BISPECIFIC ANTI-MUC16 X ANTI-CD28 ANTIBODIES AND THEIR USES

Abstract

The present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that binds specifically to human CD28, and a second antigen-binding domain that binds specifically to human MUC16. In certain embodiments, the bispecific antigen-binding molecules of the present invention can inhibit the growth of tumors expressing MUC16, such as ovarian tumors. The antibodies and bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced targeted immune response is convenient and / or therapeutically beneficial.

Description

ANTI-MUC16 X ANTI-CD28 BIASPECIAL ANTIBODIES AND THEIR USES RELATED REQUESTS This application refers to and claims priority to US Provisional Application No. 62 / 782,142, filed December 19, 2018, and US Provisional Application No. 62 / 815,861, filed March 8, 2019. All content of the above applications is expressly incorporated herein by reference. LIST OF SEQUENCES This application contains a Sequence Listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. That ASCII copy, created on April 18, 2019, is called 10493W001_118003_49320_SeqLst.txt and is 38,372 bytes in size. FIELD OF THE INVENTION The present invention relates to bispecific antigen-binding molecules that bind to CD28 and a target molecule such as MUC16, and methods of using these. L / EQnn / Lznz / E / Yi l i «αηη / Lznz / E / YiA BACKGROUND OF THE INVENTION CD28 is a type I transmembrane protein, having a single Ig-V-like extracellular domain assembled as a homodimer and expressed on the surface of T lymphocytes. CD28 is the receptor for the CD80 (B7.1) and CD86 proteins (B7.2) and is activated by CD80 or CD86 expressed on antigen presenting cells (APCs). Binding of CD28 to CD80 or CD86 provides important costimulatory signals for T cell activation and survival. T cell stimulation through CD28, in addition to the T cell receptor (TCR), provides a potent signal for T cell production. various interleukins. CD28 also potentiates cellular signals such as pathways controlled by the transcription factor NFkB after TCR activation. Simultaneous CD28 signaling is important for efficient T cell activation, such as T cell differentiation, proliferation, cytokine release, and cell death. Anti-CD28 antibodies have been proposed for therapeutic purposes involving the activation of T lymphocytes. In a clinical trial in 2006, a particular anti-CD28 antibody was used, TGN1412 (anti-CD28 superagonist). Six healthy volunteers received an intravenous dose of TGN1412 (anti-CD28 superagonist) at a dose of 0.1 mg / kg. Within two hours, all six patients had significant inflammatory responses (cytokine storm) and all patients had multiple organ failure within sixteen hours. Individuals were treated with corticosteroids and cytokine levels returned to normal levels in 2-3 days. The starting dose of 0.1 mg / kg in a Phase 1 study was based on a 500-fold multiple of the 50 mg / kg No Observed Adverse Effect Level (NOAEL) in Macaca fascicularis (Suntharalingam, et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412, NEON 355:1018-1028 (2006)). Unfortunately, the TGN1412-induced cytokine storm was not predicted by toxicology studies in Macaca fascicularis nor by ex vivo studies of human PBMCs. Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a highly glycosylated integral membrane glycoprotein. MUC16 comprises three major domains: an extracellular N-terminal domain, a sperm sea urchin interspersed large tandem repeat domain, enterokinase domains, agrin (SEA) and a carboxyl-terminal domain comprising a segment of the region. L / RQnn / Lznz / E / Yi transmembrane and a short cytoplasmic tail. Proteolytic cleavage results in the loss of the extracellular portion of MUC16 in the bloodstream. MUC16 is overexpressed in cancers, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, adenocarcinoma of the cervix, and adenocarcinoma of the digestive tract, and in diseases and conditions including inflammatory bowel disease, cirrhosis of the liver, heart failure, peritoneal infection, and abdominal surgery. (Haridas, D. et al., 2014, FASEB J., 28:4183-4199). Expression of MUC16 in cancer cells has been shown to protect cancer cells from the immune system. (Felder, M. et al., 2014, Molecular Cancer, 13:129). Methods of treating ovarian cancer through the use of antibodies against MUC16 were investigated. However, the monoclonal antibodies, oregovomab and abgovomab, have had limited success. (Felder, supra, Das, S. and Batra, SK 2015, Cancer Res. 75:4660-4674). Accordingly, there is a need in the art for improved antibodies to MUC16 to treat cancer. In addition, antigen-binding molecules Bispecific L / EQnn / Lznz / E / Yi that bind to both CD28 and a target antigen, such as MUC16, would be useful in therapeutic settings where specific targeting of tumor cells and lymphocyte-mediated cell inactivation is desirable T that express the target antigen. BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides bispecific antigen-binding molecules that bind to CD28 and MUC16, also referred to herein as anti-CD28 / anti-MUCI6 bispecific molecules. The anti-MUC16 portion of the anti-CD28 / anti-MUCI6 bispecific molecule is useful for targeting tumor cells that express MUC16 (eg, ovarian tumor cells), and the anti-CD28 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of MUC16 on a tumor cell and CD28 on a T cell facilitates targeted inactivation (cell lysis) of the target tumor cell by the activated T cell. Therefore, the antiCD28 / anti-MUC16 bispecific molecules of the invention are useful, inter alia, for treating diseases and disorders related to, or caused by, tumors that express MUGI 6 (eg, ovarian cancer). L / RQnn / Lznz / E / Yi Bispecific antigen-binding molecules according to this aspect of the present invention comprise a first antigen-binding domain that specifically binds to human CD28, and a second antigen-binding domain that specifically binds to MUC16. The present invention includes anti-CD28 / anti-MUC16 bispecific molecules (eg, bispecific antibodies), wherein each antigen-binding domain comprises a heavy chain variable region (HCVR) paired with a light chain variable region ( LCVR). In certain example embodiments of the invention, the anti-CD28 antigen binding domain and the anti-MUC16 antigen binding domain each comprise different HCVRs paired with a common LCVR. The present invention provides anti-CD28 / anti-MUC16 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises any of the HCVR amino acid sequences, as set forth in Table 3. The first domain The antigen-binding domain that specifically binds to CD28 may also comprise any of the LCVR amino acid sequences, as set forth in Table 3. According to certain embodiments, the first antigen-binding domain that binds L / RQnn / Lznz / E / Yi specifically binds CD28 comprises any of the HCVR / LCVR amino acid sequence pairs, as set forth in Table 3. The present invention also provides anti-CD28 / anti-MUCI6 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises any of the heavy chain CDR1 CDR2-CDR3 amino acid sequences, as set forth in Table 3, and / or any of the CDR1 amino acid sequences -CDR2CDR3 of the light chain, as set forth in Table 3. According to certain embodiments, the present invention provides anti-CD28 / antiMUC16 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a heavy chain variable region (HCVR) having a sequence amino acid sequence selected from the group consisting of SEQ ID NO: 18 and 42 or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The present invention also provides anti-CD28 / anti-MUCl6 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group that L / Rann / Lznz / E / Yii consists of SEQ ID NO: 10 and 34, or a sequence substantially similar thereto that has at least 90%, at least 95%, at least 98%, or at least 99% identity of sequence. The present invention also provides anti-CD28 / anti-MUCI6 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a pair of HCVR and LCVR (HCVR / LCVR) amino acid sequences selected from the group that consists of SEQ ID NO: 18 / 10 and 42 / 34. The present invention also provides anti-CD28 / anti-MUCI6 bispecific molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and 48, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40, or a sequence substantially similar thereto that is at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In certain embodiments, the first domain of L / RQnn / Lznz / B / Yi binding to the antigen that specifically binds to CD28 comprises a pair of HCDR3 / LCDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 24 / 16 and 48 / 40. The present invention also provides anti-CD28 / anti-MUCI6 bispecific antigen-binding molecules, wherein the first antigen-binding domain that specifically binds to CD28 comprises a heavy chain CDR1 (HCDR1) domain having a sequence of amino acids selected from the group consisting of SEQ ID NO: 20 and 44, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 46, or a sequence substantially similar thereto that is at least 90%, at least 95%, at less than 98% or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, or a sequence substantially similar thereto that is at least 90%, at least 95%, at less than 98% or at least 99% sequence identity; and a light chain CDR2 domain (LCDR2) that has a sequence of L / EQnn / Lznz / E / Yi amino acids selected from the group consisting of SEQ ID NO: 14 and 38, or a sequence substantially similar thereto that is at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. Certain exemplary and non-limiting anti-CD28 / anti-MUC16 bispecific antigen-binding molecules of the invention include a first antigen-binding domain that specifically binds to CD28 comprising the domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2- LCDR3 having, respectively, the amino acid sequences selected from the group consisting of: SEQ ID NO: 20-22-24-12-14-16 and 4446-48-36-38-40. The present invention also provides anti-CD28 / anti-MUC16 bispecific molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a heavy chain variable region (HCVR) having the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 26, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The present invention also provides anti-CD28 / anti-MUC16 bispecific molecules, wherein the second L / RQnn / Lznz / E / Yi antigen-binding domain that specifically binds to MUC16 comprises a light chain variable region (LCVR) having the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34 , or a sequence substantially similar to these having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The present invention also provides anti-CD28 / anti-MUC16 bispecific molecules, wherein the second antigen-binding domain that specifically binds to MUGI6 comprises a pair of HCVR and LCVR (HCVR / LCVR) amino acid sequences selected from the group that consists of SEQ ID NO: 2 / 10 and 26 / 34. The present invention also provides anti-CD28 / anti-MUC16 bispecific molecules, wherein the second antigen-binding domain that specifically binds to MUGI6 comprises a heavy chain CDR3 (HCDR3) domain having the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 32, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group that L / RQnn / Lznz / E / Yi consists of SEQ ID NO: 16 and 40, or a sequence substantially similar thereto that has at least 90%, at least 95%, at least 98%, or at least 99% identity of sequence. In certain embodiments, the second antigen-binding domain that specifically binds to MUC16 comprises a pair of HCDR3 / LCDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 8 / 16 and 32 / 40. The present invention also provides anti-CD28 / anti-MUCI6 bispecific antigen-binding molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a heavy chain CDR1 (HCDR1) domain having the sequence of amino acids selected from the group consisting of SEQ ID NO: 4 and 28, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having the amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 30, or a sequence substantially similar thereto that is at least 90%, at least 95%, at less than 98% or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group that ί / βΟΠΠ / ί7Π7 / Β / ΥΙ consisting of SEQ ID NO: 12 and 36, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38, or a sequence substantially similar thereto that is at least 90%, at least 95%, at least 98% or at least 99% sequence identity. Certain exemplary and non-limiting anti-CD28 / anti-MUC16 bispecific antigen-binding molecules of the invention include a second antigen-binding domain that specifically binds to MUGI 6 comprising the domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2 -LCDR3 having, respectively, the amino acid sequences selected from the group consisting of: SEQ ID NO: 4-6-8-12-14-16 and 28-3032-36-38-40. In a related embodiment, the invention includes anti-CD28 / anti-MUC16 bispecific antigen-binding molecules, wherein the second antigen-binding domain that specifically binds to MUGI6 comprises the heavy and light chain CDR domains contained within. heavy and light chain variable region (HCVR / LCVR) sequences selected from the group consisting of L / RQnn / Lznz / E / Yi SEQ ID NO: 2 / 10 and 26 / 34. In another aspect, the present invention provides nucleic acid molecules encoding any of the HCVR, LCVR, or CDR sequences of the anti-CD28 / anti-MUC16 bispecific antigen-binding molecules disclosed herein, including nucleic acid molecules that comprise the polynucleotide sequences shown in Table 2 and / or Table 4 hereof, as well as nucleic acid molecules comprising two or more of the polynucleotide sequences shown in Table 2 and / or the Table 4 in any combination or functional arrangement of these. Recombinant expression vectors carrying the nucleic acids of the invention, and the host cells into which such vectors were introduced, are also included in the invention as they are methods of producing the antibodies by culturing the host cells under conditions that allow the production of the antibodies, and to recover the antibodies produced. The present invention includes anti-CD28 / anti-MUCI6 bispecific antigen-binding molecules, wherein any of the aforementioned antigen-binding domains that specifically bind to CD28 is combined, linked, or otherwise associated with any of the domains The aforementioned antigen-binding L / RQnn / Lznz / E / Yi specifically bind to MUC16 to form a bispecific antigen-binding molecule that binds to CD28 and MUC16. The present invention includes anti-CD28 / anti-MUC16 bispecific antigen-binding molecules that have a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites, or an antibody that lacks a fucose portion present in the oligosaccharide chain, may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function. (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modification can be performed to modify complement-dependent cytotoxicity (CDC). In another aspect, the invention provides a pharmaceutical composition comprising an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule as disclosed herein and a pharmaceutically acceptable carrier. In a related aspect, the invention describes a composition that is a combination of an anti-CD28 / anti-MUC16 bispecific antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that combines L / EQnn / Lznz / E / Yi advantageously with an anti-CD28 / anti-MUC16 bispecific antigen-binding molecule. Exemplary agents that can be advantageously combined with an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule are discussed in detail elsewhere herein. In yet another aspect, the invention provides therapeutic methods for the targeting / inactivation of MUC16-expressing tumor cells by an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule of the invention, wherein the therapeutic methods comprise administering a therapeutically effective delivery of a pharmaceutical composition comprising an anti-CD28 / anti-MUC16 bispecific antigen-binding molecule of the invention to a subject in need thereof. The present invention also includes the use of an anti-CD28 / anti-MUC16 bispecific antigen-binding molecule of the invention in the manufacture of a medicament for the treatment of a disease or disorder related to or caused by the expression of MUC16. In yet another aspect, the invention provides therapeutic methods for the targeting / inactivation of MUC16-expressing tumor cells by an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule of the invention, L / RQnn / Lznz / E / Yi wherein the antiCD28 / anti-MUCI6 bispecific antigen-binding molecule is combined with other antitumor bispecific antigen-binding molecules that bind to CD3 (for example, anti-CD28 / anti- MUC16 combined with anti-CD3 / anti-MUCI6 antibodies). In yet another aspect, the invention provides therapeutic methods for targeting / inactivating MUGI6-expressing tumor cells by an anti-CD28 / anti-MUC16 bispecific antigen-binding molecule of the invention, wherein the antiCD28 bispecific antigen-binding molecule / anti-MUC16 is combined with a checkpoint inhibitor targeting, for example, PD-1, PD-L1, or CTLA-4 (for example, anti-CD28 / anti-MUCI6 antibodies combined with anti-PD-1 antibodies ). In certain embodiments, it is envisioned that the anti-CD28 / anti-MUC16 antibodies of the invention may be combined with agents that target PD-1, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or cemiplimab (Libtayo ®). In certain embodiments, it is envisioned that the anti-CD28 / anti-MUC16 antibodies of the invention may be combined with agents that target PD-L1, such as atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi ®). In certain embodiments, it is envisioned that the anti-CD28 / anti-MUC16 antibodies of the invention may L / Rann / Lznz / E / Yii be combined with agents that target CTLA-4, such as ipilimumab (Yervoy®). In yet another aspect, the invention provides therapeutic methods for targeting / inactivating MUGI6-expressing tumor cells by an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule of the invention, wherein the antiCD28 bispecific antigen-binding molecule / anti-MUC16 is combined with other antitumor bispecific antigen-binding molecules that bind to CD3 (for example, anti-CD28 / anti-MUCI6 bispecific antibodies combined with anti-CD3 / anti-MUCI6 bispecific antibodies) and a point inhibitor control targeting PD-1, PDL-1, or CTLA-4 (eg, anti-CD28 / anti-MUCI6 antibodies combined with anti-PD-1 antibodies). Other embodiments will become apparent upon review of the following detailed description. