and a second antigen-binding molecule that specifically binds to human CD-22. In certain embodiments, the bispecific antigen-binding molecules of the present invention can inhibit the growth of tumors that express CD-22, such as B-cell lymphomas. The antibodies and bispecific antigen-binding molecules of the present invention are useful for treating diseases and disorders in which an upregulated or induced targeted immune response is desirable and / or therapeutically beneficial.

Description

[Technical field]

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

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

[0003] The present invention relates to bispecific antigen-binding molecules that bind to CD28 and a target molecule, such as CD22, and methods of use thereof. [Background technology]

[0004] CD28 is a type I transmembrane protein expressed on the surface of T cells, with a single extracellular Ig-V-like domain organized as a homodimer. CD28 is a receptor for 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 stimulatory signal that is important for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 provides a strong signal for the production of various interleukins. CD28 also enhances cell signals, such as pathways controlled by NFκB transcription factors after TCR activation. CD28 co-signals are important for effective T cell activation, such as 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 anti-CD28 antibody, TGN1412 (anti-CD28 superagonist), was used in clinical trials in 2006. TGN1412 (anti-CD28 superagonist) was administered intravenously at a dose of 0.1 mg / kg to six healthy volunteers. Within 2 hours, there was a significant inflammatory response (cytokine storm) in all six patients, and all patients had multiple organ failure within 16 hours. The subjects were treated with corticosteroids, and cytokine levels returned to normal levels within 2-3 days. The starting dose of 0.1 mg / kg in the Phase 1 study (related to CRS) was based on 500 times the cynomolgus monkey "NOAEL" 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 that was not predicted by toxicity studies in cynomolgus monkeys or ex vivo human PBMC studies.

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

[0007] CD22 has many innate functions, including, for example, B cell homeostasis, B cell survival and migration, attenuation of TLR and CD40 signaling, and inhibition of B cell receptor (BCR) signaling via recruitment of SH2 domain-containing phosphatases by phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region, as well as promoting adhesion between B cells and other cell types.

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

[0009] Anti-CD22 antibodies have been investigated to treat B-cell lymphoma and leukemia. However, the monoclonal antibody Epratuzumab has had limited success. (Grant, et al. (2013) Cancer 119(21):10.1002 / cncr.28299) Therefore, there is a need in the art for improved anti-CD22-antibodies. There is also a need for anti-CD28 antibodies that are safe for use in pharmaceutical compositions. Furthermore, bispecific antigen-binding molecules that bind 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 is desired. [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 Summary of the Invention [Means for solving the problem]

[0011] In a first aspect, the present invention provides a bispecific antigen-binding molecule that binds CD28 and a target antigen. According to an exemplary embodiment, the bispecific antigen-binding molecule binds CD28 and CD22; such bispecific antigen-binding molecules are also referred to herein as "anti-CD28 / anti-CD22 bispecific molecules." 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 facilitates selective killing (cytolysis) of targeted cancer cells by activated T cells, for example, after TCR activation of the T cells. The anti-CD28 / anti-CD22 bispecific molecules of the invention are therefore useful, inter alia, for 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] Bispecific antigen-binding molecules according to this aspect of the invention comprise a first antigen-binding domain that specifically binds human CD28 and a second antigen-binding domain that specifically binds CD22. The invention includes anti-CD28 / anti-CD22 bispecific molecules (e.g., bispecific antibodies) 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 invention, the anti-CD28 antigen-binding domain and the anti-CD22 antigen-binding domain each comprise a different and distinct HCVR paired with a common LCVR.

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

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

[0015] The present invention also provides anti-CD28 / anti-CD22 bispecific molecules, wherein a first antigen-binding domain that specifically binds CD28 comprises a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence thereof 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, wherein a first antigen binding specifically to CD28 comprises a 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 anti-CD28 / anti-CD22 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a heavy chain CDR3 (HCDR3) domain having the amino acid sequence of SEQ ID NO: 32, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having the amino acid sequence of SEQ ID NO: 16, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0018] In certain embodiments, the first antigen-binding domain that specifically binds to CD28 comprises the HCDR3 / LCDR3 amino acid sequence pair of SEQ ID NOs: 32 / 16.

[0019] The present invention also provides anti-CD28 / anti-CD22 bispecific antigen-binding molecules, wherein 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 thereof, 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 thereof, 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 thereof, 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 thereof, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

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

[0022] The present invention also provides anti-CD28 / anti-CD22 bispecific molecules, wherein the second antigen-binding domain that specifically binds to CD22 comprises a light chain variable region (LCVR) having an amino acid sequence selected from SEQ ID NO: 10, or a substantially similar sequence thereof 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, wherein the second antigen-binding domain that specifically binds to CD22 comprises a 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 anti-CD28 / anti-CD22 bispecific molecules, wherein the second antigen-binding domain that specifically binds to CD22 comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 24, or a sequence substantially similar thereto, with 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 sequence substantially similar thereto, with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0025] In certain embodiments, 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 20, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; Also provided is an anti-CD28 / anti-CD22 bispecific antigen-binding molecule comprising a light chain CDR1 (LCDR1) domain having the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof, 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 thereof, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0027] Certain non-limiting examples of anti-CD28 / anti-CD22 bispecific antigen binding molecules of the invention include a second antigen binding domain that specifically binds to CD22, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having amino acid sequences 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 a related embodiment, the invention comprises an anti-CD28 / anti-CD22 bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to CD22 comprises heavy and light chain CDR domains contained within heavy and light chain variable region (HCVR / LCVR) sequences selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 10.

[0029] In another aspect, the 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 a polynucleotide sequence as set forth in Table 7 herein, as well as nucleic acid molecules comprising two or more of the polynucleotide sequences as set forth in Table 7, in any functional combination or arrangement thereof. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also included in the invention, as are methods of producing the antibodies by culturing host cells under conditions that permit the production of the antibody and recovering the antibody produced.

[0030] The present invention includes anti-CD28 / anti-CD22 bispecific antigen binding molecules in which any of the antigen binding domains that specifically bind CD28 specifically binds CD22 and is combined, linked or otherwise associated with any of the antigen binding domains to form a bispecific antigen binding molecule that binds both CD28 and CD22.

[0031] The present invention includes anti-CD28 / anti-CD22 bispecific antigen binding molecules with modified glycosylation patterns. In some applications, modifications may be useful to remove undesired glycosylation sites, or antibodies lacking fucose moieties present on the oligosaccharide chains, for example to increase antibody-dependent cellular cytotoxicity (ADCC) function, (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation may be performed to modify complement-dependent cytotoxicity (CDC).

[0032] In another aspect, the invention provides a pharmaceutical composition comprising an anti-CD28 / anti-CD22 bispecific antigen-binding molecule disclosed herein and a pharma- ceutically acceptable carrier. In a related aspect, the invention features a composition that 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 any agent that is advantageously combined with the anti-CD28 / anti-CD22 bispecific antigen-binding molecule. Examples of agents that are advantageously combined with the anti-CD28 / anti-CD22 bispecific antigen-binding molecule are discussed in detail elsewhere herein.

[0033] In yet another aspect, 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, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the present invention.

[0034] The present invention also includes the use of an anti-CD28 / anti-CD22 bispecific antigen-binding molecule of the invention in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by CD22 expression.

[0035] 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 molecules of the present invention, where the anti-CD28 / anti-CD22 bispecific antigen binding molecules are combined with other anti-tumor bispecific antigen binding molecules that bind CD3 (e.g. anti-CD28 / anti-CD22 combined with an anti-CD3 / anti-CD20 antibody).

[0036] In yet another aspect, the present invention provides a therapeutic method for targeting / killing cancer cells expressing CD22 using the anti-CD28 / anti-CD22 bispecific antigen binding molecules of the present invention, where the anti-CD28 / anti-CD22 bispecific antigen binding molecules are 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 certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with an agent targeting PD-1, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or cemiplimab (Libtayo®). In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the invention may be combined with an agent that targets PD-L1, such as Atezolizumab (Tecentriq®), Avelumab (Bavencio®), or Durvalumab (Imfinzi®). In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the invention may be combined with an agent that targets CTLA-4, such as Ipilimumab (Yervoy®).

[0037] In yet another aspect, the invention provides a therapeutic method for targeting / killing cancer cells expressing CD22 using the anti-CD28 / anti-CD22 bispecific antigen binding molecules of the invention, where the anti-CD28 / anti-CD22 bispecific antigen binding molecules are combined with other anti-tumor bispecific antigen binding molecules that bind CD3 (e.g. anti-CD28 / anti-CD22 combined with an anti-CD3 / anti-CD20 bispecific antibody, e.g., REGN1979 (wherein the anti-CD20 arm comprises the HCVR / LCVR amino acid pair of SEQ ID NOs: 1242 / 1258, and the anti-CD3 arm comprises the amino acid pair of SEQ ID NOs: 1250 / 1258, see U.S. Pat. No. 9,657,102)) and / or checkpoint inhibitors targeting 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 antibodies of the invention can be combined with agents that target PD-1, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or cemiplimab (Libtayo®, see U.S. Pat. No. 9,987,500, where cemiplimab comprises the HCVR / LCVR amino acid pair of SEQ ID NOs: 162 / 170). In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the invention can be combined with agents that target PD-L1, such as Atezolizumab (Tecentriq®), Avelumab (Bavencio®), or Durvalumab (Imfinzi®). In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the invention can be combined with agents that target CTLA, such as Ipilimumab (Yervoy®).

[0038] Other embodiments will become apparent from review of the following detailed description. [Brief description of the drawings]

[0039] [Figure 1]FIG. 1 is a set of graphs depicting binding of anti-CD28 / anti-CD22 bispecific antibodies to T cells expressing human CD4+CD28 and to target cells expressing human CD22 on the cell surface. [Diagram 2] Figures 2A and 2B are a set of graphs showing that anti-CD28 / anti-CD22 bispecific antibodies show increased 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 a constant 200 pM REGN1945 (hIgG4 isotype negative control) plus HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells, as assessed by luciferase production. Figure 2B is a set of graphs showing the activation of engineered reporter T cells co-incubated with a constant 200 pM REGN2281 (anti-CD20xanti-CD3) plus HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells, as assessed by luciferase production. [Diagram 3] Figures 3A and 3B are a set of graphs showing 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 showing 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 2 nM REGN1945 (hIgG4 isotype control), as assessed by IL-2 production. Figure 3B is a set of graphs showing 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 2 nM REGN2281 (anti-CD20xanti-CD3), as assessed by IL-2 production. [Figure 4]Figure 4 is a set of graphs showing that the combination of REGN5837 and cemiplimab enhances IL-2 release above 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 cemiplimab 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 cemiplimab enhances IL-2 release above REGN5837 treatment alone in RAJI cells engineered to express PD-L1. [Figure 6]Figure 6 is a graph showing that treatment of NSG mice bearing NALM-6-Luc tumors with REGN5837 in the presence of REGN1979 (anti-CD20 x anti-CD3) is associated with significant tumor inhibition. Briefly, NSG mice (n = 6-9 per group) were grafted with human PBMCs and then transplanted with NALM-6-luc B cell leukemia cells 12 days post-grafting (day 0). Mice were treated with 4 mg / kg REGN5837 + 0.04 mg / kg REGN1979 (hatched circle), 0.4 mg / kg REGN5837 + 0.04 mg / kg REGN1979 (closed up triangle), 0.04 mg / kg REGN5837 + 0.04 mg / kg REGN1979 (diamond), 4 mg / kg non-TAAxCD28 + 0.04 mg / kg REGN1979 (square), 4 mg / kg REGN5837 + 0.4 mg / kg non-TAAxCD3 (open circle), or 4 mg / kg non-TAAxCD28 + 0.4 mg / kg non-TAAxCD3 (inverted black triangle) on days 8, 15, and 22 (arrows) post-implantation. Tumor growth was monitored by bioimaging of tumor volume at days 6, 10, 14, 17, 20, and 23 post-implantation. Combined data are presented as group mean ± SEM. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc test. The following symbols were used to indicate statistical significance relative 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 to kill Nalm6 cells in a dose-dependent manner. More specifically, Figure 7A is a graph showing % survival of Nalm6 cells in the presence of the indicated antibodies. Figure 7B is a graph showing the percent of CD25 expressing (CD25+)CD8+ cells in the presence of the indicated antibodies. Figure 7C is a graph showing the proliferation of CD25+CD8+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies. [Figure 8]Figures 8A, 8B, and 8C are graphs showing that REGN1979 activates human T cells and kills WSU-DLCL2 cells in a dose-dependent manner. More specifically, Figure 8A is a graph showing the % survival of WSU-DLCL2 cells in the presence of the indicated antibodies. Figure 8B is a graph showing the percent of CD8+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Figure 8C is a graph showing the proliferation of CD8+ cells in the presence of the indicated antibodies, expressed as % division rate. [Figure 9] 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]10A-10E are graphs showing that REGN1979 activated human T cells and depleted NHL in a dose-dependent manner. Addition of fixed concentrations of CD22xCD28 bispecific antibody to REGN1979 enhanced the cytotoxic potency (EC50) of REGN1979 by 2.3 and 3.5 fold when compared to REGN1979 with one-arm CD28 control antibody or no co-stimulation control. The observed target cell lysis mediated by REGN1979 was associated with CD25 upregulation for T cell activation and proliferation as measured by CD8+ and CD4+ cells or CellTrace Violet dilution, respectively. More specifically, FIG. 10A is a graph showing the % survival of NHL cells from patient bone marrow in the presence of the indicated antibodies. FIG. 10B is a graph showing the percent of CD8+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Figure 10C is a graph showing the proliferation of CD8+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies. Figure 10D is a graph showing the percent of CD25 (CD25+)CD4+ cells in the presence of the indicated antibodies. Figure 10E is a graph showing the proliferation of CD4+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies. [Figure 11]11A-11E are graphs showing that REGN5837 concentration-dependently enhances REGN1979-mediated cytotoxicity, cell surface expression of CD25, and T cell proliferation potential. Briefly, WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMCs at a target cell to PBMC ratio of 1:5, and with anti-CD20xCD3 (REGN1979) at a range of concentrations (4.8 fM-10 nM) as single agents (i.e., no REGN5837) or in the presence of fixed concentrations of REGN5837 (ranging from 0.01 to 15 μg / mL) for 72 hours at 37°C. Conditions lacking REGN1979 contained only REGN5837 at the indicated concentrations and are plotted as 0.04 pM. Viable cells were detected by flow cytometry using a live / dead cell stain (11A). A phenotyping cocktail of Violet Cell Tracker dye and fluorophore-labeled antibodies against CD2, CD4, CD8, and CD25 was used to detect T cell activation (measured as CD25 expression; 11B, 11D) and CD4+ and CD8+ T cell proliferation by flow cytometry (11C, 11E). More specifically, FIG. 11A is a graph of percent dead cells versus the indicated concentrations of REGN5837. FIG. 11B is a graph of percent CD25+CD4+ cells versus the indicated concentrations of REGN5837. FIG. 11C is a graph of CD4+ cell proliferation assessed by CellTrace Violet dilution with the indicated concentrations of REGN5837. FIG. 11D is a graph of percent CD25+CD8+ cells. FIG. 11E is a graph of CD8+ cell proliferation assessed by CellTrace Violet dilution with the indicated concentrations of REGN5837. [Figure 12]12A-12G are graphs showing that REGN5837 concentration-dependently enhances the potential and maximum levels of REGN1979-mediated cytokine release from human T cells in the presence of WSU-DLCL2 B-cell lymphoma cells. Briefly, WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMCs at a target cell to PBMC ratio of 1:5 and anti-CD20xCD3 (REGN1979) at a range of concentrations (4.8 fM-10 nM) as a single agent (i.e., no REGN5837) or in the presence of a fixed concentration of REGN5837 (ranging from 0.01-15 μg / mL) for 72 hours at 37° C. Conditions lacking REGN1979 contained only REGN5837 at the indicated concentrations and are plotted as 0.04 pM. Supernatants were assessed 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, FIG. 12A is a graph showing the levels of IL-2 released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. FIG. 12B is a graph showing the levels of IL-4 released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. FIG. 12C is a graph showing the levels of IL-6 released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. Figure 12D is a graph showing the levels of IL-10 released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. Figure 12E is a graph showing the levels of TNF-α released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. Figure 12F is a graph showing the levels of IFN-γ released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837.FIG. 12G is a graph depicting the levels of IL-17α released from human T cells in the presence of WSU-DLCL2 cells and the indicated concentrations of REGN5837. [Figure 13] Figures 13A and 13B are graphs showing that treatment of NSG mice bearing WSU-DLCL2 tumors with REGN5837 in the presence of 0.4 or 4 mg / kg REGN1979 is associated with significant tumor inhibition. Briefly, female NSG mice (n=6-7 per group) were implanted with a 1:1 mixture of WSU-DLCL2 B-cell lymphoma cells and human PBMCs (day 0). At 1, 8, and 15 days post-implantation (arrows), mice were administered a combination of 1 mg / kg REGN5837 and 0.4 mg / kg (13A) or 4 mg / kg (13B) REGN1979 (or non-crosslinked control). Tumor growth was monitored by caliper measurements at 7, 10, 14, 16, 28, 31, 35, 38, 43, 46, 49, 53, 57, and 63 days post-implantation. Total data are presented as group mean ± SEM. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc test. 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 control, hash marks indicate significance between the combination of REGN5837 and REGN1979 and isotype control, and carets indicate significant differences between REGN1979 monotherapy and the combination of REGN5837 and REGN1979. More specifically, FIG. 13A is a graph depicting tumor growth in mice administered 1 mg / kg REGN5837 and 0.4 mg / kg REGN1979 (or non-crosslinked control, non-TAAxCD3). FIG. 13B is a graph depicting tumor growth in mice dosed with 1 mg / kg REGN5837 and 4 mg / kg (or non-crosslinked control, non-TAAxCD3). [Figure 14]FIG. 14 is a graph showing that treatment with REGN5837 in the presence of sub-efficacious doses of REGN1979 in NSG mice bearing WSU-DLCL2 tumors is associated with significantly higher survival rates than REGN5837 or REGN1979 monotherapy. Briefly, female NSG mice (n=6-7 per group) were implanted with a 1:1 mixture (day 0) of WSU-DLCL2 B cell lymphoma cells and human PBMCs. Mice were administered a combination of REGN5837 and REGN1979 or control 1, 8, and 15 days after implantation (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. Carets indicate statistical significance compared to isotype control, asterisks indicate significance compared to 0.4 mg / kg REGN1979 monotherapy, and hashes indicate significance compared to 4 mg / kg REGN1979 monotherapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Before describing the present invention, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not limiting, since the scope of the present invention is limited only by the appended claims.

