Bispecific anti-CD28 x 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 are capable of inhibiting the growth of tumors expressing CD-22, such as B-cell lymphomas. The antibodies and bispecific antigen binding molecules of the present invention can be used to treat diseases and conditions in which an upregulated or induced targeted immune response is desirable and / or therapeutically beneficial.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 781,689, filed on December 19, 2018, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated 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. Technical Field

[0005] The present invention relates to bispecific antigen binding molecules that bind to CD28 and a target molecule such as CD22 and methods of using the same. Background Art

[0006] CD28 is a type I transmembrane protein expressed on the surface of T cells, and the type I transmembrane protein has a single extracellular Ig-V-like domain assembled into a homodimer. CD28 is a receptor for CD80 (B7.1) protein and CD86 (B7.2) protein and is activated by CD80 or CD86 expressed on antigen presenting cells (APC). The combination of CD28 and CD80 or CD86 provides a co-stimulatory signal that is important for T cell activation and survival. In addition to the T cell receptor (TCR), the T cell stimulation by CD28 also provides a potent signal for the production of various interleukins. CD28 also strengthens cell signals after TCR activation, such as the path controlled by NFκB transcription factors. CD28 co-signals are important for effective T cell activation such as T cell differentiation, proliferation, cytokine release and cell death.

[0007] Anti-CD28 antibodies have been proposed for therapeutic purposes involving activation of T cells. A specific anti-CD28 antibody, TGN1412 (anti-CD28 superagonist), was used in a 2006 clinical trial. TGN1412 (anti-CD28 superagonist) was administered intravenously to six healthy volunteers at a dose of 0.1 mg / kg. Within two hours, all six patients had a significant inflammatory response (cytokine storm), and within sixteen hours, all patients had multiple organ failure. The subjects were treated with corticosteroids, and cytokine levels returned to normal levels within 2-3 days. In a Phase 1 study (associated with CRS), the starting dose of 0.1 mg / kg was based on a 500-fold multiple of the 50 mg / kg macaque "NOAEL" (Suntharalingam et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412, NEJM 355:1018-1028 (2006)). Unfortunately, TGN1412 induced a cytokine storm, which was not predicted by toxicology studies in cynomolgus macaques or by ex vivo human PBMC studies.

[0008] Siglec family member CD22 (also known as Siglec-2) specifically recognizes α2,6 sialic acid and is a transmembrane protein preferentially expressed on B lymphocytes (B cells).

[0009] CD22 has many ascribed functions including, for example, B cell homeostasis, B cell survival and migration, dampening of TLR and CD40 signaling and recruitment of SH2 domain-containing phosphatases to inhibit B cell receptor (BCR) signaling through phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region, as well as facilitating cell adhesion between B cells and other cell types.

[0010] CD22 is neither found on the surface of B cells during the early stages of development nor is it expressed in stem cells. However, 60-70% of all B cell lymphomas and leukemias express CD22.

[0011] Anti-CD22 antibodies have been studied for the treatment of B-cell lymphomas and leukemias. However, the monoclonal antibody Epratuzumab has had limited success. (Grant et al. (2013) Cancer 119(21):10.1002 / cncr.28299).

[0012] Therefore, there is a need for improved anti-CD22 antibodies in the art.Anti-CD28 antibodies that are safe for use in pharmaceutical compositions are also needed.In addition, bispecific antigen binding molecules that bind both CD28 and a target antigen (such as CD22) will be useful in such a therapeutic setting: wherein it is desired to specifically target and T cell-mediated killing of cells expressing the target antigen. Summary of the invention

[0013] In the first aspect, the present invention provides a bispecific antigen binding molecule in combination with CD28 and a target antigen. According to certain exemplary embodiments, 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 can be used to target cancer cells (e.g., cancerous B cells) expressing CD22, and the anti-CD28 portion of the bispecific molecule can be used to activate T cells. The simultaneous binding of CD22 on cancer cells and CD28 on T cells promotes the directional killing (cell lysis) of targeted cancer cells by activated T cells (e.g., after TCR activation of T cells). Therefore, the anti-CD28 / anti-CD22 bispecific molecules of the present invention are particularly useful for treating diseases and disorders (e.g., B cell proliferative disorders, e.g., B cell lymphomas, such as diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma) associated with or caused by CD22-expressing tumors.

[0014] The bispecific antigen binding molecules according to this aspect of the invention include a first antigen binding domain that specifically binds to human CD28 and a second antigen binding domain that specifically binds to CD22. The invention encompasses anti-CD28 / anti-CD22 bispecific molecules (e.g., bispecific antibodies) wherein each antigen binding domain includes 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 include a different unique HCVR paired with a common LCVR.

[0015] The present invention provides an anti-CD28 / anti-CD22 bispecific molecule, wherein the first antigen binding domain that specifically binds to CD28 comprises any one of the HCVR amino acid sequences shown in Table 6. The first antigen binding domain that specifically binds to CD28 may also comprise any one of the LCVR amino acid sequences shown in Table 6. According to certain embodiments, the first antigen binding domain that specifically binds to CD28 comprises any one of the HCVR / LCVR amino acid sequence pairs shown in Table 6. The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule, wherein the first antigen binding domain that specifically binds to CD28 comprises any one of the heavy chain CDR1-CDR2-CDR3 amino acid sequences shown in Table 6 and / or any one of the light chain CDR1-CDR2-CDR3 amino acid sequences shown in Table 6.

[0016] According to certain embodiments, the present invention provides an anti-CD28 / anti-CD22 bispecific molecule, wherein the 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.

[0017] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule, wherein the first antigen binding domain that specifically binds to CD28 comprises a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 10 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0018] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule, wherein the first antigen binding domain that specifically binds to CD28 comprises a HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NO: 28 / 10 and 26 / 10.

[0019] The present invention also provides an anti-CD28 / anti-CD22 bispecific molecule, 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 substantially similar sequence thereof 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 substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0020] In certain embodiments, the first antigen binding domain that specifically binds CD28 comprises the HCDR3 / LCDR3 amino acid sequence pair of SEQ ID NO: 32 / 16.

[0021] The present invention also provides an anti-CD28 / anti-CD22 bispecific antigen binding molecule, wherein the first antigen binding domain that specifically binds to CD28 comprises: a heavy chain CDR1 (HCDR1) domain, the 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, the 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, the 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, the LCDR2 domain having the amino acid sequence of SEQ ID NO: 13. The amino acid sequence of NO: 14 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0022] Certain non-limiting, exemplary anti-CD28 / anti-CD22 bispecific antigen binding molecules of the invention comprise a first antigen binding domain that specifically binds CD28, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having the following amino acid sequences, respectively: SEQ ID NO: 28-30-32-12-14-16.

[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 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.

[0024] 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 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.

[0025] 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 NO: 2 / 10 and 18 / 10.

[0026] 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 heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 24, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from SEQ ID NO: 16, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0027] In certain embodiments, the second antigen binding domain that specifically binds CD22 comprises a HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO: 8 / 16 and 24 / 16.

[0028] The present invention also provides an anti-CD28 / anti-CD22 bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to CD22 comprises: a heavy chain CDR1 (HCDR1) domain, the 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 having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain, the HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22, 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, the LCDR1 domain having an 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, the LCDR2 domain having an amino acid sequence of SEQ ID NO: 13. The amino acid sequence of NO: 14 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0029] Certain non-limiting, exemplary anti-CD28 / anti-CD22 bispecific antigen binding molecules of the invention comprise a second antigen binding domain that specifically binds 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.

[0030] In a related embodiment, the present invention comprises an anti-CD28 / anti-CD22 bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to CD22 comprises heavy chain and light chain CDR domains contained within a heavy chain variable region and light chain variable region (HCVR / LCVR) sequence selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 10.

[0031] In another aspect, the present invention provides a nucleic acid molecule encoding any one of the HCVR, LCVR or CDR sequences of the anti-CD28 / anti-CD22 bispecific antigen binding molecules disclosed herein, the nucleic acid molecule comprising: a nucleic acid molecule comprising a polynucleotide sequence as shown in Table 7 herein and a nucleic acid molecule comprising two or more of the polynucleotide sequences as shown in Table 7 (in any functional combination or arrangement thereof). Recombinant expression vectors carrying the nucleic acids of the present invention and host cells into which such vectors have been introduced are also encompassed by the present invention, as are methods of producing antibodies by culturing host cells under conditions that allow for the production of antibodies and recovering the produced antibodies.

[0032] The present invention comprises an anti-CD28 / anti-CD22 bispecific antigen binding molecule, wherein any one of the above-mentioned antigen binding domains that specifically binds to CD28 is combined, linked or otherwise associated with any one of the above-mentioned antigen binding domains that specifically binds to CD22 to form a bispecific antigen binding molecule that binds to CD28 and CD22.

[0033] The present invention comprises anti-CD28 / anti-CD22 bispecific antigen binding molecules with modified glycosylation patterns. In some applications, modifications for removing undesirable glycosylation sites may be useful, or, for example, antibodies lacking fucose moieties present on oligosaccharide chains for increasing 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).

[0034] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD28 / anti-CD22 bispecific antigen binding molecule as disclosed herein and a pharmaceutically acceptable carrier. In a related aspect, the present invention is characterized in that a composition 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 an anti-CD28 / anti-CD22 bispecific antigen binding molecule. Exemplary agents that can be advantageously combined with anti-CD28 / anti-CD22 bispecific antigen binding molecules are discussed in detail elsewhere herein.

[0035] 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, wherein the therapeutic method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD28 / anti-CD22 bispecific antigen binding molecules of the present invention.

[0036] The present invention also includes the use of the anti-CD28 / anti-CD22 bispecific antigen binding molecules of the present invention in the manufacture of a medicament for treating a disease or disorder associated with or caused by CD22 expression.

[0037] 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, wherein the anti-CD28 / anti-CD22 bispecific antigen binding molecules are combined with other anti-tumor bispecific antigen binding molecules that bind to CD3 (e.g., anti-CD28 / anti-CD22 in combination with anti-CD3 / anti-CD20 antibodies).

[0038] In still 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, wherein 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-PD1 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. Nivolumab or Cemiplimab In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with agents targeting PD-L1, such as atezolizumab. Avelumab or Durvalumab In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with agents targeting CTLA-4, such as Ipilimumab. combination.

[0039] In still 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, wherein the anti-CD28 / anti-CD22 bispecific antigen binding molecules are combined with other anti-tumor bispecific antigen binding molecules that bind to CD3 (e.g., anti-CD28 / anti-CD22 and anti-CD3 / anti-CD20 bispecific antibodies such as REGN1979 (see US9,657,102, wherein the anti-CD20 arm includes the HCVR / LCVR amino acid pair of SEQ ID NO: 1242 / 1258 and the anti-CD3 arm includes the amino acid pair of SEQ ID NO: 1250 / 1258)) and / or with a checkpoint inhibitor targeting PD-1, PD-L1 or CTLA-4 (i.e., anti-CD28 / anti-CD22 and anti-PD-1 antibody combination). For example, in certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with agents targeting PD-1 such as pembrolizumab Nivolumab or cemiplimab ( See, for example, US9,987,500, wherein cemiplimab comprises the HCVR / LCVR amino acid pair of SEQ ID NO: 162 / 170)) combination. In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with agents targeting PD-L1 such as atezolizumab Avelumab Durvalumab In certain embodiments, the anti-CD28 / anti-CD22 antibodies of the present invention can be combined with agents targeting CTLA-4, such as ipilimumab. combination.

[0040] Other embodiments will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a set of graphs depicting the binding of anti-CD28 / anti-CD22 bispecific antibodies to human CD4+ T cells expressing CD28 on the cell surface and target cells expressing human CD22.

[0042] Figure 2A and 2B is a set of graphs depicting anti-CD28 / anti-CD22 bispecific antibodies showing increased luciferase production in the presence of primary T cell stimulation and CD22 targeted expression. Figure 2Ais a set of graphs depicting activation of engineered reporter T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells in addition to 200 pM constant REGN1945 (negative hIgG4 isotype control) as assessed by luciferase production. Figure 2B is a set of graphs depicting activation of engineered reporter T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells in addition to 200 pM constant REGN2281 (anti-CD20 x anti-CD3) as assessed by luciferase production.

[0043] Figure 3A and Figure 3B is a set of graphs depicting the enhancement of IL-2 production by anti-CD28 / anti-CD22 bispecific antibodies in the presence of primary T cell stimulation and targeted expression of CD22. More specifically, Figure 3A Figure 5 depicts CD4 T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or Raji / CD80 and CD86 negative cells in the presence of 2 nM constant REGN1945 (hIgG4 isotype control) as assessed by IL-2 production. + A set of diagrams showing T cell activation. Figure 3B Figure 5 depicts CD4+ T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22, or Raji / CD80 and CD86 negative cells in addition to 2 nM constant REGN2281 (anti-CD20 x anti-CD3) as assessed by IL-2 production. + A set of diagrams showing T cell activation.

[0044] Figure 4 is a set of graphs showing that the combination of REGN5837 and cemiplimab enhances IL-2 release over REGN5837 treatment alone in cells engineered to express PD-L1.

[0045] 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.

[0046] Figure 5B is a set of graphs showing that the combination of REGN5837 and cemiplimab enhances IL-2 release over REGN5837 treatment alone in RAJI cells engineered to express PD-L1.

[0047] Figure 6 is a set of graphs showing that treatment of NALM-6-Luc tumor-bearing mice with REGN5837 in the presence of REGN1979 (anti-CD20 x anti-CD3) was associated with significant tumor inhibition. Briefly, NSG mice (n=6 to 9 per group) were transplanted with human PBMCs and then implanted with NALM-6-luc B cell leukemia cells 12 days after transplantation (day 0). Mice were administered 4 mg / kg REGN5837+0.04 mg / kg REGN1979 (hashed circles), 0.4 mg / kg REGN5837+0.04 mg / kg REGN1979 (closed upright triangles), 0.04 mg / kg REGN5837+0.04 mg / kg REGN1979 (diamonds), 4 mg / kg non-TAAxCD28+0.04 mg / kg REGN1979 (squares), 4 mg / kg REGN5837+0.4 mg / kg non-TAAxCD3 (open circles), or 4 mg / kg non-TAAxCD28+0.4 mg / kg non-TAAxCD3 (closed inverted triangles) at 8, 15, and 22 days after implantation (arrows). Tumor growth was monitored by bioluminescent imaging of tumor volume at 6, 10, 14, 17, 20, and 23 days after implantation. Combined data are presented as group mean ± SEM. Statistical significance was determined using two-way ANOVA and Tukey's post hoc test. The following symbols were used to indicate statistically significant differences relative to non-TAAxCD28+non-TAAxCD3 controls: * , p<0.05; ** , p<0.01; *** , p<0.001.

[0048] Figures 7A-7C is a graph showing that REGN1979 activates and directs human T cells to kill Nalm6 cells in a dose-dependent manner. More specifically, Fig. 7A is a graph depicting the percent viability of Nalm6 cells in the presence of the indicated antibodies. Figure 7B is a graph depicting the percentage of CD8+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Figure 7C is a graph depicting the proliferation of CD25+CD8+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies.

[0049] Fig. 8A , 8B8C are graphs showing that REGN1979 activates and directs human T cells to kill WSU-DLCL2 cells in a dose-dependent manner. More specifically, Fig. 8A is a graph showing the percent survival of WSU-DLCL2 cells in the presence of the indicated antibodies. Figure 8B is a graph depicting the percentage of CD8+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Figure 8C is a graph depicting proliferation of CD8+ cells in the presence of the indicated antibodies expressed as % divided.

[0050] Fig. 9 is a set of graphs 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 CD22 x CD28 compared to that induced by REGN1979 alone.

[0051] Figures 10A-10E is a graph showing that REGN1979 activates and directs human T cells to deplete NHL in a dose-dependent manner. Adding a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the cytotoxic efficacy (EC50) of REGN1979 by 2.3-fold and 3.5-fold compared to REGN1979 with a single-armed CD28 control antibody or without a co-stimulatory control. The observed target cell lysis mediated by REGN1979 was associated with T cell activation and proliferation, as measured by CD25 upregulation on CD8+ and CD4+ cells or CellTrace Violet dilution, respectively. More specifically, Fig. 10A is a graph depicting the percent survival of NHL cells from patient bone marrow in the presence of the indicated antibodies. Fig. 10B is a graph depicting the percentage of CD8+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Fig. 10C is a graph depicting the proliferation of CD8+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies. Fig. 10D is a graph depicting the percentage of CD4+ cells expressing CD25 (CD25+) in the presence of the indicated antibodies. Fig. 10E is a graph depicting the proliferation of CD4+ cells as assessed by CellTrace Violet dilution in the presence of the indicated antibodies.

[0052] Figures 11A-11EIt is a graph showing that REGN5837 enhances the efficacy of REGN1979-mediated cytotoxicity, cell surface expression of CD25, and T cell proliferation in a concentration-dependent manner. In short, WSU-DLCL2 cells and lymphocyte-enriched human PBMCs were incubated at a target cell:PBMC ratio of 1:5 and with anti-CD20xCD3 (REGN1979) in a certain concentration range (4.8fM to 10nM) as a single agent (i.e., without REGN5837) or in the presence of a fixed concentration of REGN5837 (ranging from 0.01 to 15μg / mL) at 37°C for 72 hours. The lack of REGN1979 contained only REGN5837 at the indicated concentration and was plotted as 0.04pM. Live cells were detected by flow cytometry using live / dead cell staining (11A). T cell activation (as measured by CD25 expression; 11B, 11D) and CD4 by flow cytometry) was detected using a phenotypic cocktail of Violet CellTracker dyes and fluorophore-labeled antibodies against CD2, CD4, CD8, and CD25. + and CD8 + T cell proliferation (11C, 11E).

[0053] More specifically, Fig.11A is a graph depicting the % dead cells at the indicated concentrations of REGN5837. Fig. 11B is a graph depicting the percentage of CD25+CD4+ cells at the indicated concentrations of REGN5837. Fig. 11C is a graph depicting the proliferation of CD4+ cells as assessed by CellTrace Violet dilution at the indicated concentrations of REGN5837. Fig.11D is a graph depicting the percentage of CD25+CD8+ cells. Fig.11E is a graph depicting the proliferation of CD8+ cells as assessed by CellTrace Violet dilution at the indicated concentrations of REGN5837.

[0054] Figures 12A-12Gis a graph showing the potency and maximum level of REGN1979-mediated cytokine release from human T cells enhanced by REGN5837 in the presence of WSU-DLCL2 B cell lymphoma cells in a concentration-dependent manner. Briefly, WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMCs at a 1:5 target cell:PBMC ratio and with anti-CD20xCD3 (REGN1979) at a range of concentrations (4.8 fM to 10 nM) as a single agent (i.e., without REGN5837) or in the presence of a fixed concentration of REGN5837 (ranging from 0.01 to 15 μg / mL) at 37°C for 72 hours. The absence of REGN1979 contained only REGN5837 at the indicated concentration and was plotted as 0.04 pM. Supernatants were evaluated for cytokine release of (12A) IL-2, (12B) IL-4, (12C) IL-6, (12D) IL-10, (12E) TNF-α, (12F) IFN-γ, and (12G) IL-17α using the BD Flow Cytometry Bead Array Human Th1 / Th2 / Th17 Cytokine Kit.

[0055] More specifically, Fig. 12A is a graph depicting the levels of IL-2 released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Fig. 12B is a graph depicting the levels of IL-4 released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Fig. 12C is a graph depicting the levels of IL-6 released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Fig.12D is a graph depicting the levels of IL-10 released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Fig.12E is a graph depicting the levels of TNF-α released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Fig.12F is a graph depicting the levels of IFN-γ released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837. Figure 12G is a graph depicting the levels of IL-17α released from human T cells in the presence of WSU-DLCL2 cells at the indicated concentrations of REGN5837.

[0056] Fig.13A and 13B is a graph showing that treatment of NSG mice bearing WSU-DLCL2 tumors with REGN5837 in the presence of REGN1979 at 0.4 or 4 mg / kg was associated with significant tumor inhibition. Briefly, a 1:1 mixture of WSU-DLCL2 B cell lymphoma cells and human PBMCs was implanted into female NSG mice (n=6 to 7 per group) (day 0). A combination of 1 mg / kg REGN5837 and 0.4 mg / kg (13A) or 4 mg / kg (13B) REGN1979 (or non-bridging control) was administered to mice on days 1, 8, and 15 after implantation (arrows). Tumor growth was monitored by caliper measurement on days 7, 10, 14, 16, 28, 31, 35, 38, 43, 46, 49, 53, 57, and 63 after implantation. Combined data are presented as group mean ± SEM. Statistical significance was determined using two-way ANOVA and Tukey post hoc test. The following symbols are used to indicate statistically significant differences 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 significance between REGN1979 monotherapy and the combination of REGN5837 and REGN1979.