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing inhibition of tumor growth in engineered cell lines with introduction of costimulatory ligand expression. Three tumor cell lines, B16F10.9, EL4, and MC38, were engineered to express L / RQnn / Lznz / E / Yi a costimulatory ligand, or GFP, or an empty vector as a control. The genetically engineered tumor cells were injected into C57BL / 6 mice. Data represent mean ± SEM. Data are representative of at least one experiment with five (5) mice per group. The graph shows the tumor growth as the percentage of the control calculated as Tumor volume ________________________________________ X100 Tumor volume of the control Figures 2A to 21 are schematics and graphs showing that the exemplary anti-MUCI6xCD28 of the invention enhances T cell action in the presence of TCR stimulation by anti-MUCI6xCD3 and cancer cell lines with endogenous MUC16 (PEO1). Figures 2B to 2E are graphs showing human PBMC data. Figures 2F to 2H are graphs showing the PBMC data of Macaca fascicularis. Human T cells (for Figures 2B to 2E) or Macaca fascicularis T cells (for Figures 2F to 2H) were cultured with cancer target cells with endogenous MUC16 expression (PEO-1 ovarian cancer line) and the bispecific antibodies. indicated for 96 hours. Figure 2A is a schematic of the assay setup. Figure 2B is a graph showing inactivation of tumor cells. The value on the Y-axis refers to the ί / βοπη / ίζηζ / Ε / γι percentage of viable PEO1 cell. Figure 2C is a graph showing the release of ΙΕΝγ. Figure 2D is a graph showing CD4 T cell counts and CD254 cell frequency, represented as the percentage of CD25+ cells in CD4 T cells. Figure 2E is a graph showing CD8 T cell counts and CD25+ cell frequency, represented as the percentage of CD25+ cells to CD8 T cells. Figure 2F is a graph showing the inactivation of tumor cells. The value on the Y axis refers to the percentage of viable PEO1 cells. Figure 2G is a graph showing CD4 T cell counts and CD25+ cell frequency, represented as the percentage of CD25+ cells in CD4 T cells. Figure 2H is a graph showing CD4 T cell counts and CD8 T cell counts and CD25+ cell frequency, represented as the percentage of CD25+ cells in CD4 and CD8 T cells. Figure 21 is a graph showing the binding of antibodies to cellular targets as measured by flow cytometry. L / RQnn / Lznz / E / Yi Figures 3A-3C are graphs showing that exemplary anti-MUCI6xCD28 bispecific antibodies of the present invention enhance anti-tumor immunity through anti-MUCI6xCD3-induced T cell activation. Figure 3A is a graph showing tumor burden as measured by mean glow (mean glow [p / s / cm2 / sr] over time. Values ​​represent group median plus range. Values ​​of p were calculated using the Mann Whitney test for each time point *, p<0.05 or **, p<0.01 for the comparison of MUC16xCD3 and EGFRvIIIxCD3 ##, p<0.01 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3. Human PBMC-engrafted NSG mice were implanted with OVCAR3-Luc by intraperitoneal injection.Mice were dosed IV on days 5 and 8 (arrows).Mice received 2.5 pg MUC16xCD3 or 2.5 pg EGFRvIIIxCD3. Some of the mice were also given 100 pg MUC16xCD28 Tumor burden was assessed by BLI on days 4, 8, 12, 15, 20 and 25 after tumor implantation by monitoring bioluminescence over time. N = 5 mice per group Figure 3B provides graphs showing cytokine levels in serum of blood obtained 4 hours after the first dose of the same experiments shown in Figure 3A. P values ​​were calculated with one-way ANOVA. ##, ί / βοπη / ίζηζ / Ε / γι p<0.01 or ####, ρ<0.0001 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3. @@@, p<0.005 for the comparison of MUC16xCD3 + MUC16xCD28 and MUC16xCD3.Λ, p<0.01,ΛΛ, p<0.005, p < Q.0001 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3 + MUC16xCD28. Figure 3C provides graphs showing tumor burden and correlation with serum CA-125 levels at day 26. N = 5 mice per group from the same experiments shown in Figure 3A. Figure 4A is a graph showing tumor burden as measured by mean glow (mean glow [p / s / cm2 / sr] over time. Values ​​represent group median plus range. Values ​​of p were calculated using the Mann Whitney test for each time point.**, p<0.01 for the comparison of MUC16xCD3 and EGFRvIIIxCD3.##, p<0.01 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3.p<0.05 for the comparison of MUC16xCD3 + MUC16xCD28 and MUC16xCD3. NSG mice engrafted with human PBMCs were implanted with OVCAR3-Luc by intraperitoneal injection. Mice were treated IV with 0.5 mg / kg MUC16xCD3 or 0.5 mg / kg EGFRvIIIxCD3. Some of the Mice were also given MUC16xCD28 at 0.2 mg / kg on days 5 and 8 (arrows).Tumor burden was assessed by ί / βοπη / ίζηζ / Ε / γι BLI on days 4, 8, 11, 14, 21, 28, and 34 by monitoring bioluminescence over time. N = 5 or 6 mice per group. Figure 4B provides graphs showing cytokine levels in serum of blood obtained 4 hours after the first dose of the same experiments shown in Figure 4A. P values ​​were calculated with one-way ANOVA. *, p<0.05 for the comparison of MUC16xCD3 and EGFRvIIIxCD3, ##, p<0.01 or ###, p<0.001 or ####, p<0.0001 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3. @, p<0.05 or @@@@, p<0.0001 for the comparison of MUC16xCD3 + MUC16xCD28 and MUC16xCD3.ΛΛ, p<0.001 or ΛΛΛΡ<0.001 or ΛΛΛΡ<0.0001 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD 3 + MUC16xCD28. Figure 5 is a graph showing survival over time. ID8-VEGF / hMUC16 cells were implanted into the peritoneal cavity of hCD3 / hCD28 / hMUCI6 humanized mice. Mice were treated intravenously with EGFRvIIIxCD3 or MUC16xCD3 at 1 mg / kg on days 3, 6, and 10 after tumor implantation, as indicated by the arrows. Some mice were also given MUC16xCD28 at 1 mg / kg. P values ​​were calculated using the MantelCox test for each time point. **, p<0.01 for comparison L / RQnn / Lznz / B / Yi of MUC16xCD3 and EGFRvIIIxCD3. ##, p<0.01 for the comparison of MUC16xCD3 + MUC16xCD28 and EGFRvIIIxCD3. p<0.05 for the comparison of MUC16xCD3 + MUC16xCD28 and MUC16xCD3. Figure 6A is a graph showing tumor volume over time. MC38 / hMUC16 tumor cells were implanted subcutaneously into hCD3 / hMUC16 humanized mice. Mice were treated with anti-MUCI6xCD3 at 0.01 mg / kg, exemplary anti-MUCI6xmCD28 bispecific antibody of the invention at 0.5 mg / kg, as indicated, twice weekly from day 0 (arrows). Tumor volume was monitored by caliper measurements over time. Values ​​shown are the mean ± SEM. Data are representative of three (3) experiments. N = 7 mice per group. P values ​​were calculated with 2-way ANOVA compared to isotype control (**, p<0.01 and ****, P<0.0001 for comparison of MUC16xCD3+MUC16xmCD28 and isotype control; #, p < 0.05 for the comparison of MUC16xCD3 and isotype control; $, p < 0.05 for the comparison of MUC16xmCD28 and isotype control). Figure 6B provides graphs showing cytokine levels in serum of blood obtained at the indicated time point from the same experiments shown in Figure 6A. Figures 6C and 6D are graphs showing the levels L / RQnn / Lznz / E / Yi of cytokines. Mice were bled for serum cytokines 4 hours post-dose on day 7. Statistical significance was calculated with 1-way ANOVA compared to isotype **p<0.01 and ****p<0.0001 . n = 7 mice per group. Data are representative of 3 experiments. Figure 7 is a graph showing that tethering of a MUC16xCD28 to assay plates by either a dry coating or a wet coating method does not induce T cell activation in the absence of a CD3 stimulus in contrast to the CD28 superagonist. . Figures 8A to 8C are graphs showing that MUC16xCD28 alone or in co-treatment does not induce systemic T cell activation. Macaca fascicularis received a single dose of bispecifics at 1 or 10 mg / kg (indicated in parentheses). An additional group received a total of 4 doses indicated as repeat doses. Blood was collected at the indicated times post-dose (h). Figure 8A: Serum cytokines, Figure 8B: Relative T cell counts and Figure 8C: KI67+ and ICOS+ T cell frequencies (% CD3) are shown. Data represent average + / - SEM. N = 3 animals per group. P values ​​were calculated with 2-way ANOVA compared to the L / RQnn / Lznz / E / Yi isotype control. (**, p<0.01; ***, p<0.001 and ****, p<0.0001). Figures 9A and 9B show that bispecific antibodies against MUCxCD28 and against MUC16xCD3 can bind to cells expressing MUC in the presence of soluble CA-125. OVCAR-3 cells were incubated in 8 nM of the indicated Alexa647-tagged antibodies in the presence of increasing concentrations of soluble CA-125 (Figure 9A) or MUC16 nub (Figure 9B) for 30 min at 4°C in cytometry buffer. flow (PBS + 1% FBS). After incubation, cells were washed with flow cytometry buffer and analyzed by flow cytometry. Figure 10 is a schematic of a T cell / antigen presenting cell based reporter bioassay. Figures 11A and 11B show that bs24963D (also designated REGN5668) enhances NF-κΒ signaling in engineered T cells in the presence of stimulatory antigen-presenting cells expressing MUC16. Briefly, J.RT3.T3.5 / NF-KB-Luc / lG4AB / hCD8ap / hCD28 indicator cells were incubated with bs24963D or non-bridged control bispecific antibody against CD28 (not anti-TAAxCD28) at a range of concentrations (39 p.m L / RQnn / Lznz / E / Yi at 10 nM), including a control without antibodies, in the presence of 3T3 / hp2M / HLA-A2 / NYES01p / hMUCl6 cells (Figure 11A) and 3T3 / hp2M / HLA-A2 / NYES01p in a ratio of indicator cells to 3T3 stimulator cells of 3.33:1 (Figure 11B). NF-κΒ signaling was detected as luciferase activity and measured by quantification of the luminescence signal, indicated as relative light units (RLU). Data from an assay performed in duplicate wells are represented as mean ± SD. Figure 12 shows that bs24963D (also referred to as REGN5668) mediates concentration-dependent IL-2 release from human primary T cells in the presence of REGN4018 (see WO2017 / 053856A1, BSMUC16 / CD3-001 which is REGN4018) with OVCAR target cells. -3 and REGI. Briefly, spiked human primary T cells were incubated with bs24963D or non-bridged control bispecific antibody against CD28 (not anti-TAAxCD28) at a range of concentrations (7.6 pM to 500 nM), including a control without antibodies, in the presence of a fixed concentration (5 nM) of REGN4018 or non-bridged control bispecific antibody against CD3 (not anti-TAAxCD3) and the human ovarian cancer cell lines OVCAR-3 or REGI in an effector cell to target cell ratio of 10:1 L / RQnn / Lznz / E / Yi or 4:1, respectively. Data are from triplicate well assay and are represented as mean + SD. IL-2 release was measured with a human IL-2 immunoassay according to the manufacturer's protocol. Figure 13 shows that bs24963D (also designated REGN5668) mediates the concentration-dependent enhancement of human primary T cell proliferation in the presence of REGN4018 with OVCAR-3 and PEO1 target cells. Briefly, spiked human primary T cells were incubated with bs24963D or non-bridged control bispecific antibody against CD28 (not anti-TAAxCD2 8) at a range of concentrations (7.6 pM to 500 nM), including a control without antibodies, in the presence of of a fixed concentration (5 nM) of REGN4018 or non-bridged control bispecific antibody against CD3 (not anti-TAAxCD3) and the human ovarian cancer cell lines OVCAR-3 and PEO1 in an effector cell to target cell ratio of 10:1 or 4:1, respectively. Data are from triplicate well assay and are represented as mean ± SD. T cell proliferation was measured by detection of tritium decay (from tritiated thymidine incorporated into dividing cells) and reported as CPM. Figure 14 shows that bs24963D (also called L / fiann / Lznz / E / Yii REGN5668) mediates concentration-dependent IL-2 release and the addition of cemiplimab modestly increases IL-2 release from human primary T cells with SW1990 and SW1990 / hPD-L1 target cells. Briefly, spiked human primary T cells were incubated with bs24963D or non-bridged control bispecific antibody against CD28 (not anti-TAAxCD28) at a range of concentrations (7.6 pM to 500 nM), including a control without antibodies, in the presence of a concentration binds (20 nM) cemiplimab or IgG4p control to the SW1990 and SW1990 / hPD-L1 human pancreatic cancer cell lines at a 2:1 effector to target cell ratio. Data are from triplicate well assay and are represented as mean ± SD. IL-2 release was measured with a human IL-2 immunoassay according to the manufacturer's protocol. Statistical analyzes were performed with a 2-way ANOVA. Differences were considered statistically significant when p < 0.05. bs24963D + cemiplimab demonstrated statistically significant increases in IL-2 release compared to REGN5668 + IgG4p control SW1990 / hPD-Ll cells (p < 0.0001). Figure 15 shows that bs24963D (also called L / Rann / Lznz / E / Yii REGN5668) mediates concentration-dependent enhancement of proliferation and addition of cemiplimab modestly increases proliferation of SW1990 and SW1990 / hPD-L1 human primary T cells. Briefly, spiked human primary T cells were incubated with bs24963D or non-bridged control bispecific antibody against CD28 (not anti-TAAxCD28) at a range of concentrations (7.6 pM to 500 nM), including a control without antibodies, in the presence of a fixed concentration ( 20 nM) of cemiplimab or IgG4 control p and human pancreatic cancer cell lines SW1990 and SW1990 / hPD-LL at a 2:1 effector cell to target cell ratio. Data are from triplicate well assay and are represented as mean ± SD. T cell proliferation was measured by detection of tritium decay (from tritiated thymidine incorporated into dividing cells) and reported as CPM. Statistical analyzes were performed with a 2-way ANOVA. Differences were considered statistically significant when p < 0.05. bs24963D + cemiplimab demonstrated statistically significant increases in proliferation compared to bs24963D + IgG4p control SW1990 / hPD-Ll cells (p < 0.0001). L / RQnn / Lznz / E / Yi DETAILED DESCRIPTION OF THE INVENTION Before describing the present invention, it should be noted that this invention is not limited to the particular methods and experimental conditions described, as these methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention shall be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly given to them by a person in the mid-level trade to which the present invention pertains. As used herein, the term about, when used in reference to a particular described numerical value, means that the value may vary from the described value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values ​​in between (eg, 99.1, 99.2, 99.3, 99.4, etc.). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the following L / RQnn / Lznz / E / Yi describe the preferred methods and materials. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety. Definitions The term CD28, as used herein, refers to an antigen that is expressed on T cells as a costimulatory receptor. Human CD28 comprises the amino acid sequence as set forth in SEQ ID NO: 50, and / or having the amino acid sequence as set forth in NCBI accession number NP_006130.1. The human CD28 ectodomain (N19P152) having a mouse Fe is shown in SEQ ID NO: 52. The human CD28 ectodomain (N19-P152) having a myc-myc-his tag is shown in SEQ ID NO: 53 All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless explicitly specified as being from a non-human species. Therefore, the term "CD28" means human CD28 unless it is specified that it is from a non-human species, eg, mouse CD28, monkey CD28, etc. The mouse CD28 ectodomain (accession number NP 031668.3) having a myc-myc-his tag is L / Rann / Lznz / E / Yii shown in SEQ ID NO: 54. As used herein, an antibody that binds to CD28 or an anti-CD28 antibody includes antibodies and antigen-binding fragments thereof that specifically recognize a monomeric CD28, as well as antibodies and antigen-binding fragments thereof that recognize specifically a dimeric CD28. Antibodies and antigen-binding fragments of the present invention can bind to soluble CD28 and / or CD28 expressed on the cell surface. Soluble CD28 includes natural CD28 proteins, as well as recombinant CD28 protein variants, eg, monomeric and dimeric CD28 constructs that lack a transmembrane domain or are not associated with a cell membrane. As used herein, the expression "cell surface expressed CD28" means one or more CD28 proteins that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of a CD28 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. CD28 expressed on the cell surface includes CD28 proteins contained in the context of a receptor L / RQnn / Lznz / E / Yi costimulator of T lymphocytes functional in the membrane of a cell. The expression CD28 expressed on the cell surface includes CD28 protein expressed as part of a homodimer on the surface of a cell. A cell surface expressed CD28 may comprise or consist of a CD28 protein expressed on the surface of a cell that normally expresses CD28 protein. Alternatively, cell surface expressed CD28 may comprise or consist of a cell surface expressed CD28 protein that does not normally express human CD28 on its surface, but has been artificially engineered to express CD28 on its surface. surface. As used herein, the term "anti-CD28 antibody" includes monovalent antibodies with a single specificity and bispecific antibodies comprising a first arm that binds to CD28 and a second arm that binds to a second antigen (target), wherein the arm anti-CD28 comprises any of the HCVR / LCVR or CDR sequences listed in Table 3 herein. Examples of anti-CD28 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies. L / EQnn / Lznz / E / Yi The term MUC16, as used herein, refers to the human MUGI 6 protein unless specified to be from a non-human species (eg, mouse MUGI6, monkey MUGI6, etc.). The human MUC16 protein has the amino acid sequence shown in SEQ ID NO: 49, and / or has the amino acid sequence set forth in NCBI accession number NP_07 8966. The human MUGI6 membrane proximal domain ( P13810-P14451) having a myc-myc-his tag is shown as SEQ ID NO: 51. As used herein, an antibody that binds to MUC16 or an anti-MUC16 antibody includes antibodies and antigen-binding fragments thereof that can bind to soluble MUC16 and / or MUC16 expressed on the cell surface. Soluble MUC16 includes natural MUC16 proteins, as well as recombinant MUC16 protein variants such as, for example, monomeric and dimeric MUC16 constructs that lack a transmembrane domain or are not associated with a cell membrane. As used herein, the term "anti-MUC16 antibody" includes monovalent antibodies with a single specificity and bispecific antibodies comprising a first arm that binds to MUC16 and a second arm that binds to a second (target) antigen, wherein the arm anti-MUC16 L / EQnn / Lznz / E / Yi comprises any of the HCVR / LCVR or CDR sequences listed in Table 1 herein. Examples of anti-MUC16 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies. The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (for example , CD28). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as multimers of these (eg, IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. L / RQnn / Lznz / E / Yii heavy chain. The heavy chain constant region comprises three domains, ChI, Ch2 and Ch3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or Vl) and a light chain constant region. The light chain constant region comprises a (ClI) domain. The Vh and Vl regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the RFs of the anti-CD28 and / or anti-MUC16 antibody (or the antigen-binding portion thereof) may be identical to human germline sequences, or may be modified accordingly. natural or artificial way. A consensus amino acid sequence can be defined based on a parallel analysis of two or more CDRs. The term antibody, as used herein, also includes antigen-binding fragments of whole antibody molecules. The terms antigen-binding portion of an antibody, antigen-binding fragment, L / RQnn / Lznz / E / Yi antigen of an antibody, and the like, as used herein, include any natural, enzymatically derivable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from, for example, whole antibody molecules by any suitable standard technique, such as proteolytic digestion or recombinant engineering techniques involving the manipulation and expression of DNA encoding DNA variables. antibodies and, optionally, constant domains. Such DNA is known and / or widely available through, for example, commercial sources, DNA libraries (including, for example, antibody-phage libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or through the use of molecular biological techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create tanker residues, modify, add or remove amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) Fv molecules single-chain L / RQnn / Lznz / E / Yi (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (for example, an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a FR3CDR3- peptide. FR4 restricted. 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 included within the term antigen-binding fragment, as used herein. An antigen-binding fragment of an antibody will generally comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to, or in-frame with, one or more framework sequences. In antigen-binding fragments having a Vh domain associated with a Vl domain, the Vh and Vl domains may be positioned relative to one another in any suitable arrangement. By L / RQnn / Lznz / E / Yi For example, the variable region may be dimeric and contain VH-VH, VH-VL or Vl-Vl dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric Vh or Vl domain. In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting example configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) HCV3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3 ; (vi) Vh-Ch2-Ch3; (vii) Vh_Cl,‘ (viii) Vl_Ch1,‘ (ix) Vl_Ch2; (x) Vl_Ch3; (xi) Vl_Ch1_Ch2; (xii) Vl-Ch1-Ch2-Ch3 ; (xiii) Vl-Ch2-Ch3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked together or may be linked via a partial or complete linker or hinge region. A hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that give rise to a flexible or semi-flexible link between adjacent variable and / or constant domains in a single molecule. of L / RQnn / Lznz / E / Yi polypeptides. In addition, an antigen-binding fragment may comprise a homodimer or heterodimer (or other multimer) of any of the constant and variable domain configurations listed above in non-covalent association with each other and / or with one or more Vh or Vl domains. monomeric (for example, via disulfide bridges). Like complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, each variable domain being capable of binding specifically to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using available routine techniques. in technique. The antibodies of the present invention may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) is L / fiann / Lznz / E / Yii refers to the lysis of cells expressing antigens by an antibody of the invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fe receptors (FcR) (for example, natural killer cells (NK), neutrophils, and macrophages) recognize bound antibody. in a target cell and therefore lead to lysis of the target cell. CDC and ADCC can be measured by assays that are well known and available in the art. (See, for example, US Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Nati. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in an antibody's ability to correct complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of an antibody can be selected based on whether it is suitable for the antibody to mediate cytotoxicity. In certain embodiments of the invention, the anti-CD28 and / or anti-MUC16 antibodies of the invention (monospecific or bispecific) are human antibodies. The term human antibody, as used herein, is intended to include antibodies having variable regions and L / EQnn / Lznz / E / Yi constants derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which the germline-derived CDR sequences of other mammalian species, such as mouse, were grafted onto the human framework sequences. In some embodiments, the antibodies of the invention may be recombinant human antibodies. The term recombinant human antibody, as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed with a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (eg, a mouse) that is transgenic for immunoglobulin genes human L / Rann / Lznz / E / Yii (see, for example, Taylor et al. (1992) Nucí. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means involving the splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions that are derived from human germ line immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the Vh and Vl regions of the recombinant antibodies are sequences which, while derived from and related to human germline Vh and Vl sequences, may not naturally exist in the germline repertoire of human antibodies in vivo. Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa where the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked by disulfide bonds. L / Rann / Lznz / E / Yii interchain and a molecule of around 75-80 kDa is formed, composed of covalently paired light and heavy chains (half antibody). These forms have been extremely difficult to separate, even after affinity purification. The frequency of occurrence of the second form in various intact IgG isotypes is due to, among other things, structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the use of a hinge. of human IgGl. The present invention encompasses antibodies having one or more mutations in the hinge, Ch2, or Ch3 region that may be desirable, eg, in production, to improve the yield of the desired antibody form. The antibodies of the invention may be isolated antibodies. An isolated antibody, as used herein, means an antibody that has been identified and separated, and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or eliminated from at least one component of an organism, or from a tissue or cell where L / RQnn / Lznz / E / Yi the naturally occurring or naturally occurring antibody is an isolated antibody for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other chemicals and / or cellular materials. The present invention also includes arm antibodies that bind to CD28 and / or MUC16. As used herein, a single-armed antibody means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. Antibodies to one arm of the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences as set forth in Table 1 and Table 3. The anti-CD28 and / or anti-MUC16 antibodies herein, or the antigen-binding domains thereof, may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the antibodies. heavy and light chain variable domains compared to germline sequences corresponding L / RQnn / Lznz / E / Yi from which the antigen binding proteins or antigen binding domains were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available in, for example, public antibody sequence databases. The present invention includes antibodies, and the antigen-binding domains thereof, that are 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 are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to corresponding residues of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residues (such germline changes sequence are collectively referred to herein as germline mutations). Numerous antibodies and antigen-binding fragments comprising one or more germline mutations can be readily produced from the heavy and light chain variable region sequences disclosed herein by a mid-level trade. individual or combinations of these. in certain In L / RQnn / Lznz / E / Yi embodiments, all framework and / or CDR residues within the VHy / or VL domains are mutated back to residues found in the original germline sequence from which the antibody is originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues that lie within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only those mutated residues that lie within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the sequence of the germline). germ line from which the antibody was originally derived). In addition, the antibodies, or the antigen-binding domains thereof, of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while residues other than those in the original germline sequence are maintained or mutated to the corresponding residue of L / RQnn / Lznz / E / Yi a different germ line sequence. Once obtained, antibodies, or antigen-binding fragments thereof, containing one or more germline mutations can be readily assessed for one or more desired properties, such as improved binding specificity, binding affinity increased, improved agonist or antagonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies, or antigen-binding fragments thereof, obtained in this general manner are included within the present invention. The present invention also includes anti-CD28 and / or anti-MUC16 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 that have one or more conservative substitutions. For example, the present invention includes anti-CD28 antibodies and antigen-binding molecules having HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less. , etc. conservative amino acid substitutions with respect to any of the sequences of L / EQnn / Lznz / E / Yi amino acids from HCVR, LCVR and / or CDR set forth in Table 3 hereof. 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 may have more than one epitope. Therefore, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include portions of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. The terms "substantially identical" or "substantially identical", when referring to a nucleic acid or fragment thereof, indicate that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is sequence identity. of nucleotides by at least about 95%, and, more preferably, at least about 96%, 97%, 98%, or 99% L / RQnn / Lznz / E / Yi of nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule that has substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide that has the same or substantially similar amino acid sequence to the polypeptide encoded by the reference nucleic acid molecule. . When applied to polypeptides, the terms "substantially similar" or "substantially similar" mean that two peptide sequences, when optimally aligned, for example, by the GAP or BESTFIT programs with predetermined space weights, share at least 95% sequence identity. , even more preferably, at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which one amino acid residue is substituted with another amino acid residue that has a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not change L / Rann / Lznz / E / Yii substantially the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Persons in the mid-level trade know the means to make this adjustment. See, for example, Pearson (1994) Methods Mol. Biol. 24, 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the probability matrix. L / RQnn / Lznz / E / Yi logarithmic PAM250 disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A moderately conservative replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix. Sequence similarity for polypeptides, also called sequence identity, is typically measured with sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs, such as Gap and Bestfit, that can be used with predetermined parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a protein. wild-type and a mutein thereof. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG Version 6.1. FASTA (for example, FASTA2 and FASTAS) provides alignments and percentage sequence identity of the regions of best overlap between the query sequence and the L / RQnn / Lznz / E / Yii search sequence (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention against a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using predetermined parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. bispecific antigen-binding molecules The antibodies of the present invention can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide or may contain antigen-binding domains 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 and / or anti-MUC16 antibodies of the present invention may be linked to or co-expressed with another functional molecule, eg, another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (eg, by chemical coupling, gene fusion, non-covalent association, or otherwise) to L / RQnn / Lznz / E / Yi one or more molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. The use of the term anti-CD28 antibody and / or anti-MUC16 antibody herein is intended to include monospecific anti-CD28 and / or anti-MUC16 antibodies as well as bispecific antibodies comprising either a CD28-binding arm or a CD28-binding arm. MUC16 and a second arm that binds to a target antigen. Therefore, the present invention includes bispecific antibodies in which one arm of an immunoglobulin binds to human CD28 or MUC16, and the other arm of the immunoglobulin is specific for a target antigen. The target antigen to which the other arm of the bispecific antibody against CD28 or MUC16 binds can be any antigen expressed on or near a cell, tissue, organ, microorganism or virus, against which a directed immune response is desired. The CD28 binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences as set forth in Table 3 herein. The MUGI6 binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences as set forth in Table 1 herein. In certain embodiments, the CD28-binding arm binds to human CD28. L / RQnn / Lznz / B / Yi and induces the proliferation of human T lymphocytes. In the context of the bispecific antibodies of the present invention, where one arm of the antibody binds to CD28 and the other arm binds to a target antigen, the target antigen may be a tumor associated antigen, such as MUGI6. In accordance with certain example embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind to CD28 and MUC16. These molecules may be referred to herein, for example, as anti-CD28 / anti-MUC16, or antiCD28xMUC16, or anti-CD28xMUC16, or anti-MUCI6 / anti-CD28, or anti-MUCI6xCD28, or anti-MUC16xCD28 bispecific molecules, or other similar terminology. In accordance with certain exemplary embodiments as shown in the Figures, bispecific antigen-binding molecules (eg, a bispecific antibody) may have an effector arm and a targeting arm. The effector arm may be the first antigen-binding domain (eg, an anti-CD28 antibody) that binds antigens on effector cells (eg, T cells). The targeting arm may be the second antigen-binding domain (eg, an anti-MUC16 antibody) that binds to antigens on the L / RQnn / Lznz / E / Yi target cells (eg, tumor cells). According to certain example embodiments, the effector arm is set to CD28 and the targeting arm is set to MUC16. The anti-CD28 / MUC16 bispecific molecule can provide a costimulatory signal to effector cells (eg, T cells). The effector arm has no effect in stimulating non-clustering T cells. After pooling, the effector arm alone has little effect in stimulating T cells. In combination with the targeting arm, the effector arm stimulates T cells. The tumor targeting arm may have imperfect tumor specificity. The antigen that is the target of the targeting arm (eg, MUC16) can be expressed on a fraction of tumor cells. The specificity of the tumor targeting arm can be increased by overlapping with the combination with anti-CD3 bispecific antigen-binding molecules (eg, an anti-CD3 / MUCI6 bispecific antibody). As used herein, the term "antigen-binding molecule" means a protein, polypeptide, or molecular complex that comprises or consists of at least one complementarity-determining region (CDR) that alone, or in combination with one or more CDRs and / o framework regions (FR) Additional L / EQnn / Lznz / E / Yi binds specifically to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein. As used herein, the term "bispecific antigen-binding molecule" means a protein, polypeptide, or molecular complex that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises at least one CDR that, alone or in combination with one or more additional CDRs and / or FRs, specifically binds to a particular antigen. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (for example, CD28), and the second antigen-binding domain specifically binds to a different second antigen (for example, MUC16 ). In certain example embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a molecule of L / RQnn / Lznz / E / Yi bispecific antigen-binding comprising a first and a second antigen-binding domain (eg, a bispecific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix DI and the CDRs of the second antigen-binding domain can be designated with the prefix D2. Therefore, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3. The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked together to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerization domain. Association of one multimerization domain with another multimerization domain facilitates association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a multimerization domain is any L / RQnn / Lznz / E / Yi macromolecule, protein, polypeptide, peptide or amino acid that has the ability to associate with a second multimerization domain having the same or similar structure or constitution. For example, a multimerization domain can be a polypeptide comprising an immunoglobulin Ch3 domain. A non-limiting example of a multimerization component is an Fe portion of an immunoglobulin (comprising a Ch2-Ch3 domain), for example, an Fe domain of an IgG selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group. Bispecific antigen-binding molecules of the present invention will generally comprise two multimerization domains, eg, two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerization domains can be of the same IgG isotype, eg, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, eg, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc. In certain embodiments, the multimerization domain is an Fe fragment or an amino acid sequence from 1 to about 200 amino acids in length that contains at least one cysteine ​​residue. in other ways L / EQnn / Lznz / E / Yi embodiment, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that comprise or consist of a leucine zipper, a helix loop motif, or a coiled coil motif. Any bispecific antibody format or technology can be used to make bispecific antigen-binding molecules of the present invention. For example, an antibody or a fragment thereof having a first antigen-binding specificity may be operatively linked (for example, by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities. , such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, for example, scFv or diabody-based bispecific formats, IgG-scFv fusions, Double Variable Domain (OVO)-Ig, Quadroma, Knobs-into-holes, Knobs (eg, Knobs with Knobs, etc.) ., CrossMab, CrossFab, (SEEO)body, Leucine zipper, L / Rann / Lznz / E / Yii Duobody, IgGl / IgG2, Double Effect Fab (OAF)-IgG, and Mab2 bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the aforementioned formats). In the context of bispecific antigen-binding molecules of the present invention, multimerization domains, eg, Fe domains, may comprise one or more amino acid changes (eg, insertions, deletions, or substitutions) compared to the wild-type version. wild-type Fe domain. For example, the invention includes bispecific antigen-binding molecules that comprise one or more modifications to the Fe domain that give rise to a modified Fe domain that has a modified (for example, enhanced or decreased) between Fe and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a Ch2 or Ch3 region, wherein the modification increases the affinity of the Fe domain for FcRn in an acidic environment (for example, in an endosome where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fe modifications include, for example, a modification at position 250 (eg, E or Q); 250 and 428 (eg Lo F); 252 (for example, LN / FIW or L / EQnn / Lznz / E / Yi Τ), 254 (eg S or T) and 256 (eg S / R / Q / EID or T); or a modification at position 428 and / or 433 (eg, UR / S / P / Q or K) and / or 434 (eg, H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (eg, 308F, V308F), and 434. In one embodiment, the modification comprises a modification of 428L (eg, M428L) and 434S (eg, N434S); a modification of 428L, 2591 (eg V2591) and 308F (eg V308F); a modification of 433K (eg H433K) and 434 (eg 434Y); a modification of 252, 254 and 256 (for example, 252Y, 254T and 256E); a modification of 250Q and 428L (for example, T250Q and M428L); and a modification of 307 and / or 308 (eg, 308F or 308P). The present invention also includes bispecific antigen-binding molecules comprising a first Ch3 domain and a second Ig Ch3 domain, wherein the first and second Ig Ch3 domains differ from each other by at least one amino acid, and wherein at least one The amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody that does not have the amino acid difference. In one embodiment, the first Ig Ch3 domain binds to protein A and the second Ig Ch3 domain contains a mutation that reduces or abolishes L / RQnn / Lznz / E / Yi binding to protein A, such as a modification of H95R (according to IMGT exon numbering; H435R according to EU numbering). The second Ch3 may further comprise a modification of Y96F (according to IMGT; Y436F according to EU). Other modifications that can be found within the second Ch3 include: D16E, L 18M, N44S, K52N, V57M and V821 (according to IMGT; D356E, L358M, N384S, K392N, V397M and V4221 according to EU) in the case of IgGl antibodies; N44S, K52N and V821 (according to IMGT; N384S, K392N and V4221 according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V821 (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V4221 according to EU) in the case of IgG4 antibodies. In certain embodiments, the Fe domain may be chimeric, combining the Fe sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a Ch2 sequence derived from a Ch2 region of human IgG1, human IgG2, or human IgG4, and part or all of a Ch3 sequence derived from a human IgG1, human IgG2, or human IgG4. A chimeric Fe domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge sequence, derived from a human IgG1, human IgG2, or human IgG4 hinge region, combined with a lower hinge sequence, derived from a lower hinge region. L / Rann / Lznz / E / Yii hinge of human IgGl, human IgG2 or human IgG4. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, N-terminal to C-terminal: [lgG4 ChI]-[IgG4 upper hinge]-[lower hinge of IgG2] - [Ch2 of lgG4] - [Ch3 of lgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, N-terminal to C-terminal: [IgGl ChI] [IgGl upper hinge] - [lgG2 lower hinge ] - [lgG4 Ch2] - [IgGl Ch3]. These and other examples of chimeric Fe domains that can be included in any of the antigen-binding molecules of the present invention are described in WO2014 / 022540 Al. Chimeric Fe domains having these general structural arrangements, and variants thereof, may have impaired Fe receptor binding, which in turn affects the effector function of Fe. sequence variants The antibodies and bispecific antigen-binding molecules of the present invention may comprise 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 L / RQnn / Lznz / E / Yi corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available in, for example, public antibody sequence databases. The antigen-binding molecules of the present invention may comprise antigen-binding fragments that are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and / or or CDRs are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to corresponding residues of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residues (such sequence changes are collectively referred to herein as germline mutations). Numerous antibodies and antigen-binding fragments comprising one or more germline mutations can be easily produced from the heavy and light chain variable region sequences disclosed herein by a mid-level trade. L / RQnn / Lznz / E / Yi individually or combinations of these. In certain embodiments, all framework and / or CDR residues within the Vh and / or Vl domains are mutated back to residues found in the original germline sequence from which the antigen-binding domain is originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues that lie within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only those mutated residues that lie within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the sequence of the germline). germ line from which the antigen-binding domain was originally derived). In addition, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a sequence of the particular germline sequence, while residues other than those in the original germline sequence are maintained or mutated L / EQnn / Lznz / E / Yi to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily evaluated for one or more desired properties, such as improved binding specificity, increased binding affinity, improved agonist or antagonist biological properties. (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are included within the present invention. The present invention also includes antigen-binding molecules wherein one or both of the antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes antigen-binding molecules comprising an antigen-binding domain having amino acid sequences of HCVR, LCVR, and / or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions with respect to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. A substitution of Conservative amino acid L / RQnn / Lznz / E / Yi is one in which one amino acid residue is replaced with another amino acid residue that has a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially modify the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparaginaglutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix reported in Gonnet et al. (1992) Science 256: 1443-1445. A replacement moderately Conservative L / Rann / Lznz / E / Yii is any change that has a non-negative value in the PAM250 log-likelihood matrix. The present invention also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences. disclosed herein. The term "substantially identical" or "substantially identical" when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the GAP or BESTFIT programs with predetermined gap weights, share at least 95% sequence identity, even more preferably, at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Persons in the mid-level trade know the means to make this adjustment. See, for example, Pearson L / RQnn / Lznz / E / Yi (1994) Methods Mol. Biol. 24, 307-331. Sequence similarity for polypeptides, also called sequence identity, is typically measured with sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs, such as Gap and Bestfit, that can be used with predetermined parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a protein. wild-type and a mutein thereof. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percentage sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences L / RQnn / Lznz / E / Yii from different agencies is the BLAST software, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. pH dependent fixation The present invention includes anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules with pH-dependent binding characteristics. For example, an anti-CD28 antibody of the present invention may exhibit reduced binding to CD28 at acidic pH compared to neutral pH. Alternatively, the anti-MUC16 antibodies of the invention may exhibit enhanced binding to MUGI6 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the term "neutral pH" means a pH of about 7.0 to about 7.4. The term neutral pH includes pH values ​​around 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4. In certain cases, reduced fixation... at acidic pH compared to neutral pH is expressed in terms of a L / RQnn / Lznz / E / Yi ratio of the Kd value of the binding of the antibody to its antigen at acidic pH with respect to the KD value of the binding of the antibody to its antigen at neutral pH (or vice versa). For example, an antibody, or antigen-binding fragment thereof, may be considered to exhibit reduced binding to CD28 at acidic pH compared to neutral pH for the purposes of the present invention if the antibody or antigen-binding fragment thereof it exhibits an acid / neutral Kd ratio of about 3.0 or greater. In certain example embodiments, the acid / neutral Kd for an antibody or antigen-binding fragment of the present invention 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 greater . Antibodies with pH-dependent binding characteristics can be obtained by, for example, screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH, compared to neutral pH. In addition, modifications of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by substituting one or more L / RQnn / Lznz / E / Yi amino acids from an antigen-binding domain (for example, within a CDR) with a histidine residue, an antibody with reduced antigen-binding at acidic pH relative to neutral pH can be obtained . Antibodies comprising Fe variants According to certain embodiments of the present invention, the anti-CD28 / anti-MUC16 bispecific antigen-binding molecules are provided such that they comprise an Fc domain comprising one or more mutations that increase or decrease antibody binding. to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention includes antibodies and antigen-binding molecules comprising a mutation in the Ch2 or Ch3 region of the Fe domain, wherein the mutations increase the affinity of the Fe domain to FcRn in an acidic environment (eg, in an endosome where the pH varies from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fe modifications include, for example, a modification at position 250 (eg, E or Q); 250 and 428 (eg, L or F); 252 (for example, L / Y / F / W or T), 254 (for example, S or T), and 256 (for example, S / R / Q / E / D or L / RQnn / Lznz / E / Yi Τ) ; or a modification at position 428 and / or 433 (eg H / L / R / S / P / Q or K) and / or 434 (eg H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (eg, 308F, V308F), and 434. In one embodiment, the modification comprises a modification of 428L (eg, M428L) and 434S (eg, N434S); a modification of 428L, 2591 (eg V259I) and 308F (eg V308F); a modification of 433K (eg H433K) and 434 (eg 434Y); a modification of 252, 254 and 256 (for example, 252Y, 254T and 256E); a modification of 250Q and 428L (for example, T250Q and M428L); and a modification of 307 and / or 308 (eg, 308F or 308P). For example, the present invention includes anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules comprising an Fe domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L) ; 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); and 433K and 434F (eg, H433K and N434F). All possible combinations of the above Fe domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention. L / RQnn / Lznz / B / Yi Biological Characteristics of Antibodies γ Antigen-Binding Molecules The present invention includes antibodies and antigen-binding fragments thereof that bind to human CD28 and / or MUC16 with high affinity. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD28 and / or MUC16 with medium or low affinity, depending on the therapeutic context and the particular targeting properties desired. For example, in the context of a bispecific antigen-binding molecule, where one arm binds to CD28 and the other arm binds to a target antigen (eg, MUC16), it may be desirable for the antigen-binding arm to target binds to the target antigen with high affinity while the anti-CD28 arm binds to CD28 with moderate or low affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved, while avoiding general / non-target CD28 binding and the adverse effects associated with it. According to certain embodiments, the present invention includes antibodies and antigen-binding fragments of antibodies that bind to human CD28 (for L / RQnn / Lznz / E / Yi eg at 37°C) with a Kd of less than about 165 nM as measured by surface plasmon resonance, eg with an assay format as defined in Example 4 at the moment. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind to CD28 with a Kd of less than about 150 nM, less than about 130 nM, less than about 120 nM, less than about less than about 100 nM, less than about 50 nM, less than about 80 nM, less than about 60 nM, less than about 40 nM, or less than about 30 nM, as measured by surface plasmon resonance, by example, with an assay format as defined in Example 4 herein or a substantially similar assay. The present invention also includes antibodies and antigen-binding fragments thereof that bind to CD28 with a dissociative half-life (tM) greater than about 2.1 minutes as measured by surface plasmon resonance at 37°C, e.g., with a format as defined herein in Example 4 or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind to CD28 with a tU greater than about 5 minutes, greater than about 10 minutes, greater than about L / EQnn / Lznz / E / Yi 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 greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance at 25 °C or 37°C, for example, with an assay format as defined in Example 4 herein or a substantially similar assay. The present invention includes bispecific antigen-binding molecules (eg, bispecific antibodies) that are capable of binding simultaneously to human CD2 8 and human MUGI6. According to certain embodiments, the bispecific antigen-binding molecules of the invention specifically interact with cells that express CD28 and / or MUC16. The extent to which a bispecific antigen-binding molecule binds to cells expressing CD28 and / or MUC16 can be assessed by L / EQnn / Lznz / E / Yi fluorescence activated cell sorting (FACS), as illustrated in Example 5 herein. For example, the present invention includes bispecific antigen-binding molecules that specifically bind to human or Macaca fascicularis cells that express CD28 but not MUC16 (eg, T cells), and human ovarian carcinoma cell lines that express MUC16 but not MUC16. not CD28 (eg, OVCAR-3 or PEO1). The present invention includes bispecific antigen-binding molecules that bind to any of the aforementioned cells and cell lines with an EC50 value of about 9.2x10~6 to about 2.8x10-10M, or less, as determined by a FACS assay as set forth in Example 4 or a substantially similar assay. The present invention also provides anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules that induce or enhance T-lymphocyte-mediated inactivation of tumor cells. For example, the present invention includes anti-CD28xMUCl6 antibodies that induce or enhance T-cell inactivation. T cell-mediated tumor cells with an EC50 of less than about 392 pM, as measured in an in vitro T cell-mediated tumor cell inactivation assay, e.g., with the assay format L / Rann / Lznz / E / Yii as defined in Example 7 herein (for example, to assess the degree of inactivation of PEO1 tumor cells by human or Macaca fascicularis PBMC in the presence of anti-CD28xMUC16 antibodies) or a substantially similar test. In certain embodiments, the antibodies or antigen-binding fragments of the present invention induce T cell-mediated inactivation of tumor cells (eg, PBMC-mediated inactivation of PEO1 cells) with an ECso value of less than about 200 pM, 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, less than about 1.5 pM, or less than about 1.45 pM, as measured by a in vitro T cell-mediated tumor cell inactivation assay, eg, with the assay format as defined in Example 7 herein or a substantially similar assay. The present invention also includes anti-CD28 / anti-MUC16 bispecific antigen-binding molecules that bind to human and / or Macaca fascicularis T cells that L / EQnn / Lznz / E / Yi express CD28 with an ECso value between 1.0 pM and 10 μΜ. In certain embodiments, the anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules bind to human and / or Macaca fascicularis T-lymphocytes expressing CD28 with an EC50 value of between 9.2 μΜ and 120 nM. For example, the present invention includes anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules that bind to CD28-expressing human T cells with an EC50 value of about 1 pM, about 10 pM, about 100 pM , about 500 pM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1000 nM, about 2 μΜ, about 4 μΜ, about 6 μΜ, about 8 μΜ, about 10 μΜ, or more. The present invention also includes anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) activation of T lymphocytes, induction of IL-2 release, and regulation of ascending CD25+ and PD1 in human PBMC (see, for example, Examples 6 and 7 in the L / RQnn / Lznz / E / Yi present); (b) increasing human or Macaca fascicularis T cell-mediated cytotoxicity in cell lines expressing MUC16 (see, for example, Example 7 herein); (c) inducing