[0041] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as those of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in relation to a specific listed value means that the value may vary from the listed value by 1% or less. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0042] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0043] definition The term "CD28" as used herein refers to an antigen expressed on T cells as a costimulatory receptor. Human CD28 comprises the amino acid sequence set forth in SEQ ID NO: 74 and / or has the amino acid sequence as set forth in NCBI Accession No. 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 specified as being from a non-human species. Thus, the term "CD28" refers to human CD28, unless specified as being from a non-human species, e.g., "mouse CD28", "monkey CD28", etc.

[0044] As used herein, "antibodies that bind CD28" or "anti-CD28 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize CD28, as well as antibodies and antigen-binding fragments thereof that specifically recognize dimeric CD28. The antibodies and antigen-binding fragments of the invention may bind to soluble CD28 and / or cell surface expressed CD28. Soluble CD28 includes naturally occurring CD28 protein as well as recombinant CD28 protein variants, such as monomeric and dimeric CD28 constructs, which lack a transmembrane domain or are otherwise not associated with a cell membrane.

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

[0046] As used herein, the phrase "anti-CD28 antibodies" includes both monovalent antibodies having a single specificity, as well as bispecific antibodies comprising a first arm that binds to CD28 and a second arm that binds to a second (target) antigen, where the anti-CD28 arm comprises any of the HCVR / LCVR or CDR sequences set forth in Table 1 herein. Examples of anti-CD28 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

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

[0048] As used herein, "antibodies that bind CD22" or "anti-CD22 antibodies" include antibodies and antigen-binding fragments thereof that can bind to soluble CD22 and / or cell surface expressed CD22. Soluble CD22 includes native CD22 protein as well as recombinant CD22 protein variants, e.g., CD22 constructs that lack the transmembrane domain or that are not associated with a cell membrane.

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

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

[0051] The term "antibody" as used herein refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD28). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is a region that is composed of a CDR that specifically binds to or interacts with a particular antigen (e.g., CD28). H 1. C H 2 and C H Each light chain contains three domains: a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (C L 1) is included. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), which are interspersed with regions that are further conserved, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-CD28 and / or anti-CD22 antibodies (or antigen-binding portions thereof) may be identical to human germline sequences or may be naturally or artificially modified. An 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 antigen-binding fragments of complete antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, 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 complete antibody molecules using any suitable standard techniques, for example, proteolytic or recombinant techniques, including the manipulation and expression of DNA encoding the 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. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[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) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0054] Antigen-binding antibodies typically contain at least one variable domain. A variable domain may be of any size amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, H and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H Or V L It may include a domain.

[0055] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the 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 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 H3;(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 In any configuration of the variable and constant domains, including any of the above exemplary configurations, the variable and constant domains can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provides 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 to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations non-covalently associated (e.g., by disulfide bond(s)) with respect to the domains.

[0056] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the invention using routine techniques available in the art.

[0057] The antibodies of the present invention may function by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent 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 non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells, thereby leading to lysis of the target cells. CDC and ADCC are well known and can be measured using assays available in the art. (See, for example, U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

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

[0059] The antibody of the present invention may in some embodiments be a recombinant human antibody. The term "recombinant human antibody", as used herein, is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using recombinant expression transfected vectors introduced into host cells (discussed further below), antibodies isolated from recombinant connatorial human antibody libraries (discussed further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (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 human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus determining the V H and V L The amino acid sequence of the region is H and V L These are sequences that, while derived from and related to, may not naturally exist within the human antibody germline repertoire in vivo.

[0060] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which dimers are linked by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming a molecule of approximately 75-80 kDa composed of covalently linked light and heavy chains (half antibodies). These forms have been very difficult to separate, even after affinity purification.

[0061] The frequency of occurrence of the second form in various intact IgG isotypes depends on, 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 a 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 with a human IgG1 hinge. The present invention includes antibodies with one or more mutations in the hinge, CH2 or CH3 regions, which may be desirable, for example, to improve the yield of the desired antibody form in production.

[0062] The antibody of the present invention may be an isolated antibody. "Isolated antibody" as used herein means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" in the context of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0063] The present invention also includes one-arm antibodies that bind to CD28 and / or CD22. As used herein, "one-arm antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain. The one-arm antibodies of the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences as described in Table 1.

[0064] The anti-CD28 and / or anti-CD22 antibodies, or antigen-binding domains thereof, herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains, compared to the corresponding germline sequences 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 to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies, and antigen-binding domains thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated to the original germline sequence, e.g., within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the invention, or antigen-binding domains thereof, may comprise any combination of two or more germline mutations in the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies, or antigen-binding fragments thereof, that contain one or more germline mutations can be readily tested for one or more desirable properties, such as, for example, improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed by the present invention.

[0065] The present invention also includes anti-CD28 and / or anti-CD22 antibodies, and antigen binding molecules comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect of the invention include variants 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 includes anti-CD28 antibodies and antigen binding molecules having HCVR, LCVR, and / or CDR amino acid sequences with 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in, for example, 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, known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include portions of sugars, phosphoryl groups, or sulfonyl groups on the antigen.

[0067] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity over 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 to 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] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced 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 a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the % sequence identity or similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 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 conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al (1992) Science 256:1443-1445. A "slightly conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0069] Polypeptide sequence similarity, also referred to 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 conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1, using 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) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of 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.

[0070] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation that would benefit from treatment with an anti-CD28 / anti-CD22 bispecific antigen binding molecule or method of the present invention. This includes chronic and acute disorders, including pathological conditions that predispose a mammal to the disorder in question. In one embodiment, the cell proliferative disorder is cancer, which is a physiological condition in a mammal that is typically characterized by unregulated cell growth / proliferation.

[0071] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0072] "B-cell proliferative disorder" includes, but is not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), including aggressive NHL, recurrent aggressive NHL, low-grade / follicular NHL, small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, recurrent indolent NHL, and rituximab-refractory indolent NHL. intractable NHL; refractory NHL, refractory indolent NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, lymphocyte-predominant Hodgkin lymphoma (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL); leukemias including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; and other hematological malignancies.

[0073] The term "non-Hodgkin's lymphoma" or "NHL" as used herein refers to a cancer of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphoma can generally be distinguished from non-Hodgkin's lymphoma by the presence of Reed-Sternberg cells in Hodgkin's lymphoma and the absence of said cells in non-Hodgkin's lymphoma. Examples of non-Hodgkin's lymphomas included in the term as used herein include any that are identified as such by a skilled artisan (e.g., an oncologist or pathologist) according to a classification scheme known in the art, such as the Revised European-American Lymphoma (REAL) scheme as 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 listings in Figures 11.57, 11.58 and 11.59.More specific examples include, but are not limited to, 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 prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic 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, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low grade / follicular lymphoma, intermediate grade / filtrate lymphoma, These include: follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL that relapses after or is ineffective after autologous stem cell transplant, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma. Bispecific antigen binding molecules

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

[0075] The use of the phrases "anti-CD28 antibody" and / or "anti-CD22 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. Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds human CD28 or CD22 and the other arm of the immunoglobulin is specific for a target antigen. The target antigen to which the other arm of the CD28 or CD22 bispecific antibody binds can be any antigen expressed on or near a cell, tissue, organ, microorganism or virus against which a targeted immune response is desired. The CD28-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein. The CD22-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein. In certain embodiments, the CD28 binding arm binds to human CD28 and induces human T cell proliferation.

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

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

[0078] According to an 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 an exemplary embodiment, the effector arm binds to CD28 and the targeting arm binds to CD22. The bispecific anti-CD28 / CD22 may provide a costimulatory signal for the effector cell (e.g., a T cell). The effector arm is ineffective in 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 (e.g., CD22) targeted by the targeting arm may be expressed on a fraction of tumor cells. The specificity of the tumor-targeting arm can be increased by overlapping it in combination with an anti-CD3 bispecific antigen-binding molecule (eg, an anti-CD3 / CD20 bispecific antibody).

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

[0080] As used herein, the expression "bispecific antigen-binding molecule" refers to a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. 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, different 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 a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "D1" and the CDRs of the second antigen-binding domain may be designated with the prefix "D2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; the CDRs 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 linked to a different multimerization domain. The binding of one multimerization domain to another multimerization domain promotes binding between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain can be linked to the immunoglobulin C H A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (C H 2-C H 3 domains), for example, the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotypes within each isotype group.

[0083] A bispecific antigen-binding molecule of the invention typically comprises two multimerization domains, e.g., two Fc domains, each part of an independent antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

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

[0085] Any bispecific antibody format or technology can be used to generate the bispecific antigen-binding molecules of the invention. For example, an antibody or fragment thereof with a first antigen-binding specificity can be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment with a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific examples of bispecific formats that can be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (OVO)-Ig, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEEO) body, leucine zipper, Ouobody, IgG1 / IgG2, dual acting Fab (OAF)-IgG, and Mab. 2 and bispecific formats (for a review of said formats see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0086] In the context of the bispecific antigen-binding molecules of the invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain with a modified binding interaction (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule contains a modification that increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome, where the pH ranges from about 5.5 to about 6.0). H 2 or C HNon-limiting 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 a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and a 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P).

[0087] The present invention also relates to a first C H 3 domain and second Ig C H and a bispecific antigen-binding molecule comprising a first and a second IgC domain. H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking that amino acid difference. H The 3 domain binds protein A and the second Ig C H The second C3 domain contains a mutation that reduces or abolishes protein A binding, such as, for example, the H95R modification (according to the IMGT exon numbering; H435R according to the EU numbering). H3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). Further modifications that may be found within the second CH3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V821 (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 according to EU); for IgG2 antibodies, N44S, K52N, and V821 (according to IMGT; N384S, K392N, and V4221 according to EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 according to EU).

[0088] In certain embodiments, the Fc domain can be a chimera that combines Fc sequences from multiple immunoglobulin isotypes. For example, the chimeric Fc domain can be a human IgG1, human IgG2, or human IgG4 C H C from 2 regions H 2 sequences and C derived from human IgG1, human IgG2 or human IgG4 H The chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence from a human IgG1, human IgG2 or human IgG4 hinge region combined with a "lower hinge" sequence from a 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, from the N-terminus to the C-terminus: [IgG4C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein includes, from the N-terminus to the C-terminus: [IgG1C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4C H 2]-[IgG1C H3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the invention are described in WO 2014 / 022540A1, the entire contents of which are incorporated herein by reference. Chimeric Fc domains having these general structural arrangements, and variants thereof, 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 regions 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 ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may include antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll framework and / or CDR residues within the domain are mutated to the residues found in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only those mutated residues found within the first 8 amino acids of FR1 or only those mutated residues found within the last 8 amino acids of FR4, or only those mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen binding domain was originally derived). Furthermore, the antigen binding domain may comprise any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained in this general manner are encompassed by the present invention.

[0090] The present invention also includes antigen binding molecules in which one or both of the antigen binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes antigen binding molecules comprising an antigen binding domain having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative 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 replaced 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 a protein. Examples of amino acid groups with 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 conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "slightly conservative" substitution 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 term "substantial identity" or "substantially identical," when referring to amino acid sequences, means that two amino acid sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the % sequence identity or similarity can be adjusted upwards to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331.

[0092] Polypeptide sequence similarity, also referred to 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 conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1, using 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) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of 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 anti-CD28 / anti-CD22 bispecific antigen-binding molecules with pH-dependent binding properties. For example, the anti-CD28 antibodies of the present invention may exhibit reduced binding to CD28 at acidic pH compared to neutral pH. Alternatively, the anti-CD22 antibodies of the present invention may exhibit enhanced binding to CD22 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, such as 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 term "neutral pH" refers to 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, "reduced binding of ... at acidic pH compared to neutral pH" refers to the K of antibody binding to that antigen at acidic pH. D The K value of antibody binding to its antigen at neutral pH D For example, an antibody or antigen-binding fragment thereof has an acidic / neutral K of about 3.0 or greater. D If a ratio is shown, then in the context of the present invention, the antibody or antigen-binding fragment thereof can be considered to exhibit "reduced binding to CD28 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K D The ratio can be 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 increased) binding to a particular antigen at acidic pH compared to neutral pH. Furthermore, modification of the antigen-binding domain at the amino acid level may result in antibodies with pH-dependent properties. For example, replacement of one or more amino acids in the antigen-binding domain (e.g., within the CDRs) with histidine residues may result in an antibody with decreased antigen binding at acidic pH compared to neutral pH. Antibodies Containing Fc Variants

[0096] According to certain embodiments of the invention, there are provided anti-CD28 / anti-CD22 bispecific antigen binding molecules comprising an Fc domain comprising one or more mutations that enhance or reduce antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides an Fc domain comprising a C-terminal region of the Fc domain, wherein the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome, where the pH ranges from about 5.5 to about 6.0). H 2 or C HThe present invention includes antibodies and antigen-binding molecules that contain the Fc mutations in the three regions. Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting 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., 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 positions 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 positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and a 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P).