[0057] 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-bridging control, non-TAAxCD3). Fig. 13B is a graph depicting tumor growth in mice administered 1 mg / kg REGN5837 and 4 mg / kg (or non-bridging control, non-TAAxCD3).

[0058] Fig.14is a graph showing that treatment of mice bearing WSU-DLCL2 tumors with REGN5837 in the presence of a sub-effective dose of REGN1979 is associated with significantly greater survival than REGN5837 or REGN1979 monotherapy. In brief, a 1:1 mixture of WSU-DLCL2 B cell lymphoma cells and human PBMCs was implanted into female NSG mice (n=6 to 7 per group) (Day 0). A combination of REGN5837 and REGN1979 or controls was administered to mice on days 1, 8, and 15 after implantation (arrows). Statistical significance was determined using the Mantel-Cox test. The following symbols are used to indicate statistically significant differences between groups: * , p<0.05; *** , p<0.001. The caret indicates statistical significance compared to isotype control, the asterisk indicates significance compared to 0.4 mg / kg REGN1979 monotherapy, and the hash mark indicates significance compared to 4 mg / kg REGN1979 monotherapy. DETAILED DESCRIPTION

[0059] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention is limited only by the appended claims.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. As used herein, when used to refer to a specific listed value, the term "about" means that the value may differ from the listed value by no more than 1%. 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.).

[0061] 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 in this specification are incorporated herein by reference in their entirety.

[0062] definition

[0063] As used herein, the expression "CD28" refers to an antigen expressed on T cells as a co-stimulatory receptor. Human CD28 includes an amino acid sequence as shown in SEQ ID NO:74, and / or has an amino acid sequence as shown in NCBI accession number NP_006130.1. Unless explicitly specified as from non-human species, all references to proteins, polypeptides and protein fragments herein are intended to refer to the human form of the corresponding protein, polypeptide or protein fragment. Therefore, unless specified as from non-human species such as "mouse CD28", "monkey CD28", etc., the expression "CD28" means human CD28.

[0064] As used herein, "antibodies that bind to CD28" or "anti-CD28 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize monomeric CD28 and antibodies and antigen-binding fragments thereof that specifically recognize dimeric CD28. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD28 and / or CD28 expressed on the surface of cells. Soluble CD28 includes native CD28 protein and recombinant CD28 protein variants that lack a transmembrane domain or are otherwise unassociated with a cell membrane, such as monomeric and dimeric CD28 constructs.

[0065] As used herein, the expression "CD28 expressed on the cell surface" means that one or more CD28 proteins are expressed on the surface of a cell in vitro or in vivo so that at least a portion of the CD28 protein is exposed to the extracellular side of the cell membrane and can be approached by the antigen-binding portion of the antibody. "CD28 expressed on the cell surface" includes CD28 proteins contained in the context of functional T cell co-stimulatory receptors in the membrane of the cell. The expression "CD28 expressed on the cell surface" includes CD28 proteins expressed as part of a homodimer on the cell surface. "CD28 expressed on the cell surface" may include or consist of CD28 proteins expressed on the surface of cells that normally express CD28 proteins. Alternatively, "CD28 expressed on the cell surface" may include or consist of CD28 proteins expressed on the surface of such cells: the cells do not normally express human CD28 on their surface, but have been artificially engineered to express CD28 on their surface.

[0066] As used herein, the expression "anti-CD28 antibody" encompasses monovalent antibodies with a single specificity as well as bispecific antibodies comprising a first arm that binds CD28 and a second arm that binds a second (target) antigen, wherein the anti-CD28 arm comprises any of the HCVR / LCVR or CDR sequences as shown in Table 1 herein. Examples of anti-CD28 bispecific antibodies are described elsewhere herein. The term "antigen binding molecule" encompasses antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0067] As used herein, unless specified as being from a non-human species (e.g., "mouse CD22," "monkey CD22," etc.), the term "CD22" refers to human CD22 protein. Human CD22 protein has an amino acid sequence as shown in Accession No. CAA42006. The sequence of recombinant human CD22 ecto (D20-R687) with a myc myc hexa-histidine tag ("hexa-histidine" is disclosed as SEQ ID NO: 60) is shown in Accession No. NP_001762.2 and is also shown as SEQ ID NO: 50. hCD22 extracellular domain (D20-R687).hFc can also be purchased from R&D Systems, catalog number 1968-SL-050.

[0068] As used herein, "antibodies that bind to CD22" or "anti-CD22 antibodies" include antibodies and antigen-binding fragments thereof that can bind to soluble CD22 and / or CD22 expressed on the cell surface. Soluble CD22 includes native CD22 protein and recombinant CD22 protein variants that lack a transmembrane domain or are otherwise not associated with a cell membrane, such as CD22 constructs.

[0069] As used herein, the expression "anti-CD22 antibody" encompasses monovalent antibodies with a single specificity as well as bispecific antibodies comprising a first arm that binds CD22 and a second arm that binds a second (target) antigen, wherein the anti-CD22 arm comprises any of the HCVR / LCVR or CDR sequences as shown in Table 1 herein. Examples of anti-CD22 bispecific antibodies are described elsewhere herein. The term "antigen binding molecule" encompasses antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0070] The term "antigen binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0071] As used herein, the term "antibody" means any antigen binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds or interacts with a specific antigen (e.g., CD28). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM), wherein the immunoglobulin molecules include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, C H 1. C H 2 and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes a domain (C L1) You can set V H Area and V L The V regions are further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-CD28 antibody and / or anti-CD22 antibody (or its antigen-binding portion) may be identical to human germline sequences or may be natural or artificially modified. An amino acid consensus sequence may be defined based on a juxtaposition analysis of two or more CDRs.

[0072] As used herein, the term "antibody" also includes the antigen binding fragment of a complete antibody molecule. As used herein, the term "antigen binding portion thereof", "antigen binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques that involve manipulation and expression of the antibody variable domains and optionally constant domains that encode the DNA. Such DNA is known and / or is easy to obtain or can be synthesized from, for example, commercial sources, DNA libraries (comprising, for example, phage-antibody libraries). DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration or introduce codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0073] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. 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 within the expression "antigen-binding fragment" as used herein.

[0074] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can have any size or amino acid composition and generally includes at least one CDR adjacent to or in the same frame as one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H Domain and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or V L Structural domain.

[0075] In certain embodiments, the antigen-binding fragment of an antibody may contain 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 the 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 H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -CH 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant regions (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment may include the above-listed V domains and / or one or more monomeric V domains. H or V L Homodimers or heterodimers (or other multimers) of any one of a number of variable and constant domain configurations in which the domains are non-covalently associated (e.g., via one or more disulfide bonds).

[0076] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Conventional techniques available in the art can be used to adapt any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) for use in the context of an antigen-binding fragment of an antibody of the present invention.

[0077] The antibodies of the present invention can act by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of cells expressing antigens by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to such a cell-mediated reaction: non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) expressing Fc receptors (FcR) recognize the binding antibodies on target cells and thus cause the lysis of target cells. CDC and ADCC can be measured using assays well known in the art and available. (See, e.g., U.S. Patents 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 for the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether the antibody is desired to mediate cytotoxicity.

[0078] In certain embodiments of the present invention, the anti-CD28 antibody and / or anti-CD22 antibody (monospecific or bispecific) of the present invention is a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from germline of another mammalian species such as mice have been transplanted to human framework sequences.

[0079] In some embodiments, the antibody of the present invention can be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or separated by a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies separated from a recombinant combination human antibody library (described further below), antibodies separated from human immunoglobulin gene transgenic animals (e.g., mice) (see, for example, Taylor et al., (1992), "Nucl. Acids Res.", 20: 6287-6295) or antibodies prepared, expressed, produced or separated by any other means, the other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions 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 using human Ig sequence transgenic animals, in vivo somatic cell mutagenesis is performed), and therefore the V H Area and V L The amino acid sequence of the region is such that although it is derived from human germline V H Sequence and V L sequences but may not naturally occur in the human antibody germline repertoire in vivo.

[0080] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule includes a stable four-chain construct of about 150-160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected by an interchain disulfide bond and forms a molecule of about 75-80 kDa, which is composed of covalently coupled light and heavy chains (half antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0081] The frequency of the second form in various complete IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. Single amino acid substitutions in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al., (1993), Molecular Immunology 30:105) to the level usually observed with the human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the hinge region, CH2 region or CH3 region, which mutations may be, for example, desired in production, so as to increase the yield of the desired antibody form.

[0082] The antibody of the present invention can be an isolated antibody. As used herein, "isolated antibody" 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 naturally occurring or naturally produced tissue or cell of an antibody is an "isolated antibody" for purposes of the present invention. Isolated antibodies also include in situ antibodies in recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or separation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and / or chemicals.

[0083] The present invention also includes one-armed antibodies that bind CD28 and / or CD22. As used herein, "one-armed antibody" means an antigen binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-armed antibody of the present invention may include any one of the HCVR / LCVR or CDR amino acid sequences shown in Table 1.

[0084] The anti-CD28 antibodies and / or anti-CD22 antibodies or their antigen-binding domains herein may include 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, as compared to the corresponding germline sequences from which the antigen-binding protein or antigen-binding domain is derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding domains derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to one or more corresponding residues of the germline sequence from which the antibody is derived or mutated to one or more corresponding residues of another human germline sequence or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily generate many antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, V HDomain and / or V L All frameworks and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only some residues are mutated back to the original germline sequence, such as the residues found only in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4 after mutation or the residues found only in CDR1, CDR2 or CDR3 after mutation. In other embodiments, one or more mutations in one or more frameworks and / or CDR residues are one or more corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequence from which the antibody is initially derived). In addition, the antibody of the present invention or its antigen-binding domains may contain any combination of two or more germline mutations in the framework and / or CDR region, such as, wherein some individual residues are mutated into the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence remain unchanged or mutate into the corresponding residues of different germline sequences. Once obtained, antibodies or antigen-binding fragments thereof containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed within the present invention.

[0085] The present invention also includes anti-CD28 antibodies 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, relative to any of the HCVR, LCVR and / or CDR amino acid sequences as shown in Table 6 herein, the HCVR, LCVR and / or CDR amino acid sequences have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions.

[0086] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0087] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that the nucleotide sequence identity is at least about 95% of the nucleotide bases, and more preferably, at least about 96%, 97%, 98% or 99% when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to a polypeptide encoded by the reference nucleic acid molecule.

[0088] When applied to polypeptides, the term "substantially similar" or "substantially similar" means that when the two peptide sequences are optimally aligned, such as by using the program GAP or BESTFIT with default gap weights, they share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue with a similar side chain (R group) of chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of proteins. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of amino acid groups with chemically similar side chains 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, 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 "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0089] Sequence similarity of polypeptides, also referred to as sequence identity, is usually measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications including conservative amino acid substitutions to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, for example, version 6.1 GCG. FASTA (a program in version 6.1 GCG) using default or recommended parameters can also be used to compare polypeptide sequences. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000) the same). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0090] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation that would benefit from treatment with the anti-CD28 / anti-CD22 bispecific antigen binding molecules or methods of the invention. This includes both chronic and acute disorders, including those pathological conditions that predispose a mammal to the disorder in question. In one embodiment, a cell proliferative disorder is a physiological condition, cancer, that is typically characterized by unregulated cell growth / proliferation in mammals.

[0091] As used herein, "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," and "tumor" as used herein are not mutually exclusive.

[0092] "B-cell proliferative disorders" include Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), such as aggressive NHL, relapsed 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 non-cleaved cell NHL, bulky disease NHL, indolent NHL including relapsed indolent NHL and rituximab-refractory indolent NHL; refractory NH, refractory indolent NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia. Macroglobulinemia, lymphocytic-dominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL); leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; and other hematological malignancies.

[0093] As used herein, the term "non-Hodgkin's lymphoma" or "NHL" refers to cancer of the lymphatic system rather than Hodgkin's lymphoma. Hodgkin's lymphoma can usually be distinguished from non-Hodgkin's lymphoma: Reed-Sternberg cells are present in Hodgkin's lymphoma, while the cells are not present in non-Hodgkin's lymphoma. Examples of non-Hodgkin's lymphomas as used herein include any lymphomas so identified by a person skilled in the art (e.g., an oncologist or pathologist) according to a classification scheme known in the art, such as the revised European and American lymphoma (Revised European-American Lymphoma, REAL) scheme, such as "Color Atlas of Clinical Hematology" (3rd edition), A.Victor Hoffbrand and John E.Pettit (ed.) (Harcourt Publishers Ltd., 2000) described. Specifically, see the list 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, plasma cell low-grade / follicular lymphoma, intermediate-grade / 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 relapsed or refractory after autologous stem cell implantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous (epidermal) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0094] Bispecific Antigen Binding Molecules

[0095] The antibodies of the present invention may be monospecific, bispecific or multispecific. A multispecific antibody may be specific for different epitopes of a target polypeptide or may contain an antigen binding domain that is specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD28 antibody and / or anti-CD22 antibody of the present invention may be linked or co-expressed with another functional molecule, such as 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 otherwise) with one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0096] As used herein, the expressions "anti-CD28 antibody" and / or "anti-CD22 antibody" are intended to include both monospecific anti-CD28 antibodies and / or monospecific anti-CD22 antibodies and bispecific antibodies comprising a CD28 binding arm or a CD22 binding arm and an arm that binds a target antigen. Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds to human CD28 or CD22 and the other arm of the immunoglobulin is specific for the target antigen. The target antigen bound by the other arm of the CD28 or CD22 bispecific antibody can be any antigen expressed on or near a cell, tissue, organ, microorganism or virus, for which a targeted immune response is desired. The CD28 binding arm may include any of the HCVR / LCVR or CDR amino acid sequences as shown in Table 1 herein. The CD22 binding arm may include any of the HCVR / LCVR or CDR amino acid sequences as shown in Table 1 herein. In certain embodiments, the CD28 binding arm binds to human CD28 and induces human T cell proliferation.

[0097] In the context of a bispecific antibody of the invention wherein one arm of the antibody binds CD28 and the other arm binds a target antigen, the target antigen may be a tumor associated antigen, such as CD22.

[0098] 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 "αCD22x αCD28" or "αCD28 x αCD22" or other similar terms.

[0099] According to certain exemplary embodiments, 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 certain exemplary embodiments, the effector arm is bound to CD28, and the targeting arm is bound to CD22. Bispecific anti-CD28 / CD22 may provide a co-stimulatory signal to effector cells (e.g., T cells). The effector arm has no effect of stimulating T cells in the absence of clustering. A separate effector arm has almost no effect of stimulating T cells unless combined with a targeting arm. A tumor targeting arm may have imperfect tumor specificity. An antigen (e.g., CD22) that is a target of a targeting arm may be expressed on a portion of a tumor cell. The specificity of a tumor targeting arm may be increased by overlapping a combination with an anti-CD3 bispecific antigen binding molecule (e.g., an anti-CD3 / CD20 bispecific antibody).

[0100] As used herein, the expression "antigen binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementary determining region (CDR), which specifically binds to a particular antigen either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen binding molecule is an antibody or an antibody fragment, such as those terms defined elsewhere herein.

[0101] As used herein, the expression "bispecific antigen binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a bispecific antigen binding molecule includes at least one CDR, which specifically binds to a specific antigen 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).

[0102] In certain exemplary embodiments of the present invention, bispecific antigen binding molecules are bispecific antibodies.Each antigen binding domain of a bispecific antibody includes 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) including a first antigen binding domain and a second antigen binding domain, the CDR of the first antigen binding domain can be represented by the prefix "D1", and the CDR of the second antigen binding domain can be represented by the prefix "D2".Therefore, the CDR of the first antigen binding domain can be referred to as D1-HCDR1, D1-HCDR2 and D1-HCDR3 in this article; and the CDR of the second antigen binding domain can be referred to as D2-HCDR1, D2-HCDR2 and D2-HCDR3 in this article.

[0103] The first antigen binding domain and the second antigen binding domain can be directly or indirectly connected to each other to form a bispecific antigen binding molecule of the present invention. Alternatively, the first antigen binding domain and the second antigen binding domain can each be connected to a separate multimerization domain. The association of a multimerization domain with another multimerization domain promotes the association 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 can be associated with a second multimerization domain having the same or similar structure or composition. For example, the multimerization domain can be an immunoglobulin C H A non-limiting example of a multimeric component is the Fc portion of an immunoglobulin (including the C H 2-C H 3 domains), e.g., an IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and an Fc domain of any allotype within each isotype group.

[0104] The bispecific antigen binding molecules of the present invention generally include two multimerization domains, for example, two Fc domains that are each independently a part of a separate antibody heavy chain. The first multimerization domain and the second multimerization domain can be the same IgG isotype, for example IgG1 / lgG1, IgG2 / lgG2, IgG4 / lgG4. Alternatively, the first multimerization domain and the second multimerization domain can be different IgG isotypes, for example IgG1 / lgG2, IgG1 / lgG4, IgG2 / lgG4, etc.

[0105] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short peptide containing cysteine. Other multimerization domains include peptides or polypeptides including or consisting of a leucine zipper, a spiral loop motif, or a coiled coil motif.

[0106] Any bispecific antibody form or technology can be used to prepare the bispecific antigen binding molecules of the present invention.For example, an antibody or fragment thereof with a first antigen binding specificity can be functionally connected to one or more other molecular entities such as another antibody or antibody fragment with a second antigen binding specificity (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to produce a bispecific antigen binding molecule. Specific exemplary bispecific forms that can be used in the context of the present invention include, but are not limited to, for example, based on scFv or double antibody bispecific forms, IgG-scFv fusion, dual variable domains (OVO)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Ouobody, IgG1 / lgG2, dual-acting Fab (OAF)-IgG and Mab 2 Bispecific formats (for review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein).

[0107] In the context of the bispecific antigen binding molecules of the present invention, a multimerization domain, such as an Fc domain, may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of an Fc domain. For example, the present invention comprises bispecific antigen binding molecules comprising one or more modifications in an Fc domain, thereby producing a modified Fc domain with a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, a bispecific antigen binding molecule comprises a C H Zone 2 or C HA modification in region 3, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., modification at position 250 (e.g., E or Q); modification at position 250 and position 428 (e.g., L or F); modification at position 252 (e.g., LN / FIW or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / EID or T); or modification at position 428 and / or position 433 (e.g., UR / S / P / Q or K) and / or position 434 (e.g., H / F or V); or modification at position 250 and / or position 428; or modification at position 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V2591) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0108] The present invention also includes a bispecific antigen binding molecule comprising a first C H 3 domain and the second Ig C H 3 domains, the first and second Ig C H The three domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. H 3 domains bind protein A, and the second Ig C H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). H3 may further include modifications Y96F (by IMGT; Y436F by EU). Additional modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M and V4221 by EU) in the case of IgG1 antibodies; N44S, K52N and V821 (IMGT; N384S, K392N and V4221 by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V4221 by EU) in the case of IgG4 antibodies.

[0109] In certain embodiments, the Fc domain can be chimeric, thereby combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can include Fc sequences derived from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence and a portion or all of a C sequence derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may include a combination of an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region and a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any of the antigen-binding molecules described herein include, from N-terminus to C-terminus: [IgG4 C H 1]-[lgG4 upper hinge]-[lgG2 lower hinge]-[lgG4CH2]-[lgG4 C H 3]. Another example of a chimeric Fc domain that can be included in any of the antigen binding molecules set forth herein includes, from N-terminus to C-terminus: [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 C H 2]-[IgG1 C H 3]. These and other examples of chimeric Fc domains that may be included in any of the antigen binding molecules of the present invention are described in WO2014 / 022540A1, the entire contents of which are incorporated herein by reference. Chimeric Fc domains and variants thereof having these general structural arrangements may have altered Fc receptor binding, which in turn affects Fc effector function.