naïve primate T cell-mediated cytotoxicity in cell lines expressing MUC16 (see, eg, Example 7 herein); (e) decrease tumor cells in mice (eg Example 8 herein); (f) enhance tumor clearance in mice (eg Example 8 herein); (g) not inducing systemic T cell activation in Macaca fascicularis. The present invention includes anti-CD28 / anti-MUC16 bispecific antigen-binding molecules that are capable of depleting tumor cells in a subject (see, eg, Example 9). For example, according to certain embodiments, anti-CD28 / anti-MUCI6 bispecific antigen-binding molecules are provided, wherein dual administrations of the bispecific antigen-binding molecule to a subject (eg, in a dose of about 5.0 mg / kg, about 2.5 mg / kg, about 1.0 mg / kg, about 0.5 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.05 mg / kg, about of 0.02mg / kg, about 0.Olmg / kg or less) causes a reduction in the number of tumor cells in the subject. According to certain forms In embodiment L / RQnn / Lznz / E / Yi, anti-CD28 / anti-MUCl6 bispecific antigen-binding molecules are provided, wherein double administrations of the bispecific antigen-binding molecule to a subject (eg, in one dose about 2500 mg, about 1000 mg, about 500 mg, about 200 mg, about 100 mg, about 50 mg / kg, about 25 mg / kg or less) causes a reduction in the number of cells tumors in the subject. Epitope mapping and related technologies The epitope on CD28 and / or MUC16 to which the antigen-binding molecules of the present invention bind may consist of a single contiguous sequence of 3 or more (for example, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CD28 protein or a MUC16 protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) from CD28 or MUC16. Antibodies of the invention can interact with amino acids contained within a CD28 monomer or can interact with amino acids on two different CD28 chains of a CD28 dimer. The term epitope, as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region. L / RQnn / Lznz / E / Yi of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include portions of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. Various techniques known to those of the mid-level trade can be used to determine whether the 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 a particular antibody epitope or binding domain or antigen binding domain include, for example, a routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Coid Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (eg, alanine scanning mutagenesis, L / Rann / Lznz / E / Yii arginine scanning etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248: 443-463), protease protection and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens may be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the deuterium / hydrogen exchange method involves deuterium-labeling the protein of interest, then fixing the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues, with the exception of antibody-protected residues (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, which reveals deuterium-tagged residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; engen and smith L / RQnn / Lznz / E / Yi (2001) Anal. Chem. 73:256A-265A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antibody interacts. The present invention also includes anti-CD28 and anti-MUC16 antibodies that bind to the same epitope as any of the exemplary specific antibodies described herein (for example, antibodies comprising any of the amino acid sequences as set forth in Tables 1 and 3 hereof). Similarly, the present invention also includes anti-CD28 and / or anti-MUC16 antibodies that compete for binding to CD28 and / or MUC16 with any of the exemplary specific antibodies described herein (eg, antibodies comprising any of the amino acid sequences as set forth in Table 1 hereof). The present invention also includes 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 MUC16, wherein the first antigen-binding domain antigen binding binds to the same epitope on CD28 as any of the antigen binding domains exemplary CD28-specific L / RQnn / Lznz / E / Yi described herein, and / or wherein the second antigen-binding domain binds to the same epitope on MUGI6 as either of the anti-specific antigen-binding domains. Specific exemplary MUC16s described herein. Similarly, the present invention also includes 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 MUC16, wherein the where the first antigen-binding domain competes for binding to CD28 with any of the specific exemplary anti-CD28 antigen-binding domains described herein, and / or wherein the second antigen-binding domain competes for binding to MUC16 with any of the specific exemplary MUC16-specific antigen-binding domains described herein. One can readily determine whether a particular antigen-binding molecule (eg, antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the antibody. present invention by L / EQnn / Lznz / E / Yi routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CD28 (or MUC16) as a reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first allowed to bind to a protein. CD28 (or MUC16 protein). Next, the ability of a test antibody to bind to the CD28 (or MUC16) molecule is assessed. If the test antibody can bind to CD28 (or MUC16) after saturation binding with the reference bispecific antigen binding molecule, it can be concluded that the test antibody binds to an epitope on CD28 (or MUC16) that it is different from the one that binds to the reference bispecific antigen binding molecule. On the other hand, if the test antibody cannot bind to the CD28 (or MUC16) molecule after saturation binding with the reference bispecific antigen-binding molecule, then the test antibody can bind to the same epitope of CD28 ( or MUC16) than the epitope to which the reference bispecific antigen binding molecule of the invention binds. Additional routine experimentation (eg, peptide mutation and binding assays) can then be carried out to confirm whether the observed lack of antibody binding of L / RQnn / Lznz / E / Yii test is indeed due to binding to the same epitope as the reference bispecific antigen-binding molecule or if spherical blocking (or other phenomenon) is responsible for the observed lack of binding. Experiments of this type can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antigen-binding proteins bind to the same epitope (or overlap) if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen-binding protein binding to the antigen inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al. ., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or abolish binding of one antigen-binding protein reduce or abolish binding of the other. Two antigen-binding proteins are considered to have overlapping epitopes if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. L / RQnn / Lznz / E / Yi To determine whether an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the binding methodology described above is carried out in two orientations: in a first orientation, the The reference antigen binding molecule binds to a CD28 protein (or MUC16 protein) under saturation conditions, followed by an assessment of the binding of the test antibody to the CD28 (or MUC16) molecule. In a second orientation, the test antibody is allowed to bind to a CD28 (or MUC16) molecule under saturation conditions, followed by an assessment of the binding of the reference antigen-binding molecule to the CD28 (or MUC16) molecule. ). If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the CD28 (or MUC16) molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for each other. binding to CD28 (or MUC16). As the mid-level trader will appreciate, an antibody that competes for binding with a reference antigen binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block the reference antibody from binding. when looking at a L / Rann / Lznz / E / Yii overlapping or adjacent epitope. Preparation of antigen-binding domains and construction of bispecific molecules Antigen binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigen-binding domains, specific for two different antigens (eg, CD28 and MUC16), can be suitably arranged with one another to produce a bispecific antigen-binding molecule of the present invention by routine methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present invention is provided elsewhere herein). In certain embodiments, one or more of the individual components (eg, heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the present invention can be prepared with L / RQnn / Lznz / E / Yi VELOCIMMUNE™ technology. Using the VELOCIMMUNE™ technology (or any other technology for generating human antibodies), high affinity chimeric antibodies to a particular antigen (eg, CD28 or MUGI6) that have a human variable region and a mouse constant region are initially isolated. Antibodies are characterized and selected based on desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a 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 present invention. Genetically engineered animals can be used to make human bispecific antigen-binding molecules. For example, a genetically engineered mouse that is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence can be used, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically engineered mice can be used to L / RQnn / Lznz / E / Yi produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain comprising a variable domain derived from one of two variable region gene segments. different human light chain. (See, for example, US 2011 / 0195454 for a detailed discussion of such genetically engineered mice and the use of these to produce bispecific antigen-binding molecules). bioequivalent The present invention encompasses antigen-binding molecules that have amino acid sequences that vary from those of the disclosed antibodies, but that retain the ability to bind to CD28 and / or MUC16. Such variant molecules comprise one or more amino acid additions, deletions, or substitutions when compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the disclosed antigen-binding molecules. Similarly, DNA sequences encoding antigen-binding molecules of the present invention encompass sequences that comprise one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequence, but that encode a L / RQnn / Lznz / E / Yi antigen-binding molecule that is essentially bioequivalent to the described antigen-binding molecules of the invention. Examples of such variant amino acid and DNA sequences are discussed above. The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and degree of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either unit doses or multiple doses. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in degree of absorption but not in rate of absorption, and yet can be considered bioequivalent because such differences in rate of absorption are intentional and reflected in the labeling, are not essential for the achievement of effective drug concentrations in the body, for example, in chronic use, and are considered medically significant for the drug product studied in question. L / RQnn / Lznz / E / Yi In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency. In one embodiment, two antigen-binding proteins are bioequivalent if a patient can interchange the reference product with the biologic product one or more times without an expected increase in the risk of adverse effects, including a significant change from baseline. clinically in immunogenicity, or decreased efficacy, compared to continued treatment without such switching. In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism(s) of action for the condition(s) of use, to the extent such mechanisms are known. Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) an in vivo test in humans or other mammals, where the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of in vivo bioavailability data in humans; (cradle L / RQnn / Lznz / E / Yi in vivo testing in humans or other mammals where the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of an antibody. Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed by, for example, making various residue or sequence substitutions or by deleting terminal or internal sequences or residues that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bond formation after renaturation. In other contexts, bioequivalent antibodies may include the exemplary bispecific antigen-binding molecules set forth herein, comprising amino acid changes that modify the glycosylation characteristics of the antibodies, eg, mutations that abolish or remove glycosylation. Species selectivity and cross-species reactivity The present invention, according to certain forms of The L / EQnn / Lznz / E / Yi embodiment provides antigen-binding molecules that bind to human CD28, but not CD28 from other species. The present invention also provides antigen-binding molecules that bind to human MUGI6, but not to MUGI6 from other species. The present invention also includes antigen-binding molecules that bind to human CD28 and CD28 from one or more non-human species; and / or antigen-binding molecules that bind to human MUGI6 and MUGI6 from one or more non-human species. In accordance with certain example embodiments of the invention, antigen-binding molecules are provided which bind to human CD2 8 and / or human MUGI 6 and may or may not bind, as the case may be, to one or more of CD28 and / or MUC16 from mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, Macaca fascicularis, marmoset, Macaca mulatta, or chimpanzee. For example, in a particular exemplary embodiment of the present invention, bispecific antigen-binding molecules are provided comprising a first antigen-binding domain that binds to human CD28 and Macaca fascicularis CD28, and a second antigen-binding domain. binding to the antigen that specifically binds to human MUC16. L / EQnn / Lznz / E / Yi immunoconjugates 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 detrimental to cells. Examples of cytotoxic agents and chemotherapeutic agents suitable for forming immunoconjugates are known in the art (see, for example, WO 05 / 103081). Formulation and therapeutic administration The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and the like. A multitude of suitable formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as L1POFECTIN™, Life Technologies, L / Rann / Lznz / E / Yii Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (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 52:238311. The dose of the antigen-binding molecule to be administered to a patient may vary depending on the age and size of the patient, the target disease, conditions, the route of administration, and the like. The preferred dose is generally calculated according to body weight or body surface area. When a bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in an adult patient, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously, usually in a single dose of about 0.01 to about 20 mg / kg of body weight, more preferably, from about 0.02 to about 7, from about 0.03 to about 5, or from about 0.05 to about 3 mg / kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The doses and the L / EQnn / Lznz / E / Yi Effective schedules for administering a bispecific antigen-binding molecule can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dose can be adjusted accordingly. In addition, interspecies dose escalation can be accomplished by methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351). Various delivery systems are known that can be used to deliver the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the 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, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. A pharmaceutical composition of the present invention is L / RQnn / Lznz / B / Yi 101 can be administered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous administration, a pen delivery device has applications in the administration of a pharmaceutical composition of the present invention. Said pen delivery device may be reusable or disposable. A reusable pen delivery device generally uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition is dispensed into the cartridge and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Then the pen delivery device can 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 contained in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen and autoinjector delivery devices have applications in L / EQnn / Lznz / E / Yi 102 subcutaneous administration of a pharmaceutical composition of the present invention. Examples include but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, RN), DISETRONIC™ Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ Pen, HUMALOG™ Pen, HUMALIN 70 / Pen 30™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ Pen (Becton Dickinson, Franklin Lakes, NJ) , OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices that have applications in the subcutaneous administration of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™. (Eli Lilly), the SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name a few. In certain situations, the pharmaceutical composition can be administered via a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. L / RQnn / Lznz / E / Yi 103 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (ed.), 1974, CRC Fres., Boca Raton, Florida. In yet another embodiment, a controlled-release system can be placed in close proximity to the target of the composition, requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, volume 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533. Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, 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 antibody, or its salt described above, in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there is, for example, a physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a solubilizing agent. L / RQnn / Lznz / E / Yi 104 suitable, such as an alcohol (for example, ethanol), a polyol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene adduct (50 mol) of oil of hydrogenated castor)], etc. As the oily medium, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc., are used. The injection thus prepared is preferably filled into a suitable ampoule. Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a suitable unit dose to suit a dosage of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody is generally from about 5 to about 500 mg per dosage form in a unit dose; especially in the injection form, it is preferred that the aforementioned antibody is contained in about 5 to about 100 mg and about 10 to about 250 mg for the other dosage forms. Therapeutic uses of antigen-binding molecules L / RQnn / Lznz / E / Yi 105 The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds to CD28 and a target antigen (eg, MUC16). The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the term "a subject in need thereof" means a human or non-human animal exhibiting one or more symptoms or indications of cancer (for example, a subject expressing a tumor or having any of the types of cancer discussed hereinafter), or would otherwise benefit from an inhibition or reduction of MUC16 activity or a decrease in MUC16+ cells. The antibodies and bispecific antigen-binding molecules of the invention (and therapeutic compositions comprising them) are useful, inter alia, for the treatment of any disease or disorder where the stimulation, activation and / or targeting of a immune response would be beneficial. In particular, bispecific antigen-binding molecules anti L / RQnn / Lznz / E / Yi 106 CD28 / anti-MUC16 of the present invention can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by the expression of MUC16 or the activity or proliferation of MUC16+ cells. The mechanism of action by which the therapeutic methods of the invention are achieved include inactivation of MUC16-expressing cells in the presence of effector cells, eg, T-lymphocytes. MUC16-expressing cells that can be inhibited or inactivated by the molecules Bispecific antigen-binding agents of the invention include, for example, tumorigenic ovarian cells. The antigen-binding molecules of the present invention can be used to treat, for example, primary and / or metastatic tumors arising in colon, lung, breast, kidney, and bladder cancer subtypes. In accordance with certain exemplary embodiments, the bispecific antigen-binding molecules of the present invention are used to treat ovarian cancer. The present invention also includes methods for the treatment of residual cancer in a subject. As used herein, the term "residual cancer" means the existence or persistence of one or more cancer cells in a subject after treatment with a treatment. L / RQnn / Lznz / E / Yi 107 antineoplastic. According to certain aspects, the present invention provides methods of treating a disease or disorder associated with the expression of MUC16 (for example, cancer expressing MUGI 6 such as ovarian cancer) comprising administering one or more of the binding molecules to the bispecific antigens described elsewhere herein to a subject after it is determined that the subject is unresponsive to other types of antineoplastic treatments. For example, the present invention includes methods of treating ovarian cancer comprising administering a bispecific anti-CD28 / anti-MUC16 antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days , 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received the standard treatment for patients suffering from cancer, e.g. cancer of ovary. In other aspects, a bispecific antigen-binding molecule of the invention (an anti-CD28 / anti-MUCI6 bispecific antigen-binding molecule) comprising an IgG4 Fe domain is initially administered to a subject at one or more time points ( for example, to provide a robust initial depletion of ovarian cancer cells), followed by administration of a molecule L / RQnn / Lznz / E / Yii Equivalent bispecific antigen-binding 108 comprising a different IgG domain, such as an IgGl Fe domain, at later time points. It is envisioned that the anti-CD28 / anti-MUC16 antibodies of the invention may be used in conjunction with other bispecific antigen-binding molecules, such as with an anti-MUCI6 / anti-CD3 bispecific antibody. It is also envisioned that the bispecific antibodies of the invention will be used in conjunction with checkpoint inhibitors, eg, those that target PD-1 and CTLA-4, and other targets. It may be advantageous to combine two bispecific antibodies that target the same tumor antigen (eg, MUC16), but where one bispecific targets CD3 on T lymphocytes and the other bispecific targets a costimulatory molecule such as CD28. This combination can be used alone to increase tumor cell inactivation or can be used in combination with a checkpoint inhibitor. Exemplary MUC16-expressing cancers include, but are not limited to, ovarian cancer, breast cancer, endometrial cancer, pancreatic cancer, non-small cell lung cancer, mass-forming type intrahepatic cholangiocarcinoma, adenocarcinoma of the cervix and adenocarcinoma of the digestive tract. L / Rann / Lznz / E / Yii 109 Joint treatments and formulations The present invention includes therapeutic compositions and formulations comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutically active components, and methods of treatment comprising administering such combinations to subjects who need them. Additional exemplary therapeutic agents that may be combined or administered in combination with an antigen-binding molecule of the present invention include, for example, chemotherapy, radiation therapy, checkpoint inhibitors that target PD-1 (for example, a antiPD-1 antibody such as pembrolizumab, nivolumab, or cemiplimab (see US9,987,500), CTLA-4, LAG3, TIM3, and others, costimulatory agonist bivalent antibodies that target molecules such as GITR, 0X40, 4-1BB, and others ), bispecific antibodies against CD3x (see, for example, WO2017 / 053856A1, WO2014 / 047231A1, WO2018 / 067331A1, and WO2018 / 058001A1), other antibodies that target MUC16 X CD3 (see, for example, WO2017 / 053856A1), and others bispecific antibodies against costimulatory CD28. Other agents that may be beneficially administered L / RQnn / Lznz / E / Yi 110 in combination with 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, IL13, IL-17, IL-18 or their respective receptors. The pharmaceutical compositions of the present invention (for example, pharmaceutical compositions comprising an anti-CD28 / antiMUC16 bispecific antigen-binding molecule as disclosed herein) may also be administered as part of a therapeutic regimen comprising one or more combinations. selected ICE therapeutics: ifosfamide (eg, Ifex®), carboplatin (eg, Paraplatin®), etoposide (eg, Etopophos®, Toposar®, VePesid®, VP-16); DHAP: dexamethasone (eg Decadron®), cytarabine (eg Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (eg Platinol®-AQ); and ESHAP: etoposide (eg, Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (eg, Medrol®), high-dose cytarabine, cisplatin (eg, Platinol®AQ). The present invention also includes therapeutic combinations comprising any of the molecules of L / RQnn / Lznz / E / Yi 111 binding to the antigen mentioned herein and an inhibitor of 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 of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, a siRNA, a peptibody, a nanobody, or a fragment of antibody (eg, Fab fragment; F(ab')2 fragment,' Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units ) . The antigen-binding molecules of the invention may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation treatment and / or conventional chemotherapy, or treatment with a biological agent, including checkpoint inhibitors or other bispecific antibodies. . The present invention includes therapeutic compositions and formulations comprising any of the L / RQnn / Lznz / E / Yi 112 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 (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards, such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucin, doxorubicin, epirubicin, esorubicin, idarubicin , marcelomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, L / RQnn / Lznz / E / Yi 113 tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcine; Diaziquone; eflornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicuone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; L / EQnn / Lznz / E / Yi 114 thiotepa; taxanes, for example, paclitaxel (Taxol™, BristolMyers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbina; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); Retinoic acid; esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are antihormonal agents that act to regulate or inhibit hormonal action in tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazole aromatase inhibitors, 4-hydroxytamoxifen, trioxifen, keoxifene, LY 117018, onapristone and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Additional therapeutically active components may be administered immediately before, consecutively, or L / RQnn / Lznz / E / Yi 115 shortly after administration of an antigen-binding molecule of the present invention; (For the purposes of the present disclosure, such administration regimens are considered the administration of the antigen-binding molecule in combination with an additional therapeutically active component). The present invention includes pharmaceutical compositions wherein an antigen-binding molecule of the present invention is formulated together with one or more additional therapeutically active components, as described elsewhere herein. Administration regimes In accordance with certain embodiments of the present invention, multiple doses of an antigen-binding molecule (eg, an anti-CD28 antibody or a bispecific antigen-binding molecule that specifically binds to MUC16 and CD28) may be administered. to a subject for a defined period of time. Methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an antigen-binding molecule of the invention. As used herein, sequential administration means that each dose of an antigen-binding molecule is administered to the subject. L / Rann / Lznz / E / Yii 116 at a different time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks, or months). The present invention includes methods comprising sequentially administering to the patient a single initial dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and, optionally, followed by one or more doses. tertiaries of the antigen-binding molecule. The terms initial dose, secondary dose and tertiary dose refer to the temporal sequence of administration of the antigen-binding molecule of the invention. Therefore, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the reference dose); secondary doses are the doses given after the initial dose; and the tertiary doses are the doses that are administered after the secondary doses. The initial, secondary and tertiary doses may contain the same amount of the antigen-binding molecule, but in general they may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of an antigen-binding molecule contained in the initial, secondary, and / or tertiary dose varies between them (eg, adjusts toward L / EQnn / Lznz / E / Yi 117 up or down as appropriate) during the course of treatment. In certain embodiments, two or more doses (eg, 2, 3, 4, or 5) are administered at the beginning of the treatment regimen as a loading dose, followed by later doses that are administered less frequently (eg, maintenance dose). In an exemplary embodiment of the present invention, each secondary and / or tertiary dose of 1 to 26 (eg, 1, Ib, 2, 2b, 3, 3b, 4, 4b, 5, 5b, 6 , 6b, 7, 7b, 8, 8b, 9, 9b, 10, 10b, 11, 11b, 12, 12b, 13, 13b, 14, 14b, 15, 15b, 16, 16b, 17, 17b, 18, 18b , 19, 19b, 20, 20b, 21, 21b, 22, 22b, 23, 23b, 24, 24b, 25, 25b, 26, 26b, or more) weeks after the immediately preceding dose. The phrase immediately preceding dose, as used herein, means, in a sequence of multiple administrations, the dose of antigen-binding molecule that is administered to a patient prior to administration of the next dose in the sequence without doses. intermediate. Methods according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of an antigen-binding molecule (for example, an anti-CD28 antibody or a bispecific antigen-binding molecule that notices L / EQnn / Lznz / E / Yi 118 specifically to MUC16 and CD28). For example, in certain embodiments, only a secondary dose is administered to the patient. In other embodiments, the patient is administered two or more secondary doses (eg, 2, 3, 4, 5, 6, 7, 8 or more). Similarly, in certain embodiments, only a tertiary dose is administered to the patient. In other embodiments, two or more tertiary doses (eg, 2, 3, 4, 5, 6, 7, 8, or more) are administered to the patient. In embodiments that include multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose can be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to a patient may vary during the course of the treatment regimen. The frequency of administration can also be adjusted during the L / EQnn / Lznz / E / Yi 119 course of treatment by a physician, based on the needs of the individual patient after a clinical examination. In one embodiment, the antigen-binding molecule (eg, a bispecific antigen-binding molecule that specifically binds to MUC16 and CD28) is administered to a subject as a weight-based dose. A weight-based dose (eg, a dose in mg / kg) is a dose of the antibody or antigen-binding fragment thereof or bispecific antigen-binding molecule that will change according to the subject's weight. In another embodiment, an antibody or the antigen-binding fragment thereof or a bispecific antigen-binding molecule is administered to a subject as a fixed dose. A fixed dose (for example, a dose in mg) means that a dose of the antibody or its antigen-binding fragment or bispecific antigen-binding molecule is used for all subjects regardless of any specific factors related to the subject. , such as weight. In a particular embodiment, a fixed dose of an antibody or the antigen-binding fragment thereof or a bispecific antigen-binding molecule of the invention is based on a predetermined weight or age. L / RQnn / Lznz / B / Yi 120 In general, a suitable dose of the antigen-binding molecule of the invention may be in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient, generally in the range of about 1 to 50 mg per kilogram of body weight. For example, the antibody or the antigen-binding fragment thereof or the bispecific antigen-binding molecule can be administered at about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg per single dose. Intermediate values ​​and ranges to the enumerated values ​​are also intended to be a part of this invention. In some embodiments, the antigen-binding molecule of the invention is administered as a fixed dose of between about 25 mg and about 2500 mg. In some embodiments, the antigen-binding molecule of the invention is administered as 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 L / Rann / Lznz / E / Yii 121 about 250mg, about 275mg, about 300mg, about 325mg, about 350mg, about 375mg, about 400mg, about 425mg, about 450mg, about 475mg, about from 500mg, around 525mg. about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg. about 925 mg, about 950 mg, about 975 mg, about L / Rann / Lznz / E / Yii of 1000mg, about 1500mg, about 2000mg, or about 2500mg. Intermediate values ​​and ranges to the enumerated values ​​are also intended to be a part of this invention. Diagnostic uses of antibodies The bispecific antibodies of the present invention can also be used to detect and / or measure CD28 or MUC16, or CD28-expressing or MUC16-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-CD28 x MUC16 antibody, or fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression (eg, overexpression, underexpression, non-expression, etc.) of CD28 or 122 MUC16. Exemplary diagnostic assays for CD28 or MUC16 may comprise, for example, contacting a sample, obtained from a patient, with an antibody of the invention, wherein the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled antibody can be used in diagnostic applications in combination with a secondary antibody that is detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Exemplary specific assays that can be used to detect or measure CD28 or MUC16 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). Samples that can be used in diagnostic assays with CD28 or MUC16 in accordance with the present invention include any tissue or fluid sample that can be obtained from a patient that contains detectable amounts of CD28 or MUC16 protein, or fragments thereof, in normal conditions or pathological conditions. In general, CD28 or MUC16 levels will be measured on a L / RQnn / Lznz / E / Yi 123 particular sample obtained from a healthy patient (for example, a patient who does not have a disease or condition associated with abnormal CD28 or MUC16 levels or activity) to initially establish a baseline, or standard, CD28 or MUC16 level. This baseline CD28 or MUC16 value can be compared to CD28 or MUC16 levels measured in samples obtained from individuals believed to have a CD28 or MUC16-related disease or condition. EXAMPLES The following examples are set forth to provide the mid-level trade person with a complete disclosure and description of how the methods and compositions of the invention are made and used, and are not intended to limit the scope of what the inventors consider to be their invention. An attempt has been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. L / RQnn / Lznz / E / Yi 124 BACKGROUND OF THE INVENTION T cell activation is initiated upon binding of the T cell receptor (TCR) / CD3 complex to peptide-MHC complexes (signal 1); activation is then increased by the coupling of a second costimulatory receptor, such as the CD28 receptor on T cells that binds its cognate ligands on the target cell (signal 2). The recently described CD3-based bispecific antibodies work by replacing the conventional signal 1, binding T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific antibody, and binding the TCR / CD3 with the CD3-based bispecific antibody. other. Although some of these so-called TSAxCD3 bispecifics have shown promising antitumor efficacy in cancer patients, their activity has not yet been optimized. As described elsewhere herein, a novel class of bispecific antibodies that mimic signal 2, by binding a second TSA to the costimulatory CD28 receptor on T cells, is introduced herein. TSAxCD28. As described herein, an exemplary antibody of the present invention is specific for ovarian cancer antigens (eg, MUC16). Unlike the L / RQnn / Lznz / E / Yi 125 CD28 superagonists, extensively activating T cells and resulting in profound toxicity in early clinical trials, these bispecifics against TSAxCD28 show limited activity and no toxicity when used alone in primate or genetically humanized immunocompetent mouse models . However, when combined with TSAxCD3 bispecifics, the exemplary antibody of the invention enhanced the artificial synapse between a T cell and its target cell, enhanced T cell activation, and markedly enhanced the antitumor activity of the CD3 bispecifics. in a variety of xenogeneic and syngeneic tumor models. Combining this novel class of costimulatory CD28 bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide available and well-tolerated antibody treatments with potentially enhanced antitumor efficacy. The ability of T lymphocytes to recognize and inactivate their cellular targets, such as virus-infected cells or tumor cells, depends on a coordinated