[0097] For example, the invention includes anti-CD28 / anti-CD22 bispecific antigen binding molecules comprising an Fc domain that comprises 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). All possible combinations of the above Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated within the scope of the invention.

[0098] Biological properties of antibodies and antigen-binding molecules The present invention includes antibodies and antigen-binding fragments thereof that bind to human CD28 and / or CD22 with high affinity. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD28 and / or CD22 with moderate or low affinity depending on the therapeutic situation and the particular targeting properties desired. For example, in the context of a bispecific antigen-binding molecule in which one arm binds to CD28 and another 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 only moderate or low affinity. In this way, selective targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding global / non-targeted CD28 binding and the resulting adverse side effects associated therewith.

[0099] According to certain embodiments, the present invention provides a method for the treatment of cancer, comprising administering to a patient a therapeutic agent having a K of less than about 15 nM, as measured by surface plasmon resonance with human CD22 (e.g., at 25° C.), for example using an assay format as defined in Example 5 herein. D In certain embodiments, the antibodies or antigen-binding fragments of the invention have a K of less than about 15 nM, less than about 14 nM, less than about 13 nM, less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance using, for example, an assay format as defined in Example 5 herein, or a substantially similar assay. D Combine with.

[0100] According to certain embodiments, the present invention provides a method for the treatment of monkey CD22 (e.g., at 25° C.) with a K of less than about 60 μM as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein. DIn certain embodiments, the antibodies or antigen-binding fragments of the invention bind to monkey CD22 at 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, or less than about 60 μM, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein, or a substantially similar assay. K 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, less than about 15 μM, or less than about 10 μM D Combine with.

[0101] According to certain embodiments, the present invention provides a method for the production of antibodies that bind to human CD28 (e.g., at 25° C.) and have a K of less than about 45 μM as measured by surface plasmon resonance using an assay format, e.g., as defined in Example 5 herein. D In certain embodiments, the antibodies or antigen-binding fragments of the invention have 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, or less than about 10 μM with human CD28 as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein, or a substantially similar assay. D Combine with.

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

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

[0104] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD28 with a dissociation half-life (t1 / 2) of greater than about 2.3 minutes at 25°C as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein, or a substantially similar assay. In certain embodiments, an antibody or antigen-binding fragment of the invention binds to CD28 with a t1 / 2 of greater than about 2 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using an assay format as defined in the Examples herein, or a substantially similar assay.

[0105] The present invention also includes bispecific antigen-binding molecules (e.g., bispecific antibodies) capable of binding 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 a bispecific antigen-binding molecule binds to cells expressing CD28 and / or CD22 can be assessed by fluorescence-activated cell sorting (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 human cell lines or cynomolgus monkey cells (e.g., B cells or Nalm6 cells) that express CD22 but not CD28. The present invention provides a method for the production of bispecific antigen-binding molecules that specifically bind to any of the above cells and cell lines, as measured using a FACS assay as described in Example 6 or a substantially similar assay, at about 1.3×10 -6 ~Approx. 2.3×10 -8 M or lower EC 50 The present invention includes bispecific antigen-binding molecules that bind at different levels.

[0106] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) capable of binding to human CD28 and / or human 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 a bispecific antigen-binding molecule binds to cells expressing CD28 and / or CD22 can be assessed by flow cytometry, as shown in Example 7 herein. For example, the present invention includes bispecific antigen-binding molecules that specifically bind to human cells (e.g., T cells) that express CD28 but not CD22, and human cell lines that express CD22 but not CD28 (e.g., HEK293 cells transduced with human CD22 and Raji B cells genetically modified to lack CD80 and CD86). The present invention provides a method for the production of a bispecific antigen-binding molecule that specifically binds to any of the above cells and cell lines, as measured by flow cytometry or a substantially similar assay as described in Example 7, at about 1.14×10 -8 ~Approx. 9.76×10 -9 M or lower EC 50 The present invention includes bispecific antigen-binding molecules that bind at different levels.

[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 provides an anti-CD28 / anti-CD22 bispecific antigen binding molecule that induces or enhances the efficacy of CD20xCD3 T cell-mediated tumor cell killing of approximately 1.48×10 T cells, as measured in an in vitro T cell-mediated tumor cell killing assay, for example, using an assay format as defined herein in Example 8 (e.g., assessing the extent of Raji cell killing by human PBMCs in the presence of anti-CD20xCD3 and anti-CD28xCD22 antibodies), or a substantially similar assay. -10 EC below M 50In certain embodiments, the antibodies or antigen-binding fragments of the invention have 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, as measured by an in vitro T cell-mediated tumor cell killing assay, e.g., PBMC-mediated killing of Raji cells, e.g., using an assay format as defined in Example 8 herein, or a substantially similar assay. 50 Guided by value.

[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 activating T cells, inducing IL-2 release, inducing CD25+ upregulation in human PBMCs; and enhancing human T cell-mediated cytotoxicity for CD22-expressing cell lines (see, e.g., Example 9 herein). The present invention also includes anti-CD28 / anti-CD22 bispecific antigen-binding molecules that enhance killing of tumor cells expressing CD22 when combined with a bispecific antibody that binds CD20 and CD3, such as, but not limited to, REGN1979. The present invention also includes anti-CD28 / anti-CD22 bispecific antigen-binding molecules that enhance killing of tumor cells expressing CD22 when combined with an antibody that binds PD-1, such as, but not limited to, cemiplimab (see, e.g., Examples 10-15).

[0109] Epitope mapping and related techniques The epitope on CD28 or CD22 to which the antigen binding molecule of the present invention binds may consist of a single contiguous 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-contiguous amino acids (or amino acid sequences) of CD28 or CD22. The antibodies 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. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. An epitope may be either conformational or linear. A conformational epitope is generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is one generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on an antigen.

[0110] A variety of techniques known to those of skill in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within 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., Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), 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 the amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, in the hydrogen / deuterium exchange method, the protein of interest is deuterium-labeled, and then an antibody is bound 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 dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, which reveals the deuterium-labeled residues that correspond to the specific amino acids that the antibody interacts with.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 in the polypeptide that the antibody interacts with.

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

[0112] According to certain embodiments, the present invention provides antibodies and antigen-binding fragments of antibodies that bind to an epitope on human CD22 comprising 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 (e.g., antibodies comprising an amino acid sequence set forth in Table 6 herein) that compete with any of the specific exemplary antibodies described herein for binding to CD28 and / or CD22.

[0114] The present invention also includes bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds human CD28 and a second antigen binding fragment that specifically binds human CD22, where 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 bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds human CD28 and a second antigen binding fragment that specifically binds human CD22, where 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 can be easily determined whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as a reference antigen-binding molecule of the present invention or competes for binding by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CD28 (or CD22) as a reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first bound to CD28 protein (or CD22 protein). The ability of the test antibody to bind to CD28 (or CD22) molecule is then evaluated. If the test antibody can bind to CD28 (or CD22) after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of CD28 (or CD22) than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CD28 (or CD22) molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody can bind to the same epitope of CD28 (or CD22) 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 confirm whether the observed lack of binding of the test antibody is indeed 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. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the invention, two antigen binding proteins bind to the same (or overlapping) epitope if a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antigen binding protein inhibits binding of the other by at least 50%, preferably 75%, 90% or even 99%, e.g., as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antigen binding proteins are considered to have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other. Two antigen binding proteins are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other.

[0117] To determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the binding method is carried out in two directions: in the first direction, the reference antigen-binding molecule is allowed to bind to CD28 protein (or CD22 protein) under saturating 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 allowed to bind to CD28 (or CD22) molecules under saturating conditions, and then the binding of the reference antigen-binding molecule to the CD28 (or CD22) molecule is evaluated. If only the first (saturating) antigen-binding molecule can bind to the CD28 (or CD22) molecule in both directions, 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 that competes for binding with a reference antigen-binding molecule does not necessarily have 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 for a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD28 and CD22) can be appropriately positioned relative to each other to produce the bispecific antigen-binding molecules of the invention using routine methods. (Exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the invention are discussed elsewhere herein). In certain embodiments, one or more individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the 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 molecules of the invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technique), high affinity chimeric antibodies against a particular antigen (e.g., CD28 or CD22) with human variable regions and mouse constant regions are first isolated. Antibodies are characterized and selected for desirable properties including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0119] Genetically modified animals can be used to generate human bispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically modified mice can be used to generate fully human bispecific antigen-binding molecules that contain two different heavy chains that bind to the same light chain that contains a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, U.S. Application No. 2011 / 0195454, the entire contents of which are incorporated herein by reference, for a detailed discussion of such recombinant mice and their use to generate bispecific antigen-binding molecules.)

[0120] Bioequivalent The present invention encompasses antigen-binding molecules having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to CD28 and / or CD22. Such variant molecules contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described antigen-binding molecules. Similarly, the antigen-binding molecule-encoding DNA sequences of the present invention encompass sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antigen-binding molecules that are essentially biologically equivalent to the described antigen-binding molecules of the present invention. Examples of such variant amino acid 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, for example, if they are pharmaceutical equivalents or pharmaceutical substitutes and do not show significant differences in their absorption rates and extents when administered under similar experimental conditions, at the same molar dose, either in a single dose or multiple doses. Some antibodies may be considered equivalent or pharmaceutical substitutes if they have a comparable degree of absorption but not a comparable rate of absorption, but still be considered bioequivalent because such differences in absorption rates are intentional, reflected in the label, are not essential to achieve effective body drug concentrations, e.g., in chronic use, and are not medically significant for the particular pharmaceutical agent being investigated.

[0122] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0123] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without a clinically significant change in immunogenicity, or an expected increased risk of side effects, including a decrease in efficacy, when compared to continued treatment without such switching.

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

[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 that measure the concentration of the antibody or its metabolites 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 that measure the relevant acute pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0126] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by removing terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antibodies can include the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

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

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

[0129] immune complex The present invention encompasses antigen-binding molecules conjugated to a therapeutic moiety, such as, for example, a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope ("immunoconjugate"). Cytotoxic drugs include agents that are detrimental to cells. Examples of cytotoxic and chemotherapeutic drugs suitable for forming immunoconjugates are known in the art (see, for example, WO 05 / 103081, the entire contents of which are incorporated herein by reference).

[0130] Therapeutic Formulations and Administration The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. Many suitable formulations can be found in a formulary known to any pharmacist: 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., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. 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 age and physique of the patient, the target disease, the condition, the route of administration, and the like. A preferred dose is 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 an adult patient, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously at a single dose of usually about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering the bispecific antigen-binding molecule can be determined empirically; for example, the progress of the patient can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0132] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. BioI. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.

[0133] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been fully administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can be reused. A disposable pen delivery device does not have a replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0134] A number of reusable pen and autoinjector delivery devices are adapted for the immediate delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, the AUTOPEN™, the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN™ JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, ... Examples of disposable pen delivery devices applicable to subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR Pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLlCK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA Pen (Abbott Labs, Abbott Park IL), to name just a few.

[0135] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric 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 placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0136] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections and drip infusions. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by dissolving, suspending or emulsifying the antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Thus, the aqueous medium for injection includes, for example, saline, isotonic solutions containing glucose and other auxiliary agents, which can be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct) of hydrogenated castor oil], etc. As the oily medium, for example, sesame oil, soybean oil, etc. can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injections thus prepared are preferably filled into suitable ampoules.

[0137] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with a unit dose suitable for one dose of active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; in particular, in the form of injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, about 10 to about 250 mg.

[0138] Therapeutic Uses of Antigen-Binding Molecules The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds CD28 and a target antigen (e.g., CD22). The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules disclosed herein and a pharma- ceutically acceptable carrier or diluent. As used herein, the phrase "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that develops a tumor or is afflicted with 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 (as well as therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder in which stimulating, activating and / or targeting an 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 amelioration of any disease or disorder associated with or mediated by CD22 expression or activity or proliferation of CD22+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved includes the killing of cells expressing CD22 in the presence of effector cells, e.g., T cells. Cells expressing CD22 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 invention can be used to treat primary and / or metastatic tumors occurring, for example, in blood, bone marrow, lymph nodes (e.g., thymus, spleen), colon, liver, lung, breast, rectal cancer, central nervous system, and bladder cancer. According to an exemplary embodiment, the bispecific antigen binding molecules of the 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" refers to the presence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0142] According to certain aspects, the present invention provides methods of treating a disease or disorder associated with CD22 expression (e.g., a B cell proliferative disorder) comprising administering to a subject one or more of the bispecific antigen binding molecules described elsewhere herein after the subject has been shown to be non-responsive to other types of anti-cancer therapy. For example, the present invention encompasses methods of treating a B cell proliferative disorder comprising administering to the patient an anti-CD28 / anti-CD22 bispecific antigen binding molecule 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 more after the subject has received a standard of care treatment for patients suffering from cancer, e.g., a B cell proliferative disorder. In other embodiments, a bispecific antigen-binding molecule of the invention comprising an IgG4 Fc domain (anti-CD28 / anti-CD22 bispecific antigen-binding molecule) is first administered to a subject at one or more time points (e.g., to provide robust initial depletion of prostate cancer cells), followed by an equivalent bispecific antigen-binding molecule comprising a different IgG domain, e.g., an IgG1 Fc domain, at a later time point. It is contemplated that the anti-CD28 / anti-CD22 antibodies of the invention may be used in combination with other bispecific antigen-binding molecules, e.g., anti-CD20 / anti-CD3 bispecific antibodies. It is also contemplated that the bispecific antibodies of the invention are used in combination with checkpoint inhibitors, e.g., PD-1 and CTLA-4, as well as those targeting other targets. It may be advantageous to combine two bispecific antibodies that target the same tumor antigen (e.g., CD22), but where one of the bispecific antibodies targets CD3 on T cells and the other bispecific antibody targets the costimulatory molecule-like CD28. This combination may be used alone or in combination with a checkpoint inhibitor to enhance tumor cell killing.

[0143] Combination Therapies and Formulations 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 therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0144] Exemplary additional therapeutic agents that can be administered in conjunction or in combination with the antigen binding molecules of the invention include, for example, chemotherapy, radiation therapy, checkpoint inhibitors that target PD-1 (e.g., anti-PD-1 antibodies, e.g., pembrolizumab, nivolumab, or cemiplimab, see U.S. Pat. No. 9,987,500, HCVR / LCVR of SEQ ID NO: 162 / 170), co-targeting molecules such as CTLA-4, LAG3, TIM3, etc., GITR, OX40, 4-1BB, etc. Stimulatory agonist bivalent antibodies, CD3x bispecific antibodies (see, e.g., U.S. Pat. No. 9,657,102 (REGN1979), WO 2017 / 053856A1, WO 2014 / 047231A1, WO 2018 / 067331A1 and WO 2018 / 058001A1), other antibodies targeting CD22XCD3, CD22XCD28 or targeting CD20XCD3 and other co-stimulatory CD28x bispecific antibodies.

[0145] Other agents that may be beneficially administered in combination with the antibodies of the invention include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors, as well as antibodies that bind to 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 their respective receptors. Pharmaceutical compositions of the invention (e.g., pharmaceutical compositions comprising the anti-CD28 / anti-CD22 bispecific antigen binding molecules disclosed herein) may also be administered in combination with other agents, such as "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 (registered trademark)), cyclosporine (e.g., cyclosporine ... and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0146] The present invention also encompasses therapeutic combinations comprising any of the antigen binding molecules described herein and 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 one or more inhibitors of any of the above cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen-binding molecules of the present invention can also be administered in combination and / or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs. The antigen-binding molecules of the present invention can also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy, or treatment with biologics, including checkpoint inhibitors or other bispecific antibodies.