[0110] Sequence variants

[0111] As compared to the corresponding germline sequences derived from a single antigen binding domain, the antibodies and bispecific antigen binding molecules of the present invention may include 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. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, a public antibody sequence database. The antigen binding molecules of the present invention may include an antigen binding fragment derived from any one of the exemplary amino acid sequences disclosed herein, wherein one or more amino acid mutations in one or more frameworks and / or CDR regions are mutated to one or more corresponding residues of the germline sequence derived from the antibody or mutated to one or more corresponding residues of another human germline sequence or mutated to one or more corresponding germline residues. Conservative amino acid substitutions (such sequence changes are collectively referred to herein as "germline mutations")). Starting from the heavy and light chain variable region sequences disclosed herein, a person of ordinary skill in the art can easily produce many antibodies and antigen binding fragments including one or more single germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V L All frameworks and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence that initially derived antigen-binding domains. In other embodiments, only some residues are mutated back to the original germline sequence, such as the residues found only in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4 after mutation or the residues found only in CDR1, CDR2 or CDR3 after mutation. In other embodiments, one or more mutations in one or more frameworks and / or CDR residues are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequences that initially derived antigen-binding domains). In addition, antigen-binding domains can contain any combination of two or more germline mutations in framework and / or CDR regions, for example, wherein some individual residues are mutated into the corresponding residues of specific germline sequences, and some other residues different from the original germline sequence remain unchanged or mutate into the corresponding residues of different germline sequences. Once obtained, antigen binding domains containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen binding molecules comprising antigen binding domains obtained in this general manner are encompassed within the invention.

[0112] The present invention also includes antigen binding molecules, wherein one or two antigen binding domains include 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, which include antigen binding domains having HCVR, LCVR and / or CDR amino acid sequences, relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein, the HCVR, LCVR and / or CDR amino acid sequences have, for example, 10 or less, 8 or less, 6 or less, 4 or less conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) similar to a chemical property (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of amino acid groups with chemically similar side chains 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science, 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0113] The present invention also comprises antigen binding molecules, which include antigen binding domains having HCVR, LCVR and / or CDR amino acid sequences substantially identical to any one of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. When referring to amino acid sequences, the term "substantially identical" or "substantially identical" means that when the best alignment is performed by programs such as GAP or BESTFIT using default gap weights, the two amino acid sequences share at least 95% sequence identity, and even more preferably, at least 98% or 99% sequence identity. Preferably, the non-identical residue positions differ due to conservative amino acid substitutions. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the sequence identity percentage or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods in Molecular Biology 24: 307-331.

[0114] Sequence similarity of polypeptides, also referred to as sequence identity, is usually measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications including conservative amino acid substitutions to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, for example, version 6.1 GCG. FASTA (a program in version 6.1 GCG) using default or recommended parameters can also be used to compare polypeptide sequences. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000) the same). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Molecular Biology 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0115] pH-dependent binding

[0116] 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, as compared to neutral pH. Alternatively, the anti-CD22 antibodies of the present invention may exhibit enhanced binding to CD22 at acidic pH, as compared to neutral pH. The expression "acidic pH" includes 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 pH values. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" encompasses pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0117] In some cases, "reduced binding at acidic pH, as compared to neutral pH" means that the K of an antibody binding to its antigen at acidic pH is D The K value of the antibody binding to its antigen at neutral pH D For example, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D For purposes of the present invention, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to CD28 at acidic pH as compared to neutral pH" if the ratio of acidic pH to neutral pH is greater than or equal to 1.5. D The ratio may 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.

[0118] Antibodies with pH-dependent binding properties can be obtained, for example, by screening for an antibody population that has reduced (or enhanced) binding to a specific antigen at an acidic pH compared to a neutral pH. In addition, modifications of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by replacing one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced binding to an antigen at an acidic pH relative to a neutral pH can be obtained.

[0119] Antibodies including Fc variants

[0120] According to certain embodiments of the present invention, there is provided an anti-CD28 / anti-CD22 bispecific antigen binding molecule comprising an Fc domain, wherein the Fc domain comprises one or more mutations that enhance or attenuate the binding of the antibody to the FcRn receptor at an acidic pH, such as compared to a neutral pH. H Zone 2 or C H Region 3 includes mutated antibodies and antigen-binding molecules, wherein one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). Such mutations can 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, e.g., modification at position 250 (e.g., E or Q); modification at position 250 and position 428 (e.g., L or F); modification at position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modification at position 428 and / or position 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y); or modification at position 250 and / or position 428; or modification at position 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0121] For example, the present invention comprises an anti-CD28 / anti-CD22 bispecific antigen binding molecule comprising an Fc domain comprising one or more mutation pairs or groups 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 foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the present invention.

[0122] Biological properties of antibodies and antigen-binding molecules

[0123] 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 medium or low affinity, depending on the treatment context and the desired specific targeting properties. For example, in the context of a bispecific antigen binding molecule in which one arm binds CD28 and the other arm binds to a target antigen (e.g., CD22), it may be desirable that the target antigen binding arm binds to the target antigen with high affinity, while the anti-CD28 arm only binds to CD28 with medium or low affinity. In this way, preferential targeting of cells expressing target antigens by antigen binding molecules can be achieved while avoiding general / non-targeted CD28 binding and the adverse side effects associated therewith.

[0124] According to certain embodiments, the invention comprises antibodies and antigen-binding fragments of antibodies that have a K of less than about 15 nM. D Binding to human CD22 (e.g., at 25°C), as measured, for example, by surface plasmon resonance using the assay format defined in Example 5 herein. 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. D Binding to human CD22 as measured, for example, by surface plasmon resonance using the assay format defined in Example 5 herein or a substantially similar assay.

[0125] According to certain embodiments, the invention comprises antibodies and antigen-binding fragments of antibodies that have a K of less than about 60 μM. DBinding to monkey CD22 (e.g., at 25°C) as measured, for example, by surface plasmon resonance using the assay format defined in Example 5 herein. In certain embodiments, the antibodies or antigen-binding fragments of the invention are less than about 60 μM, less than about 59 μM, less than about 58 μM, less than about 57 μM, less than about 56 μM, less than about 55 μM, less than about 54 μM, less than about 53 μM, less than about 52 μM, less than about 51 μM, less than about 50 μM, less than about 49 μM, less than about 48 μM, less than about 47 μM, less than about 46 μM, less than about 45 μM, less than about 46 μM, less than about 47 μM, less than about 48 μM, less than about 49 μM, less than about 49 μM, less than about 48 μM, less than about 49 μM, less than about 48 μM, less than about 48 μM, less than about 49 ... The K of 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 Binding to monkey CD22 as measured, for example, by surface plasmon resonance using the assay format defined in Example 5 herein, or a substantially similar assay.

[0126] According to certain embodiments, the invention comprises antibodies and antigen-binding fragments of antibodies that have a K of less than about 45 μM. D Binding to human CD28 (e.g., at 25°C), as measured, for example, by surface plasmon resonance using an assay format as defined in Example 5 herein. In certain embodiments, an antibody or antigen-binding fragment of the invention has 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. D Binding to human CD28 as measured, for example, by surface plasmon resonance using the assay format defined in Example 5 herein or a substantially similar assay.

[0127] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD22, wherein the dissociation half-life (t 1 / 2) is greater than about 7.5 minutes, as measured, for example, by surface plasmon resonance at 25°C using the assay format defined in Example 5 herein or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention are subjected to a t 1 / 2 binding to human CD22: 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, for example, by surface plasmon resonance at 25°C using an assay format defined in Example 5 herein or a substantially similar assay.

[0128] The present invention also comprises antibodies and antigen-binding fragments thereof that bind to monkey CD22, wherein the dissociation half-life (t 1 / 2) is greater than about 4.3 minutes, as measured, for example, by surface plasmon resonance at 37°C using an assay format defined in the Examples herein or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention are 1 / 2 binding to CD28: 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 using an assay format defined in Example 5 herein or a substantially similar assay.

[0129] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD28, wherein the dissociation half-life (t 1 / 2) is greater than about 2.3 minutes, as measured, for example, by surface plasmon resonance at 25°C using the assay format defined in Example 5 herein or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention are subjected to a t 1 / 2 binding to CD28: 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, e.g., by surface plasmon resonance at 25°C or 37°C using an assay format as defined in the Examples herein or a substantially similar assay.

[0130] The present invention also includes bispecific antigen binding molecules (e.g., bispecific antibodies) that can bind to human CD28 and human and monkey CD22. According to certain embodiments, the bispecific antigen binding molecules of the present invention specifically interact with cells expressing CD28 and / or CD22. The degree to which bispecific antigen binding molecules bind 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 macaque cells that express CD28 but do not express CD22 (e.g., T cells) and human cell lines or macaque cells that express CD22 but do not express CD28 (e.g., B cells or Nalm6 cells). The present invention includes bispecific antigen binding molecules that bind to any one of the above cells and cell lines, wherein EC 50 The value is about 1.3x10 -6 To about 2.3x10 -8 M or less as determined using a FACS assay as set forth in Example 6 or a substantially similar assay.

[0131] The present invention includes bispecific antigen binding molecules (e.g., bispecific antibodies) that can bind 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 degree to which bispecific antigen binding molecules bind 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 expressing CD28 but not CD22 (e.g., T cells) and human cell lines expressing 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 includes bispecific antigen binding molecules that bind to any one of the above-mentioned cells and cell lines, wherein EC 50 The value is about 1.14x10 -8 To about 9.76x10 -9 M or less as determined by flow cytometry as set forth in Example 7 or a substantially similar assay.

[0132] The present invention also provides an anti-CD28 / anti-CD22 bispecific antigen binding molecule that induces or enhances the efficacy of CD20xCD3 T cell-mediated killing of tumor cells. For example, the present invention comprises an anti-CD28xCD22 antibody that induces or enhances the efficacy of CD20xCD3 T cell-mediated killing of tumor cells, wherein EC 50 Less than about 1.48x10 -10M, as measured, for example, in an in vitro T cell-mediated tumor cell killing assay using an assay format defined in Example 8 herein (e.g., assessing the extent of killing of Raji cells by human PBMCs in the presence of anti-CD20xCD3 antibodies and anti-CD28xCD22 antibodies) or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention induce T cell-mediated tumor cell killing (e.g., PBMC-mediated killing of Raji cells), wherein EC 50 A value 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 using, for example, an assay format defined in Example 8 herein or a substantially similar assay.

[0133] The present invention also includes anti-CD28 / anti-CD22 bispecific antigen binding molecules that exhibit one or more properties selected from the group consisting of: activated T cells, induced IL-2 release, induced CD25+ upregulation in human PBMCs, and increased human T cell-mediated cytotoxicity to 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 CD22-expressing tumor cells when combined with bispecific antibodies that bind 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 CD22-expressing tumor cells when combined with antibodies that bind PD-1 (such as, but not limited to, Cimiprilimab). (See Examples 10-15).

[0134] Epitope mapping and related technologies

[0135] The epitope on CD28 or CD22 to which the antigen binding molecules of the present invention are combined can be composed of a single continuous sequence of 3 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 CD28 protein or CD22 protein. Alternatively, the epitope can be composed of multiple non-continuous amino acids (or amino acid sequences) of CD28 or CD22. The antibody of the present invention can interact with the amino acids contained in the CD28 monomer or can interact with the amino acids on two different CD28 chains of the CD28 dimer. As used herein, 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 a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. An epitope can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl or sulfonyl group on the antigen.

[0136] Various techniques known to those of ordinary 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, conventional cross-blocking assays, such as those described in: Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY); site mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.); peptide blot analysis (Reineke, 2004, Methods in Molecular Biology 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 used (Tomer, 2000, Protein Science Science)》9:487-496). Another method that can be used to identify amino acids in polypeptides that interact with antibodies is hydrogen / deuterium exchange detected by mass spectrometry. In general, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest and then binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the antibody-protected residues (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids that interact with the antibody. See, for example, Ehring (1999) 《Analytical Biochemistry (Analytical Biochemistry)》267(2):252-259; Engen and Smith (2001) 《Analytical Chemistry (Anal. Chem.)》73:256A-265A. X-ray crystal structure analysis can also be used to identify amino acids in polypeptides that interact with antibodies.

[0137] The invention further encompasses anti-CD28 and anti-CD22 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences shown in Table 6 herein).

[0138] According to certain embodiments, the present invention provides antibodies and antigen-binding fragments of antibodies that bind to an epitope on human CD22, wherein the epitope includes 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 shown in Examples 3 and 4.

[0139] Likewise, the present invention also encompasses anti-CD28 and / or anti-CD22 antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences shown in Table 6 herein) for binding to CD28 and / or CD22.

[0140] The present invention also encompasses bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds to human CD28 and a second antigen binding fragment that specifically binds to human CD22, wherein the first antigen binding domain binds to the same epitope on CD28 as any of the specific exemplary CD28-specific antigen binding domains described herein, and / or wherein 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.

[0141] Likewise, the present invention also encompasses bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds to human CD28 and a second antigen binding fragment that specifically binds to human CD22, wherein the first antigen binding domain competes for binding to CD28 with any one of the specific exemplary CD28-specific antigen binding domains described herein, and / or wherein the second antigen binding domain competes for binding to CD22 with any one of the specific exemplary CD22-specific antigen binding domains described herein.

[0142] By using conventional methods known in the art, it is easy to determine whether a specific antigen binding molecule (e.g., antibody) or its antigen binding domain is combined with or competes with the reference antigen binding molecule of the present invention to bind to the same epitope as the reference antigen binding molecule of the present invention. For example, in order to determine whether the test antibody is combined with the same epitope as the reference bispecific antigen binding molecule of the present invention on CD28 (or CD22), first allow the reference bispecific molecule to bind to CD28 protein (or CD22 protein). Next, assess the ability of the test antibody to bind to CD28 (or CD22) molecules. 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 is combined with an epitope different from the reference bispecific antigen binding molecule in CD28 (or CD22). On the other hand, if the test antibody cannot be combined with the CD28 (or CD22) molecule after saturation binding with the reference bispecific antigen binding molecule, then the test antibody can be combined with the epitope identical to the epitope combined with the reference bispecific antigen binding molecule of the present invention in CD28 (or CD22). Then other routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of test antibody binding is actually due to binding to the same epitope as the reference bispecific antigen binding molecule or whether steric blocking (or another phenomenon) is the reason for the observed lack of binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assays available in the art. According to certain embodiments of the present invention, if, for example, 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antigen-binding protein inhibits at least 50% of another antigen-binding protein, but preferably 75%, 90%, or even 99% of the binding (as measured in a competitive binding assay), then two antigen-binding proteins are combined with the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen binding proteins are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen binding protein also reduce or eliminate binding of the other antigen binding protein. Two antigen binding proteins are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other antigen binding protein.

[0143] In order to determine whether the antibody or its antigen binding domain competes with the reference antigen binding molecule for binding, the above-mentioned binding method is performed in two orientations: in the first orientation, the reference antigen binding molecule is allowed to bind to the CD28 protein (or CD22 protein) under saturation conditions, and then the binding of the test antibody to the CD28 (or CD22) molecule is evaluated. In the second orientation, the test antibody is allowed to bind to the CD28 (or CD22) molecule under saturation conditions, and then the binding of the reference antigen binding molecule to the CD28 (or CD22) molecule is evaluated. If only the first (saturated) antigen binding molecule can bind to the CD28 (or CD22) molecule in two orientations, it can be concluded that the test antibody and the reference antigen binding molecule compete for binding to CD28 (or CD22). As understood by those of ordinary skill in the art, the antibody that competes for binding with the reference antigen binding molecule may not necessarily bind to the same epitope as the reference antibody, but can spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0144] Preparation of antigen binding domains and construction of bispecific molecules

[0145] Antigen binding domains with specificity for a particular antigen can be prepared by any antibody generation technology known in the art. Once obtained, two different antigen binding domains with specificity for two different antigens (e.g., CD28 and CD22) can be appropriately arranged relative to each other to produce the bispecific antigen binding molecules of the present invention using conventional methods. (Discussion of exemplary bispecific antibody forms that can be used to construct bispecific antigen binding molecules of the present invention is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy chain and light chain) of the multispecific antigen binding molecules of the present invention are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used TM One or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the invention are prepared using VELOCIMMUNE TM The invention relates to a method for producing antibodies of the invention that is directed to a human antigen (e.g., CD28 or CD22) using a human variable region and a mouse constant region. The antibodies are characterized and selected to obtain the desired properties including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0146] Human bispecific antigen binding molecules can be prepared using genetically engineered animals.For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein mice only express one or two human light chain variable domains encoded by human immunoglobulin sequences, and the human immunoglobulin sequences are operably connected to the mouse κ constant genes at the endogenous mouse κ locus.This genetically modified mouse can be used to produce fully human bispecific antigen binding molecules including two different heavy chains, and the heavy chain is associated with the same light chain including the variable domain derived from one of two different human light chain variable region gene segments.(For a detailed discussion of this engineered mouse and its use for producing bispecific antigen binding molecules, see, for example, US2011 / 0195454, the entire contents of the document are incorporated herein by reference).

[0147] Bioequivalence

[0148] The present invention encompasses antigen binding molecules with amino acid sequences different from the described antibodies but retaining the ability to bind CD28 and / or CD22. Compared with the parent sequence, such variant molecules include one or more additions, deletions or substitutions of amino acids, but show biological activities substantially equivalent to the biological activities of the described antigen binding molecules. Similarly, the DNA sequence of the present invention encoding the antigen binding molecules encompasses such sequences: compared with the disclosed sequence, it includes one or more additions, deletions or substitutions of nucleotides, but encodes antigen binding molecules substantially bioequivalent to the described antigen binding molecules of the present invention. Examples of such variant amino acids and DNA sequences are discussed above.

[0149] The present invention includes any bioequivalent antigen binding molecule with the exemplary antigen binding molecules shown herein.If for example two antigen binding proteins or antibodies are to be used with the same molar dose (single dose or multiple doses) under similar experimental conditions, absorption rate and absorption extent do not show the drug equivalent or drug substitute of significant difference, then it is considered that the two antigen binding proteins or antibodies are bioequivalent.If some antibodies are equivalent in their absorption extent but not equivalent in their absorption rate, then it is considered that the antibody is an equivalent or drug substitute, and it can be considered that the antibody is bioequivalent, because this absorption rate difference that is intended and is reflected in the labeling is not necessary for reaching effective body drug concentration when for example long-term use and is considered to be medically insignificant for the specific drug product studied.

[0150] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in safety, purity, and potency of the two antigen binding proteins.

[0151] In one embodiment, two antigen binding proteins are bioequivalent if patients can switch between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or reduced effectiveness, compared to continued therapy in the absence of switching between the reference product and the biological product.

[0152] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use (provided such mechanisms are known).

[0153] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence metrics include, for example: (a) in vivo testing in humans or other mammals, where the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum or other biological fluids; (b) in vitro testing that correlates with and is reasonably predictive of bioavailability data in humans; (c) in vivo testing in humans or other mammals, where appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) in well-controlled clinical trials to establish the safety, efficacy or bioavailability or bioequivalence of the antibody.

[0154] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed by, for example, making various substitutions to residues or sequences or deleting terminal or internal residues or sequences required for non-biological activity. For example, cysteine ​​residues required for non-biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies can include the exemplary bispecific antigen-binding molecules described herein that include amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0155] Species selectivity and cross-species reactivity

[0156] 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 bind to CD28 from one or more non-human species; and / or antigen binding molecules that bind to human CD22 and bind to CD22 from one or more non-human species.

[0157] According to certain exemplary embodiments of the present invention, antigen binding molecules are provided, which bind to human CD28 and / or human CD22 and may bind to or not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, macaque, marmoset, rhesus monkey or chimpanzee CD28 and / or CD22, as appropriate. For example, in a specific exemplary embodiment of the present invention, a bispecific antigen binding molecule is provided, which includes a first antigen binding domain that binds to human CD28 and macaque CD28 and a second antigen binding domain that specifically binds to human CD22.

[0158] Immunoconjugates

[0159] The present invention encompasses antigen binding molecules conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of suitable cytotoxic and chemotherapeutic agents for forming immunoconjugates are known in the art (see, e.g., WO 05 / 103081, the entire contents of which are incorporated herein by reference).

[0160] Therapeutic formulations and administration

[0161] The present invention provides pharmaceutical compositions comprising antigen binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated together with suitable carriers, excipients and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cations or anions) containing vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous adsorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0162] The dosage of the antigen binding molecules applied to the patient can vary according to the patient's age and body type, target disease, symptom, route of administration, etc. The preferred dosage is usually calculated according to body weight or body surface area. When the bispecific antigen binding molecules of the present invention are used in adult patients for therapeutic purposes, it is usually advantageous to administer the bispecific antigen binding molecules of the present invention intravenously with a single dose of 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 symptom, the frequency and duration of treatment can be adjusted. The effective dose and schedule for administering the bispecific antigen binding molecules can be determined empirically; for example, patient progress can be monitored by regular assessment, and the dosage can be adjusted accordingly. In addition, interspecific analogies of dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, "Pharmaceut. Res.", 8: 1351).

[0163] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other bioactive agents. Administration can be systemic or local.

[0164] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered, and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0165] Many reusable pen delivery devices and auto-injector delivery devices are useful for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Co., Indianapolis, IN), I, II and III NOVOPEN TM (NovoNordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM PEN (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPENPRO TM 、OPTIPEN STARLET TM and OPTICLK TM (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (Sanofi), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLlCK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TMPen (Abbott Labs, Abbott Park IL), to name a few.