set of interactions. The most important of these is the recognition and fixation of the target cell by the TCR complex (which includes the CD3 chains and, L / RQnn / Lznz / E / Yii 126 δ, ε, ζ associated); this interaction has been termed signal 1 for T cell activation. The TCR can recognize viral or tumor peptides presented in the groove of an MHC protein expressed on the surface of target cells. Typically, this binding is of low affinity; therefore, for successful activation of signal 1, it is necessary to cluster many TCR complexes along the interface between a T lymphocyte and its target cell, and this interface has been termed the immune synapse (J. B. Huppa, Μ. M. Davis, Tcell-antigen recognition and the immunological synapse, Nat Rev Immunol 3, 973-983 (2003). T cell activation and proliferation are further promoted by additional interactions with costimulatory receptors such as CD28 (signal 2) (J. H. Esensten, Y. A. Helou, G. Chopra, A. Weiss, J. A. Bluestone, CD28 Costimulation: From Mechanism to Therapy Immunity 44, 973-988 (2016)). When a T lymphocyte recognizes a target cell through the TCR complex, and activates signal 2 through binding of CD28 to its cognate ligands (CD80 / B7.1 and / or CD86 / B7.2) on a cell presenting professional antigen or the target cell, T cell activation is enhanced. As with signal 1, CD28-mediated signal 2 is believed to be produced by co-clustering at the immune synapse. L / Rann / Lznz / E / Yii 127 Conventional monoclonal antibodies directed against tumor-specific antigens (TSAs) have been used as antitumor therapeutic agents for the past two decades (G. Salles et al., Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience. Adv Ther 34, 2232-2273 (2017); Μ. V. Mateos et al., Daratumumab plus Bortezomib, Melphalan, and Prednisone for Untreated Myeloma. N Engl J Med 378, 518-528 (2018) : W. Eiermann, G International Herceptin Study, Trastuzumab combined with chemotherapy for the treatment of HER2-positive metastatic breast cancer: pivotal trial data Ann Oncol 12 Suppl 1, S57-62 (2001), J. M. Connors et al., Brentuximab Vedotin with Chemotherapy for Stage III or IV Hodgkin's Lymphoma N Engl J Med 378, 331-344 (2018) V. Dieras et al., Trastuzumab emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive advanced breast cancer (EMILIA) : a descriptive analysis of final overall survival results from a randomized, open-label, phase 3 trial. Lancet Oncol 18, 732-742 (2017)). However, this class of antibodies had limited ability to induce T cell-mediated cytotoxicity and instead acted by promoting antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), L / RQnn / Lznz / E / Yi 128 or by delivering a toxin to tumor cells. Recently, a new class of bispecific antibodies (against TSAxCD3) has emerged that can effectively trigger T-lymphocyte-mediated inactivation of tumor cells by binding a lymphocyte to a tumor cell and activating the CD3 / TCR complex (usually through the CD3 chain). e of CD3) through a surrogate mechanism, thus mimicking signal 1. An early version of such a bispecific (one arm binds to CD19 on leukemia cells, while the other binds to CD3) recently received regulatory approval for B-cell acute lymphoblastic leukemia (R. Bargou et al., Tumor regression in cancer patients by very low doses of a T cell engaging antibody. Science 321, 974-977 (2008); H. Kantarjian et al., Blinatumomab versus Chemotherapy for Advanced Acute Lymphoblastic Leukemia. N Engl J Med. 376, 836847 (2017)). More advanced versions of the bispecifics have recently been shown to have good activity against non-Hodgkin lymphomas, targeting CD20 in these lymphomas (E.J. Smith et al., A novel, native-format bispecific antibody triggering T-cell killing of B cells is robustly active in mouse tumor models and cynomolgus monkeys. Sel Rep 5, 17943 (2015); L. L. Sun et al., Anti-CD20 / CD3 T celldependent bispecific antibody for the treatment of B cell L / Rann / Lznz / E / Yii 129 malignancies. Scí Transí Med Ί, 287ra270 (2015); M. Bacac et al., CD20-TCB with Obinutuzumab Pretreatment as Next Generation Treatment of Hematologic Malignancies. Clin Cancer Res 24, 4785-4797 (2018); R. Bannerji et al., Emerging Clinical Activity of REGN1979, an Anti-CD20 x Anti-CD3 Bispecific Antibody, in Patients with Relapsed / Refractory Follicular Lymphoma (FL), Diffuse Large B-Cell Lymphoma (DLBCL) , and Other B- Cell Non-Hodgkin Lymphoma (B-NHL) Subtypes. American Society of Hematology, (2018); L. Budde et al., Mosunetuzumab, a FullLength Bispecific CD20 / CD3 Antibody, Displays Clinical Activity in Relapsed / Refractory B-Cell Non-Hodgkin Lymphoma (NHL) : Interim Safety and Efficacy Results from a Phase 1 Study. American Society of Hematology, (2018)). However, although TSAxCD3 bispecifics are emerging as an important new class of immunotherapy in haematological malignancies, cross-study comparisons (E. A. Zhukovsky, R. J. Morse, Μ. V. Maus, Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection Curr Opin Immunol 40, 24-35 (2016)) suggest that in some cases they may not be achieving the level of efficacy seen with personalized chimeric antigen receptor T (CAR-T) cell therapies. One of the reasons for the great effectiveness of the treatments L / RQnn / Lznz / E / Yi 130 with CAR-T is that the chimeric antigen receptor (CAR) is engineered to provide both signal 1 (through a portion of the CD3z cytodomain) and signal 2 (for example, through a portion of the CD3z cytodomain). CD28 cytodomain) after binding to its target in a tumor cell. Two CAR-T cell therapies have recently received FDA approval for B cell malignancies, which work by binding to and targeting the CD19 antigen (S. S. Neelapu et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B Cell Lymphoma. N Engl J Med 377, 2531-2544 (2017); S. J. Schuster et al., Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas. N Engl J Med 377, 25452554 (2017)). CAR-T lymphocyte approaches may be associated with serious adverse effects such as cytokine release syndrome (CRS) and neurotoxicity (S. S. Neelapu et al., Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol 15, 47-62 (2018); J. Gust et al., Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells. Cancer Discov 7, 1404-1419 (2017); A. Shimabukuro-Vornhagen et al., Cytokine release syndrome J Immunother Cancer 6, 56 (2018)); and due to highly customized manufacturing processes and the requirement for L / RQnn / Lznz / E / Yi 131 preconditioning chemotherapy regimens (S.S. Neelapu et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B Cell Lymphoma. N Engl J Med 377, 2531-2544 (2017); S. J. Schuster et al., Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas N Engl J Med 377, 25452554 (2017) P. Salmikangas, N. Kinsella, P. Chamberlain, Chimeric Antigen Receptor T-Cells (CART-Cells) for Cancer Immunotherapy - Moving Target for Industry? Pharm Res 35, 152 (2018)), many patients are not considered suitable candidates. The advantages of TSAxCD3 bispecifics as relatively well-tolerated therapeutic solutions available to broader patient populations would be enhanced if their antitumor activity could be further optimized, especially if this could be done without sacrificing tolerability, or perhaps even increasing, specificity. by tumor cells compared to normal cells. To this end, it was hypothesized that the combination of bispecifics against TSAxCD3 with a novel class of bispecifics that independently activate signal 2 might provide potential increased efficacy, as well as an opportunity for increased specificity. Therefore, a second class of bispecifics was designed. These L / RQnn / Lznz / B / Yi 132 bispecifics could couple a second epitope on the same tumor-specific antigen, or a separate second tumor antigen, with the costimulatory receptor CD28 (bispecific against TSAxCD28) expressed on T cells. It was reasoned that the combination of TSAlxCD3 with a TSA2xCD28 should allow activation targeted and improved surrogate for T cells by activating both signal 1 and signal 2, with specificity directed only against tumor cells expressing both epitopes or both antigens, allowing for increased antitumor activity along with an opportunity for increased specificity. Described herein is the generation and evaluation of costimulatory bispecific antibodies against TSAxCD28 directed against ovarian cancer (MUC16xCD28, which binds to MUC16, a large integral membrane glycoprotein expressed at high levels in certain types of cancer (H. Suh, K Pillai, D. L. Morris, Mucins in pancreatic cancer: biological role, implications in carcinogenesis and applications in diagnosis and therapy.Am J Cancer Res 7, 1372-1383 (2017), and which is cleaved to release the ovarian tumor biomarker CA-125 (I. Mylonas et al., Immunohistochemical expression of the tumor marker CA-125 in normal, hyperplastic and malignant L / EQnn / Lznz / E / Yi endometrial tissue. Antlcancer Res 23, 1075-1080 (2003)). The 133 Toxicology studies in genetically humanized immunocompetent mice, as well as in Macaca fascicularis, show that these bispecifics exhibit limited activity and do not exhibit toxicity as individual agents. However, these novel costimulatory bispecifics can be effectively combined with the emerging class of anti-TSAxCD3 bispecifics to enhance antitumor responses in xenogeneic and syngeneic tumor models. Taken together, these data suggest that the combination of this novel class of CD28-based bispecifics (against TSAxCD28) with CD3-based bispecifics (against TSAxCD3) may provide available and well-tolerated biologic solutions with markedly enhanced and synergistic antitumor activity. Example 1. Construction of anti-MUC16xCD28 antibodies Generation of anti-CD28 antibodies Anti-CD28 antibodies were obtained by immunizing a VELOCIMMUNE® mouse (i.e., a genetically engineered mouse comprising DNA encoding the human immunoglobulin kappa light and heavy chain variable regions) with human CD28 protein fused with the Fe portion of mouse IgG2a, or with cells expressing CD28 or with DNA encoding CD28. The answer L / RQnn / Lznz / E / Yi 134 immune response of the antibody was monitored by a specific immunoassay against CD28. When a desired immune response was achieved, the splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce specific antibodies against CD28. Several chimeric anti-CD28 antibodies (ie, antibodies possessing human variable domains and mouse constant domains) were obtained by this technique. In addition, several fully human anti-CD28 antibodies were isolated directly from antigen-positive B lymphocytes without fusion with myeloma cells, as described in US 2007 / 0280945A1. Certain biological properties of the exemplary anti-CD28 antibodies generated according to the methods of this Example are described in detail in the Examples set forth below. Generation of anti-MUC16 antibodies Anti-MUC16 antibodies were obtained by immunizing a genetically modified mouse with a human MUGI6 antigen or by immunizing a genetically modified mouse comprising DNA L / EQnn / Lznz / E / Yi 135 encoding the variable regions of the human immunoglobulin kappa heavy and light chain with a human MUGI6 antigen. The engineered mice were immunized with hMUC16.nub (a truncated format comprising the last five SEA domains of mucin-16 (SEQ ID: 49)), or were immunized with a cell line expressing hMUC16, such as cells OVCAR-3. After immunization, splenocytes were harvested from each mouse and (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for specificity for MUC16, or (2) lymphocytes sorted. B cells (as described in US 2007 / 0280945A1) by using a human MUGI6 fragment as a sorting reagent that binds to and identifies reactive antibodies (antigen-positive B cells). Chimeric antibodies to MUC16 having a human variable region and a mouse constant region were initially isolated. Antibodies were characterized and selected based on desirable characteristics, including affinity, selectivity, etc. If necessary, the mouse constant regions were replaced with a desired human constant region, for example, the constant region of L / EQnn / Lznz / E / Yi 136 Wild-type or modified IgG1 or IgG4, to generate a fully human anti-MUC16 antibody. While the selected constant region may vary according to specific use, high affinity antigen-binding characteristics and target specificity reside in the variable region. Certain biological properties of the exemplary anti-MUC16 antibodies generated according to the methods of this Example are described in detail in the Examples set forth below. Generation of bispecific antibodies that bind to CD28 and MUGI 6 Bispecific antibodies comprising an anti-MUC16 specific binding domain and an anti-CD28 specific binding domain were constructed via standard methodologies, where the anti-MUC16 antigen binding domain and the anti-MUC16 antigen binding domain antiCD28 each comprise different HCVRs paired with a common LCVR. In some cases, bispecific antibodies were constructed with an anti-CD28 antibody heavy chain, an anti-MUC16 antibody heavy chain, and a common light chain (see Table 5). The bispecific antibodies created in accordance with the present Example comprise two domains binding to the L / Rann / Lznz / E / Yii 137 separate antigens (ie, binding arms). The first antigen-binding domain comprises a heavy chain variable region derived from an anti-CD28 antibody (CD28VH), and the second antigen-binding domain comprises a heavy chain variable region derived from an anti-MUC16 antibody ( MUGI6-VH). Both anti-MUC16 and anti-CD28 share a common light chain. The CD28-VH / MUC16-VH pairing creates antigen-binding domains that specifically recognize CD28 on T cells and MUC16 on tumor cells. Example 2. Amino Acid and Nucleic Acid Sequences of the Variable Region of the Heavy γ Light Chain Table 1 lists the amino acid sequence identifiers of the CDRs and variable regions of the heavy and light chain of selected anti-MUC16 antibodies of the invention. The corresponding sequence identifiers L / RQnn / Lznz / E / Yi of nucleic acids are listed in Table 2. L / Rann / Lznz / E / Yi Table 1: Amino acid sequence identifiers of antibodies against SEQ ID NO: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb8799P2 2 4 6 8 10 12 14 16 mAb8794P2 26 28 30 32 34 36 38 40 Table 2: Nucleic acid sequence identifiers of antibodies against MUC16 SEQ ID NO: Antibody Designation HCVR HCDRl HCDR2 HCDR3 LCVR LCDRl LCDR2 LCDR3 mAb8799P2 1 3 5 7 9 11 13 15 mAb8794P2 25 27 29 31 33 35 37 39 Table 3 lists the amino acid sequence identifiers of the CDRs and heavy and light chain variable regions (HCVR and LCVR) of selected anti-CD28 antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 4. Table 3: Amino acid sequence identifiers of antibodies against CD28 SEQ ID NO: Designation of the antibody HCVR HCDRl HCDR2 HCDR3 LCVR LCDRl LCDR2 LCDR3 mAbl4226P2 18 20 22 24 10 12 14 16 mAbl4216P2 42 44 46 48 34 3 6 38 40 Table 4: Nucleic Acid Sequence Identifiers of Antibodies against CD28 SEQ ID NO: Antibody Designation HCVR HCDRl HCDR2 HCDR3 LCVR LCDRl LCDR2 LCDR3 mAbl4226P2 17 19 21 23 9 11 13 15 mAbl4216P2 41 43 45 47 33 35 37 39 In Table 5, a summary of the component parts of the various anti-MUC16xCD3 bispecific antibodies constructed is set forth. Tables 6 and 7 list the sequence identifiers for HCVR, LCVR, CDR, and the heavy and light chain of the bispecific antibodies. Table 5: Summary of the component parts of the anti-MUC16 x anti-CD28 bispecific antibodies Bispecific antibody identifier Anti-MUC16 Anti-CD28 Common light chain variable region Antigen-binding domain Antigen-binding domain Heavy chain variable region Heavy chain variable region bs24963D mAb8799P2 mAbl4226P2 ULC3-20 bs32897D mAb8794P2 mAbl4216P2 ULC1- 39 139 Table 6 shows the amino acid sequence identifiers for the bispecific anti-MUC16 x anti-CD28 antibodies exemplified herein. The corresponding nucleic acid sequence identifiers are listed in Table 7. L / RQnn / Lznz / E / Yi L / Rann / Lznz / E / Yi Table 6: Amino acid sequences of the antibodies Bispecific antibody identifier Anti-CD28 First antigen-binding domain (DI) Anti-MUC16 Second antigen-binding domain (D2) Common light chain variable region HCV R HCD R1 HCD R2 HCD R3 HCV R HCD R1 HCD R2 HC DR 3 L C V R LCDR 1 LC DR 2 LCD R3 bs24963 D 18 20 22 24 2 4 6 8 1 0 12 14 16 bs32897 D 42 44 46 48 26 28 30 32 3 4 36 38 40 Table 7: Nucleic acid sequences of anti-MUC16 x anti-CD28 bispecific antibodies Bispecific antibody identifier Anti-CD28 First antigen-binding domain (DI) Anti-MUC16 Second antigen-binding domain (D2) Common light chain variable region HC VR HCD R1 HCD R2 HCD R3 HC VR HCD R1 HCD R2 HCD R3 L C V R LC DR 1 LCDR 2 LCDR 3 bs2496 3D 17 19 21 23 1 3 5 7 9 11 13 15 bs3289 7D 41 43 45 47 25 27 29 31 3 3 35 37 39 Example 3. CD28 is a potent costimulatory receptor To determine costimulatory receptors that are effective in providing the costimulatory signal important for T cell activation, a blinded screen for costimulatory pathways was performed by forced expression of costimulatory ligands in a panel of syngeneic tumors. (Table 8 and Figure 1) re-established CD28 as one of the most potent costimulatory receptors along with 4-1BB. Table 8 summarizes the number of tumor-free mice in the blinded screen. Assays were performed in three different tumor cell lines that were engineered to express seven different costimulatory ligands. The number in each cell represents the number of tumor-free mice out of a total of 5 mice. Table 8: Inhibition of tumor growth in genetically engineered cell lines with introduction of costimulatory ligand expression Costimulatory Ligand Costimulatory Receptor Lymphoma (EL4) Carcinoma (MC38) Melanoma (B16F10.9) 4-1BBL 4-BB 3 4 1 CD80 (B7.1) CD28 2 2 2 CD86 (B7.2) CD28 1 0 2 CD70 CD27 5 0 OX40L 0X4 0 0 0 2 CD40 CD40L 0 1 0 ICOSL ICOS 0 0 0 Empty vector 0 0 0 Non-transfected parental cells 0 0 141 Example 4. Binding affinities and kinetic constants of anti-MUC16xCD28 bispecific antibodies derived from surface plasmon resonance In order to determine the binding kinetics of the exemplary anti-MUCI6xCD28 bispecific monoclonal antibodies of the invention, the binding affinities and kinetic constants derived from surface plasmon resonance of the antiMUCI 6xCD28 bispecific antibodies were determined for MUC16 and / or CD28. Equilibrium dissociation constants (Ko values) were determined for the binding of hMUC16.mmh (SEQ ID NO: 51), hCD28.mmh (SEQ ID NO: 53) and mCD28.mmh (murine CD28.mmh; SEQ ID NO: 54) to the exemplary purified anti-MUCI6xCD28 bispecific monoclonal antibody of the invention using a real-time surface plasmon resonance biosensor with a Biacore T-200 instrument. The Biacore CM5 sensor surface was derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody to capture exemplary purified anti-MUCI6xCD28 bispecific antibodies of the invention. Two exemplary bispecific antibodies, bs24963D and REGN4615, were tested. REGN4615 is an antibody / scFv that recognizes human MUC16 and murine CD28 and is sometimes referred to as anti-MUCI6xmsCD28 antibody. the arm of L / RQnn / Lznz / E / Yi 142 MUC16 in REGN4615 uses the VH and VK-ULC 1-39 sequences as shown above in Table 1 for mAb8794P2. mCD28 (PV-1) is described in US2004 / 0116675, with a light chain of SEQ ID NO: 11 (see also Figure 15A in US2004 / 0116675) and a heavy chain of SEQ ID NO: 13 (see Figure 15 ), which was reformatted as a scFv for the experiments described herein. This SPR fixation study was performed in a buffer composed of 0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% v / v P20 surfactant at pH 7.4 (HBS-ET running buffer). Different concentrations of hMUC16 with C-terminal myc-myc-6xHis tag (hMUCI6.mmh), hCD28 with C-terminal myc-myc-6xHis tag (hCD28.mmh) and mCD28 with C-terminal myc-myc-6xHis tag were prepared. (mCD28.mmh) in HBS-ET running buffer, ranging from 3.33 nM to 90 nM (for hMucl6) or 22.2 nM to 600 nM (for hCD2 8 or mCD2 8) as 3-fold serial dilutions, for determination of the affinity on anti-MUCl6xCD28 bispecific antibodies and anti-MUCl6xmCD28 bispecific antibodies. The surface of the MASS2 high capacity amine sensor was first derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody to capture approximately 500-900 RU of monoclonal antibodies. L / RQnn / Lznz / E / Yii bispecific anti-MUCl6xCD28 or anti-MUCl6xmCD28. 