[0147] The present invention encompasses compositions and therapeutic formulations comprising any of the antigen binding molecules described herein in combination with one or more chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (Cytoxan™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenediamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolmelamine; methylamelamine; nitrogen mustards such as Chlorambucil, Chlornaphazine, Chlorophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan, Novemvitin, Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard; nitrosureas such as Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine antibiotics, such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, Peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenoceptors such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as folinic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;amsacrine;bestravcil;bisantrene;edalaxate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate;etoglu cido;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;pirarubicin;podophyllic acid;2-ethylhydrazide;procarbazine;PSK(trademark);razoxane;sizofiran;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosid ("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, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine;and pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included within this definition are antihormonal agents that act to modulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharma-ceutically acceptable salts, acids, or derivatives of any of the foregoing.;

[0148] The additional therapeutically active ingredient may be administered immediately prior to, simultaneously with, or immediately following administration of the antigen binding molecule of the present invention; (in the context of the present disclosure, such an administration regimen is considered administration of the antigen binding molecule "in combination with" the additional therapeutically active ingredient).

[0149] The present invention encompasses pharmaceutical compositions in which the antigen-binding molecules of the invention are co-formulated with one or more of the additional therapeutically active ingredient(s) described elsewhere herein. Dosing regimen

[0150] According to certain embodiments of the 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 period of time. The method according to this aspect of the invention comprises sequentially administering multiple doses of the antigen-binding molecule of the invention to a subject. As used herein, "sequentially administering" means administering each dose of the antigen-binding molecule to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention encompasses methods comprising sequentially administering a single initial dose of the antigen-binding molecule, followed by one or more second doses of the antigen-binding molecule, optionally followed by one or more third doses of the antigen-binding molecule.

[0151] The terms "first dose", "second dose", and "third dose" refer to the temporal order of administration of the antigen-binding molecules of the present invention. Thus, the "first dose" is the dose administered at the beginning of a treatment regimen (also called the "baseline dose"); the "second dose" is the dose administered after the first dose; and the "third dose" is the dose administered after the second dose. The first dose, the second dose, and the third dose may all contain the same amount of antigen-binding molecule, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecule contained in the first dose, the second dose, and / or the third dose varies from each other during the course of treatment (e.g., adjusted up or down as necessary). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0152] In an exemplary embodiment of the invention, each of the second and / or third doses is administered 1 to 26 (e.g., 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) weeks after the immediately preceding dose. The term "immediately preceding dose" as used herein means a dose of an antigen-binding molecule administered to a patient in a series of multiple doses prior to administration of the next dose in the sequence without any intervening doses.

[0153] The method according to this aspect of the invention may include administering 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 CD22 and CD28) to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, 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 with the same frequency as the other second doses. For example, each second dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple third doses, each third dose may be administered with the same frequency as the other third doses. For example, each third dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the second and / or third doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted over the course of treatment by a physician according to the needs of an individual patient after clinical testing.

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

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

[0157] In general, suitable doses of the antigen-binding molecules of the present invention can be in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight, and generally in the range of about 1 to 50 mg per kilogram of body weight. For example, the antibody or antigen-binding fragment thereof, or the bispecific antigen-binding molecule can be administered at 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, about 50 mg / kg per single dose. Intermediate values ​​and ranges of the recited values ​​are also contemplated as part of the present invention.

[0158] In some embodiments, the antigen-binding molecule of the present invention is administered at a fixed dose of about 25 mg to about 2500 mg. In some embodiments, the antigen-binding molecule of the present invention is administered at a fixed dose 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, about 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, and administered as a fixed dose of about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0159] Diagnostic Uses of Antibodies The bispecific antibodies of the invention can also be used to detect and / or measure CD28 or CD22, or CD28- or CD22-expressing cells, in a sample, e.g., 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 aberrant expression (e.g., overexpression, underexpression, underexpression, etc.) of CD28 or CD22. An exemplary diagnostic assay for CD28 or CD22 can include, for example, contacting a sample obtained from a patient with an antibody of the 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 can be a radioisotope, e.g., 3 H, 14 C. 32 P, 35 S, or 125I; fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or enzyme, such as alkaline phosphatase, beta-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 assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Samples that can be used in the CD28 or CD22 diagnostic assay according to the present invention include any tissue or fluid sample that can be obtained from a patient that contains a detectable amount of CD28 or CD22 protein, or fragments thereof, under normal or pathological conditions. In general, the level of CD28 or CD22 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal CD28 or CD22 levels or activity) is measured to first establish a baseline or standard level of CD28 or CD22. 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 being suffering from a CD28- or CD22-associated disease or condition. EXAMPLES

[0160] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of the inventors' contemplated invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0161] Example 1 Construction of anti-CD22xCD28 antibodies Generation of anti-CD28 antibodies Anti-CD28 antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., genetically engineered mice containing DNA encoding human immunoglobulin heavy and kappa 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. Antibody immune responses were monitored by CD28-specific immunoassays. 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. The hybridoma cell lines were screened and selected to identify cell lines producing CD28-specific antibodies. Using this technique, several anti-CD28 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) 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 US Application No. 2007 / 0280945A1.

[0162] Certain biological properties of exemplary anti-CD28 antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0163] Generation of anti-CD22 antibodies Anti-CD22 antibodies were obtained by immunizing genetically modified mice (VELOCIMMUNE® mice, see above) with human CD22 antigen (see, e.g., hCD22 ecto (D20-R687).hFc, R&D Systems, Catalog# 1968-SL-050; Accession# CAA42006 (see also FIG. 3)) or by immunizing genetically modified mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions with human CD22 antigen.

[0164] After immunization, spleen cells were collected from each mouse and either (1) fused with mouse myeloma cells to preserve their viability, forming hybridoma cells, and screened for CD22 specificity, or (2) sorted for B cells using human CD22 fragments as sorting reagents that bind and identify reactive antibodies (antigen-positive B cells) (as described in U.S. Application No. 2007 / 0280945A1).

[0165] Chimeric antibodies against CD22 with human variable regions and mouse constant regions were first isolated. The antibodies were characterized and selected for desired properties including affinity, selectivity, etc. If necessary, the mouse constant regions were replaced with the desired human constant regions, e.g., wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-CD22 antibodies. The constant region selected can vary depending on the particular application, but the high affinity antigen binding and target specificity properties reside in the variable regions.

[0166] Generation of a bispecific antibody that binds both CD28 and CD22 Bispecific antibodies comprising 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 comprise a different distinct HCVR paired with a common LCVR. In some cases, the bispecific antibodies were constructed utilizing a heavy chain from an anti-CD28 antibody, a heavy chain from an anti-CD22 antibody and a common light chain (see Table 1).

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

[0168] Example 2 Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-CD22 antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. Table 1: Amino acid sequence identifiers of CD22 antibodies [Table 1] Table 2: Nucleic acid sequence identifiers for CD22 antibodies [Table 2]

[0169] Table 3 lists the amino acid sequence identifiers of the heavy and light chain variable regions (HCVR and LCVR), CDRs of selected anti-CD28 antibodies of the 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 for CD28 antibodies [Table 4]

[0170] A summary of the component parts of the various anti-CD22xanti-CD28 bispecific antibodies that were constructed is set forth in Table 5. Tables 6 and 7 set forth the HCVRs, LCVRs, CDRs, and heavy and light chain sequence identifiers of the bispecific antibodies. Table 5: Summary of components of anti-CD22xanti-CD28 bispecific antibodies [Table 5]

[0171] Table 6 shows the amino acid sequence identifiers of the bispecific anti-CD22xanti-CD28 antibodies described herein. The corresponding nucleic acid sequence identifiers are set forth in Table 7. Table 6: Amino acid sequences of anti-CD22xanti-CD28 bispecific antibodies [Table 6] Table 7: Nucleic acid sequences of anti-CD22xanti-CD28 bispecific antibodies [Table 7] Example 3: Epitope Mapping of REGN5837 Binding to CD22 by Hydrogen-Deuterium Exchange

[0172] H / D exchange epitope mapping with mass spectrometry (HDX-MS) 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 SEQ ID NO: 2 / 10) (anti-hCD22 monoclonal antibody; parental anti-hCD22 antibody of REGN5837). Overviews of the H / D exchange methodology are described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0173] 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.

[0174] Labeling solutions were prepared as PBS buffer in D2O at pD7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent 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) at a 1:0.6 molar ratio was incubated in duplicate with 44 μl of D2O labeling solution at 20°C for various time points (e.g., non-deuterated control = 0 s; deuterium labeling for 5 and 10 min). The deuteration reaction was quenched by adding 55 μl of pre-chilled quenching buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample and incubated at 20°C for 5 min. The quenched samples were then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were separated on a C8 column (1.0 mm x 50 mm, NovaBioassays) using a 13 min 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] The Byonic search engine (Protein Metrics) was used to search the LC-MS / MS data of non-deuterated CD22 samples for databases containing CD22 and its randomized sequences. Search parameters (in ELN units) were set using non-specific enzymatic digestion as default and human glycosylation as a common variable modification. The list of identified peptides was then imported into HDX Workbench software (version 3.3) to calculate the deuterium uptake of each peptide detected by LC-MS from all deuterated samples. For a given peptide, the centroid mass (intensity-weighted average mass) at each time point was used to calculate deuterium uptake (D) and percentage of deuterium uptake (%D) (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 in complex with H4sH33037P2 (HCVR / LCVR pair of SEQ ID NO:2 / 10) samples, resulting in 92.0% sequence coverage of hCD22. Peptides showing a D incorporation percentage difference of more 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 binding to CD22 by hydrogen-deuterium exchange

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

[0178] 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 at pD7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent 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 premixed with H4sH33041P2 at 1:0.6 molar ratio (Ag-Ab complex) was incubated in duplicate with 44 μl of D2O labeling solution at 20°C for various time points (e.g., non-deuterated control = 0 s; deuterium labeling for 5 and 10 min). The deuteration reaction was quenched by adding 55 μl of pre-chilled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample and incubated at 20°C for 5 min. The quenched samples were then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were separated on a C8 column (1.0 mm x 50 mm, NovaBioassays) using a 13 min 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] The Byonic search engine (Protein Metrics) was used to search the LC-MS / MS data of non-deuterated CD22 samples for databases containing CD22 and its randomized sequences. Search parameters (units ELN) were set using non-specific enzymatic digestion as default and human glycosylation as common variable modification. The list of identified peptides was then imported into 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 centroid mass (intensity-weighted average mass) at each time point was used to calculate deuterium incorporation (D) and percentage of deuterium incorporation (%D) as described below.

number

[0181] A total of 454 peptides from hCD22.mmH (SEQ ID NO:50) were identified from both hCD22.mmH alone and hCD22.mmH in complex with H4sH33041P2 samples, resulting in 90.5% sequence coverage of hCD22. Peptides showing a percent difference in D incorporation value of greater than 5% were defined as 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 kinetic constants of CD22xCD28 bispecific antibodies derived by surface plasmon resonance

[0182] The equilibrium dissociation constants (K D Values) (see Table 1, mAB33037P2; HCVR / LCVR: SEQ ID NO:2 / 10) and mAb33041P2; HCVR / LCVR: SEQ ID NO:18 / 10) were determined using real-time surface plasmon resonance biosensors using Biacore T-200 or Biacore 4000 instruments. CM5 Biacore sensor surfaces were 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). The Biacore binding studies were performed in a buffer composed of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 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 90nM to 3.33 or 0.37nM, 3-fold dilutions) bearing a C-terminal myc.myc hexahistidine tag ("hexahistidine" disclosed as SEQ ID NO: 60) prepared in HBS-EP running buffer were injected over the mAb capture surface at a flow rate of 30μl / min. Binding of CD22.mmH (SEQ ID NO: 50) to the captured monoclonal antibody was monitored for 5 minutes and dissociation of CD22.mmH in HBS-EP running buffer was monitored for 10 minutes. All binding kinetics experiments were performed at 25°C. Kinetic association (k a ) and dissociation (k dThe binding / dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows: K D (M)=k d / k a , and t1 / 2(min)=0.693 / k d / 60

[0183] The binding kinetic parameters of human and cynoCD22 binding to purified mAbs at 25° C. are shown below in Tables 10-12.

[0184] hCD28.mmH (SEQ ID NO: 54) Equilibrium dissociation constant (K DValues) were determined using a real-time surface plasmon resonance biosensor using 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 parental mAb (see above). The Biacore binding studies were performed in a buffer consisting of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Different concentrations of hCD28 (ranging from 600 nM to 2.47 nM, 3-fold dilutions) with a C-terminal myc.myc hexahistidine tag ("hexahistidine" disclosed as SEQ ID NO: 60) prepared in HBS-EP running buffer were injected over the mAb capture surface at a flow rate of 50 μl / min. Binding of CD28.mmH (SEQ ID NO: 54) to the captured monoclonal antibody was monitored for 5 min, and dissociation of CD28.mmH in HBS-EP running buffer was monitored for 10 min. All binding kinetic experiments were performed at 25°C. Kinetic association (ka) and dissociation (kd) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows: K D (M)=k d / k a , and t1 / 2(min)=0.693 / k d / 60

[0185] The binding kinetic parameters of human CD28 binding to purified mAbs at 25° C. are shown below in Table 13. [Table 10] Table 10: Human CD22.mmH binding kinetics to anti-CD22xCD28 bispecific mAb at 25°C. Table 11: Monkey CD22.mmH (XP_005588899.1) binding kinetics to anti-CD22xCD28 bispecific mAb at 25°C [Table 11] Table 12: Monkey CD22.mmH (EHH59463.1) binding kinetics to anti-CD22xCD28 bispecific mAb at 25°C. [Table 12-1] [Table 12-2] Table 13: Human CD28.mmH binding kinetics to anti-CD22xCD28 bispecific mAb at 25°C. [Table 13] Example 6 Binding specificity of anti-CD28 and anti-CD22xCD28 bispecific antibodies to a target cell line (Nalm6), an effector cell line (Jurkat), and cynomolgus monkey T and B cells using flow cytometry

[0186] Flow cytometry analysis was used to determine the binding of CD22xCD28 bispecific antibodies to human CD22-expressing Nalm6 cells and human CD28-expressing Jurkat T cells, as well as cynomolgus monkey T (CD28+) and B (CD22+) cells. Briefly, 1×10 5Cells / well were incubated for 30 min at 4° C. with serial dilutions of CD22xCD28 bispecific antibody or H4sH15260P (isotype control human IgG4 antibody that binds human antigens without cross-reactivity to human or cynomolgus CD28 or CD22) ranging from 133 nM to 61 pM for Jurkat and Nalm6 cells. Cynomolgus PBMCs were incubated with a single antibody at 67 nM concentration. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS and PE-conjugated anti-human secondary antibody was added to the cells and incubated for an additional 30 min. Additional phenotyping antibody cocktails (anti-CD2, anti-CD20, anti-CD16, anti-CD14) were added to wells containing cynomolgus PBCM. Wells containing no antibody or only secondary antibody were used as controls.

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

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

[0189] REGN5837 binds to Nalm6 cells and ECs 50 REGN5838 bound to Nalm6 cells with an EC50 value of 1.8E-08M. The isotype control antibody showed no binding to cell lines expressing CD22.

[0190] REGN5837 binds to Jurkat T cells and ECs50 Value 2.1E-08M. REGN5838 bound to Jurkat T cells. 50 Value 2.3E-08M. The isotype control antibody showed no binding to cell lines expressing CD28.