[0166] In some cases, 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: Critical Review of Bioengineering (CRC Crit. Ref Biomed. Eng.) 14: 201). In another embodiment, a polymer material can be used; see "Medical Applications of Controlled Release", Langer and Wise (ed.), 1974, CRC Press, Boca Raton, Florida (CRC Pres., Boca Raton, Florida). In yet another embodiment, a controlled release system can be placed near the target of the composition, so only a portion of the systemic dose is required (see, for example, Goodson, 1984, "Medical Applications of Controlled Release" supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review of Langer, 1990, Science 249: 1527-1533.

[0167] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, instillations, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or a conventional oily medium for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose, and other adjuvants, etc., which can be used in combination with suitable solubilizers such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc. As an oily medium, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection prepared in this way is preferably filled in a suitable ampoule.

[0168] Advantageously, the pharmaceutical composition for oral or parenteral use is prepared into a unit dosage form suitable for matching a certain dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is usually about 5 mg to about 500 mg per unit dosage form; especially in the form of injection, it is preferred that the content of the above-mentioned antibody is about 5 mg to about 100 mg and about 10 mg to about 250 mg for other dosage forms.

[0169] Therapeutic uses of antigen binding molecules

[0170] The present invention includes methods of administering a therapeutic composition to a subject in need thereof, the 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 include any of the antibodies or bispecific antigen binding molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that exhibits a tumor or suffers from any of the cancers mentioned herein below) or otherwise benefits from inhibition or reduction of CD22 activity or depletion of CD22+ cells.

[0171] The antibodies and bispecific antigen binding molecules of the present invention (and therapeutic compositions comprising the same) are particularly useful for treating any disease or condition in which stimulation, activation and / or targeting of an immune response would be beneficial. Specifically, the anti-CD28 / anti-CD22 bispecific antigen binding molecules of the present invention can be used to treat, prevent and / or improve any disease or condition associated with or mediated by CD22 expression or activity or CD22+ cell proliferation. The mechanism of action of the therapeutic method of the present invention is included in the presence of effector cells such as T cells that kill cells expressing CD22. 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.

[0172] The antigen binding molecules of the present invention can be used to treat primary and / or metastatic tumors occurring in, for example, blood, bone marrow, lymph nodes (e.g., thymus, spleen), colon, liver, lung, breast, kidney cancer, central nervous system, and bladder cancer. According to certain exemplary embodiments, the bispecific antigen binding molecules of the present invention are used to treat B cell proliferative disorders.

[0173] The present invention also encompasses methods for treating residual cancer in a subject.As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0174] According to certain aspects, the present invention provides a method for treating a disease or condition associated with CD22 expression (e.g., B cell proliferative disorders), the method being included in the application of one or more of the bispecific antigen binding molecules described elsewhere herein to a subject after showing that the subject is unresponsive to other types of anti-cancer therapies. For example, the present invention includes a method for treating B cell proliferative disorders, the method being included in the application of anti-CD28 / anti-CD22 bispecific antigen binding molecules to a patient after the subject has received standard care for a patient with cancer, such as a B cell proliferative disorder, 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. In other aspects, initially at one or more time points, a subject is administered a bispecific antigen binding molecule (anti-CD28 / anti-CD22 bispecific antigen binding molecule) of the present invention including an IgG4 Fc domain (e.g., to provide a robust initial depletion of prostate cancer cells), followed by administration of an equivalent bispecific antigen binding molecule including different IgG domains such as an IgG1 Fc domain at a subsequent time point. It is conceivable that the anti-CD28 / anti-CD22 antibodies of the present invention can be used in conjunction with other bispecific antigen binding molecules such as anti-CD20 / anti-CD3 bispecific antibodies. It is also conceivable that the bispecific antibodies of the present invention are used in conjunction with checkpoint inhibitors such as checkpoint inhibitors targeting PD-1 and CTLA-4 and other targets. Combining two bispecific antibodies targeting the same tumor antigen (e.g., CD22) can be advantageous, but one of the bispecific antibodies targets CD3 on T cells, while the other bispecific antibody targets costimulatory molecules such as CD28. This combination can be used alone to enhance tumor cell killing or can be used in combination with checkpoint inhibitors.

[0175] Combination therapies and formulations

[0176] The 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 components, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0177] Additional exemplary therapeutic agents that can be combined or administered in combination with the antigen binding molecules of the invention include, for example, chemotherapy, radiotherapy, checkpoint inhibitors targeting PD-1 (e.g., anti-PD-1 antibodies such as pembrolizumab, nivolumab or cemiplimab, see US9,987,500, HCVR / LCVR of SEQ ID NO 162 / 170), CTLA-4, LAG3, TIM3, etc., co-stimulatory agonist bivalent antibodies targeting molecules such as GITR, OX40, 4-1BB, etc., CD3x bispecific antibodies (see, e.g., US9,657,102 (REGN1979), WO2017 / 053856A1, WO2014 / 047231A1, WO2018 / 067331A1 and WO2018 / 058001A1), targeting CD22xCD3, CD22xCD28 or targeting CD20xCD3. Other antibodies to CD3 and other co-stimulatory CD28x bispecific antibodies.

[0178] Other agents that can 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 and 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. The pharmaceutical compositions of the invention (e.g., pharmaceutical compositions comprising anti-CD28 / anti-CD22 bispecific antigen binding molecules as disclosed herein) can also be administered as part of a therapeutic regimen that includes one or more therapeutic combinations selected from the following: "ICE": ifosfamide (e.g., ), carboplatin (e.g., ), etoposide (e.g., VP-16); "DHAP": dexamethasone (e.g., ), cytarabine (e.g., cytosine arabinoside, ara-C), cisplatin (e.g., ); and "ESHAP": etoposide (e.g., VP-16), methylprednisolone (e.g. ), high-dose cytarabine, cisplatin (e.g. ).

[0179] The present invention also comprises a therapeutic combination, the therapeutic combination comprising any one of the antigen binding molecules mentioned herein and one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvlll, cMet, IGF1 R, B-raf, PDGFR-o, PDGFR-I3, FOLH1, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin or any inhibitor of the above cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules, such as double antibodies, three antibodies, four antibodies, mini antibodies and minimum recognition units). The antigen binding molecules of the present invention can also be administered in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs and / or co-formulated. The antigen binding molecules of the invention may 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).

[0180] The present invention includes compositions and therapeutic formulations comprising any of the antigen binding molecules described herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards, such as thiamethoxam, ... mustards, such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, methoxychlor, melphalan, nembixin, phenylephrine, prednimustine, trolofosamide, uramustine; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclarubicin, actinomycin, anthramycin, azaserine , bleomycin, cactinomycin, calicheamicin, caramidocin, carmomycin, chromomycin, actinomycin D, daunorubicin, detoximum, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins, mycophenolic acid, nogamycin, olivemycins, peplomycin, potfiromycin, puromycin, triferon-doxorubicin, rhodorubicin, streptomycin, streptozotocin, tuberculocin, ubenamide , chlortetracycline, doxycycline, oxazolidinone, chlortetracycline, oxazolidinone, doxy ... leucoderma; mitotane, trilostane; folic acid supplements such as folinic acid; acetoglucosalactone; aldehyde phosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclofenac; diazocine; eflornithine; elliptonium acetate; etogluconate; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; diamine nitracrine; pentostatin; methamine nitrogen mustard; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK TM; aprotinin; sizoran; spirogermanium; tenuazonic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel TM , 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; noantor; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition also includes antihormonal agents used to modulate or inhibit hormonal effects on tumors, such as: antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, troxifene, keoxifene, LY 117018, onapristone and toremifene (Falutamide); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0181] One or more additional therapeutically active components may be administered prior to, simultaneously with, or immediately after administration of the antigen binding molecules of the invention; (for purposes of this disclosure, such administration regimens are considered administration of the antigen binding molecules "in combination with" additional therapeutically active components).

[0182] The invention encompasses pharmaceutical compositions in which the antigen binding molecules of the invention are co-formulated together with one or more additional therapeutically active ingredients as described elsewhere herein.

[0183] Administration regimen

[0184] According to certain embodiments of the present invention, multiple doses of antigen binding molecules (e.g., anti-CD28 antibodies or bispecific antigen binding molecules specifically in combination with CD22 and CD28) can be administered to a subject within a limited time course. The method according to this aspect of the present invention includes sequentially administering multiple doses of the antigen binding molecules of the present invention to a subject. As used herein, "sequential administration" means administering each dose of antigen binding molecules to a subject at different time points, for example, on different dates separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes the following method: sequentially administering a single initial dose of antigen binding molecules to a patient, followed by one or more second doses of antigen binding molecules and optionally then one or more third doses of antigen binding molecules.

[0185] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time series of the antigen binding molecules of the present invention. Therefore, "initial dose" is the dosage (also referred to as "baseline dose") applied at the beginning of the treatment regimen; "secondary dose" is the dosage applied after the initial dose; and "tertiary dose" is the dosage applied after the second dose. Initial dose, second dose and third dose can all contain the same amount of antigen binding molecules, but may generally be different from each other in terms of frequency of application. However, in certain embodiments, during the course of treatment, the amount of antigen binding molecules contained in initial dose, second dose and / or third dose is different from each other (for example, appropriately adjusted up or down). In certain embodiments, two or more (for example, 2, 3, 4 or 5) dosages are applied as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses (for example, "maintenance doses") applied based on lower frequency.

[0186] In an exemplary embodiment of the invention, 1 to 26 (e.g., 1, 1 1 / 2,2,2 1 / 2,3,3 1 / 2,4,4 1 / 2,5,5 1 / 2,6,6 1 / 2,7,7 1 / 2,8,8 1 / 2,9,9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 Each second dosage and / or the 3rd dosage is administered in 1 / 2 or more weeks. As used herein, phrase "previous dosage" means in a sequence of multiple administrations, in the absence of an intermediate dose, before the next dosage in the sequence is administered, the dosage of the antigen binding molecules administered to the patient.

[0187] The method according to this aspect of the invention may include administering any number of second doses and / or third doses of antigen binding molecules (e.g., anti-CD28 antibodies or bispecific antigen binding molecules that specifically bind 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.

[0188] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administering the second dose and / or the third dose to the patient can vary during the course of the treatment regimen. The frequency of administration can also be adjusted according to the needs of individual patients after clinical examination during the course of treatment by a physician.

[0189] In one embodiment, the antigen binding molecule (e.g., a bispecific antigen binding molecule that specifically binds CD22 and CD28) is administered to a subject at a dose based on body weight. 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 is altered according to the subject's body weight.

[0190] In another embodiment, the antibody or its Fab or bispecific antigen binding molecule is administered to the subject in a fixed dose. "Fixed dose" (e.g., in mg) means that a dose of the antibody or its Fab or bispecific antigen binding molecule is used for all subjects, regardless of any specific subject-related factors such as body weight. In a specific embodiment, the fixed dose of the antibody or its Fab or bispecific antigen binding molecule of the present invention is based on a predetermined body weight or age.

[0191] Typically, the scope of the suitable dose of antigen binding molecules of the present invention can be about 0.001 to about 200.0 milligrams per kilogram of recipient body weight, typically in the range of about 1 to 50 mg per kilogram of body weight. For example, antibody or its Fab or bispecific antigen binding molecules can be applied by 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. The intermediate value and scope of stated values ​​are also intended to be a part of the present invention.

[0192] In some embodiments, the antigen binding molecules of the present invention are administered at a fixed dose of about 25 mg to about 2500 mg. In some embodiments, the antigen binding molecules of the present invention are 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, 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 600 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2500 mg, about 2750 mg, about 3000 mg, about 3250 mg, about 3500 mg, about 3750 mg, about 4000 mg, about 4250 mg, about 4500 mg, about 4750 mg, about 50 ... In some embodiments, the present invention relates to an oral dosage form of at least about 100 mg of the present invention. The oral dosage form of at least about 100 mg of the present invention is preferably about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 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 stated values ​​are also intended to be part of the present invention.

[0193] Diagnostic Uses of Antibodies

[0194] The bispecific antibodies of the invention can also be used, for example, for diagnostic purposes to detect and / or measure CD28 or CD22 or CD28-expressing or CD22-expressing cells in a sample. For example, anti-anti-CD28 x anti-CD22 antibodies or fragments thereof can be used to diagnose a condition or disease characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of CD28 or CD22. Exemplary diagnostic assays for CD28 or CD22 can include, for example, contacting a sample obtained from a patient with an antibody of the invention, wherein the antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled antibody can be combined with a second antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule can be: a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125 I; fluorescent or chemiluminescent moieties, such as fluorescein isothiocyanate or rhodamine; or enzymes, 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 CD28 or CD22 diagnostic assays according to the present invention include any tissue or fluid sample obtained from a patient containing a detectable amount of CD28 or CD22 protein or fragment thereof under normal or pathological conditions. Typically, the CD28 or CD22 level in a specific sample obtained from a healthy patient (e.g., a patient who does not suffer from a disease or condition associated with abnormal CD28 or CD22 levels or activity) is measured to preliminarily establish a baseline or standard CD28 or CD22 level. This baseline CD28 or CD22 level can then be compared to the CD28 or CD22 level measured in a sample obtained from an individual suspected of having a CD28 or CD22-related disease or condition.

[0195] Examples

[0196] The following examples are presented to provide a complete disclosure and description of how to prepare and use the methods and compositions of the present invention to those of ordinary skill in the art, and the following examples are not intended to limit the scope of the inventors' invention. Efforts have been made to ensure accuracy with respect to the numerals used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is or is close to atmospheric pressure.

[0197] Example 1: Construction of anti-CD22xCD28 antibody

[0198] Generation of anti-CD28 antibodies

[0199] The cells were fused with human CD28 protein fused to the Fc portion of mouse IgG2a, or with cells expressing CD28, or with DNA encoding CD28. Mice (i.e., engineered mice comprising DNA encoding human immunoglobulin heavy chains and kappa light chain variable regions) are immunized to obtain anti-CD28 antibodies. The antibody immune response is monitored by CD28-specific immunoassay. When the desired immune response is achieved, splenocytes are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines are screened and selected to identify cell lines that produce CD28-specific antibodies. Several anti-CD28 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) have been obtained using this technology. In addition, as described in US 2007 / 0280945A1, several fully human anti-CD28 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells.

[0200] Certain biological properties of exemplary anti-CD28 antibodies produced according to the methods of this example are described in detail in the Examples presented below.

[0201] Generation of anti-CD22 antibodies

[0202] By using human CD22 antigen (e.g., see hCD22 ecto (D20-R687).hFc, R&D Systems, Catalog No. 1968-SL-050; Accession No. CAA42006) (see also FIG. 3 ), the genetically modified mice ( Anti-CD22 antibodies are obtained by immunizing mice (e.g., mice, see above) or by immunizing engineered mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human CD22 antigen.

[0203] After immunization, spleen cells are harvested from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells and screened for their CD22 specificity or (2) subjected to B cell sorting using human CD22 fragments as sorting agents to bind and identify reactive antibodies (antigen-positive B cells) (as described in US 2007 / 0280945A1).

[0204] Initially, chimeric antibodies against CD22 with human variable regions and mouse constant regions were isolated. The antibodies were characterized and selected to obtain desired properties including affinity, selectivity, etc. If necessary, the mouse constant region was replaced with a desired human constant region, such as a wild-type or modified IgG1 or IgG4 constant region, to generate fully human anti-CD22 antibodies. Although the selected constant region can vary depending on the specific use, high affinity antigen binding and target specificity properties are present in the variable region.

[0205] Generation of bispecific antibodies binding CD28 and CD22

[0206] Bispecific antibodies comprising an anti-CD22 specific binding domain and an anti-CD28 specific binding domain are constructed using standard methods, wherein the anti-CD22 antigen binding domain and the anti-CD28 antigen binding domain each comprise a different unique HCVR paired with a common LCVR. In some cases, the bispecific antibodies are constructed using a heavy chain from an anti-CD28 antibody, a heavy chain from an anti-CD22 antibody, and a common light chain (see Table 1).

[0207] The bispecific antibody produced according to this example includes two separate antigen binding domains (i.e., binding arms). The first antigen binding domain includes a heavy chain variable region derived from an anti-CD28 antibody ("CD28-VH"), and the second antigen binding domain includes a heavy chain variable region derived from an anti-CD22 antibody ("CD22-VH"). Anti-CD22 and anti-CD28 both share a common light chain. The CD28-VH / CD22-VH pairing produces an antigen binding domain that specifically recognizes CD28 on T cells and CD22 on tumor cells.

[0208] Example 2: Heavy and light chain variable region amino acid and nucleic acid sequences

[0209] Table 1 lists the amino acid sequence identifiers of the heavy chain and light chain variable regions and CDRs of selected anti-CD22 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0210] Table 1: Amino Acid Sequence Identifiers of CD22 Antibodies

[0211]

[0212] Table 2: Nucleic acid sequence identifiers of CD22 antibodies

[0213]

[0214]

[0215] Table 3 lists the amino acid sequence identifiers of the heavy chain and light chain variable regions (HCVR and LCVR), CDRs of selected anti-CD28 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 4.

[0216] Table 3: Amino acid sequence identifiers of CD28 antibodies

[0217]

[0218] Table 4: Nucleic acid sequence identifiers of CD28 antibodies

[0219]

[0220] A partial summary of the components of the various anti-CD22xanti-CD28 bispecific antibodies constructed is listed in Table 5. Tables 6 and 7 list the HCVR, LCVR, CDR, and heavy and light chain sequence identifiers of the bispecific antibodies.

[0221] Table 5: Summary of components of anti-CD22 x anti-CD28 bispecific antibodies

[0222]

[0223] Table 6 shows the amino acid sequence identifiers for the bispecific anti-CD22 x anti-CD28 antibodies exemplified herein. The corresponding nucleic acid sequence identifiers are listed in Table 7.

[0224] Table 6: Amino acid sequences of anti-CD22 x anti-CD28 bispecific antibodies

[0225]

[0226] Table 7: Nucleic acid sequences of anti-CD22 x anti-CD28 bispecific antibodies

[0227]

[0228] Example 3: Epitope mapping of REGN5837 binding to CD22 by hydrogen-deuterium exchange

[0229] Mass spectrometry H / D exchange epitope mapping (HDX-MS) was performed to identify amino acid residues in CD22 (recombinant human CD22, SEQ ID NO: 50) that interact with H4sH33037P2 (see Table 1, HCVR / LCVR pair of SEQ ID NO: 2 / 10) (anti-hCD22 monoclonal antibody; parent anti-hCD22 antibody of REGN5837). A general description of the H / D exchange method is listed in, for example, Ehring (1999) Analytical Biochemistry 267(2): 252-259; and Engen and Smith (2001) Analytical Chemistry 73: 256A-265A.

[0230] 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 (μ binary solvent manager) for resolving gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0231] The labeling solution was prepared as PBS buffer (10 mM phosphate buffer, 140 mM NaCl and 3 mM KCl, corresponding to pH 7.4, temperature 25° C.) in D 2 O at pD 7.0. For deuterium labeling, at various time points, 11 μL CD22.mmH (REGN5140 (SEQ ID NO: 50) or 56.7 μM CD22.mmH premixed with H4sH33037P2 (see above) at a 1:0.6 molar ratio (Ag-Ab complex) was incubated with 44 μL D2O labeling solution at 20°C in duplicate (e.g., undeuterated control = 0 seconds; deuterium labeled 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 55 μL pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample for 5 minutes at 20°C. The quenched samples were then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The 50 μL DNase was passed through a C8 column (1.0 mm x 100 μL). 50 mm, Novabioassays) from 10%-32% B (mobile phase A: 0.5% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile) in a 13-minute gradient. The eluted peptides were analyzed by QExactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0232] The LC-MS / MS data of the undeuterated CD22 samples were searched against a database containing CD22 and its randomized sequences using the Byonic search engine (Protein Metrics). The search parameters (in ELN) were set to default values ​​using nonspecific enzyme digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into the 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 the deuterium uptake (D) and the deuterium uptake percentage (%D) (see below).

[0233]

[0234] A total of 427 peptides from hCD22.mmH (SEQ ID NO: 50) were identified from both hCD22.mmH alone and hCD22.mmH complexed with H4sH33037P2 (HCVR / LCVR pair of SEQ ID NO: 2 / 10) samples, representing a sequence coverage of 92.0% for hCD22. Any peptide that exhibited a percentage difference in D uptake values ​​greater than 5% was defined as being 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.