[0191] Table 15 presents the binding data 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 from 12 of 12 cynomolgus monkeys and to T cells from 11 of 12 monkeys tested. Binding to CD20+ B cells ranged from 12.6-30.3-fold higher than secondary cells, with a median of 15.7-fold higher. Binding to CD2+ / CD16- T cells was 1.2-5.2-fold higher than secondary cells, with a median of 3.5-fold higher. Positive binding was defined as >1.2-fold higher than secondary cells. REGN5838 bound to B cells from 12 of 12 cynomolgus monkeys and to T cells from 11 of 12 monkeys tested. Binding to CD20+ B cells ranged from 6.5-13.5-fold higher than secondary cells, with a median of 9.3-fold higher. Binding to CD2+ / CD16- T cells was 1.2-4.7-fold higher than secondary cells, with a median of 3.8-fold higher. Positive binding was defined as >1.2-fold over secondary cells. Isotype control antibodies showed no binding to cynomolgus T or B cells. Table 14: Binding and fold binding results of flow cytometry experiments on recombinant target and effector cells. [Table 14] Table 15: Fold binding results of 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 Flow cytometry analysis was used to identify effector cells expressing human CD28 for the CD22xCD28 bispecific (REGN5837; REGN5838) and control antibodies (human CD4 + Binding to human CD22-expressing target cells (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 in D-PBS + 2% FBS. Subsequent steps were performed according to the SepMate manufacturer's protocol. CD4 + T cells were then isolated from PBMCs using the Miltenyi Biotec human CD4 Microbead kit according to the manufacturer's instructions. + T cells, 5 x 10 per vial 6 The cells were frozen in FBS containing 10% DMSO at a concentration.

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

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

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

[0198] Cells were stained as follows.

[0199] Briefly, human CD4+ T cells, HEK293 / hCD20, HEK293 / hCD20 / hCD22 and Raji / CD80 and CD86 negative cells were resuspended in staining buffer containing D-PBS + 2% FBS. Raji cells were incubated with mouse IgG (625 mg / mL final) to block endogenous Fcγ receptors. Briefly, 2 × 10 5Cells / well were incubated with serial dilutions of antibodies ranging from 6.1 pM to 100 nM at 4 °C for 30–60 min. A negative control sample without antibody was included. Cells were washed once with cold staining buffer and incubated with Allophycocyanin (APC)-conjugated anti-human secondary antibody for 30–45 min. 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 the manufacturer's instructions to distinguish between live and dead cells. Cells were then fixed in BD Cytofix Buffer according to the manufacturer's instructions, washed, resuspended in staining buffer, and analyzed by flow cytometry on an iQue Screener flow cytometer. EC 50 To determine geometric mean fluorescence intensity (MFI) values, analysis was performed using a four-parameter logistic equation on a nine-point response curve using GraphPad Prism. Fold binding was calculated using the following formula: Binding fold = maximum geometric mean within the dose range tested MFI value Background geometric MFI value [0 nM]

[0200] The ability of the CD22xCD28 bispecific antibody to bind human CD22 and CD28 was examined by immunohistochemistry using primary human CD4+ cells that endogenously overexpress CD22 (HEK293 / hCD20 / hCD22) or endogenously (Raji / CD80 and CD86 negative). + T cells and engineered cells were assessed by flow cytometry. A negative cell line was included as a control (HEK293 / hCD20).

[0201] EC 50 The / fold binding values ​​are summarized in FIG. Table 16: Human CD4 + EC of flow cytometry experiments on T cells and engineered target cells 50 and multiple combination results: [Table 16] Abbreviations: NC = not calculable (denoted for curves where binding did not reach saturation); ND = not determined Table 16 Human CD4 + EC of antibodies against T cells and engineered cell lines, e.g., HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative B cells 50 and a table of multiple binding values.

[0202] As expected, none of the CD28 antibodies, parental (REGN5705; HCVR / LCVR SEQ ID NO:35 / 36) or its bispecific format (REGN5837, REGN5838 and the one-arm CD28 control (SEQ ID NO:48) bound to negative HEK293 / hCD20 cells. Weak binding of approximately 1.8x at the highest concentration was detected with the isotype control antibody due to non-specific binding (Figure 1 and Table 16).

[0203] Binding of anti-CD22xanti-CD28 antibodies was 16.44x for REGN5837 and 34.9x for REGN5838, EC 50 11.4 nM) and Raji / CD80 and CD86 negative cells (38.35x for REGN5837, EC 50 Approximately 9.76nM, 81.74x for REGN5838, EC 50 Approximately 14.9 nM) was observed. No significant binding was detected with the one-arm CD28 and isotype controls (Figure 1 and Table 16).

[0204] Binding of antibodies targeting human CD28 is inhibited by primary human CD4 + The parent CD28 antibody, REGN5705, had an EC of approximately 4.13 nM over background. 50 REGN5837, REGN5838, and the one-arm control bound 37.48x, while the bispecific antibodies REGN5837, REGN5838, and the one-arm control bound 9.25x, 10.63x, and 10.97x, respectively. As expected, the isotype control did not bind to the cells (Figure 1 and Table 16). Example 8 Anti-CD22xCD28 Bispecific Antibody Costimulation Enhances Anti-CD20xCD3 Bispecific Antibody-Induced Targeted Cytotoxicity, T Cell Activation, and Cytokine Release

[0205] CD22xCD28 enhancement of CD20xCD3-targeted killing was assessed in a 96-h cytotoxicity assay targeting Raji cells engineered to lack expression of CD80 and CD86 (Raji-80 / 86DKO). Briefly, human PBMCs were cultured at 1 × 10 6 Lymphocytes were enriched for by plating 1000 cells / mL and incubating overnight at 37° C. to remove adherent macrophages, dendritic cells, and some monocytes. The following day, Raji-80 / 86DKO cells were labeled with 1 uM of the fluorescent tracking dye CFDA-SE and adherent cell-depleted naïve PBMCs were labeled with 1 uM of the fluorescent tracking dye CellTrace Violet. Labeled target cells and PBMCs (effector / target cell 10:1 ratio) were co-incubated with serial dilutions (concentration range: 5nM-0.64pM) of CD20xCD3 bispecific antibody REGN1979, with one heavy chain arm composed of SEQ ID NO: 42, the other heavy chain arm composed of SEQ ID NO: 43, and a light chain of SEQ ID NO: 44, and fixed concentrations of CD22xCD28 costimulatory molecules REGN5837 or REGN5838, one-arm control CD28 bispecific (REGN5678), or IgG4s isotype control (H4sH10154P3, isotype control with HCVR / LCVR pair of SEQ ID NO: 37 / 38) at 2.5ug / ml (16.7nM) for 96 hours at 37°C. Cells were harvested from the plates and analyzed by FACS on a FACS BD LSRFortessa-X20. For FACS analysis, cells were stained with Fixable Live / Dead Far Red reactive (Invitrogen) dye. 20,000 counting beads were added to each well immediately prior to FACS analysis, and 10,000 beads were collected per sample. To assess specificity of killing, cells were gated on the live CFDA-SE labeled population. The percent of 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 proliferate, CellTraceViolet is diluted and the MFI measured by FACS decreases. Thus, 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 Values ​​were calculated using four-parameter nonlinear regression analysis in Prism software.

[0207] Supernatants from this assay were collected for analysis of cytokine levels. Concentrations of IL17a, IFNγ, TNFα, IL-10, IL-6, IL-4, and IL-2 were analyzed using Cytometric Bead Array (CBA) kits according to the manufacturer's instructions. Cytokine levels were interpolated from curves generated by kit standards and reported as pg / mL. Maximum cytokine levels were calculated using a four-parameter nonlinear regression analysis in Prism software.

[0208] The results of an assay evaluating the ability of the anti-CD20xCD3 bispecific antibody REGN1979 (see above) to induce unstimulated human T cells to kill target cells expressing human CD20 and CD22 in combination with a costimulatory CD22xCD28 antibody or a one-arm CD28 or isotype control antibody were examined.

[0209] REGN1979 activated human T cells and dose-dependently depleted Raji cells that lacked CD80 and CD86 expression. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 reduced the cytotoxic potency (EC 50 ) was enhanced 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 on CD8+ cells or CellTrace Violet dilution, respectively. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced REGN1979-induced T cell activation and proliferation potential by 2.1-2.6-fold and 7.4-8.4-fold, respectively, when compared to REGN1979 with one-arm CD28 or isotype control antibody (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 a fixed concentration of CD22xCD28 costimulatory molecule with a fixed concentration of one-arm CD28 or an isotype control antibody (Table 18).

[0212] Taken together, costimulation increased the potential for targeted cytotoxicity, T cell activation, and cytokine release compared to that observed with CD20xCD3 in combination with a control antibody. Table 17: EC of cytotoxicity and T cell activation 50 Values ​​(average of three experiments) [Table 17-1] [Table 17-2] Table 18: Cytokine release (pg / mL) [Table 18]

[0213] Example 9: Bioassay of CD22 Bispecific Antibodies T cell activation is achieved by stimulating the T cell receptor (TCR), which recognizes specific peptides presented by tumor histocompatibility complex class I or II (MHCI or MHCII) proteins on antigen-presenting cells (APCs) (Goldrath et al. 1999). The activated TCR then initiates a cascade of signaling events that can be monitored by various transcription factors, such as activator-protein 1 (AP-1), nuclear factor of activated T cells (NFAT) or nuclear factor kappa-light-chain enhancer of activated B cells (NFκB). The T cell response is then further refined by the engagement of co-receptors, such as CD28, CTLA-4 (cytotoxic T lymphocyte protein 4), PD-1 (programmed cell death protein 1), LAG-3 (lymphocyte-activation gene 3) or other molecules that are constitutively or inducibly expressed on T cells (Sharpe et al. 2002). The costimulatory molecule CD28 is activated by its endogenous ligands, CD80 or CD86, expressed on APCs. CD28 enhances cell signals, such as pathways controlled by NFκB transcription factors, after TCR activation. CD28 costimulatory signals are important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death (Smeets et al. 2012).

[0214] Anti-CD22xCD28 bispecific antibodies were used in luciferase-based reporter bioassays and primary human CD4 + T cells were characterized using an IL-2 functional assay.

[0215] Luciferase-based reporter assays : A T cell / APC (antigen presenting cell) luciferase-based reporter assay was developed to assess the effect of CD28 activation on NFκB activity through the engagement of an anti-CD28xanti-CD22 bispecific antibody.

[0216] Engineering reporter T cells: Clonal reporter T cell lines were engineered 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 supplemented with 1 μg / mL puromycin.

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

[0218] Human Raji B cells (ATCC#CCL-86), which endogenously express CD20, CD22, Fcγ receptors (FcγR), CD80 and CD86 on the cell surface, were genetically engineered by deletion of CD80 and CD86 using CRIPSR technology. CD80 and CD86 are known ligands for CD28. 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 by two bispecific antibodies. The first stimulus is delivered by T cell activating bispecific antibody REGN2281 (anti-CD20xanti-CD3 antibody with one heavy chain arm composed of SEQ ID NO: 39, one heavy chain arm composed of SEQ ID NO: 40, and a light chain arm of SEQ ID NO: 41) and targets CD3 molecules on engineered reporter T cells and CD20 on HEK293 or Raji / CD80 and CD86 negative B cells. The first stimulus avoids the need for activation of TCRs by their ligands, specific peptides presented on MHC molecules. The second stimulus is driven by a CD28 bispecific antibody (i.e., anti-CD28xanti-CD22 bispecific antibody). This antibody mimics CD28 activation of T cells by its ligands CD80 / CD86 expressed on APCs. It interacts with CD28 on T cells and CD22 on HEK293 cells or Raji / CD80 and CD86-negative B-cells, driving activation of CD28 on engineered reporter T cells. TCR and CD28 coactivation leads to enhanced transcriptional activity of NFκB, which increases production of the reporter gene, luciferase.

[0220] Luciferase assay setup: RPMI1640 supplemented with 10% FBS and P / S / G was used as assay medium to prepare cell suspensions and antibody dilutions for screening of anti-CD22xanti-CD28 bispecific antibodies.

[0221] One day before screening, engineered reporter T cells were cultured in cell culture medium at 0.5 × 10 6 Cells / mL. Serially diluted 1:3 anti-CD28xanti-CD22 bispecific antibodies and controls 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 of SEQ ID NO:45 / 46). 10-point dilutions ranged from 15 pM to 100 nM, with the final dilution containing no CD28 antibody.

[0222] Reagents were added in the following order: 1) a fixed concentration of 200 pM final of REGN2281 (anti-CD20 x anti-CD3, see above) or REGN1945 (hIgG4 isotype control, see above) 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 resuspended in 1000 mM NaCl (2×10 cells / well) and 2×10 6 cells / mL (final cell concentration 5 x 10 4 3) serially diluted antibodies were added to the corresponding wells; 4) 2 × 10 reporter T cells cultured overnight were added to the corresponding wells; 6 Resuspend at a final concentration of 5 x 10 / mL. 4 Cells / well were added to the plates. Plates were incubated for 4-6 hours at 37°C / 5% CO2, after which 100 μL of ONE-Glo™ (Promega) reagent was added to lyse the cells and detect luciferase activity. Emitted light was captured in relative light units (RLU) on a multilabel plate reader Envision (PerkinElmer). All serial dilutions were tested in duplicate. Tested.

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

number

[0224] Primary human CD4 + IL-2 functional assay using T cells : Primary CD4 + A T cell / APC functional assay was developed to assess the effect of CD28 activation on IL-2 production upon engagement with an anti-CD22xanti-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 following 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 leukocytes diluted 1:2 in D-PBS + 2% FBS. Subsequent steps were performed according to the SepMate manufacturer's protocol. CD4 + T cells were then isolated from PBMCs using the Miltenyi Biotec human CD4 Microbead kit according to the manufacturer's instructions. + T cells, 5 x 10 per vial 6 The cells were frozen in FBS containing 10% DMSO at a 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 of CD3 on their surface using an anti-CD20xanti-CD3 bispecific antibody (REGN2281, see above) in combination with either HEK293 cells engineered to express human CD20 or Raji cells expressing endogenous CD20, where CD80 and CD86 have been silenced using CRISPR technology (Raji / CD80 and CD86 negative cells). Binding of the CD20 arm of REGN2281 to CD20-expressing cells drives clustering of the CD3 receptor, providing the first signal required for T cell stimulation. Importantly, in some cases, co-culturing primary leukocytes with genetically distinct cells can result in mismatch of allogeneic determinants, resulting in activation of T cells. This allows for sufficient primary stimulation to be provided without exogenously added anti-CD20xanti-CD3 bispecific antibody. Regardless of the primary stimulation, costimulation, which can be provided by crosslinking CD28 molecules, is required to detect quantifiable IL-2 release. Here, a bispecific CD28 antibody (i.e., an anti-CD28x anti-CD22 bispecific antibody) is used to bind to CD4 + It interacts with CD28 on T cells and CD22 on HEK293 / hCD20 or RAJI / CD80 and CD86 negative cells, driving CD28 clustering-activation. Combined TCR and CD28 engagement enhances IL-2 production and release into the cell culture medium. IL-2 is detected and quantified from cell supernatants using a homogenous no-wash AlphaLisa kit from PerkinElmer.

[0227] Pre-isolated and frozen human CD4 + T cells were thawed on the day of the assay in stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME containing 50 U / ml benzonase nuclease). Cells were centrifuged at 1200 rpm for 10 min, resuspended in stimulation medium, and plated at 1 × 10 in 96-well round-bottom plates. 5 HEK293 cells engineered to express human CD20 alone or in combination with human CD22 were plated at a concentration of 10 × 10 cells / well in primary stimulation medium.6 The Raji / CD80 and CD86 negative cells were treated with 15 μg / mL mitomycin C at a concentration of 10 × 10 6 After incubation at 37°C and 5% CO2 for 1 h, HEK293 and Raji cells were washed three times with D-PBS containing 2% FBS and then incubated for 1 h at 37°C for 1 h at 37°C. After incubation at 37°C and 5% CO2 for 1 h at 37°C ... + Add 1 × 10 to wells containing T cells 4 5 x 10 cells / well for HEK293 cells or Raji / CD80 and CD86 negative cells 4 The plates were added at a final concentration of 1000 ng / cell. Anti-CD28xanti-CD22 bispecific or control antibodies serially diluted 1:3 ranging from 15 pM to 100 nM were then added to the wells in the presence of 2 nM REGN2281 (anti-CD20xanti-CD3) or REGN1945 (negative hIgG4 isotype control, see above). The final 10-point dilutions did not contain CD28 antibodies. The plates were incubated for 72 hours at 37°C and 5% CO2, then centrifuged to pellet the cells and 40 μL of media supernatant was collected. From this, 5 μL was tested in the human IL-2AlphaLISA assay according to the manufacturer's protocol. Measurements were acquired on a multilabel plate reader Envision and raw RFU (relative fluorescence units) values ​​were plotted. All serial dilutions were tested in duplicate.

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

number

[0229] 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 was assessed in a reporter cell-based bioassay using luciferase activity as readout.

[0230] Activation curves are shown in Figure 2 (A and B) 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), and EC 50 and the fold induction 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 line used in the assay. An increase in luciferase activation was only observed with the parental CD28 antibody (REGN5705) and HEK293 / hCD20 cells (2.18x) and HEK293 / hCD20 / hCD22 cells (2.05x). One-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-CD22 x anti-CD28 bispecific antibodies (REGN5837 and REGN5838) suppressed EC 50 and induced strong luciferase activity in CD22-expressing HEK293 cells as indicated by increased fold induction values. The one-arm CD28 control antibody and the parental CD28 antibody (REGN5705; see HCVR / LCVR SEQ ID NO:35 / 36) showed similar activity against both HEK293 lines. The isotype control antibody 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 were treated with 200 pM REGN1945, both anti-CD22xanti-CD28 bispecific antibodies (REGN5837 and REGN5838) as well as the parental CD28 antibody induced luciferase activity, whereas the one-arm CD28 control antibody and the isotype control showed no activity (Table 19 and Figure 2).

[0234] When reporter T cells and Raji / CD80 and CD86 negative were treated with 200 pM REGN2281, all CD28 bispecific antibodies (REGN5837 and REGN5838), including the parental CD28 antibody, and the one-arm CD28 control antibody induced luciferase activity. EC 50 Values ​​could be determined for the one-arm CD28 control as saturation levels were not reached. No activation was detected with the isotype control (Table 20 and Figure 2). Primary human CD4 + IL-2 functional assay using T cells:

[0235] The ability of the anti-CD22xanti-CD28 bispecific antibody to provide costimulation through CD28 on T cells in the absence or presence of CD22 target expression was assessed using functional primary CD4 T cells measuring IL-2 cytokine production. + was assessed by T cell assay.

[0236] The activation curves are shown in Figure 3 (A and B), and EC 50 and fold induction values ​​were HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80. and CD4+ cells co-incubated with CD86-negative cells in the presence of either 2 nM REGN1945 (hIgG4 isotype control) or REGN2281 (anti-CD20 x anti-CD3). + T cells are summarized in Table 21.

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

[0238] When a constant 2 nM concentration of REGN2281 and the parental 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, IL2 release was not dramatically enhanced when anti-CD22xanti-CD28 bispecific mAb was added to wells containing HEK293 / hCD20 cells and REGN2281. 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 to 22 are set forth below.

[0240] Table 19 shows the EC20 luciferase activity in HEK293 / hCD20, HEK293 / hCD20 / hCD22 or RAJI / CD80 and CD86 negative cells and engineered T cells co-incubated with a constant 200 pM REGN1945 (isotype control). 50 A table of values, maximum values ​​and fold induction values ​​is presented.

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

[0242] Table 21 shows the CD4+ / CD86+ expression levels of HEK293 / hCD20, HEK293 / hCD20 / hCD22, or RAJI / CD80 and CD86 negative cells co-incubated with a constant 2 nM REGN1945 (isotype control). + ECs of IL-2 release from T cells 50 A table of values, maximum values ​​and fold induction values ​​is presented.

[0243] Table 22 HEK293 / hCD20, HEK293 / hCD20 / hCD22, Raji / CD80 and CD86 negative cells and CD4 co-incubated with 2 nM REGN2281 (anti-CD20 x anti-CD3) + ECs of IL-2 release from T cells 50 A table of values, maximum values ​​and fold induction values ​​is presented. Table 19: EC of luciferase activity in engineered reporter T cells in the absence of TCR stimulation with 200 pM REGN1945 (isotype control) 50 Values, maximum values ​​and derivatives: [Table 19] Abbreviations: ND = not determined Table 20: EC of luciferase activity in engineered reporter T cells in the presence of TCR stimulation with 200 pM REGN2281 (αCD20xαCD3) 50 Values, maximum values ​​and derivatives: [Table 20] Abbreviations: NC = not calculable (denoted for curves where the response did not reach saturation); ND = not determined. Table 21: Primary human CD4 in the presence of 2 nM REGN1945 (isotype control) + EC of IL-2 release from cells 50 Value, maximum value and induction factor value [Table 21] Abbreviations: NC = not calculable (denoted for curves where the response did not reach saturation); ND = not determined. Table 22: Primary human CD4 in the presence of 2 nM REGN2281 (αCD20xαCD3) + ECs of IL-2 release from T cells 50 Value, maximum value and induction factor value [Table 22] Abbreviations: NC = not calculable (denoted for curves where the response did not reach saturation); ND = not determined. Example 10: Effect of the combination of anti-CD22X anti-CD28 antibody + cemiplimab 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 (ATCC® CCL-86™). RAJI is maintained in RPMI+10% FBS+P / S / G+NaPyr+HEPES.

[0245] RAJI CD80 and CD86 negative Expression of CD80 and CD86 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 effusion 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 engineered to stably express human PD-L1 (amino acids M1-T290 of accession 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 supplemented with 0.5 μg / mL puromycin for RAJI / CD80-CD86- and 1 μg / mL puromycin for NALM-6 / PD-L1 and WSU-DLCL2 / PD-L1 cells.

[0249] T cell activation assays for T cell proliferation and IL-2 release The effect of REGN5837 on IL-2 release was evaluated in the presence of a fixed concentration of cemiplimab 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 dissimilar cells may result in mismatch of allogeneic determinants and consequent T cell activation. For assays using NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1) cells, T cell activation assays were performed with enriched human primary T cells from three donors, whereas assays utilizing WSU-DLCL2(+ / -PD-L1) cells used T cells from one donor.

[0250] Isolation of T cells for use in T cell activation assays to test combination treatment of REGN5837+REGN2810. For experiments utilizing NALM-6 and RAJI / CD80-CD86- cells, CD3+ T cells were isolated from PBMCs of three donors (555109, 555130, and 555131), and for assays with 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 overlaying with 30 ml of blood diluted 1:1 in PBS with 2% FBS. After centrifugation at 400xg for 30 min without brake, the mononuclear cell layer was transferred to a new tube, diluted 5x in PBS with 2% FBS, and centrifuged at 300xg for 8 min. For donors 555130, 555131, and 555175, PBMCs were isolated from peripheral blood of healthy donors using the EasySep Direct Human PBMC Isolation Kit from Stem Cell Technologies according to the manufacturer's protocol. Isolated PBMCs were frozen in FBS containing 10% DMSO. For CD3+ T cell isolation, frozen vials of PBMCs were thawed in a 37°C water bath and diluted in stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / ml benzonase nuclease. Cells were centrifuged at 1200 rpm for 10 min, resuspended in EasySep buffer, and isolated using the StemCell 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 stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) were plated in 96-well round-bottom plates at 1 × 10 5 NALM-6, RAJI / CD80-CD86-, and WSU-DLCL2 cells with or without PD-L1 (+ / -PD-L1) were treated with mitomycin C at 20 μg / mL (RAJI) or 15 μg / mL (NALM-6 and WSU-DLCL2) to stop proliferation. After 1 h incubation at 37°C and 5% CO2, mitomycin C-treated cells were washed three times with D-PBS containing 2% FBS and then resuspended in stimulation medium. NALM-6, RAJI / CD80-CD86-, and WSU-DLCL2 cells (+ / -PD-L1) cells were plated at a concentration of 10 x 10 for RAJI and WSU-DLCL2 cells, 2.5 x 10 for RAJI and WSU-DLCL2 cells, and 2.5 x 10 for NALM-6 and WSU-DLCL2 cells. 4 cells / well, 5 x 10 for NALM-6 cells 4 A fixed concentration of cemiplimab or non-binding IgG4 was added to wells containing CD3+ T cells at a final concentration of cells / well. P Controls (20 nM) were added to the wells. For assays using WSU-DLCL2 (+ / -PD-L1) cells, a fixed concentration of belatacept (hCTLA4.hIgG1) or a non-binding IgG1 control (50 nM) was added to the wells. REGN5837 or non-TAAxCD28 control antibodies were then titrated in a 1:4 dilution series from 3.1 pM to 200 nM for NALM-6 (+ / -PD-L1) cells and in a 1:6 dilution series from 0.6 pM to 1000 nM for WSU-DLCL2 and RAJI / CD80-CD86- (+ / -PD-L1) cells and added to the wells. The end points of the 10-point concentration curve did not include REGN5837 or non-TAAxCD28 control antibodies. Plates were incubated for 72 hours (WSU-DLCL2(+ / -PD-L1)) or 96 hours (NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1)) at 37°C, 5% CO2, after which 50 μL of culture supernatant was collected to measure IL-2 release.

[0252] For IL-2 release, 5 μL of supernatant was tested using the Human IL-2 Alpha LISA kit according to the manufacturer's protocol. IL-2 measurements were taken on a Perkin Elmer multilabel plate reader Envision. 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. Antibody EC50 values ​​were determined from a four-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism™ software. Maximum levels of IL-2 release are given as the average maximum response detected within the dose range tested. Additionally, data reported for assays using WSU-DLCL2 cells include IL-2 produced in the absence or presence of 1000 nM titrating antibody to capture the decrease in IL-2 observed with increasing concentrations of non-TAAxCD28 antibody.

[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 that endogenously express CD22 was assessed in a functional primary CD3+ T cell assay measuring IL-2 cytokine production.

[0255] The 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 ​​were determined using a constant 20 nM hIgG4 PTable 23A summarizes CD3+ T cells incubated with NALM-6 (+ / -PD-L1) cells and Table 23B summarizes CD3+ T cells incubated with RAJI / CD80-CD86- (+ / -PD-L1) cells in the presence of either isotype control or cemiplimab. Activation curves are shown in Figure 4 for CD3+ T cells incubated with WSU-DLCL2 (+ / -PD-L1) cells. Constant 20 nM hIgG4 P For T cells incubated with WSU-DLCL2 (+ / -PD-L1) cells in the presence of either isotype control or cemiplimab, and in the presence of either a constant 50 nM hIgG1 isotype control or the CTLA-4 receptor, belatacept, EC50 and IL-2 values ​​(reported for 0 nM or 1000 nM REGN5837 or non-TAAxCD28) are summarized in Table 24.

[0256] In the presence of human primary T cells and the allogeneic B cell lymphoid cell lines RAJI / CD80-CD86- and NALM-6, REGN5837 increased IL-2 release in a concentration-dependent manner. A non-TAAxCD28 control antibody slightly increases 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 cemiplimab has no effect on IL-2 release. In the presence of RAJI / CD80-CD86- or NALM-6 cells expressing PD-L1, the maximum IL-2 released in response to treatment with REGN5837 alone is reduced compared to T cells incubated with non-PD-L1 expressing cells. The addition of cemiplimab slightly enhanced REGN5837-mediated IL-2 release in conditions with NALM-6 / PD-L1 cells, but in the presence of RAJI / CD80-CD86- / PD-L1 cells significantly enhanced IL-2 release to levels observed in conditions with PD-L1-deficient, RAJI / CD80-CD86- cells.

[0257] No concentration-dependent increase in IL-2 release was observed in the presence of human primary T cells and the allogeneic B cell lymphocyte cell line WSU-DLCL2. Conversely, the non-TAAxCD28 control antibody was observed to reduce IL-2 release in a concentration-dependent manner. Unlike NALM-6 and RAJI / CD80-CD86- cells, which express little or no CD28 ligand, the WSU-DLCL2 cell line is known to express CD28 ligand. Since the CD28 binding arm of REGN5837, and thus 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 and reduces IL-2 release. Unlike the non-TAAxCD28 control, IL-2 is not reduced by REGN5837 due to its ability to anchor to WSU-DLCL2 cells via its CD22 binding arm, making it behave similarly to the CD28 ligand and essentially displacing them. 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. Addition of REGN5837 in the absence of cemiplimab slightly enhances IL-2 release. Addition of 20 nM cemiplimab enhances basal activity, which can be further enhanced slightly with dose escalation of REGN5837, with max IL-2 release being higher for the combination of REGN5837 and cemiplimab than either treatment alone. As observed with WSU-DLCL2 cells, incubation of WSU-DLCL2 / PD-L1 cells in the presence of non-TAAxCD28 enhances IL-2 release in response to cemiplimab or hIgG4. PIL-2 levels are reduced whether or not the isotype control is present. To further explore the effect of CD28 ligand expression on masking the effects of REGN5837, soluble CTLA-4 receptor belatacept, or hIgG1-matched isotype control, was added at 50 nM. Belatacept binds with high affinity to CD28 ligands, CD80 and CD86, blocking their interaction and thus the activation of CD28. In the presence of 50 nM belatacept, basal IL-2 release is dramatically reduced due to the inability of CD28 ligand to bind CD28 and provide costimulatory signaling. Even under these conditions, REGN5837 is able to bind CD28 and provide costimulation, as seen by the dose-dependent enhancement of IL-2 release. The addition of 20 nM cemiplimab alone does not enhance IL-2 release in the presence of belatacept, but combining cemiplimab with increasing doses of REGN5837 increases max IL-2 release compared to REGN5837 alone in the presence of cells engineered to overexpress PD-L1. Table 23A: Combination of REGN5837 with cemiplimab enhances IL-2 release above 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 and therefore not determined. Table 23B: REGN5837 in combination with cemiplimab inhibits RAJI / CD80 - CD86 - In cells, it enhances IL-2 release above REGN5837 treatment alone. [Table 23B-1] [Table 23B-2] NC: not calculated as data could not be fitted with a four-parameter logistic equation. Table 24: Combination of REGN5837 with cemiplimab enhances IL-2 release above REGN5837 treatment alone in WSU-DLCL2 cells engineered to express PD-L1. [Table 24] ND: No concentration-dependent response was observed and therefore not determined. NC: not calculated as data could not be fitted with a four-parameter logistic equation.

[0258] Example 11 Antitumor Efficacy of REGN5837 Administration in the Presence and Absence of REGN1979 introduction REGN5837 is a CD22 + B cells and CD28 + REGN5837 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., deliver a signal via TCR or CD3 clustering upon primary T-cell stimulation), such as the CD20xCD3 bispecific antibody (bsAb) REGN1979, can provide amplified T-cell activation and T-cell-mediated killing of B-cell NHL, deepening the response to CD20xCD3. Additionally, REGN5837 may increase efficacy in patients who do not respond to CD20xCD3 monotherapy.