[0235] Table 8: Selected CD22.mmH peptides with significant protection when bound to H4sH33037P2

[0236]

[0237] Example 4: Epitope mapping of H4sH33041P2 binding to CD22 by hydrogen-deuterium exchange

[0238] Mass spectrometry H / D exchange peptide epitope mapping (HDX-MS) was performed to identify 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 of SEQ ID NO: 18 / 10) (parent anti-hCD22 of REGN5838). A general description of the H / D exchange method is listed in, for example, Ehring (1999) Analytical Biochemistry 267(2): 252-259; and Engen and Smith (2001) Analytical Chemistry 73: 256A-265A.

[0239] 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 (μ binary solvent manager) for resolving gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0240] The labeling solution was prepared as PBS buffer (10 mM phosphate buffer, 140 mM NaCl and 3 mM KCl, corresponding to pH 7.4, temperature 25° C.) in D 2 O at pD 7.0. For deuterium labeling, 11 μL CD22.mmH (REGN5140 (SEQ ID NO: 50) or 56.7 μM CD22.mmH premixed with H4sH33041P2 at a 1:0.6 molar ratio (Ag-Ab complex) was incubated with 44 μL D2O labeling solution at 20°C in duplicate at various time points (e.g., undeuterated control = 0 seconds; deuterium labeled for 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 55 μL pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample for 5 minutes at 20°C. The quenched samples were then injected into a Waters HDX manager for online pepsin / protease XIII digestion. The 100 μL DNase was added to the column and the quenching buffer was added to the column. The quenched samples were then injected into a Waters HDX manager for online pepsin / protease XIII digestion. The DNase was then added to the column and the DNase was added to the column. 50 mm, Novartis Bioassays) from 10%-32% B (mobile phase A: 0.5% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile) in a 13 min gradient. The eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0241] The LC-MS / MS data of the undeuterated CD22 samples were searched against a database containing CD22 and its randomized sequences using the Byonic search engine (Protein Metrics). The search parameters (in ELN) were set to default values ​​using nonspecific enzyme digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into the 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 deuterium uptake (D) and deuterium uptake percentage (D%) were calculated using the center of mass mass (intensity-weighted average mass) at each time point, as shown below.

[0242]

[0243] A total of 454 peptides from hCD22.mmH (SEQ ID NO: 50) were identified from both hCD22.mmH alone and hCD22.mmH complexed with H4sH33041P2 samples, representing a sequence coverage of 90.5% for hCD22. Any peptide that exhibited a percentage difference in D uptake values ​​greater than 5% was defined as being significantly protected. For hCD22.mmH, the peptide corresponding to amino acids 246-277 (CEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNL; SEQ ID NO: 59) was significantly protected by H4sH33041P2. Table 9 provides results from selected peptides that were significantly protected when combined with H4sH33041P2.

[0244] Table 9: Selected CD22.mmH peptides with significant protection when bound to H4sH33041P2

[0245]

[0246]

[0247] Example 5: Binding affinity and kinetic constants obtained by surface plasmon resonance for CD22xCD28 bispecific antibodies

[0248] The equilibrium dissociation constants (K) of hCD22.mmH (SEQ ID NO: 50) and mfCD22.mmH (SEQ ID NO: 51) binding to purified anti-CD22xCD28 bispecific mAbs or anti-CD22 bivalent parental mAbs (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 a Biacore T-200 or Biacore 4000 instrument. D The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567; HCVR / LCVR: SEQ ID NO: 33 / 34) to capture purified anti-CD22xCD28 bispecific or anti-CD22 parent mAbs (see Tables 1 and 2 for mAb33037P2 and mAb33041P2). This Biacore binding study was performed in a buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM 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) with a C-terminal myc.myc hexa-histidine tag ("hexa-histidine" as disclosed in SEQ ID NO: 60) prepared in HBS-EP running buffer (ranging from 90 nM to 3.33 nM or 0.37 nM, 3-fold dilution) were injected over the mAb capture surface at a flow rate of 30 microliters / minute. The association of CD22.mmH (SEQ ID NO: 50) with the captured monoclonal antibody was monitored for 5 minutes, and the dissociation of CD22.mmH in HBS-EP running buffer was monitored for 10 minutes. All binding kinetic experiments were performed at 25°C. The kinetic association (k) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. a ) and dissociation (k d ) rate constant. Calculate the binding and dissociation equilibrium constant (K) from the kinetic rate constant D ) and dissociation half-life (t 1 / 2) is:

[0249] K D (M) = k d / k a and t 1 / 2(minutes)=0.693 / k d / 60

[0250] The binding kinetic parameters for human and cyno CD22 binding to purified mAbs at 25°C are shown below in Tables 10-12.

[0251] The equilibrium dissociation constants (K) of hCD28.mmH (SEQ ID NO: 54) purified anti-CD22xCD28 bispecific mAb or anti-CD28 bivalent parental mAb (for mAb14226P2, see Tables 3 and 4) were determined using a real-time surface plasmon resonance biosensor using a Biacore T-200 instrument. D Values). The CM4 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567; HCVR / LCVR SEQ ID NO: 33 / 34) to capture purified anti-CD22xCD28 bispecific or anti-CD28 parent mAb (see above). This Biacore binding study was 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 with a C-terminal myc.myc hexa-histidine tag ("hexa-histidine" as disclosed in SEQ ID NO: 60) prepared in HBS-EP running buffer (ranging from 600nM to 2.47nM, 3-fold dilution) were injected over the mAb capture surface at a flow rate of 50 microliters / minute. The association of CD28.mmH (SEQ ID NO: 54) with the captured monoclonal antibody was monitored for 5 minutes, and the dissociation of CD28.mmH in HBS-EP running buffer was monitored for 10 minutes. All binding kinetic experiments were performed at 25°C. The kinetic association (k) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. a ) and dissociation (k d ) rate constant. Calculate the binding and dissociation equilibrium constant (K) from the kinetic rate constant D ) and dissociation half-life (t 1 / 2) is:

[0252] K D (M) = k d / k a and t 1 / 2(minutes)=0.693 / k d / 60

[0253] The binding kinetic parameters for human CD28 binding to purified mAbs at 25°C are shown below in Table 13.

[0254] Table 10: Binding kinetics of human CD22.mmH to anti-CD22xCD28 bispecific mAbs at 25°C

[0255]

[0256] Table 11: Binding kinetics of monkey CD22.mmH (XP_005588899.1) to anti-CD22xCD28 bispecific mAbs at 25°C

[0257]

[0258] Table 12: Binding kinetics of monkey CD22.mmH (EHH59463.1) to anti-CD22xCD28 bispecific mAbs at 25°C

[0259]

[0260]

[0261] Table 13: Binding kinetics of human CD28.mmH to anti-CD22xCD28 bispecific mAbs at 25°C

[0262]

[0263] Example 6: Binding specificity of anti-CD28 and anti-CD22xCD28 bispecific antibodies to target cell line (Nalm6), effector cell line (Jurkat), and cynomolgus monkey T cells and B cells using flow cytometry

[0264] Flow cytometry was used to determine the binding of the CD22xCD28 bispecific antibody to human CD22 expressing Nalm6 cells and human CD28 expressing Jurkat cells, as well as to cynomolgus monkey T (CD28+) and B (CD22+) cells. Briefly, 1x10 of Jurkat and Nalm6 cells were cultured in 1% paraformaldehyde (P < 0.05) of the cynomolgus monkey. 5 Serial dilutions of CD22xCD28 bispecific antibodies or H4sH15260P (isotype control human IgG4 antibody that binds human antigens to human or macaque CD28 or CD22 without cross-reactivity) were incubated together at 4 ° C for 30 minutes. Cynomolgus monkey PBMCs were incubated with a single 67nM concentration of antibody. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS, and PE-conjugated anti-human second antibodies were added to the cells and incubated for another 30 minutes. Additional phenotypic antibody mixtures (anti-CD2, anti-CD20, anti-CD16, anti-CD14) were added to the wells with cynomolgus monkey PBMCs. Wells without antibodies or only containing second antibodies were used as controls.

[0265] After incubation with the secondary antibody, cells were washed, resuspended in 200 μL of 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 as CD20+. EC values ​​for FACS binding were calculated in Prism software using 4-parameter nonlinear regression analysis. 50 value.

[0266] Table 14 provides binding data of the CD22xCD28 bispecific antibodies to the surface of cell lines expressing CD22, as determined by flow cytometry. Table 14 also provides binding data of the CD22xCD28 bispecific antibodies to the surface of cell lines expressing human CD28, as determined by flow cytometry.

[0267] REGN5837 interacted with Nalm6 cells with an EC of 1.3E-08M 50 REGN5838 bound to Nalm6 cells with an EC50 value of 1.8E-08 M. The isotype control antibody did not show any binding to cell lines expressing CD22.

[0268] REGN5837 interacted with Jurkat cells with an EC of 2.1E-08M 50 REGN5838 binds to Jurkat cells with an EC value of 2.3E-08M 50 The isotype control antibody did not show any binding to the cell lines expressing CD28.

[0269] Table 15 provides data on the binding of the CD22xCD28 bispecific antibodies to the surface of cynomolgus monkey (Cambodian origin) T cells and B cells as determined by flow cytometry.

[0270] REGN5837 bound to B cells of 12 of 12 cynomolgus monkeys tested and to T cells of 11 of 12 cynomolgus monkeys tested. Binding to CD20+ B cells ranged from 12.6-30.3 times that of the second antibody, with a median of 15.7 times. Binding to CD2+ / CD16- T cells ranged from 1.2-5.2 times that of the second antibody, with a median of 3.5 times. Positive binding was defined as 1.2-fold greater than the secondary antibody. REGN5838 bound to B cells of 12 of 12 cynomolgus monkeys tested and to T cells of 11 of 12 cynomolgus monkeys tested. Binding to CD20+ B cells ranged from 6.5-13.5 times that of the second antibody, with a median of 9.3 times. Binding to CD2+ / CD16- T cells ranged from 1.2-4.7 times that of the second antibody, with a median of 3.8 times. Positive binding was defined as 1.2-fold greater than the secondary antibody. The isotype control antibody did not show any binding to cynomolgus T or B cells.

[0271] Table 14: Binding and fold binding results of flow cytometry experiments on engineered target and effector cells.

[0272]

[0273] Table 15: Fold binding results from flow cytometry experiments on macaque (Cambodian origin) T and B cells.

[0274]

[0275] Example 7: Binding specificity of anti-CD28 and anti-CD22xCD28 bispecific antibodies to human CD4+ T cells and engineered target cells using flow cytometry

[0276] Flow cytometry analysis was used to investigate the relationship between CD22 x CD28 bispecific (REGN5837; REGN5838) and control antibodies and antibodies expressing human CD28 (human CD4 + The binding of effector cells (T cells) and target cells expressing human CD22 (HEK293 / hCD20 / hCD22 and Raji / CD80 and CD86 negative B cells). HEK293 / hCD20 cells were included as a negative cell line for CD28 and CD22.

[0277] Human CD4 + T cells. Use 50 mL of SepMate according to the manufacturer's recommended protocol. TMPBMC isolation was accomplished by density gradient centrifugation in 50 mL SepMate tubes. Briefly, 15 mL Ficoll-Paque PLUS was layered in 50 mL SepMate tubes, and then 30 mL of leukocytes diluted 1:2 with D-PBS + 2% FBS were added. Subsequent steps were performed according to the SepMate manufacturer's protocol. CD4 was then isolated from PBMC using a human CD4 microbead kit from Miltenyi Biotec according to the manufacturer's instructions. + T cells. The isolated CD4 + T cells were frozen in FBS containing 10% DMSO at a concentration of 5 × 10 per vial. 6 cells.

[0278] Target cells including HEK293 cell line and human Raji B cells were prepared as follows.

[0279] A stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (amino acids M1 to A847 of accession number NP_001762.2) was transduced with human CD22 (amino acids M1 to P297 of accession number NP_068769.2). Human CD22 positive cells were isolated and monocloned by fluorescence activated cell sorting (FACS). The resulting clonal cell line (HEK293 / hCD20 / hCD22 clone E4) was maintained in DMEM+10%+P / S / G+NEAA supplemented with 500 g / mL G418.

[0280] Human Raji B cells (ATCC # CCL-86) were genetically modified by deleting CD80 and CD86 using CRIPSR technology, which endogenously express CD20, CD22, Fcγ receptors (FcγR), CD80 and CD86 on the cell surface. CD80 and CD86 are known CD28 ligands. Engineered Raji / CD80 and CD86 negative cells were maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine supplemented with HEPES and sodium pyruvate.

[0281] Cells were stained as follows.

[0282] 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 (final concentration of 625 mg / mL) to block endogenous Fcγ receptors. Briefly, 2x10 5Cells / well were incubated at 4°C with serial dilutions of antibodies ranging from 6.1 pM to 100 nM for 30-60 minutes. Negative cell samples were included without antibodies. The cells were washed once with cold staining buffer and incubated with anti-human secondary antibodies labeled with allophycocyanin (APC) for 30-45 minutes. After incubation, the 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 live and dead cells. The cells were then fixed in BD Cytofix buffer, washed, resuspended in staining buffer and analyzed by flow cytometry on an iQue Screener flow cytometer according to the manufacturer's instructions. In order to determine EC 50 Geometric mean fluorescence intensity (MFI) values ​​were analyzed using a four-parameter logistic equation on a 9-point response curve using GraphPad Prism. Binding fold was calculated using the following equation:

[0283]

[0284] Flow cytometry analysis of primary human CD4 + The ability of the CD22 x CD28 bispecific antibody to bind to human CD22 and CD28 was assessed on T cells and on engineered cells that overexpress (HEK293 / hCD20 / hCD22) or endogenously (Raji / CD80 and CD86 negative) CD22. A negative cell line was included as a control (HEK293 / hCD20).

[0285] Figure 1 and EC are summarized in Table 16 50 / Combination multiple value.

[0286] Table 16: Human CD4 + Flow cytometry experiments on ECs of T cells and engineered target cells 50 And the combined multiple results:

[0287]

[0288] Abbreviations: NC = cannot be calculated (indicated for curves where binding did not reach saturation); ND = not determined

[0289] Table 16. Antibodies and human CD4 + Listed ECs of T cells and engineered cell lines such as HEK293 / hCD20, HEK293 / hCD20 / hCD22 or Raji / CD80 and CD86 negative B cells 50and combined multiple values.

[0290] As expected, none of the CD28 antibodies (parental (REGN5705; HCVR / LCVR SEQ ID NO:35 / 36) or their bispecific forms (REGN5837, REGN5838 and single-armed CD28 control (SEQ ID NO:48)) bound to negative HEK293 / hCD20 cells. Approximately 1.8-fold weaker binding (0.05 fold) was detected at the highest concentration using the isotype control antibody due to non-specific binding. Figure 1 and Table 16).

[0291] In HEK293 / hCD20 / hCD22 (16.44-fold for REGN5837 and 34.9-fold for REGN5838), EC 50 is about 11.4 nM) and on Raji / CD80 and CD86 negative cells (REGN5837 is 38.35 times, where EC 50 is about 9.76 nM; and REGN5838 is 81.74 times, with EC 50 Binding of anti-CD22 x anti-CD28 antibodies was observed with the single-arm CD28 control and isotype control ( Figure 1 and Table 16).

[0292] In primary human CD4 + Binding of antibodies targeting human CD28 was detected on T cells. The parental CD28 antibody REGN5705 had an EC of approximately 4.13 nM relative to background. 50 The isotype control did not bind to the cells ( Figure 1 and Table 16).

[0293] Example 8: Enhanced targeted cytotoxicity, T cell activation and cytokine release by anti-CD22xCD28 bispecific antibody co-stimulation with anti-CD20xCD3 bispecific antibodies

[0294] CD22xCD28 enhancement of CD20xCD3 targeted killing was evaluated in a 96-hour cytotoxicity assay targeting Raji cells engineered to lack CD80 and CD86 expression (Raji-80 / 86DKO). 6Cells / mL were plated in supplemented RPMI medium and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells and some monocytes. The next day, Raji-80 / 86DKO cells were labeled with 1uM fluorescent tracking dye CFDA-SE, and primary PBMCs depleted of adherent cells were labeled with 1uM fluorescent tracking dye CellTrace Violet. Labeled target cells and PBMCs (effector cell / target cell ratio of 10:1) were incubated with serial dilutions (concentration range: 5nM to 0.64pM) of CD20xCD3 bispecific antibody REGN1979 (having one heavy chain arm comprising SEQ ID NO:42, another heavy chain arm comprising SEQ ID NO:43, and a light chain of SEQ ID NO:44) and a fixed concentration of 2.5ug / ml (16.7nM) of CD22xCD28 co-stimulatory molecules REGN5837 or REGN5838, a single-arm control CD28 bispecific (REGN5678), or an IgG4 isotype control (H4sH10154P3, an isotype control with a HCVR / LCVR pair of SEQ ID NO:37 / 38) 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 using a fixable live / dead far-red reactive (Invitrogen) dye. 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected for each sample. In order to assess the specificity of killing, cells were gated on the live CFDA-SE-labeled population. The live population percentage was recorded and used to calculate survival.

[0295] 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. In addition, as T cells proliferate, CellTraceViolet is diluted, which results in a lower MFI as measured by FACS. T cell proliferation is therefore reported as a decrease in the MFI of CellTraceViolet on CD8+ T cells. The EC of target Raji cells lacking CD80 and CD86 expression and binding was calculated in Prism software using 4-parameter nonlinear regression analysis. 50 value.

[0296] The supernatant from this assay is collected for the analysis of cytokine levels. The concentration of IL 17a, IFNγ, TNFα, IL-10, IL-6, IL-4 and IL-2 is analyzed using a flow cytometry bead array (CBA) test kit according to the manufacturer's instructions. Cytokine levels are inserted from the curve generated by the test kit standard and cytokine levels are reported as pg / mL. In Prism software, 4 parameter nonlinear regression analysis is used to calculate the maximum cytokine level.

[0297] Results of an assay to evaluate the ability of the anti-CD20xCD3 bispecific antibody REGN1979 (see above) in combination with a co-stimulatory CD22xCD28 antibody or a single-armed CD28 or isotype control antibody to induce killing of target cells expressing human CD20 and CD22 by unstimulated human T cells were tested.

[0298] REGN1979 activated and directed human T cells to deplete Raji cells lacking CD80 and CD86 expression in a dose-dependent manner. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 increased the cytotoxic efficacy (EC 50 ) was enhanced by 3.5-6.4 times (Table 17).

[0299] The observed target cell lysis mediated by REGN1979 correlated 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 the potency of REGN1979 to induce T cell activation and proliferation by 2.1 to 2.6-fold and 7.4-8.4-fold, respectively, compared to REGN1979 and single-armed CD28 or isotype control antibodies (Table 17).

[0300] REGN1979 induces release of human cytokines. The released cytokines observed with REGN1979 in combination with the CD22xCD28 bispecific antibody were enhanced in the presence of a fixed concentration of CD22xCD28 co-stimulatory molecules and a fixed concentration of single-armed CD28 or isotype control antibody (Table 18).

[0301] In summary, co-stimulation increased the efficacy of targeted cytotoxicity, T cell activation, and cytokine release compared to that observed with CD20xCD3 in combination with a control antibody.

[0302] Table 17: ECs for cytotoxicity and T cell activation 50 Value (average of 3 experiments)

[0303]

[0304] Table 18: Cytokine release (pg / ml)

[0305] REGN5837 REGN5838 Single-arm CD28 IgG4 isotype IL-4 46 50 33 29 IL-6 907 810 248 283 IL-2 531 270 36 39 IL-10 1917 2555 739 375 TNFa 277 339 100 66 IFNg 1847 1956 267 160 IL-17A 154 172 41 35

[0306] Example 9: Bioassay of CD22 Bispecific Antibodies

[0307] T cell activation is achieved by stimulating the T cell receptor (TCR), which recognizes specific peptides presented by class I or class II major histocompatibility complex (MHCI or MHCII) proteins on antigen presenting cells (APCs) (Goldrath et al. 1999). The activated TCR then initiates a series of signal transduction events that can be monitored by reporter genes driven by various transcription factors, such as activating 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-associated protein 4), PD-1 (programmed cell death protein 1), LAG-3 (lymphocyte activation gene 3), or other molecules) expressed constitutively or inducibly on T cells (Sharpe et al. 2002). The co-stimulatory molecule CD28 is activated by its endogenous ligands CD80 or CD86 expressed on APCs. CD28 enhances cell signaling following TCR activation, such as pathways controlled by the NFκB transcription factor. CD28 co-signaling is important for efficient T cell activation, such as T cell differentiation, proliferation, cytokine release, and cell death (Smeets et al. 2012).

[0308] Use primary human CD4 + T cell characterization of the anti-CD22xCD28 bispecific antibody in a luciferase-based reporter bioassay and an IL-2 functional assay.

[0309] Luciferase-based reporter gene assay :

[0310] A T cell / APC (antigen presenting cell) luciferase-based reporter assay was developed to evaluate the effect of CD28 activation on NFκB activity when engaged with an anti-CD28 x anti-CD22 bispecific antibody.

[0311] Reporter T cell engineering:

[0312] The cloned reporter T cell line was engineered by transducing immortalized human Jurkat T cells (ATCC #TIB-152) with NFκB-luciferase (NFκB-Luc) lentiviral reporter (from Qiagen) according to the manufacturer's instructions. The cloned reporter line (Jurkat / NFκB-Luc clone 1C11) was maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine supplemented with 1 μg / mL puromycin.

[0313] APC Engineering:

[0314] A stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (amino acids M1 to A847 of accession number NP_001762.2) was transduced with human CD22 (amino acids M1 to P297 of accession number NP_068769.2). Human CD22-positive cells were isolated and monocloned by fluorescence activated cell sorting (FACS). 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.

[0315] Human Raji B cells (ATCC # CCL-86) were genetically modified by deleting CD80 and CD86 using CRIPSR technology, which endogenously express CD20, CD22, Fcγ receptors (FcγR), CD80 and CD86 on the cell surface. CD80 and CD86 are known CD28 ligands. Engineered Raji / CD80 and CD86 negative cells were maintained in RPMI + 10% FBS + penicillin + streptomycin + glutamine supplemented with HEPES and sodium pyruvate.

[0316] T cell / APC stimulation:

[0317] In this experiment, engineered reporter T cells were stimulated by two bispecific antibodies. The first stimulus was delivered by targeting the CD3 molecules on engineered reporter T cells and the T cell activation bispecific antibody REGN2281 (anti-CD20 x anti-CD3 antibody with a heavy chain arm comprising SEQ ID NO:39, a heavy chain arm comprising SEQ ID NO:40 and a light chain arm comprising SEQ ID NO:41) on HEK293 or Raji / CD80 and CD86 negative B cells. The first stimulus bypassed the need for TCR to be activated by its natural ligand, which is a specific peptide displayed on the MHC molecule. The second stimulus was driven by a CD28 bispecific antibody (i.e., anti-CD28 x anti-CD22 bispecific antibody). This antibody mimics CD28 activation on T cells by its ligand CD80 / CD86 expressed on APC. The antibody interacts with CD28 on T cells and CD22 on HEK293 cells or Raji / CD80 and CD86 negative B cells and drives the activation of CD28 on engineered reporter T cells. Simultaneous activation of TCR and CD28 leads to enhanced transcriptional activity of NFκB, which increases the production of the reporter gene luciferase.

[0318] Luciferase assay setup:

[0319] RPMI1640 supplemented with 10% FBS and P / S / G was used as assay medium for preparing cell suspension and antibody dilutions for screening of anti-CD22 x anti-CD28 bispecific antibodies.

[0320] One day before screening, culture engineered reporter T cells to 0.5 × 10 6 Cells / mL. 1:3 serial dilutions of anti-CD28 x anti-CD22 bispecific antibodies and controls were tested in the presence of a constant 200 pM REGN2281 (anti-CD20 x anti-CD3, see above) or REGN1945 (hIgG4 isotype control with HCVR / LCVR pair of SEQ ID NO:45 / 46). The 10-point dilution ranged from 15 pM to 100 nM, with the final dilution containing no CD28 antibody.

[0321] Reagents were added in the following order: 1) a fixed concentration of 200 pM final 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) the cells were resuspended to 4 × 10 5 cells / mL (the final cell concentration was 1×10 4HEK293 cells (cells / well) or resuspended to 2×10 6 cells / mL (the final cell concentration was 5×10 4 1) Add Raji / CD80 and CD86 negative B cells (cells / well) to the corresponding plates; 2) Add serially diluted antibodies to the corresponding wells; 3) Resuspend the reporter T cells cultured overnight to 2×10 6 / mL and added to the plate, where the final concentration was 5×10 4 The plate was incubated at 37°C / 5% CO2 for 4 to 6 hours, after which 100 μL ONE-Glo TM (Promega) reagent to lyse cells and detect luciferase activity. Emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer). All serial dilutions were tested in duplicate.

[0322] By using GraphPad Prism TM The software fits the data to a four-parameter logistic equation on a 10-point dose-response curve to determine the EC of the antibody. 50 The induction fold was calculated using the following equation:

[0323]

[0324] Use primary human CD4 + IL-2 functional assay for T cells:

[0325] Development of primary CD4 + T cell / APC functional assay to evaluate the impact of CD28 activation on IL-2 production when engaged with an anti-CD22 x anti-CD28 bispecific antibody.

[0326] Isolation of primary human CD4+ T cells:

[0327] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using a 50 mL SepMate 400 RT-PCR kit according to the manufacturer's recommended protocol. TM PBMC isolation was accomplished by density gradient centrifugation in 50 mL SepMate tubes. Briefly, 15 mL FicollPaquePLUS was layered in a 50 mL SepMate tube, and then 30 mL of leukocytes diluted 1:2 with D-PBS + 2% FBS was added. Subsequent steps were performed according to the SepMate manufacturer's protocol. CD4 was then isolated from PBMC using the Human CD4 Microbead Kit from Miltenyi Biotech according to the manufacturer's instructions. + T cells. The isolated CD4 +T cells were frozen in FBS containing 10% DMSO at a concentration of 5 × 10 per vial. 6 cells.

[0328] From primary CD4 + IL-2 release by T cells:

[0329] In this assay, an anti-CD20 x anti-CD3 bispecific antibody (REGN2281, see above) was used in combination with either HEK293 cells engineered to express human CD20 or endogenous CD20 expressing Raji cells to induce CD4 + Crosslinking CD3 on the surface of T cells to activate primary CD4 + T cells in which 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 CD3 receptors, providing the first signal necessary for T cell stimulation. Importantly, in some cases, co-culture of primary leukocytes with genetically different cells causes incompatibility of allogeneic determinants and leads to T cell activation. This can provide sufficient primary stimulation in the absence of exogenous addition of anti-CD20xanti-CD3 bispecific antibodies. Regardless of the primary stimulation, co-stimulation that can be provided by cross-linking CD28 molecules is necessary to detect quantifiable IL-2 release. Here, the bispecific CD28 antibody (i.e., anti-CD28 x anti-CD22 bispecific antibody) is co-cultured with CD4 + CD28 on T cells and CD22 on HEK293 / hCD20 or RAJI / CD80 and CD86 negative cells interact and drive clustered activation of CD28. Combined TCR and CD28 engagement results in enhanced IL-2 production released into the cell culture medium. IL-2 is detected and quantified from cell supernatants using the homogenous no-wash AlphaLisa kit from PerkinElmer.

[0330] On the day of the assay, previously isolated and frozen human CD4 + T cells. The cells were centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium and cultured at 1×10 5 Cells were plated at a concentration of 10 × 10 cells / well in a 96-well round-bottom plate. 6HEK293 cells engineered to express human CD20 alone or in combination with human CD22 were treated with 20 μg / mL mitomycin C at a concentration of 10 × 10 6 After incubation for 1 hour at 37°C, 5% CO2, HEK293 and Raji cells were washed three times with D-PBS containing 2% FBS and added to a final concentration of 1×10 4 HEK293 cells or 5 × 10 4 cells per well containing CD4 Raji / CD80 and CD86 negative cells + The wells of T cells. Subsequently, the anti-CD28 x anti-CD22 bispecific or control antibody with a range of 15pM to 100nM was added to the wells in which 2nM REGN2281 (anti-CD20 x anti-CD3) or REGN1945 (negative hIgG4 isotype control, see above) was present. The final point of the 10-point dilution did not contain CD28 antibody. After the flat plate was incubated at 37°C, 5% CO2 for 72 hours, it was centrifuged to precipitate cells, and 40 μL of culture supernatant was collected. Thus, 5 μL was tested in the human IL-2AlphaLISA assay according to the manufacturer's protocol. The measurement of the multi-label plate reader Envision and the original RFU (relative fluorescence unit) value drawn was obtained. All serial dilutions were tested in duplicate.

[0331] By using GraphPad Prism TM The software fits the data to a four-parameter logistic equation on a 10-point dose-response curve to determine the EC of the antibody. 50 The induction fold was calculated using the following equation:

[0332]

[0333] Summary of results and conclusions:

[0334] Luciferase-based reporter assay:

[0335] The ability of the anti-CD22 x anti-CD28 bispecific antibodies to provide co-stimulation 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 a readout.

[0336] Activation curves are shown in Figure 2 (A and B), and the EC values ​​of engineered reporter T cells co-incubated with HEK293 / hCD20 or HEK293 / hCD20 / hCD22 cells in addition to 200 pM constant REGN1945 (hIgG4 isotype control) or REGN2281 (anti-CD20 x anti-CD3) are summarized in Tables 19 and 20. 50 and induction fold values.

[0337] When reporter T cells and HEK293 derived APCs were treated with 200pM REGN1945, in the absence of TCR stimulation, CD28 bispecific antibodies did not show an increase in luciferase activity, regardless of the HEK293 system used in the assay. Observation of luciferase activation increase of parental CD28 antibodies (REGN5705) of only HEK293 / hCD20 cells (2.18 times) and HEK293 / hCD20 / hCD22 cells (2.05 times). Single-arm CD28 and isotype control antibodies did not produce luciferase responses in this case (Table 19 and Fig. 2).

[0338] 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) induced strong luciferase activity with CD22-expressing HEK293, which was confirmed by EC 50 The single-armed CD28 control antibody and the parental CD28 antibody (REGN5705; see HCVR / LCVR SEQ ID NO: 35 / 36) showed similar activity on both HEK293 lines. The isotype control antibody did not generate a luciferase response in this case (Table 20 and Figure 2).

[0339] When reporter T cells and Raji / CD80 and CD86 negative were treated with 200 pM REGN1945, both anti-CD22 x anti-CD28 bispecific antibodies (REGN5837 and REGN5838) and parental CD28 antibody induced luciferase activity, while the one-armed CD28 control antibody and isotype control showed no activity (Table 19 and Figure 2).

[0340] When reporter T cells and Raji / CD80 and CD86 negative were treated with 200 pM REGN2281, all CD28 bispecific antibodies (REGN5837 and REGN5838) and the one-armed CD28 control antibody (including the parental CD28 antibody) induced luciferase activity. EC values ​​of anti-CD22 x anti-CD28 and parental CD28 antibodies only 50 The EC values ​​of the single-arm CD28 antibody can be determined.50 Values ​​could not be determined since saturation levels were not reached. No activation was detected using isotype controls (Table 20 and Figure 2).

[0341] Use primary human CD4 + IL-2 functional assay for T cells:

[0342] Measuring IL-2 cytokine production in functional primary CD4 + In a T cell assay, the anti-CD22 x CD28 bispecific antibodies were evaluated for their ability to provide co-stimulation via CD28 on T cells in the absence or presence of CD22 target expression.

[0343] Activation curves are shown in Figure 3 (A and B) and CD4+ T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or Raji / CD80 and CD86 negative cells in the presence of 2 nM constant REGN1945 (hIgG4 isotype control) or REGN2281 (anti-CD20 x anti-CD3) are summarized in Table 21. + T cell EC 50 and induction fold values.

[0344] In wells containing HEK293 / hCD20 or HEK293 / hCD20 / CD22 cells with 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 with a constant amount of REGN1945, IL-2 release was detected, due to a significant allogeneic response providing sufficient primary stimulation, even in the absence of antibody-mediated CD3 clustering (Figure 3 and Table 21).

[0345] 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, there was no significant enhancement of IL2 release when the anti-CD22 x anti-CD28 bispecific mAb was added to wells containing HEK293 / hCD20 cells and REGN2281. The anti-CD22 x anti-CD28 bispecific mAb significantly enhanced IL-2 release only in the presence of HEK293 / hCD20 / CD22 cells and REGN2281 (Figure 3 and Table 22).

[0346] Tables 19-22 are presented below.

[0347] Table 19 presents the tabulated EC values ​​for luciferase activity in engineered T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or RAJI / CD80 and CD86 negative cells and 200 pM constant REGN1945 (isotype control). 50 value, maximum value and induction fold value.

[0348] Table 20 presents the tabulated EC values ​​for luciferase activity in engineered T cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or Raji / CD80 and CD86 negative cells and 200 pM constant REGN2281 (anti-CD20 x anti-CD3). 50 value, maximum value and induction fold value.

[0349] Table 21 presents CD4 T cells from cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or RAJI / CD80 and CD86 negative cells and 2 nM constant REGN1945 (isotype control). + List of ECs released by T cells 50 value, maximum value and induction fold value.

[0350] Table 22 presents CD4+ T cells from cells co-incubated with HEK293 / hCD20, HEK293 / hCD20 / hCD22 or Raji / CD80 and CD86 negative cells and 2 nM constant REGN2281 (CD20 x anti-CD3). + List of ECs released by T cells 50 value, maximum value and induction fold value.

[0351] Table 19: ECs for luciferase activity in engineered reporter T cells in the absence of TCR stimulation with 200 pM REGN1945 (isotype control) 50 Value, maximum value and induction multiple value:

[0352]

[0353] Abbreviation: ND = Not Determined

[0354] Table 20: EC of luciferase activity in engineered reporter T cells in the presence of TCR stimulation using 200 pM REGN2281 (αCD20 x αCD3) 50 Value, maximum value and induction multiple value:

[0355]

[0356] Abbreviations: NC = cannot be calculated (indicated for curves where the response did not reach saturation); ND = not determined.

[0357] Table 21: Primary human CD4 T cells in the presence of 2 nM REGN1945 (isotype control) + IL-2 release from T cells in EC 50 value, maximum value and induction fold value.

[0358]

[0359] Abbreviations: NC = cannot be calculated (indicated for curves where the response did not reach saturation); ND = not determined.

[0360] Table 22: Primary human CD4 in the presence of 2 nM REGN2281 (αCD20 x αCD3) + IL-2 release from T cells in EC 50 value, maximum value and induction fold value.

[0361]

[0362] Abbreviations: NC = cannot be calculated (indicated for curves where the response did not reach saturation); ND = not determined.

[0363] Example 10: Effect of a combination of anti-CD22 x anti-CD28 antibodies plus cemiplimab on IL-2 release from cells engineered to express PD-L1

[0364] Materials and Methods

[0365] APC Engineering:

[0366] RAJI cells

[0367] RAJI was a 11-year-old male ( CCL-86 TM ) Isolated B lymphocyte cell line. RAJI was maintained in RPMI+10% FBS+P / S / G+NaPyr+HEPES.

[0368] RAJI / CD80 and CD86 negative

[0369] The CRISPR / Cas9 system was used to eliminate the expression of CD80 and CD86 in RAJI cells.

[0370] NALM6 Clone G5

[0371] 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 were maintained in RPMI+10% FBS+P / S / G.

[0372] WSU-DLCL2

[0373] WSU-DLCL2 is a human DLBCL cell line isolated from the pleural effusion of a 41-year-old Caucasian male (Leibnitz Institute-DSMZ, catalog number ACC 575).

[0374] PD-L1 engineered cell lines

[0375] NALM-6, RAJI CD80 and CD86 negative (RAJI / CD80-CD86-) and WSU-DLCL2 cell lines were genetically engineered to stably express human PD-L1 (amino acids M1-T290 of 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.

[0376] T cell activation assay for T cell proliferation and IL-2 release

[0377] Human primary T cells and allogeneic 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]) were used to evaluate the effect of REGN5837 on IL-2 release in the presence of a fixed concentration of cimiprilimumab. Co-culture of primary leukocytes with genetically different cells causes incompatibility of allogeneic determinants and may lead to T cell activation. Regarding assays using NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1) cells, T cell activation assays were performed using enriched human primary T cells from 3 donors, while assays using WSU-DLCL2(+ / -PD-L1) cells used T cells from 1 donor.

[0378] Isolation of T cells used in T cell activation assays for testing REGN5837+REGN2810 combination treatments.

[0379] About the experiment utilizing NALM-6 and RAJI / CD80-CD86-cells, CD3+T cells are separated from 3 donor PBMCs (555109, 555130 and 555131), and PBMCs from a donor (555175) are used for the determination utilizing WSU-DLCL2 cells.About donor 555109, PBMCs are separated from peripheral blood using density gradient centrifugation.In brief, 15ml Ficoll-Paque PLUS is added to a 50ml conical tube, and then 30ml blood diluted 1:1 with PBS containing 2% FBS is layered at the top.After centrifugation at 400x g for 30 minutes when the brake is turned off, the mononuclear cell layer is transferred to a fresh tube, diluted 5 times with PBS containing 2% FBS and centrifuged at 300x g for 8 minutes. For donors 555130, 555131 and 555175, PBMCs were isolated from peripheral blood from healthy donors using the EasySep Direct Human PBMC Isolation Kit from Stem Cell Technologies according to the manufacturer's protocol. The isolated PBMCs were frozen in FBS containing 10% DMSO. For CD3+ T cell separation, PBMC cryovials were thawed in a 37°C water bath and diluted in a stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA and 0.01mM BME) containing 50U / ml all-purpose nuclease. According to the manufacturer's protocol, cells were centrifuged at 1200rpm for 10 minutes, resuspended in EasySep buffer and separated using the EasySep T cell separation kit of Stem Cell Technologies.

[0380] From primary CD3 treated with CD28 antibody + IL-2 release by T cells:

[0381] T cell activation assay using human OVCAR-3, PEO1, NALM-6, RAJI / CD80-CD86- and WSU-DLCL2 cells (+ / - PD-L1)

[0382] 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 cultured at 1×10 5The concentration of cells / well was plated in a 96-well round-bottom plate. NALM-6, RAJI / CD80-CD86-, WSU-DLCL2 cells with or without PD-L1 (+ / -PD-L1) were treated with 20 μg / mL (RAJI) or 15 μg / mL (NALM-6 and WSU-DLCL2) mitomycin C to inhibit proliferation. After incubation for 1 hour at 37°C and 5% CO2, the cells treated with mitomycin C were washed 3 times with D-PBS containing 2% FBS and then resuspended in stimulation medium. NALM-6, RAJI / CD80-CD86- and WSU-DLCL2 cells (+ / -PD-L1) were added to a 4% paraformaldehyde-containing flask containing a final concentration of 2.5x10 4 cells / well and for NALM-6 cells the final concentration was 5x10 4 A constant concentration of cemiplimab or non-binding IgG4 P Control (20nM) was added to the wells. In the assay using WSU-DLCL2 (+ / -PD-L1) cells, a constant concentration of belatacept (hCTLA4.hIgG1) or a non-binding IgG1 control (50nM) was added to the wells. Subsequently, REGN5837 or a non-TAAxCD28 control antibody was titrated from 3.1pM to 200nM in a 1:4 dilution series for NALM-6 (+ / -PD-L1) cells and from 0.6pM to 1000nM in a 1:6 dilution series for WSU-DLCL2 and RAJI / CD80-CD86- (+ / -PD-L1) cells and added to the wells. The final point of the 10-point concentration curve did not contain REGN5837 or a non-TAAxCD28 control antibody. After incubation of the plates at 37°C, 5% CO2 for 72 hours (WSU-DLCL2(+ / -PD-L1)) or 96 hours (NALM-6 and RAJI / CD80-CD86-(+ / -PD-L1)), 50 μL of culture supernatant was collected to measure IL-2 release.

[0383] For IL-2 release, 5 μL of supernatant was tested using a human IL-2α LISA kit according to the manufacturer's protocol. IL-2 measurements were acquired on a multilabel plate reader Envision from PerkinElmer. A standard curve of known IL-2 concentrations was included and used to derive pg / ml values.

[0384] All serial dilutions were tested for IL-2 release in triplicate. GraphPad Prism TMThe software determines the EC50 value of the antibody from a 4-parameter logistic equation on a 10-point dose-response curve. The maximum level of IL-2 release is given as the average maximum response detected over the dose range tested. In addition, data reported for assays using WSU-DLCL2 cells include IL-2 values ​​generated in the absence or presence of 1000 nM titrated antibody to capture the IL-2 reduction observed with increasing concentrations of non-TAAxCD28 antibodies.

[0385] Summary of results and conclusions:

[0386] IL-2 functional assay using primary human CD3+ T cells:

[0387] The ability of the anti-CD22 x anti-CD28 bispecific antibodies to provide co-stimulation via CD28 on T cells in the presence of a B cell lymphocyte line that endogenously expresses CD22 was evaluated in a functional primary CD3+ T cell assay measuring IL-2 cytokine production.