[0259] The studies described below evaluated the antitumor efficacy of CD22xCD28 bsAb REGN5837 in the presence or absence of a subeffective amount of CD20xCD3 bsAb (REGN1979) administered to immunodeficient NSG mice bearing day 8 established B cell leukemia tumors.

[0260] Briefly, mice (n=6-9 per group) were engrafted intraperitoneally (IP) with human peripheral blood mononuclear cells (PBMCs) and intravenously (IV) 12 days later with human NALM-6 B-cell leukemia cells engineered to express luciferase to enable bioluminescence imaging (NALM-6-luc). The antitumor efficacy of 0.04, 0.4, and 4 mg / kg REGN5837 in combination with a fixed dose of 0.04 mg / kg REGN1979 was compared with REGN5837 and REGN1979 monotherapy as well as with non-crosslinked IgG4. P-PVA Mice were administered the antibodies by intraperitoneal (IP) injection 8, 15, and 22 days after implantation of NALM-6-luc cells. Tumor burden was assessed twice weekly for the duration of the experiment.

[0261] Materials and Methods Human 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 imaging of tumor cell growth in vivo.

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

[0263] Experimental design Testing Systems Female NSG mice (8-9 weeks old) were used in all experiments. All mice were engrafted IP with human PBMCs and then IV transplanted with NALM-6-luc B cell leukemia cells 12 days post-engraftment. The experimental design is detailed in Table 25. Tumor growth was monitored by bioluminescence imaging twice weekly over the study period. For all experiments, mice were housed under standard conditions in the Regeneron animal facility. All experiments were performed in accordance with the guidelines of the Regeneron Animal Care and Use Committee.

[0264] Engraftment of NSG mice 4 × 10 7 Human PBMCs were engrafted IP. Retro-orbital collection of blood and flow cytometry revealed human CD45 expression in total viable cells in whole blood. + T cell levels were checked 11 days after evaluation of the percentage of cells; engraftment levels ranged from 0.16 to 16% hCD45 + PBMC-engrafted NSG mice were subsequently transplanted with NALM-6-luc cells.

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

[0266] NALM-6-luc cells were collected by centrifugation and diluted to 2.5 × 10 in PBS. 7 Twelve days after PBMC engraftment, NSG mice were administered 200 μl (5 × 10 6 cells) were injected IV with NALM-6-luc cells.

[0267] Antibody administration for tumor measurement Prior to administration of test articles or controls, mice were assigned to groups stratified according to tumor burden and T cell engraftment levels. Prior to administration of test articles or controls, mice were assigned to groups stratified according to tumor size and T cell engraftment levels. 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 at 8, 15, and 22 days (for in vivo efficacy) post-implantation at the doses listed in the table. Table 25: Experimental design for assessing tumor growth [Table 25] a One mouse in this group died early during the study and was excluded, this death was not due to tumor burden and was unlikely to be related to test article dosing as one mouse had died in the control group.

[0268] Tumor Measurements and Specified Endpoints Mice implanted with NALM-6-luc tumors were imaged twice weekly using an IVIS Spectrum instrument, and data were analyzed using Living Image software. Prior to imaging, mice were IP injected with luciferin substrate. Ten minutes later, mice were anesthetized with isoflurane, and bioluminescence (total flux, expressed as photons per second [p / s]) was quantified. According to IACUC criteria, experiments were terminated when mice began to show signs of graft-versus-host disease (GVHD) (assessed as ≥20% weight loss).

[0269] Statistical analysis of tumor growth The time course of tumor volume results were analyzed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons. Differences were considered statistically significant when p<0.05. Statistical analysis was performed using GraphPad Prism software (version 8). result Antitumor efficacy of REGN5837 administered in the presence and absence of REGN1979 Immunodeficient NSG mice bearing NALM-6-luc tumors were injected IP with antibodies as above or non-crosslinked controls.

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

[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 post-dose on day 7, 1 hour pre-dose and 4 hours post-dose on days 14 and 21, and once on day 29. During the dosing period, trough concentrations of antibodies in serum were measured 1 hour pre-dose on days 14 and 21. During the dosing period, trough concentrations of antibodies in serum were measured 1 hour pre-dose on days 14 and 21. Administration of REGN5837 doses of 0.04, 0.4, and 4 mg / kg in the presence of REGN1979 at 0.04 mg / kg 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. Serum trough concentrations of REGN1979 were at BLQ in all cases (data not shown).

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

[0273] Example 12: FACS-based cytotoxicity against CD22 cells + human PBMC + / - CD22xCD28 costimulatory bispecific antibody (fixed CD22xCD28, titrated CD20xCD3) Materials and Methods CD22xCD28 enhancement of CD20xCD3-targeted killing was assessed in a 96-hour cytotoxicity assay targeting Nalm6 or WSU-DLCL2 cells. Briefly, human PBMCs were cultured at 1 × 10 6Lymphocytes were enriched for by plating 1000 cells / mL and incubating overnight at 37°C to remove adherent macrophages, dendritic cells, and some monocytes. The following day, Nalm6 or WSU-DLCL2 cells were labeled with 1 uM of the fluorescent tracking dye CFDA-SE and adherent cell-depleted naive PBMCs were labeled with 1 uM of the fluorescent tracking dye CellTrace Violet. Labeled target cells and PBMCs (effector / target cell 4:1 ratio for Nalm6 and 5:1 for WSU-DLCL2) were co-incubated with serial dilutions of the CD20xCD3 bispecific antibody REGN1979 (concentration range: 5 nM-0.64 pM) with or without CD22xCD28 REGN5837 at a fixed concentration of 2.5 ug / ml (16.7 nM). In assays targeting Nalm6 cells, a fixed amount of CD22xCD28REGN5838, 2.5ug / ml (16.7nM) of one-arm control CD28 bispecific (REGN5678) or IgG4s isotype control (H4sH10154P3) was added. After 96 hours of incubation at 37°C, cells were harvested from the plate and analyzed by FACS on a FACS BD LSRFortessa-X20. For FACS analysis, cells were stained with 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 killing, cells were gated on the 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, decreasing the MFI measured by FACS. T cell proliferation was therefore reported as a decrease in the MFI of CellTraceViolet in CD8+ T cells, or as the percentage of CD8+ cells that decreased the CellTraceViolet MFI.

[0275] Supernatants from this assay were collected for analysis of cytokine levels. Concentrations of IL17a, 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 generated by 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 a four-parameter nonlinear regression analysis in Prism software.

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

[0277] REGN1979 activated human T cells to kill Nalm6 (Figure 7) or WSU-DLCL2 (Figure 8) cells in a dose-dependent manner. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the cytotoxic potency (EC50) of REGN1979 by 4.7-5.2-fold against Nalm6 cells when compared to REGN1979 and one-arm CD28 or isotype control antibody (Table 26) or by 17.5-fold against WSU-DLCL2 cells when compared to REGN1979 alone (Table 27).

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

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

[0280] Taken together, costimulation increased the likelihood of targeted cytotoxicity, T cell activation, and cytokine release compared to that observed with CD20xCD3 in combination with a control antibody or alone. Data summary table: Table 26: EC50 values ​​for cytotoxicity and T cell activation on Nalm6 targets (single experiment) [Table 26] Table 27: EC50 values ​​for cytotoxicity and T cell activation on WSU-DLCL2 targets (average of two experiments) [Table 27-1] [Table 27-2] Table 28: Cytokine release from WSU-DLCL2 cytotoxicity assay (average of two experiments) [Table 28]

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

[0282] T cells were gated as live CD45+ Violet labeled CD4+ or CD8+ populations. The percentage of CD8+ and CD4+ cells expressing CD25 was reported as a measure of T cell activation. Furthermore, as T cells proliferate, CellTraceViolet is diluted and the MFI measured by FACS decreases. T cell proliferation was therefore reported as a decrease in the MFI of CellTraceViolet on CD8+ and CD4+ T cells.

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

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

[0285] REGN1979 activated human T cells and depleted NHL in a dose-dependent manner. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the cytotoxic potency (EC50) of REGN1979 by 2.3 and 3.5 fold when compared to REGN1979 plus one-arm CD28 control antibody or no costimulation control (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 on CD8+ and CD4+ cells, respectively. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the REGN1979-induced T cell activation and proliferation potential by 2.8-4.8-fold and 2.8-4.2-fold, respectively, when compared to REGN1979 with one-arm CD28 or isotype control antibody (Table 29 and FIG. 10).

[0287] Taken together, costimulation increased the potential for targeted cytotoxicity and T cell activation compared to that observed with CD20xCD3 in combination with a control antibody. Data summary table: Table 29: EC50 values ​​for cytotoxicity and T cell activation [Table 29-1] [Table 29-2] Example 14 In Vitro Characterization and In Vivo Evaluation 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 of the study and summary of results In vitro and in vivo studies were performed to evaluate:

[0288] (1) CD28 + T cells expressing CD22 + The ability of REGN5837 to enhance activation of primary T cells by cross-linking 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 performed in the presence or absence of CD20xCD3bsAb, which crosslinks target cells and leads to T cell activation.

[0289] (2) Antitumor efficacy of CD22xCD28bsAb REGN5837 in the presence of 0.4 or 4 mg / kg REGN1979 administered to immunodeficient NSG mice bearing DLBCL tumors.

[0290] REGN5837 and REGN1979 were tested in combination at various concentrations to evaluate the effect of REGN5837 on REGN1979-mediated T cell cytotoxicity against a human DLBCL cell line (WSU-DLCL2), upregulation of a marker of late T cell activation (CD25), T cell proliferation, and cytokine release from primary human T cells. REGN5837 inhibited T cell cytotoxicity, CD4 + and CD8 + CD25 cell-surface expression on T cells, as well as CD4 + and CD8 +REGN5837 concentration-dependently enhanced the potential of REGN1979 to mediate T cell proliferation. Similarly, REGN5837 concentration-dependently enhanced the potential of REGN1979 to mediate cytokine release. At concentrations ranging from 77.2 pM to 100 nM, REGN5837 increased the potential of REGN1979-mediated T cell cytotoxicity against target cells; at concentrations ranging from 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 enhance the potential of REGN1979 (Table 30). Addition of REGN5837 did not substantially increase the maximum amount of REGN1979-mediated target cell killing and T cell proliferation, but 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 implanted subcutaneously (SC) with a 1:1 ratio of WSU-DLCL2 cells and human PBMCs. The antitumor efficacy of REGN5837 at 1 mg / kg in combination with REGN1979 at doses of 0.4 or 4 mg / kg was compared with REGN5837 and REGN1979 monotherapy, as well as with non-crosslinked IgG4 P-PVA Mice were administered antibodies by intraperitoneal (IP) injection 1, 8, and 15 days after implantation of WSU-DLCL2 cells. Treatment with 1 mg / kg REGN5837 in the presence of 0.4 or 4 mg / kg REGN1979 resulted in statistically significant inhibition of WSU-DLCL2 tumor growth by 28 days after implantation compared to REGN5837 or REGN1979 monotherapy and non-crosslinked control bsAb. REGN1979 monotherapy resulted in modest inhibition of tumor growth, while REGN5837 monotherapy was ineffective compared to the non-crosslinked control.

[0292] In summary, when REGN5837 and REGN1979 were tested in combination at various concentrations in vitro, REGN5837 inhibited CD22 +Enhanced the potential of REGN1979 to mediate human T cell activation in the presence of WSU-DLCL2 cells. Maximal levels of REGN1979-mediated cytokine release, but not cytotoxicity, T cell activation, or proliferation, were increased in the presence of REGN5837. In vivo, REGN5837 at 1 mg / kg in the presence of either 0.4 or 4 mg / kg REGN1979 was more effective at 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 on 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 fold change in EC 50 (Without REGN5837) / EC 50 ([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 Both in vitro and in vivo studies were performed to evaluate the antitumor efficacy of human CD22xCD28 bsAb REGN5837 in the presence or absence of sub-effective doses of human CD20xCD3 bsAb (REGN1979) by measuring the following parameters in NSG mice after transplantation with human PBMC and WSU-DLCL2 cells: a) CD22 + T-cell cytotoxicity against target cells; b) CD4 +and CD8 + upregulation of CD25, a marker of T cell activation, on the cell surface of T cells; 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 effusion of a 41-year-old Caucasian male (Leibnitz Institute-DSMZ, Cat.#ACC575).

[0295] Human PBMCs For cytotoxicity, T cell activation, T cell proliferation, and cytokine release assays, leukopaks from a human donor were obtained from the New York Blood Center (donor #1500A).

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

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

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

[0299] In Vitro Assessment of the Effects of REGN5837 on the Potential of REGN1979 to Mediate T Cell Activation 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 following 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 tubes were centrifuged at 1200xg for 10 min at room temperature with the brake removed. 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 collected in 5 mL cryovials at 250 x 10 6 PBMCs were frozen in FBS containing 10% DMSO at a concentration of 100 cells / mL. PBMCs were thawed in a 37°C water bath, resuspended in stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / mL benzonase nuclease at 10 mL / 100 million PBMCs, and centrifuged at 300 x g for 10 min. CD3 + T cells were obtained from StemCell Technologies' EasySep™ human CD3 + T cells were isolated from pelleted PBMCs using a T cell isolation kit following the manufacturer's recommended instructions. PBMC 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 test substances and control antibodies as described herein. Flow cytometry was performed to evaluate upregulation of markers of T cell cytotoxicity, proliferation, and T cell activation as described herein. In addition, the effect of REGN5837 on REGN1979-mediated cytokine release was evaluated as described herein. A non-TAAxCD28 bsAb (containing the same CD28 binding arm as REGN5837, and a non-binding arm) was tested as a non-crosslinking control for REGN5837.

[0301] Lymphocyte enrichment of PBMCs Human PBMCs were cultured at 1 × 10 in complete medium (RPMI cell culture medium supplemented with 10% FBS, penicillin-streptomycin-glutamine). 6 The cells were plated at 1000 cells / mL and incubated overnight at 37° C. to enrich for lymphocytes by removing adherent cells such as macrophages, dendritic cells, and some monocytes.

[0302] Incubation of PBMCs and target cells with test substances Lymphocyte-enriched PBMCs were collected and labeled with 1 μM Violet Cell Tracker fluorescent tracking dye. WSU-DLCL2 target cells were labeled with 1 μM of the fluorescent dye Vybrant CFDA-SE.

[0303] The dye-labeled PBMCs were then mixed with dye-labeled target cells (5 × 10 3 Target cells / well (WSU-DLCL2) were plated in 96-well round-bottom plates.

[0304] Plated PBMCs and target cells were incubated for 72 hours at 37° C. with test substances 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 test articles and controls, dye-labeled cells were stained with a cocktail of LIVE / DEAD stains and fluorophore-labeled antibodies against CD2, CD4, CD8, and CD25. Counting beads (20 μl / well) were added immediately prior to sample analysis on a BD Celesta flow cytometer. Flow cytometry data were used to measure upregulation of markers of target cell survival, T cell proliferation, and T cell activation. EC 50 Values ​​were determined from a four-parameter logistic equation on a nine-point dose-response curve using GraphPad Prism software. Maximal responses for cytotoxicity, T cell activation (CD25 upregulation), and proliferation were determined as the maximum response plateau obtained by fitting the Prism curves. EC 50 The relative change in wa is the EC50 No REGN5837 / EC50 [M] REGN5837 The relative change in maximum cytokine release was calculated as Max [M] REGN5837 / Max No REGN5837 It was calculated as:

[0306] target cell survival The percentage of viable target cells in each experimental condition was calculated as the number of live CFDA-SE-labeled target cells / well normalized to the number of beads collected / well. The percentage of target cell viability was determined as the ratio of the number of viable target cells in any experimental condition to the number of viable target cells in the no-antibody control condition (target cells in the presence of PBMCs only).