[0388] Figure 5A and 5B The results are shown in Figure 2 for NALM-6 (+ / - PD-L1) ( Figure 5A ) or RAJI / CD80-CD86-(+ / -PD-L1)( Figure 5B ) activation curve of T cells incubated with 20 nM constant hIgG4 P EC50 and maximum IL-2 values ​​for CD3+ T cells incubated with NALM-6 (+ / - PD-L1) cells are summarized in Table 23A and for CD3+ T cells incubated with RAJI / CD80-CD86-(+ / - PD-L1) in the presence of isotype control or cemiplimab. EC50 and maximum IL-2 values ​​for CD3+ T cells incubated with WSU-DLCL2 (+ / - PD-L1) cells are summarized in Table 23B. Figure 4 The activation curves are shown in Table 24 for constant hIgG4 at 20 nM. P EC50 and IL-2 values ​​(reported for 0 nM or 1000 nM REGN5837 or non-TAAxCD28) were pooled for T cells incubated with WSU-DLCL2 (+ / - PD-L1) cells in the presence of isotype control or cemiplimab and in the presence of 50 nM constant hIgG1 isotype control or CTLA-4 receptor belatacept.

[0389] REGN5837 mediated a concentration-dependent increase in IL-2 release in the presence of human primary T cells and the allogeneic B-cell lymphocyte cell lines RAJI / CD80-CD86- and NALM-6. A non-TAAxCD28 control antibody slightly increased IL-2 at high antibody concentrations. In the absence of PD-L1 on RAJI / CD80-CD86- or NALM-6 cells, the addition of 20 nM cemiplimab had 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 was reduced compared to T cells incubated with non-PD-L1 expressing cells. In the presence of NALM-6 / PD-L1 cells, the addition of cemiplimab minimally enhanced REGN5837-mediated IL-2 release, whereas in the presence of RAJI / CD80-CD86- / PD-L1 cells, it significantly enhanced IL-2 release to levels observed in the presence of RAJI / CD80-CD86- cells (lacking PD-L1).

[0390] In the presence of human primary T cells and the allogeneic B cell lymphocyte cell line WSU-DLCL2, no concentration-dependent increase in IL-2 release was observed. In contrast, a concentration-dependent reduction in IL-2 release was observed with the non-TAAxCD28 control antibody. Unlike NALM-6 and RAJI / CD80-CD86- cells, which express little to no CD28 ligand, the WSU-DLCL2 cell line is known to express CD28 ligand. While it is known that the CD28 binding arm of REGN5837, and therefore the CD28 arm of the non-TAAxCD28 antibody, competes 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, which results in reduced IL-2 release. Unlike non-TAAxCD28 controls, IL-2 was not reduced by REGN5837 because its ability to anchor to WSU-DLCL2 cells through its CD22 binding arm allows it to behave similarly to the CD28 ligand, essentially replacing the ligand. In the presence of WSU-DLCL2 / PD-L1 cells, basal IL-2 release was reduced compared to WSU-DLCL2 cells that do not express PD-L1. Addition of REGN5837 alone in the absence of cemiplimab resulted in a slight enhancement of IL-2 release. When 20nM cemiplimab was added, basal activity was enhanced and could be further enhanced slightly using dose titration of REGN5837, resulting in a higher maximum IL-2 release for the combination of REGN5837 and cemiplimab than for either treatment alone. As observed with WSU-DLCL2 cells, incubation of WSU-DLCL2 / PD-L1 cells in the presence of non-TAAxCD28 resulted in reduced IL-2 levels, regardless of cemiplimab or hIgG4 PIsotype control exists or not. In order to further explore the effect of CD28 ligand expression in masking the effect of REGN5837, 50nM soluble CTLA-4 receptor Belatacept or hIgG1-matched isotype control was added. Belatacept binds to CD28 ligands, CD80 and CD86 with high affinity and blocks their interaction and thus blocks the activation of CD28. In the presence of 50nM Belatacept, basal IL-2 release is significantly reduced because CD28 ligands cannot bind CD28 and provide costimulatory signaling. Under these conditions, REGN5837 is still able to engage CD28 and provide costimulation, which is indicated by the dose-dependent enhancement of IL-2 release. Although the addition of 20nM Cimiprili monoclonal antibody itself does not enhance the IL-2 release in the presence of Belatacept, the combination of Cimiprili monoclonal antibody and increased doses of REGN5837 increases the maximum IL-2 release in the presence of cells engineered to overexpress PD-L1 compared to REGN5837 alone.

[0391] Table 23A: Combination of REGN5837 and cemiplimab enhances IL-2 release over REGN5837 treatment alone in NALM-6 cells engineered to express PD-L1

[0392]

[0393]

[0394] ND: Not determined, as no concentration-dependent response was observed.

[0395] Table 23B: RAJI / CD80 engineered to express PD-L1 - In CD86 cells, the combination of REGN5837 and cemiplimab enhanced IL-2 release over REGN5837 treatment alone

[0396]

[0397] NC: Not calculated because the data were not fit to a 4-parameter logistic equation.

[0398] Table 24: Combination of REGN5837 and cemiplimab enhances IL-2 release over REGN5837 treatment alone in WSU-DLCL2 cells expressing PD-L1

[0399]

[0400]

[0401] ND: Not determined, as no concentration-dependent response was observed.

[0402] NC: Not calculated because the data were not fit to a 4-parameter logistic equation.

[0403] Example 11: Antitumor efficacy of REGN5837 administered in the presence or absence of REGN1979

[0404] introduction

[0405] REGN5837 is designed to be used to bridge CD22 + B cells and CD28 + T cells to target B cell NHL (e.g., DLBCL) based on human IgG4 bispecific antibodies (bsAb). The "signal 2" provided by REGN5837 and other agents providing "signal 1" (e.g., by stimulating primary T cells via TCR or CD3 clustering to deliver signals) such as CD20xCD3 bispecific antibody (bsAb) REGN1979 combination can provide amplified T cell activation and T cell-mediated killing of B cell NHL, thereby deepening the response to CD20xCD3. In addition, REGN5837 can provide increased efficacy for patients who do not respond to CD20xCD3 monotherapy.

[0406] The studies described below evaluated the anti-tumor efficacy of CD22xCD28 bsAb REGN5837 administered to immunodeficient NSG mice bearing established B cell leukemia tumors for 8 days in the presence or absence of a sub-effective dose of CD20xCD3 bsAb (REGN1979).

[0407] Briefly, mice (n=6 to 9 per group) were transplanted intraperitoneally (IP) with human peripheral blood mononuclear cells (PBMCs) and 12 days later were implanted intravenously (IV) with human NALM-6 B cell leukemia cells engineered to express luciferase for bioluminescent imaging (NALM-6-luc). The antitumor efficacy of 0.04 mg / kg, 0.4 mg / kg, and 4 mg / kg of REGN5837 combined with 0.04 mg / kg fixed dose of REGN1979 was comparable to that of REGN5837 and REGN1979 monotherapy and to that of non-bridging IgG4 P-PVA Control bsAb was used for comparison. Mice received multiple doses of antibody by intraperitoneal (IP) injection 8, 15, and 22 days after implantation of NALM-6-luc cells. Tumor burden was assessed twice a week throughout the experiment.

[0408] Materials and Methods

[0409] Human-derived cell lines

[0410] NALM-6-luc: The NALM-6 cell line is an acute lymphoblastic leukemia cell line isolated from a 19-year-old male patient (DSMZ, catalog number ACC 128); this line was modified with EF1a-luciferase-2A-GFP-Puro (GenTarget) to facilitate in vivo tumor cell growth imaging.

[0411] PBMC: Human PBMC were obtained from ReachBio, Inc. Catalog No. 0500-401, donor No. 0180905 (tumor growth assay) and 0180621 (serum antibody assay).

[0412] Experimental design

[0413] Test system

[0414] Female NSG mice (8-9 weeks old) were used in all experiments. Human PBMCs were transplanted into all mice IP and then NALM-6-luc B cell leukemia cells were implanted IV 12 days after transplantation. The experimental design is detailed in Table 25. During the entire study, tumor growth was monitored by bioluminescent imaging twice a week. For all experiments, mice were housed in the animal facility of Regeneron under standard conditions. All experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Regeneron.

[0415] Transplantation of NSG mice

[0416] 4x10 cells were transplanted IP into female immunodeficient NSG mice 7 Human PBMCs were collected by retro-orbital bleeding and assessed by flow cytometry for human CD45 in all viable cells in whole blood. + As a percentage of cells, T cell levels were examined 11 days after transplantation; engraftment levels ranged from 0.16% to 16% hCD45 + PBMC-transplanted NSG mice were then implanted with NALM-6-luc cells.

[0417] NALM-6-Luc culture conditions and tumor implantation

[0418] The NALM-6 cell line was modified with EF1a-luciferase-2A-GFP-Puro lentivirus (Gene Target) to facilitate in vivo tumor cell growth imaging. The cell line was maintained in RPMI supplemented with 10% FBS with PSG (penicillin, streptomycin and glutamine) under puromycin selection.

[0419] NALM-6-luc cells were collected by centrifugation and cultured at 2.5 × 10 7 12 days after PBMC implantation, NSG mice were injected IV with 200 μl (5×10 6 cells)NALM-6-luc cells.

[0420] Antibody administration for tumor measurement

[0421] Prior to dosing with the test article or control, mice were assigned to groups stratified by tumor burden and T cell engraftment level. Antibodies (REGN5837, REGN1979, REGN5671 [non-TAAxCD28 non-bridging control bsAb] or H4sH17664D [non-TAAxCD3 non-bridging control bsAb]) were administered by IP injection as monotherapy or in combination on days 8, 15, and 22 after implantation (for in vivo efficacy) according to the doses set forth in Table 25.

[0422] Table 25: Experimental design for assessing tumor growth

[0423]

[0424] a One mouse from this group died early during the experiment and was excluded. The deaths of these mice were not due to tumor burden and could not have been related to administration of the test article, as one mouse also died in the control group.

[0425] Tumor Measurements and Specified End Points

[0426] Mice implanted with NALM-6-luc tumors were imaged twice a week using IVIS Spectrum Instruments, and data were analyzed using Living Image software. Prior to imaging, luciferin substrate was injected into mice IP. Ten minutes later, mice were anesthetized with isoflurane and quantitative bioluminescence (total flux, expressed as photons per second [p / s]). According to IACUC standards, the experiment ended when mice began to show signs of graft-versus-host disease (GVHD) (assessed as weight loss ≥ 20%).

[0427] Statistical analysis of tumor growth

[0428] Results of tumor volume over time 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).

[0429] result

[0430] Antitumor efficacy of REGN5837 administered in the presence or absence of REGN1979

[0431] Immunodeficient NSG mice bearing NALM-6-luc tumors received IP injections of antibodies as described herein or non-bridging controls.

[0432] In tumor-bearing mice, treatment with 0.04 mg / kg, 0.4 mg / kg, and 4 mg / kg REGN5837 in the presence of 0.04 mg / kg REGN1979 resulted in statistically significant inhibition of tumor growth (p<0.05, p<0.01, and p<0.001, respectively) on day 23 post-implantation compared to non-bridging control bsAbs (non-TAAxCD28 and non-TAAxCD3 bsAbs) ( Figure 6 ). At day 20 after implantation, significant inhibition of tumor growth was observed for the 0.4 mg / kg and 4 mg / kg groups (p<0.05 for both groups). Neither REGN5837 (4 mg / kg) nor REGN1979 (0.04 mg / kg) monotherapy significantly reduced tumor growth compared to the non-bridging control bsAb. The difference between any REGN5837+REGN1979 combination dose and bsAb monotherapy did not reach statistical significance. During the entire dosing period, rapid tumor growth was observed when administered with the non-bridging control bsAb, and on day 23, all mice were euthanized. In all groups, GVHD (assessed as a weight loss of ≥20%) was observed in at least one mouse at the end of the experiment.

[0433] In independent experiments using different groups of mice, blood was collected at the following time points to determine serum antibody concentrations: 1 hour, 4 hours after dosing on day 7, 1 hour before and 4 hours after dosing on days 14 and 21, and once on day 29. The trough concentration of antibody in serum during the dosing period was determined 1 hour before dosing on days 14 and 21. Administration of 0.04 mg / kg, 0.4 mg / kg, and 4 mg / kg REGN5837 doses in the presence of 0.04 mg / kg REGN1979 was associated with trough concentrations of REGN5837 in serum ranging from below the limit of quantitation (BLQ) to 0.1 μg / mL, 1.6 μg / mL to 2.3 μg / mL, and 16.5 μg / mL to 21.1 μg / mL, respectively. In all cases, the trough concentration of REGN1979 in serum was BLQ (data not shown).

[0434] in conclusion

[0435] Doses of 0.04 mg / kg, 0.4 mg / kg and 4 mg / kg REGN5837 effectively inhibited NALM-6 B cell leukemia tumor growth in mice in the presence of 0.04 mg / kg REGN1979. No significant tumor inhibition was observed with 4 mg / kg REGN5837 or 0.04 mg / kg REGN1979 monotherapy relative to controls.

[0436] Example 12: FACS-based cytotoxicity on CD22 cells + human PBMC + / - CD22xCD28 co-stimulatory bispecific antibody (immobilized CD22xCD28, titrated CD20xCD3)

[0437] Materials and Methods

[0438] CD22xCD28 enhancement of CD20xCD3-targeted killing was evaluated in a 96-hour cytotoxicity assay targeting Nalm6 or WSU-DLCL2 cells. Briefly, human PBMCs were plated at 1 × 10 6Cells / ml were plated in supplemented RPMI medium and incubated overnight at 37°C to enrich lymphocytes by exhausting adherent macrophages, dendritic cells and some monocytes. The next day, Nalm6 or WSU-DLCL2 cells were labeled with 1uM fluorescent tracking dye CFDA-SE, and the original PBMCs with adherent cell exhaustion were labeled with 1uM fluorescent tracking dye CellTrace Violet. With or without 2.5ug / ml (16.7nM) fixed concentration of CD22xCD28 REGN5837, the labeled target cells and PBMCs (the effector cell / target cell ratio of Nalm6 was 4:1, and the effector cell / target cell ratio of WSU-DLCL2 was 5:1) were incubated with a serial dilution of CD20xCD3 bispecific antibody REGN1979 (concentration range: 5nM to 0.64pM). In the determination of targeting Nalm6 cells, 2.5ug / ml (16.7nM) constant amount of CD22xCD28 REGN5838, single-arm control CD28 bispecific (REGN5678) or IgG4 isotype control (H4sH10154P3) were added. After incubation at 37°C for 96 hours, cells were harvested from the plate and analyzed by FACS on FACSBD LSRFortessa-X20. Regarding FACS analysis, cells were stained using fixable live / dead far-red reactive (Invitrogen) dyes. 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected for each sample. In order to assess the specificity of killing, cells were gated on the colony of live CFDA-SE markers. The percentage or quantity of live target cells was recorded and used to calculate survival rate.

[0439] T cell activation is assessed by incubating cells with antibodies directly conjugated to CD2, CD4, CD8, and CD25. The percentage of CD8+ cells expressing CD25 is reported as a T cell activation metric. In addition, as T cells proliferate, CellTraceViolet is diluted, which results in a lower MFI as measured by FACS. Therefore, cell proliferation is reported as a decrease in CellTraceViolet MFI on CD8+ T cells or the percentage of CD8+ cells that have reduced CellTraceViolet MFI.

[0440] The supernatant from this assay is collected for the analysis of cytokine levels. Flow cytometry bead array (CBA) test kit is used to analyze the concentration of IL 17a, IFNγ, TNFα, IL-10, IL-6, IL-4 and IL-2 according to the manufacturer's instructions. Cytokine levels are inserted from the curve generated by the test kit standard and cytokine levels are reported as pg / mL. In Prism software, 4 parameter nonlinear regression analysis is used to calculate the EC50 values ​​of target cell killing, T cell activation, proliferation and cytokine levels and maximum cytokine levels.

[0441] Results, Summary and Conclusions:

[0442] The anti-CD20xCD3 bispecific antibody REGN1979 was tested for its ability to induce naive human T cells to kill target cells expressing human CD20 and CD22 in combination with a co-stimulatory CD22xCD28 antibody or a single-armed CD28 or isotype control antibody.

[0443] REGN1979 activates and directs human T cells to kill Nalm6 (Figure 7) or WSU-DLCL2 (Figure 8) cells in a dose-dependent manner. Adding a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhances the cytotoxicity (EC50) of REGN1979 against Nalm6 cells by 4.7-5.2 times compared to REGN1979 using a single-arm CD28 or isotype control antibody (Table 26) or enhances 17.5 times compared to REGN1979 alone against WSU-DLCL2 cells (Table 27).

[0444] As measured by CD25 upregulation on CD8+ cells or CellTrace Violet dilution, the observed target cell lysis mediated by REGN1979 was associated with T cell activation and proliferation (Figure 7, Figure 8). The addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the efficacy 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 compared to REGN1979 using single-arm CD28 or isotype control antibodies (Table 26) and by 8.2-fold and 16.1-fold, respectively, in the presence of WSU-DLCL2 cells compared to REGN1979 alone (Table 27).

[0445] In assays using human PBMCs and WSU-DLCL2 cells, REGN1979 induced the release of human cytokines. The release of cytokines observed with REGN1979 in the presence of a fixed concentration of CD22xCD28 was enhanced compared to the cytokine release induced by REGN1979 alone (Table 28, Fig. 9 ).

[0446] In summary, co-stimulation increased the efficacy of targeted cytotoxicity, T cell activation, and cytokine release compared to that observed with CD20xCD3 alone or in combination with control antibodies.

[0447] Summary of tabular data:

[0448] Table 26: EC50 values ​​for cytotoxicity and T cell activation using Nalm6 target (1 experiment)

[0449]

[0450] Table 27: EC50 values ​​for cytotoxicity and T cell activation using WSU-DLCL2 target (average of 2 experiments)

[0451]

[0452] Table 28: Cytokine release from WSU-DLCL2 cytotoxicity assay (average of 2 experiments)

[0453]

[0454]

[0455] Example 13: FACS-based cytotoxicity on NHL+ human PBMC + / - CD22xCD28 stimulation (fixed CD22xCD28, titrated CD20xCD3)

[0456] Experimental Procedure

[0457] CD22xCD28 enhancement of CD20xCD3 targeted killing was evaluated in a 96-hour cytotoxicity assay targeting NHL cells isolated from primary NHL patient biopsies with autologous PBMCs in the presence of human stromal cells (HS-5). Briefly, HS-5 cells were plated in flat-bottom 96-well plates at 5000 cells per well and incubated overnight. The next day, PBMCs from NHL patients were labeled with 1uM fluorescent tracking dye CellTrace Violet. Bone marrow and labeled PBMC (effector cell / target cell ratio of 10: 1) were plated in wells with stromal cells and incubated at 37°C with serial dilutions of CD20xCD3 bispecific antibody REGN1979 (concentration range: 6.7nM to 10.7pM) and 2.5ug / ml (16.7nM) fixed concentrations of CD22xCD28 co-stimulatory molecule REGN5837 or single-arm control CD28 bispecific (REGN5678) for 96 hours. Cells were harvested from the plate and analyzed by FACS on FACS BD LSRFortessa-X20. For FACS analysis, cells were stained with antibody mixtures (CD45, CD19, CD4, CD8, CD25) and fixable live / dead near IR reactive dyes (Invitrogen). 20,000 counting beads were added to each well immediately before FACS analysis, and 10,000 beads were collected for each sample. To assess specificity of killing, cells were gated on the live CD45+Violet negative CD19+ population. Survival was calculated based on the number of target cells in treated wells normalized to the number of target cells in untreated wells.

[0458] T cells are gated as live CD4+ or CD8+ populations marked by CD45+Violet. The percentage of CD8+ and CD4+ cells expressing CD25 is reported as a measure of T cell activation. In addition, as T cells proliferate, CellTraceViolet is diluted, which results in a lower MFI as measured by FACS. Therefore, T cell proliferation is reported as a reduction in the MFI of CellTraceViolet on CD8+ and CD4+T cells.

[0459] EC50 values ​​for targeted killing as well as T cell activation and proliferation were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0460] Summary of results and conclusions:

[0461] 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 co-stimulatory CD22xCD28 antibody or a one-armed CD28 control antibody.

[0462] REGN1979 activated and directed human T cells to deplete NHL in a dose-dependent manner. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 increased the cytotoxic efficacy (EC50) of REGN1979 by 2.3 and 3.5 fold compared to REGN1979 with a single-armed CD28 control antibody or without a co-stimulatory control (Table 29).

[0463] The observed target cell lysis mediated by REGN1979 correlated with T cell activation and proliferation as measured by CD25 upregulation on CD8+ and CD4+ cells or CellTrace Violet dilution, respectively. Addition of a fixed concentration of CD22xCD28 bispecific antibody to REGN1979 enhanced the potency of REGN1979-induced T cell activation and proliferation by 2.8 to 4.2-fold and 2.8-4.8-fold, respectively, compared to REGN1979 with single-arm CD28 or no co-stimulation controls (Table 29 and Figure 10).

[0464] In summary, co-stimulation increased the efficacy of targeted cytotoxicity and T cell activation compared to that observed with CD20xCD3 in combination with a control antibody.

[0465] Summary of tabular data:

[0466] Table 29: EC50 values ​​for cytotoxicity and T cell activation

[0467]

[0468] Example 14: In vitro characterization and in vivo evaluation of the anti-tumor efficacy of REGN5837 alone and in combination with REGN1979 in a diffuse large B-cell lymphoma (DLBCL) model

[0469] Materials and methods for study introduction and summary of results

[0470] In vitro and in vivo studies were performed to evaluate:

[0471] (1) REGN5837 bridges CD28 + T cells and CD22 + The ability of primary T cells to be activated is enhanced by targeting target cells. T cell activation is assessed using cytotoxicity against target cells, expression of cell surface markers of T cell activation, T cell proliferation, and cytokine release levels as readouts. + The experiments were performed in the presence or absence of CD3 molecules on target cells and the CD20xCD3 bsAb REGN1979 that leads to T cell activation.

[0472] (2) Antitumor efficacy of CD22xCD28 bsAb REGN5837 administered to immunodeficient NSG mice bearing DLBCL tumors in the presence or absence of 0.4 mg / kg or 4 mg / kg REGN1979.

[0473] REGN5837 and REGN1979 were tested in combination over a range of concentrations to evaluate the effect of REGN5837 on REGN1979-mediated T cell cytotoxicity against a human DLBCL cell line (WSU-DLCL2), upregulation of late T cell activation markers (CD25), T cell proliferation, and cytokine release from primary human T cells. REGN5837 enhanced REGN1979-mediated T cell cytotoxicity, CD4 + and CD8 + CD25 cell surface expression on T cells and CD4 + and CD8 + The effect of T cell proliferation. Similarly, REGN5837 enhances the effect of REGN1979-mediated cytokine release in a concentration-dependent manner. At a concentration ranging from 77.2pM to 100nM, REGN5837 increases the effect of REGN1979-mediated T cell cytotoxicity against target cells; At a concentration ranging from 77.2pM to 2.78nM, REGN5837 increases the effect of REGN1979-mediated T cell activation and proliferation, but a higher concentration of REGN5837 does not further increase the effect of REGN1979 (Table 30). When adding REGN5837, the maximum amount of REGN1979-mediated target cell killing and T cell proliferation did not increase significantly, but REGN5837 enhanced the maximum level of REGN1979-mediated IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17α release in a concentration-dependent manner.

[0474] Immunodeficient NSG mice (n=6 to 7 per group) were implanted subcutaneously (SC) with WSU-DLCL2 cells and human PBMCs at a ratio of 1:1. The antitumor efficacy of 1 mg / kg REGN5837 combined with 0.4 mg / kg or 4 mg / kg doses of REGN1979 was comparable to that of REGN5837 and REGN1979 monotherapy and with non-bridging IgG4 P-PVAControl bsAb was compared. 1 day, 8 days and 15 days after implantation of WSU-DLCL2 cells, mice received multiple doses of antibodies by intraperitoneal (IP) injection. On day 28 after implantation, treatment with 1 mg / kg REGN5837 in the presence of 0.4 mg / kg or 4 mg / kg REGN1979 resulted in statistically significant inhibition of WSU-DLCL2 tumor growth compared to REGN5837 or REGN1979 monotherapy and non-bridging control bsAb. REGN1979 monotherapy resulted in moderate inhibition of tumor growth, however REGN5837 monotherapy had no effect relative to non-bridging control.

[0475] In conclusion, when REGN5837 and REGN1979 were tested in combination in vitro over a range of concentrations, REGN5837 enhanced the activation of REGN1979 on CD22 + The efficacy of REGN1979 in mediating human T cell activation in the presence of WSU-DLCL2 cells was enhanced in the presence of REGN5837. The maximum level of REGN1979-mediated cytokine release, but not cytotoxicity, T cell activation, or proliferation, was increased. In vivo, 1 mg / kg REGN5837 in the presence of 0.4 mg / kg or 4 mg / kg REGN1979 effectively inhibited WSU-DLCL2 B cell lymphoma tumor growth in mice relative to REGN5837 or REGN1979 monotherapy alone.

[0476] Table 30: Summary of the effects of REGN5837 on REGN1979-mediated T cell activation (measured by cytotoxicity against target cells, CD25 expression, and T cell proliferation) using human PBMCs

[0477]

[0478] a. REGN1979 was tested at concentrations ranging from 4.8 fM to 10 nM

[0479] b. EC 50 The fold change was calculated as EC 50 (without REGN5837) / EC 50 ([M]REGN5837)

[0480] Example 15: Evaluation of the effect of REGN5837 on REGN1979-mediated human T cell activation and testing of REGN5837 and REGN1979 in combination over a range of concentrations in the presence of WSU-DLCL2 cells.

[0481] Both in vivo and in vitro studies were performed to evaluate the antitumor efficacy of the human CD22xCD28 bsAb REGN5837 in the presence or absence of a sub-effective dose of a human CD20xCD3 bsAb (REGN1979) in NSG mice implanted with human PBMCs and WSU-DLCL2 cells by measuring the following parameters: a) CD22 + T cell cytotoxicity to target cells; b) T cell activation marker CD25 in CD4 + and CD8 + Upregulation of T cells on the cell surface; c) T cell proliferation; d) cytokine release (IL-4, IL-2, IL-6, IL-10, TNF-α, IFN-γ and IL-17Α)

[0482] Materials and Methods

[0483] Cell lines

[0484] WSU-DLCL2: WSU-DLCL2 is a human DLBCL cell line isolated from the pleural effusion of a 41-year-old Caucasian male (Leibniz Institute-DSMZ, catalog number ACC 575).

[0485] Human PBMC

[0486] For cytotoxicity, T cell activation, T cell proliferation and cytokine release, leukopak cells from a human donor were obtained from the New York Blood Center (donor number 1500A).

[0487] For in vivo mouse experiments, human PBMCs were obtained from ReachBio (Catalog No. 0500-401).

[0488] Experimental design

[0489] REGN5837 and REGN1979 were tested in combination over a range of concentrations to assess the effect of REGN5837 on REGN1979-mediated T cell cytotoxicity against WSU-DLCL2 cells, T cell proliferation, cell surface expression of a late T cell activation marker (CD25), and cytokine release from human T cells. The percentages of target cell killing, T cell activation, and T cell proliferation were determined as described herein.

[0490] As described herein, the anti-tumor efficacy of REGN5837 alone and in combination with REGN1979 was evaluated in a DLBCL model using WSU-DLCL2 cells and PBMCs. See Table 31.

[0491] In vitro evaluation of the effect of REGN5837 on the potency of REGN1979-mediated T cell activation

[0492] Human primary T cell isolation

[0493] Use 50 mL of SepMate according to the manufacturer's recommended protocol. TM Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs by density gradient centrifugation. Briefly, 15 mL of FicollPaque PLUS was layered into a 50 mL SepMate tube, and then 30 mL of whole blood diluted 1:2 with D-PBS was added. The tube was centrifuged at 1200 x g for 10 minutes at room temperature with the brake turned off. The top layer containing plasma and PBMCs was poured into a fresh tube. Subsequent steps were performed according to the SepMate manufacturer's protocol. The isolated PBMCs were centrifuged at 250 x 10 6 Cells were frozen in 5 mL cryovials at a concentration of 10 cells / mL in FBS containing 10% DMSO. PBMCs were thawed in a 37°C water bath and resuspended in 10 mL of stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA and 0.01 mM BME) containing 50 U / mL universal nuclease per 100 million PBMCs and centrifuged at 300 x g for 10 minutes. EasySep from Stem Cell Technologies was used according to the manufacturer's recommended instructions. TM Human CD3 + T Cell Isolation Kit isolates CD3 from pelleted PBMCs + T cells.

[0494] Flow cytometry-based T cell activation assay using PBMCs

[0495] Using WSU-DLCL2 target cells and human PBMC as effector cells, the ability of REGN5837 to enhance T cell activation mediated by allogeneic primary stimulation or by "signal 1" provided by REGN1979 was calculated. As described herein, PBMC was enriched for lymphocytes. As described herein, target cells and effector cells were incubated with test articles and control antibodies. As described herein, flow cytometry was performed to assess the upregulation of T cell cytotoxicity, proliferation and T cell activation markers. In addition, as described herein, the effect of REGN5837 on REGN1979-mediated cytokine release was assessed. Non-TAAxCD28 bsAb (containing the same CD28 binding arm and non-binding arm as REGN5837) was tested as a non-bridging control for REGN5837.

[0496] Lymphocyte enrichment of PBMCs

[0497] 1x10 6 Human PBMCs were plated in complete medium (RPMI cell culture medium supplemented with 10% FBS, penicillin-streptomycin-glutamine) at 10 cells / ml and incubated overnight at 37°C to enrich lymphocytes by depleting adherent cells such as macrophages, dendritic cells and some monocytes.

[0498] Incubation of PBMCs and target cells with test articles

[0499] Lymphocyte-enriched PBMCs were harvested and labeled with 1 μM Violet Cell Tracker fluorescent tracking dye. WSU-DLCL2 target cells were labeled with 1 μM fluorescent dye Vybrant CFDA-SE.

[0500] Subsequently, dye-labeled PBMCs and dye-labeled target cells were plated in a 5:1 ratio in a round-bottom 96-well plate (5×10 3 target cells / well).

[0501] Plated PBMCs and target cells were incubated with test articles or 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) for 72 hours at 37°C.

[0502] Flow cytometric analysis

[0503] After incubation with the test article and control, dye-labeled cells were stained with a live / dead stain and with a mixture of fluorophore-labeled antibodies to CD2, CD4, CD8, and CD25. Counting beads (20 μL per well) were added, followed by sample analysis on a BD Celesta flow cytometer. Flow cytometry data were used to determine target cell viability, T cell proliferation, and upregulation of T cell activation markers. EC was determined from a four-parameter logistic equation on a 9-point dose-response curve using GraphPad Prism software. 50 The maximum responses for cytotoxicity, T cell activation (CD25 upregulation), and proliferation were determined as the maximum response plateau generated by Prism curve fitting. 50 The relative change relative to the control was calculated as EC50 No REGN5837 / EC50 [M]REGN5837 , and the relative change in maximal cytokine release was calculated as the maximum [M]REGN5837 / maximum No REGN5837 .

[0504] Target cell survival rate

[0505] The percentage of live target cells under 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 target cell viability percentage was determined as the ratio of the number of live target cells under any experimental condition to the number of live target cells under the no antibody control condition (target cells in the presence of PBMCs alone).

[0506] Where reported in this manner, the percent cytotoxicity against target cells for each experimental condition was determined as 100 minus the percent viability (calculated as described above).

[0507] CD4 + and CD8 + CD25 expression on T cells

[0508] By using live CD2 + and CD4 + or CD8 + Cells were gated to assess upregulation of CD25, a marker for late activated T cells. The percentage of activated T cells expressing CD25 relative to the total number of T cells expressing CD4 or CD8 is reported.

[0509] CD4 + and CD8 + Proliferation of T cells

[0510] By counting the number of dividing cells, CD4 + or CD8 + The percentage of total T cells was assessed using flow cytometry for primary CD4 + and CD8 + T cell proliferation. The fluorescence intensity of cells stained with Violet Cell Tracker was used as a readout of cell division, because the fluorescence intensity of each cell decreased by 2-fold with each round of division.

[0511] Cytokine release analysis

[0512] The levels of cytokines (IL-4, IL-2, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A) in the cell culture supernatant were quantified using the BD Flow Cytometry Bead Array Human Th1 / Th2 / Th17 Cytokine Kit according to the manufacturer's instructions.

[0513] In vivo model of DLBCL using WSU-DLCL2 cell xenografts

[0514] Female NSG mice were used in all experiments. All mice were SC implanted with WSU-DLCL2 B cell lymphoma cells and IP administered with antibodies. Tumor volume was measured several times a week using a caliper throughout the study. For all experiments, mice were housed in a Regeneron animal facility under standard conditions. All experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Regeneron.

[0515] WSU-DLCL2 cell culture conditions and tumor implantation

[0516] The WSU-DLCL2 cell line was obtained from the Leibniz Institute-DSMZ and maintained in RPMI-1640 medium with 10% FBS supplemented with penicillin, streptomycin, glutamine and 1 mM HEPES.

[0517] Collect WSU-DLCL2 cells (3x10 6 cells) and compared them with 5x10 5 PBMCs were mixed and resuspended in a 1:1 mixture of PBS and GFR Matrigel (Matrigel). Female NSG mice were injected SC with 100 μL of the cell mixture into the right flank.

[0518] Antibody administration for tumor measurement

[0519] Prior to dosing with test article or control, mice were assigned to groups stratified according to tumor burden.

[0520] Antibodies (REGN5837, REGN1979, REGN5671 [non-TAAxCD28 non-bridging control bsAb] or H4sH17664D [non-TAAxCD3 non-bridging control bsAb]) were administered by IP injection as monotherapy or in combination on days 1, 8 and 15 after implantation at the doses stated in Table 31.

[0521] Table 31: Experimental design for assessing tumor growth and survival

[0522]

[0523]

[0524] a. One mouse died during the experiment and was excluded.

[0525] Tumor Measurements and Specified End Points

[0526] Tumor growth was monitored over time using caliper measurements of the tumor X and Y diameters (length and width measured perpendicularly). Tumor volume was calculated (X*Y*[X / 2], where X is the shorter diameter). Mice were euthanized when tumors reached the specified tumor endpoint (tumor diameter>20 mm or tumor ulceration). This endpoint met IACUC criteria.

[0527] Statistical analysis of tumor growth and survival

[0528] Results for tumor volume 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 the Mantel-Cox test across all groups, and the Mantel-Cox test was run for comparisons of individual groups one by one. Differences were considered statistically significant when p < 0.05. Statistical analysis was performed using GraphPad Prism 8 software.

[0529] result

[0530] Effect of REGN5837 on the ability of REGN1979 to mediate T cell cytotoxicity against WSU-DLCL2 target cells and cytokine release from human PBMCs

[0531] REGN5837 and REGN1979 were tested in combination over a range of concentrations to assess the effects of REGN5837 on REGN1979-mediated cytotoxicity, T cell activation, T cell proliferation, and cytokine release from T cells of human PBMCs against WSU-DLCL2 cells, as previously described. Table 30 shows the effects of REGN5837 on REGN1979-mediated cytotoxicity, T cell activation, and T cell proliferation against WSU-DLCL2 cells. Numerical results from 2 human donors demonstrating the effects of REGN5837 on cytokine release are summarized in Table 32.

[0532] Effects of REGN5837 on REGN1979-mediated cytotoxicity and proliferation of human T cells

[0533] We evaluated REGN1979-mediated cytotoxicity against WSU-DLCL2 target cells, REGN1979-mediated T cell activation, and REGN1979-mediated activation of human CD4 T cells from human PBMCs. + and CD8 + The proliferation of T cells was used to evaluate the efficacy of increasing concentrations of REGN5837 on REGN1979 (EC 50REGN5837 enhanced REGN1979-mediated cytotoxicity, T cell activation (measured as CD4 + and CD8 + CD25 expression on T cells) and CD4 + and CD8 + Efficacy of T cell proliferation. At concentrations ranging from 77.2 pM to 100 nM, REGN5837 increased the efficacy of REGN1979-mediated T cell cytotoxicity against target cells; at concentrations ranging from 77.2 pM to 2.78 nM, REGN5837 increased the efficacy of REGN1979-mediated T cell activation and proliferation, but higher concentrations of REGN5837 did not further increase the efficacy of REGN1979. These data are graphically represented in Figure 11 and Table 30.

[0534] REGN1979-mediated cytokine release from human PBMCs in the presence of REGN5837

[0535] The effect of increasing concentrations of REGN5837 on the efficacy and maximum response of REGN1979-mediated cytokine release from human PBMCs was evaluated. In the presence of human PBMCs and WSU-DLCL2 cells, increasing concentrations of REGN5837 enhanced the maximum levels of REGN1979-mediated IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17Α release in a concentration-dependent manner (Figure 12). In addition, increasing concentrations of REGN5837 showed a trend in enhancing the efficacy of REGN1979-mediated cytokine release. Figure 12 and Table 32 summarize the EC 50 Values, maximum cytokine levels, and relative increases above background cytokine levels mediated by REGN1979 (without REGN5837).

[0536] Table 32

[0537]

[0538]

[0539] Antitumor efficacy of REGN5837 administered in the presence or absence of a sub-effective dose of REGN1979

[0540] Immunodeficient NSG mice bearing WSU-DLCL2 tumors received IP injections of antibodies or non-bridging controls as previously described herein.

[0541] In tumor-bearing mice, treatment with 1 mg / kg REGN5837 in the presence of 0.4 mg / kg or 4 mg / kg REGN1979 resulted in statistically significant inhibition of tumor growth at day 28 (6 days after the last antibody dose) compared with non-bridging control bsAbs (non-TAAxCD28 and non-TAAxCD3 bsAbs). Fig.13A and 13B On day 46, the combination of 1 mg / kg REGN5837 and 0.4 mg / kg REGN1979 resulted in a significant reduction in tumor volume relative to REGN1979 monotherapy.

[0542] On day 28, 0.4 mg / kg and 4 mg / kg REGN1979 monotherapy resulted in modest tumor inhibition relative to non-bridging controls, whereas REGN5837 monotherapy had no effect. Rapid tumor growth was observed upon dosing with the non-bridging control bsAb throughout the dosing period, and on day 125, all mice were euthanized.

[0543] The Mantel-Cox test detected statistically significant differences in survival across all groups (p=0.0001), and additional Mantel-Cox tests were performed for group-by-group comparisons. Fig.14 ), a significant increase in the survival rate of mice dosed with 1 mg / kg REGN5837 in combination with 0.4 mg / kg or 4 mg / kg REGN1979 was observed (85% and 70% survival, respectively).

[0544] Furthermore, a significant increase in the survival rate of mice dosed with 1 mg / kg REGN5837 in combination with 0.4 mg / kg or 4 mg / kg REGN1979 was observed compared to mice dosed with REGN5837 or REGN1979 monotherapy.

[0545] The present invention should not be limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will also become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. An isolated human bispecific antibody or antigen-binding fragment thereof, comprising: a) a first antigen binding domain (D1) that binds to human CD28 and comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) pair comprising the amino acid sequence of SEQ ID NO: 26 / 10; and b) a second antigen binding domain (D2) that specifically binds to human CD22 on target tumor cells and comprises a HCVR / LCVR pair comprising SEQ ID NO: 2 / 10.

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

58.

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

4. The isolated human bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the bispecific antibody or antigen-binding fragment thereof has a K of 10 μM to 45 μM as measured by surface plasmon resonance at 25°C. D Binds human CD28.

5. The isolated human bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the bispecific antibody or antigen-binding fragment thereof has a binding affinity of 1.3 x 10 -6 Up to 2.3x10 -8 EC of M 50 Binds to the surface of cells expressing human CD28.

6. The isolated human bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the bispecific antibody or antigen-binding fragment thereof has a binding affinity of 1.3 x 10 -6 Up to 2.3x10 -8 EC of M 50 Binds to the surface of cells expressing human CD22.

7. The isolated human bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the bispecific antibody or antigen-binding fragment thereof exhibits a co-stimulatory effect when used in conjunction with an anti-CD20xCD3 bispecific antibody.

8. The isolated human bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the co-stimulatory effect is selected from the group consisting of: activation of T cells, induction of IL-2 release, induction of CD25+ upregulation in human PBMCs, and increase in human T cell-mediated cytotoxicity on CD22-expressing cell lines, and combinations thereof. 9 . A pharmaceutical composition comprising the human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or diluent.

10. 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 8; or a group of nucleic acid molecules comprising a nucleotide sequence encoding the HCVR of the first antigen-binding domain, the HCVR of the second antigen-binding domain, and the LCVRs of the first and second antigen-binding domains of the human bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, respectively.

11. An expression vector comprising the nucleic acid molecule according to claim 10; or a group of expression vectors each comprising the group of nucleic acid molecules according to claim 10.

12. A host cell comprising the nucleic acid molecule or a group of nucleic acid molecules according to claim 10; or the expression vector or a group of expression vectors according to claim 11.

13. A method for producing an anti-CD22 x anti-CD28 human bispecific antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 12 under conditions that allow production of the human bispecific antibody or the antigen-binding fragment thereof, and recovering the human bispecific antibody or the antigen-binding fragment thereof so produced.

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