[0307] The percentage of cytotoxicity against target cells in each experimental condition thus reported was determined as 100 minus the percent viability (calculated as above).

[0308] CD4 + and CD8 + CD25 expression on T cells Upregulation of CD25 (a marker of late activated T cells) is a marker of live, CD2 + , and CD4 + or CD8 + The percentage of activated T cells expressing CD25 among total T cells expressing either CD4 or CD8 was reported.

[0309] CD4 + and CD8 + T cell proliferation Primary CD4 + and CD8 + T cell proliferation was determined by total CD4 + and CD8 + The percentage of divided T cells was assessed using flow cytometry by calculating the percentage of divided cells. The fluorescence intensity of cells stained with Violet Cell Tracker was used as a readout for cell division, since the fluorescence intensity of each cell decreases by 2-fold with each division.

[0310] Cytokine release analysis Cytokine (IL-4, IL-2, IL-6, IL-10, TNF-α, IFN-γ, and IL-17α) levels in cell culture supernatants 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 implanted SC with WSU-DLCL2 B cell lymphoma cells and administered IP with antibodies. Tumor growth was measured using calipers several times a week for the duration of the study. For all experiments, mice were housed in the Regeneron animal facility under standard conditions. All experiments were performed at Regeneron in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

[0312] WSU-DLCL2 cell culture conditions and tumor implantation The WSU-DLCL2 cell line was obtained from the 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 Cells) were collected and 5 x 10 5 PBMCs were mixed and resuspended in a 1:1 mixture of PBS and GFR Matrigel. 100 μl of the cell mixture was injected SC into the right flank of female NSG mice.

[0314] Antibody administration for tumor measurement Prior to administration of test substances or controls, mice were assigned to groups stratified according to tumor burden.

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

[0316] Tumor Measurements and Specified Endpoints Tumor growth was monitored over time using tumor caliper measurements X and diameter Y (perpendicular measurement of length and width). Tumor volume was calculated (X*Y*[X / 2], where X is the minor diameter). Mice were euthanized when tumors reached the designated tumor endpoint (tumor diameter >20 mm or tumor ulceration). This endpoint was in accordance with IACUC criteria.

[0317] Statistical analysis of tumor growth and survival Tumor volume results over time were analyzed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons. Survival results over time were analyzed using Mantel-Cox test across all groups and compared with Mantel-Cox test for each group. Differences were considered statistically significant when p<0.05. Statistical analysis was performed using GraphPad Prism 8 software.

[0318] result The effect of REGN5837 on the ability of REGN1979 to mediate T cell cytotoxicity against WSU-DLCL2 target cells and cytokine release from human PBMCs REGN5837 and REGN1979 were tested in combination at various concentrations to evaluate the effect of REGN5837 on REGN1979-mediated cytotoxicity against WSU-DLCL2 cells, T cell activation, T cell proliferation, and cytokine release from T cells derived from human PBMCs, as previously described. Table 30 shows the effect of REGN5837 on REGN1979-mediated cytotoxicity against WSU-DLCL2 cells, T cell activation, and T cell proliferation. Table 32 summarizes the numerical results from two human donors showing the effect of REGN5837 on cytokine release.

[0319] Effect of REGN5837 on REGN1979-mediated cytotoxicity and human T cell proliferation REGN1979-mediated cytotoxicity against WSU-DLCL2 target cells, REGN1979-mediated T cell activation, and human CD4 from human PBMCs + and CD8 + The efficacy of REGN1979 (EC 50 The effect of increasing concentrations of REGN5837 on cytotoxicity against WSU-DLCL2 cells, T cell activation (CD4 + and CD8 + T cells), as well as CD4 + and CD8 + Concentration-dependent enhancement of the potential of REGN1979 to mediate T cell proliferation. At concentrations ranging from 77.2 pM to 100 nM, REGN5837 increased the potential of REGN1979-mediated T cell toxicity against target cells; at concentrations ranging from 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 of FIG. 11 and Table 30.

[0320] REGN1979-mediated cytokine release from human PBMCs in the presence of REGN5837 The effect of increasing concentrations of REGN5837 on the potential and maximum response of REGN1979-mediated cytokine release from human PBMCs was evaluated. Increasing concentrations of 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-17A in the presence of human PBMCs and WSU-DLCL2 cells (FIG. 12). Furthermore, increasing concentrations of REGN5837 tended to enhance the potential of REGN1979 to mediate cytokine release. The EC 50Values, maximum cytokine levels, and relative increase over background cytokine levels (without REGN5837) are summarized in FIG. 12 and Table 32. Table 32 [Table 32-1] [Table 32-2] Antitumor efficacy of REGN5837 administered in the presence and absence of sub-effective doses of REGN1979

[0321] Immunodeficient NSG mice bearing WSU-DLCL2 tumors were injected IP with antibodies previously described herein or a non-crosslinking control.

[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 inhibition of tumor growth compared to non-crosslinked control bsAbs (non-TAAxCD28 and non-TAAxCD3 bsAbs) by day 28 (6 days after the last antibody dose) (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 compared to REGN1979 monotherapy by day 46.

[0323] REGN1979 monotherapy at both 0.4 and 4 mg / kg resulted in modest tumor inhibition compared to non-crosslinked controls by day 28, whereas REGN5837 monotherapy was ineffective. Rapid tumor growth was observed upon administration of non-crosslinked control bsAb throughout the dosing period, and all mice were euthanized at day 125.

[0324] A Mantel-Cox test detected a statistically significant difference in survival across all groups (p=0.0001), and additional Mantel-Cox tests were performed for group comparisons. A significant increase in survival was observed for mice receiving 1 mg / kg REGN5837 in combination with either 0.4 or 4 mg / kg REGN1979 (85% and 70% survival, respectively) compared to mice receiving a non-crosslinking control antibody (no survival) (FIG. 14).

[0325] Furthermore, a significant increase in survival was observed in mice receiving 1 mg / kg REGN5837 in combination with either 0.4 or 4 mg / kg REGN1979 compared to mice receiving either REGN5837 or REGN1979 monotherapy.

[0326] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become 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) a first antigen-binding domain (D1) that binds to human CD28; b) a second antigen-binding domain (D2) that specifically binds to human CD22 on target tumor cells; 1. An isolated bispecific antigen-binding molecule comprising: (Item 2) 2. The isolated bispecific antigen-binding molecule of item 1, wherein the second antigen-binding domain (D2) binds to an epitope on human CD22 comprising 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 has a K of less than about 15 nM as measured by surface plasmon resonance at 25° C. D 3. The isolated bispecific antigen-binding molecule of item 1 or 2, which binds to human CD22 at (Item 4) The bispecific antigen-binding molecule has a K of less than about 60 μM as measured by surface plasmon resonance at 25° C. D 4. The isolated bispecific antigen-binding molecule of any one of items 1 to 3, which binds to Macaca fascicularis CD22 at 100 nm. (Item 5) The bispecific antigen-binding molecule has a K of less than about 45 μM as measured by surface plasmon resonance at 25° C. D 5. The isolated bispecific antigen-binding molecule of any one of items 1 to 4, which binds to human CD28 at (Item 6) The bispecific antigen-binding molecule has a binding affinity of about 1×10 -8 EC below M 50 6. The isolated bispecific antigen-binding molecule of any one of items 1 to 5, which binds to the surface of a cell expressing human CD28 at 100 .mu.m. (Item 7) The bispecific antigen-binding molecule has a binding affinity of about 1×10 -8 EC below M 50 7. The isolated bispecific antigen-binding molecule of any one of items 1 to 6, which binds to the surface of a cell expressing human CD22 at 100 .mu.m. (Item 8) 8. The isolated bispecific antigen-binding molecule of any one of items 1 to 7, wherein the bispecific antigen-binding molecule exhibits a costimulatory effect when combined with an anti-CD20xCD3 bispecific antibody. (Item 9) The costimulatory effects were: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in human PBMCs; and inducing CD22-expressing cell lines. Seki Human T cell-mediated cytotoxicity Increase 9. The isolated bispecific antigen-binding molecule of item 8, wherein the bispecific antigen-binding molecule is one or more of the following: (Item 10) wherein the first antigen-binding domain (D1) is: a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26; b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10; 10. The isolated bispecific antigen-binding molecule of any one of items 1 to 9, comprising: (Item 11) a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 11. The isolated bispecific antigen-binding molecule of item 10, comprising: (Item 12) a) LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 13. The isolated bispecific antigen-binding molecule of item 12, comprising: (Item 13) the first antigen-binding domain comprising: 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; 11. The isolated bispecific antigen-binding molecule of item 10, comprising: (Item 14) 11. The isolated bispecific antigen-binding molecule of claim 10, wherein the first antigen-binding domain comprises an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 26 / 10. (Item 15) the second antigen-binding domain comprising: a) three HCDRs contained within the HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18; b) three LCDRs contained within the LCVR comprising the amino acid sequence of SEQ ID NO: 10; 15. The isolated bispecific antigen-binding molecule of any one of items 1 to 14, comprising: (Item 16) the second antigen-binding domain comprising: a) an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 20; b) an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; and c) an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 24. (Item 17) the second antigen-binding domain comprising: a) LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 17. The isolated bispecific antigen-binding molecule of item 16, comprising: (Item 18) the second antigen-binding domain comprising: a) a set of HCVR CDRs (HCDR1, HCDR2, HCDR3) comprising the 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) comprising the amino acid sequences of SEQ ID NOs: 12, 14, 16; 18. The isolated bispecific antigen-binding molecule of item 17, comprising: (Item 19) a) a first antigen-binding domain comprising an HCVR CDR comprising an amino acid sequence of SEQ ID NO: 28, 30, or 32, and an LCVR CDR comprising an amino acid sequence of SEQ ID NO: 12, 14, or 16; b) a second antigen-binding domain comprising an HCVR CDR comprising the amino acid sequence of SEQ ID NO: 4, 6, 8 and an LCVR CDR comprising the amino acid sequence of SEQ ID NO: 12, 14, 16; 19. The isolated bispecific antigen-binding molecule of any one of items 1 to 18, comprising: (Item 20) a) a first antigen-binding domain comprising an HCDR comprising an amino acid sequence of SEQ ID NO: 28, 30, or 32 and an LCDR comprising an amino acid sequence of SEQ ID NO: 12, 14, or 16; b) a second antigen-binding domain comprising an HCDR comprising an amino acid sequence of SEQ ID NO: 20, 22, 24 and an LCDR comprising an amino acid sequence of SEQ ID NO: 12, 14, 16; 19. The isolated bispecific antigen-binding molecule of any one of items 1 to 18, comprising: (Item 21) a) a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 26 / 10; b) a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 2 / 10; 19. The isolated bispecific antigen-binding molecule of any one of items 1 to 18, comprising: (Item 22) a) the first antigen-binding domain comprises an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 26 / 10; b) The isolated bispecific antigen-binding molecule of any one of items 1 to 18, wherein the second antigen-binding domain comprises 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 a reference antibody, wherein the reference antibody comprises a first antigen-binding domain having an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 26 / 10 and a second antigen-binding domain having an HCVR / LCVR pair comprising 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 comprising the amino acid sequence of SEQ ID NO: 26 / 10 and a second antigen-binding domain having an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 2 / 10 or 18 / 10. (Item 25) 25. A pharmaceutical composition comprising the bispecific antigen-binding molecule according to any one of items 1 to 24 and a pharma- ceutically acceptable carrier or diluent. (Item 26) 25. A nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody according to any one of items 1 to 24. (Item 27) 27. An expression vector comprising the nucleic acid according to item 26. (Item 28) A host cell comprising the expression vector according to item 27. (Item 29) (Item 30) A method for inhibiting a B cell proliferation disorder in a subject, comprising administering to the subject the isolated bispecific antibody according to any one of Items 1 to 24 or the pharmaceutical composition according to Item 25, thereby inhibiting the growth of the B cell lymphoma in the subject. 30. The method of claim 29, further comprising administering a second therapeutic agent to the subject. (Item 31) 31. The method of item 30, wherein the second therapeutic agent comprises an anti-tumor agent, radiation therapy, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof. (Item 32) 26. A method for treating a patient suffering from a B-cell proliferative disorder, or another CD22-expressing cell malignancy, comprising administering to said subject the isolated bispecific antibody according to any one of items 1 to 24 or the pharmaceutical composition according to item 25, thereby treating said patient suffering from a B-cell lymphoma or another CD22-expressing cell malignancy. (Item 33) 33. The method of claim 32, further comprising administering a second therapeutic agent to the subject. (Item 34) 34. The method of item 33, wherein the second therapeutic agent comprises an anti-tumor agent, radiation therapy, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof. (Item 35) 35. The method of any one of items 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 a T cell.

Claims

1. 1. An isolated human bispecific antibody, or antigen-binding fragment thereof, comprising: a) a first antigen-binding domain (D1) that binds to human CD28, The first antigen-binding domain (D1) comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR); the HCVR comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 28, a HCDR2 comprising SEQ ID NO: 30, and a HCDR3 comprising SEQ ID NO: 32; a first antigen-binding domain (D1), wherein the LCVR comprises a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 12, a LCDR2 comprising SEQ ID NO: 14, and a LCDR3 comprising SEQ ID NO: 16; b) a second antigen-binding domain (D2) that specifically binds to human CD22 on a target tumor cell, the second antigen-binding domain (D2) comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR); the HCVR comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 6, and a HCDR3 comprising SEQ ID NO: 8; a second antigen-binding domain (D2), wherein the LCVR comprises a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 12, a LCDR2 comprising SEQ ID NO: 14, and a LCDR3 comprising SEQ ID NO: 16; 1. An isolated human bispecific antibody or antigen-binding fragment thereof comprising:

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

58.

3. The bispecific antibody or antigen-binding fragment thereof has a K of less than 15 nM as measured by surface plasmon resonance at 25° C. Ｄ 3. The isolated human bispecific antibody or antigen-binding fragment thereof of claim 1 or 2, which binds to human CD22 at

4. The bispecific antibody or antigen-binding fragment thereof has a K of less than 60 μM as measured by surface plasmon resonance at 25° C. Ｄ 4. The isolated human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which binds to Macaca fascicularis CD22 at

5. The bispecific antibody or antigen-binding fragment thereof has a K of less than 45 μM as measured by surface plasmon resonance at 25° C. Ｄ 5. The isolated human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to human CD28 at the

6. The bispecific antibody or antigen-binding fragment thereof has a cell viability of 2.1 x 10 as measured by an in vitro FACS binding assay. －８ EC below M ５０ 6. The isolated human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to the surface of a cell expressing human CD28 at a specific binding site.

7. The bispecific antibody or antigen-binding fragment thereof has a cell viability of 1.3 x 10 as measured by an in vitro FACS binding assay. －８ EC below M ５０ 7. The isolated human 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 CD22 at a specific binding site.

8. 8. The isolated human bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the bispecific antibody or antigen-binding fragment thereof exhibits a costimulatory effect when combined with an anti-CD20xCD3 bispecific antibody.

9. 9. The isolated human bispecific antibody or antigen-binding fragment thereof of claim 8, wherein the costimulatory effect is one or more of: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in human PBMCs, and augmenting human T cell-mediated cytotoxicity for CD22 expressing cell lines.

10. a) a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 26 / 10; b) a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 2 / 10; 10. The isolated human bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 9, comprising:

11. A pharmaceutical composition comprising the human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharma- ceutically acceptable carrier or diluent.

12. A nucleic acid molecule comprising a nucleotide sequence encoding the human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

13. An expression vector comprising the nucleic acid of claim 12.

14. A host cell comprising the expression vector of claim 13.

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