1 RU (response unit) represents 1 pg of protein per mm2, as defined by the manufacturer. Different concentrations of hMUC16 with C-terminal myc-myc-6xHis tag (hMUCl6.mmh), hCD28 with C-terminal myc-myc-6xHis tag (hCD28.mmh) and mCD28 with C-terminal myc-myc-6xHis tag were prepared. (mCD28.mmh) in HBS-ET running buffer, ranging from 3.33 nM to 90 nM (for hMucl6) or 22.2 nM to 600 nM (for hCD28 or mCD28) as 3-fold serial dilutions and injected onto the surfaces of anti-MUCI6xCD28 or anti-MUCI6xmCD28 bispecific monoclonal antibodies captured with anti-human Fc for 5 minutes at a flow rate of 50 pL / minute. Dissociation of fixed hMUC16, hCD28 and mCD28 reagents was monitored for 10 min in HBS-ET running buffer. Association (ka) and dissociation (kd) rate constants were determined by fitting real-time binding sensorgrams to a 1:1 binding model with limited mass transport using Scrubber version 2.0c evaluation software. . Equilibrium dissociation constants for binding (Kd) and dissociative half-lives (th) were calculated from kinetic rate constants as follows: L / Rann / Lznz / E / Yii kd ln(2) KD(M)=-, and rA(min>= — 144 l i «ann / ίζηζ / Ε / γΐΛ The binding kinetic parameters of exemplary bispecific antibodies that bind to purified hMUC16, hCD28, mCD28 recombinant proteins at 37°C are shown below in Tables 9-12. Table 9: Biacore binding affinities of antibodies AbPID mAb Capture (RU) 90 nM hMUC16.mmh Binding (RU) Ka (1 / Ms) Kd (1 / s) Kd (M) TH (min) bs24963D (Experiment 1) 987.1±9.9 365.0 4.41E+ 05 4.12E04 9.33E-10 28.0 bs24963D (Experiment 2) 211.7+1.2 120.0 2.38E+05 2.18E04 9.12E-10 53.1 Table 10: Biacore binding affinities of anti-MUCl 6xCD28 antibodies to hCD28 AbPID mAb Capture (RU) 600 nM hCD28.mmh Binding (RU) Ka (1 / Ms) Kd (1 / s) KD (M) TH (min) bs24963D 985.4+2.7 88.9 3.27E+04 5.38E03 1.65 E07 2.1 Table 11: Biacore binding affinities of anti-MUC16xmsCD28 antibodies to hMUC16 AbPID mAb Capture (RU) 90 nM hMUC16.mmh Binding (RU) Ka (1 / Ms) Kd (1 / s) KD (M) TU (min) REGN4615 1041.0+10.0 513.3 6.29E+05 4.72E04 7.49 E-10 24.5 Table 12: Biacore binding affinities of anti-MUC16xmsCD28 antibodies to mCD28 AbPID mAb Capture (RU) 90 nM hMUC16.mmh Binding (RU) Ka (1 / Ms) Kd (1 / s) Kd (M) TU (min) REGN4615 1021.8±4.0 25.8 2.07E+04 7.77E- 05 3.76E-09 148.6 145 Example 5. Binding of anti-MUC16xCD28 bispecific monoclonal antibodies to T lymphocytes and target cells In order to determine the binding of the exemplary bispecific antibodies of the present invention to human and Macaca fascicularis T lymphocytes and target cells, flow cytometric analysis was used to determine the binding of bispecific antibodies against MUGI6xCD28 to OVACR3 cells. PEO1, negative control Raj i, human and Macaca fascicularis T cells, followed by detection with a phycoerythrin (PE)-labeled or Alexa-647-labeled anti-human IgG antibody. Briefly, Ix 105 cells / well were incubated for 30 minutes at 4°C with a serial dilution of the exemplary MUC16xCD28 bispecific antibodies or an IgG4 isotype control that binds to a human antigen without cross-reactivity with human or Macaca CD28. fascicularis, ranging from 133 nM to 32.6 pM for human and Macaca fascicularis T cells, and ranging from 133 nM to 8.14 pM for Mucl6-expressing cells and negative control Raji cells. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS and a PE-conjugated or Alexa-647-conjugated anti-human secondary antibody was added to cells expressing MUC16 or human / growth T cells. Macaca fascicularis, respectively, L / RQnn / Lznz / E / Yi 146 and incubated for an additional 30 minutes. Live / dead cell dye was added to the Macaca fascicularis and human T-lymphocyte incubations. Wells containing no antibody or containing only a secondary antibody were used as controls. After incubation with cells expressing MUC16, cells were washed, resuspended in 200 µL of cold PBS containing 1% filtered FBS, and analyzed by flow cytometry on a BD FACS Canto II. After incubation with human or Macaca fascicularis T cells, cells were washed and stained with a cocktail of anti-CD2, anti-CD16, anti-CD4 and antiCD8 antibodies in Brilliant staining buffer for an additional 20 min incubation. at 4°C. After washing, cells were resuspended in 200 pL of cold PBS containing 1% filtered FBS, sorted as live / CD2+ / CD4+ / CD16- or live / CD2+ / CD8+ / CD16- and analyzed by cytometry. flow on a BD FACS LSR-Fortessa-X20. The binding of the exemplary MUC16xCD28 bispecific antibodies to the surface of human T cells was assessed by flow cytometry. bs24963D bound to CD4+ T cells with an EC50 value of 2.61 x 10_7M. It bound to CD8+ T cells with an EC50 value of 2.53 x 10~7M. L / RQnn / Lznz / B / Yi 147 bs32897D weakly bound to CD4+ T cells with an ECso value of 9.16 x 10~6M. It also bound weakly to CD8 + T cells, with an EC50 value of 7.58 x 10~6M. The results were summarized in Table 13. Table 13: Binding of anti-MUC16xCD28 to human T cells L / Rann / Lznz / E / Yii Antibody PiD Human CD4+ T cell EC50 by FACS [M] Human CD8+ T cell EC50 by FACS [M] bs24963D 2.61E-07M 2.53E-07M bs32897D 9.16E-06M 7.58E-06M Isotype control No fixation No fixation The binding of the exemplary MUC16xCD28 bispecific antibodies to the surface of Macaca fascicularis T lymphocytes was assessed by flow cytometry. The example bs24963D bound to CD4+ T cells with an EC50 value of 2.03 x 10~7M. It bound to CD8+ T cells with an EC50 value of 1.22 x 10~7M. Example bs32897D bound to OVCAR-3 and PEO1 cells with EC50 values ​​of 2.87 x ~10M and 5.96 x 10~10M, respectively. Example bs24963D did not exhibit any binding to negative control RAJI cells expressing MUGI6. The results are summarized in Table 14 . 148 lj AQnn / Lznz / E / Yi Table 14: Binding of anti-MUC16xCD28 to Macaca T lymphocytes PiD of the ECso antibody of Macaca fascicularis CD4+ T lymphocytes according to FACS [M] EC50 of Macaca fascicularis CD8+ T lymphocytes according to FACS [M] bs24963D 2.03E-07M 1.22E-07M bs32897D 5.70E-06M 3.02E-06M Isotype control Without fixation Without fixation The binding of the exemplary MUC16xCD28 bispecific antibodies to the surface of cell lines expressing IVIUC16 was assessed by flow cytometry. bs24963D was bound to OVCAR-3 and PEO1 cells with ECso values ​​of 6.09 x 10'10M and 4.67 x 10-10M, respectively. bs32897D did not exhibit any binding to negative control RAJI cells expressing MUC16. bs24963D was bound to OVCAR-3 and PEO1 cells with ECso values ​​of 2.87 x 10 -10M and 5.96 x 10 -10M, respectively. bs24963D did not exhibit any binding to negative control RAJI cells expressing MUC16. The isotype control antibody did not exhibit any binding to human or Macaca fascicularis T cells, nor did it bind to cell lines expressing MUC16. The results were summarized in Table 15. Table 15: Binding of anti-MUC16xCD28 to cells expressing MUC16 PiD of antibody EC50 of OVCAR-3 cells according to FACS [M] EC50 of PEO1 cells according to FACS [M] EC50 of Raji cells according to FACS [M] bs24963D 6.09E-10M 4.67E-10M Non-binding bs32897D 2.87E-10M 5.96E -10M No fixation Isotype control No fixation No fixation No fixation 149 Example 6. Primary bioassay for bispecific antibodies against MUC16xCD28 T cell activation is achieved by stimulating T cell receptors (TCRs) that recognize specific peptides presented by major histocompatibility complex class I or II (MHCI or MHCII) proteins on antigen presenting cells (APCs) ( Goldrath et al., Selecting and maintaining a diverse T-cell repertoire, Nature 402, 255-262 (1999)). An activated TCR in turn initiates a cascade of signaling events, which can be controlled 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 lymphocytes (NFkB). The T cell response is then further refined through the coupling of co-receptors constitutively or inducibly expressed on T cells such as CD28, CTLA-4 (cytotoxic T cell-associated protein 4), PD-1 ( programmed cell death protein 1), LAG-3 (lymphocyte activation gene 3) or other molecules (Sharpe et al., The B7-CD28 Superfamily, Nat. Rev. Immunol., 2(2): 116-26 ( 2002)). The costimulatory molecule, CD28, is activated by its L / Rann / Lznz / E / Yii 150 endogenous CD80 or CD86 ligands expressed on APCs. CD28 potentiates cellular signals such as pathways controlled by the transcription factor NFkB after TCR activation. The simultaneous CD28 signal is important for efficient T cell activation, such as T cell differentiation, proliferation, cytokine release, and cell death (Smeets et al., NFkB activation induced by T cell receptor / CD28 costimulation is mediated by protein kinase C-θ, PNAS, 97(7):3394-3399 (2012). In order to identify antibodies that enhance T cell activity in the presence of both primary stimulation and expression of the MUC16 target, exemplary anti-MUCI6xCD28 bispecific antibodies of the invention were characterized in cell-based assays with primary T cells. humans. The assays evaluate the behavior of the anti-MUC16 / CD28 bispecific antibody in the presence and absence of primary stimulation and in the presence and absence of expression of the target. IL-2 functional assay with primary human CD4+ T cells: A functional primary CD4+ T cell / APC assay was developed to assess the effect of CD28 activation on IL-2 production following coupling with L / EQnn / Lznz / E / Yi 151 anti-MUC16 x CD28 bispecific antibodies. a) Isolation of human primary CD4+ T lymphocytes: Human peripheral blood mononuclear cells (PBMC) were isolated from a concentrate of leukocytes from a healthy donor. PBMC isolation was achieved by density gradient centrifugation using 50 mL SepMate™ tubes according to the manufacturer's recommended protocol. Briefly, 15 mL of FicollPaque PLUS was layered into 50 mL SepMate tubes, followed by the addition of 30 mL of leukocytes diluted 1:2 with D-PBS. Subsequent steps were followed according to the SepMate manufacturer's protocol. CD4+ T cells were subsequently isolated from PBMC with microsphere kits of Human CD4 from Miltenyi Biotec according to the manufacturer's instructions. Isolated CD4+ T cells were frozen in FBS containing 10% DMSO at a concentration of 5 χ 106 cells per vial. b) Release of IL-2 from primary CD4+ T lymphocytes treated with antibodies against CD28: In this assay, human primary CD4+ T cells are activated by cross-linking CD3 molecules, in complex with T cell receptors (TCRs), using L / RQnn / Lznz / E / Yi of a bispecific antibody against otMucl6 x aCD3 (REGN4018) 152 incubated with human target cells, OVCAR3 or PEO-1, expressing Mucl6 on the cell surface. Binding of the Mucl6 arm of REGN4018 to target cells expressing Mucl6 drives clustering of CD3 molecules and provides the first signal, necessary for stimulation of T cells in the absence or addition of an allogeneic response. However, in this assay, in order to complete T cell activation and increase levels of IL-2 release, costimulation provided by cross-linking of CD28 molecules is necessary. Here, bispecific anti-CD28 antibodies interact with CD28 on CD4+ T cells and Mucl6 on OVCAR3 or PEO-1 cells and drive clustering activation of the costimulatory molecule, CD28. The combined coupling of TCR and CD28 leads to increased production of IL-2, which is released into cell culture media. IL-2 is detected and quantitated from the cell supernatant using a homogeneous, no-wash AlphaLisa kit from PerkinElmer. Previously isolated and frozen human CD4+ T cells from Donor 104 were thawed on the day of the assay in stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing it contained 50 U / mL of Benzonase nuclease. The cells will L / EQnn / Lznz / E / Yi 153 were centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium, and plated in 96-well round bottom plates at a concentration of 1 x 105 cells / well. OVCAR3 and PEO-1 cells were treated with mitomycin C in primary stimulation medium with 25 pg / mL mitomycin C for OVCAR3 cells and 10 pg / mL for PEO-1 cells. After incubation for 1 hour at 37 °C, 5% CO2, target cells were washed 3 times with washing buffer (PBS+2% FBS) and added to wells containing CD4+ T cells at a final concentration of 1*104 OVCAR3 cells or 2.5 χ 104 PEO-1 cells per well. Subsequently, serially diluted 1:4 anti-CD28 antibodies, ranging from 3 pM to 200 nM, were added to the wells in the presence of a constant 5 nM of either a REGN4018 antibody (against aMucl6 x aCD3) or a negative control antibody (hIgG4 isotype control = H4sH). The end point of the 10 point dilution did not contain antibody against CD28, which is the background signal. After incubating the plates for 72 hours at 37°C, 5% CO2, they were centrifuged to pellet the cells and 20 pL of medium supernatant was collected. From this, 5 pL was tested in an AlphaLISA human IL-2 assay according to the manufacturer's protocol. The measurements were acquired at the L / RQnn / Lznz / E / Yi 154 Envision Multi-Label Reader and raw RLU (relative light units) values ​​were plotted. All serial dilutions were tested in duplicate. Antibody EC50 values ​​were determined by fitting the data to a four parameter logistic equation on a 10 point dose-response curve using GraphPad Prism™ software. The maximum induction factor is calculated with the following equation: L / Rann / Lznz / E / Yu Mean of the highest RLU value within the evaluated dose range Induction factor =-------------——---------------———---------Average of values from IL — 2 (rondo) Activation of CD4+ T cells (as measured by IL-2 release) was enhanced by hMUC16xhCD28 in the presence of primary stimulation (anti-MUCI6xCD3) and MUC16 expressed on the target cells. c) Result of the IL-2 functional assay with primary human CD4+ T cells: The ability of anti-Mucl6 x anti-CD28 bispecific antibodies to provide costimulation via CD28 in isolated CD4+ T lymphocytes in the absence or presence of a TCR-stimulating bispecific antibody (REGN4018 = anti-Mucl6 x anti-CD3) was evaluated. in a release trial 155 of functional IL-2 with isolated human TCD4+ lymphocytes incubated with endogenously expressing Mucl6 target cells (OVAR3 and PEO-1 cells) on the cell surface. Induction factor values ​​are summarized in Table 16 and 17 for CD4+ T cells coincubated with OVCAR3 or PEO-1 cells plus a constant 5 nM hIgG4 H4sH or REGN4018 anti-Mucl6 x anti-CD3 isotype control. . When isolated CD4+ T cells are incubated with OVCAR3 or PEO-1 target cells in the absence of REGN4018-directed TCR stimulation with a constant amount of H4sH isotype control, the amounts of IL-2 detected are similar between antibodies. against parental CD28, the anti-Mucl6 x anti-CD28 antibodies (bs32897D and bs24963D) and the negative H4sH isotype control antibody. (Table 16) In contrast, higher levels of IL-2 are detected in samples treated with anti-Mucl6 x anti-CD3 (REGN4018). Under these conditions, if human CD4+ T cells were coincubated with OVCAR3 or PEO-1 cells, both bispecific CD28 antibodies increase IL-2 levels more than their respective parental CD28 antibodies. As expected, minimal IL-2 release is not observed with the isotype control. (Table 17) If OVCAR3 are used as target cells, a L / Rann / Lznz / E / Yii 156 similar dose-dependent IL-2 release for both bispecific CD28 antibodies (bs32897D: 5.63x and ECso = 606 pM) and bs24963D: 5.32x and EC50 = 401 pM). While with PEO-1 cells, a difference in the induction factor of IL-2 levels could be observed between both bispecific molecules. Here, bs24963D (10.94x and EC50 = 996 pM) gives rise to higher IL-2 values ​​than bs32897D (5.22x and EC50 could not be determined, because the dose-response curve did not reach saturation). In the absence of TCR stimulation, either through an allogeneic or anti-MUCI6xCD3-driven response, no measurable IL-2 release is observed with anti-CD28 antibodies in wells containing constant amounts of isotype control in the presence of cells. OVCAR3 or PEO-1 (Table 16). Table 16 summarizes the EC50 values ​​and factor induction of IL-2 release from CD4+ T cells coincubated with OVCAR3 or PEO-1 and a 5 nM isotype control constant. Table 16: Results of EC50 γ factor induction for IL-2 release from primary human CD4+ T cells in the presence of 5 nM IgG4 isotype control L / fiann / Lznz / E / Yii human: 157 Antibodies OVCAR3 PEO-1 EC50 [M] Induction factor EC50 [M] Induction factor bs32897D N / A 1.06 N / A 1.12 bs24963D N / A 1.33 N / A 1.12 hCD28 parental 1 (for bs32897D) N / A 1.10 N / C 1.30 hCD28 parental 2 (for bs24963D) N / C 1.12 N / C 1.03 H4sH isotype control N / C 1.12 N / C 1.48 L / Rann / Lznz / E / Yii Table 16. Tabulated EC50 values ​​and peak induction factor of IL-2 release over background signal from CD4+ T cells coincubated with OVCAR3 or PEO-1 and a 5 nM constant H4sH isotype control. N / C = not calculated In contrast, measurable IL-2 levels (RLU) are detected in samples treated with anti-MUCI6xCD3. Under these conditions, if human CD4+ T cells were coincubated with OVCAR3 or PEO-1 cells, both bispecific CD28 antibodies increase IL-2 levels more than their respective parental CD28 antibody. as it was from 158 expected, no IL-2 release is observed with the isotype control (Table 17). Table 17 summarizes the ECso values ​​and factor induction of IL-2 release from CD4+ T cells coincubated with OVCAR3 or PEO-1 and a 5 nM anti-MUCI6xCD3 constant. Table 17: ECso and factor induction results for IL-2 release from primary human CD4+ T cells in the presence of 5 nM REGN4018 (anti-Mucl6 x antiCD3): L / RQnn / Lznz / B / Yi OVCAR3 antibodies PEO-1 ECso [M] ECso induction factor [M] Induction factor bs32897D 6.07E-10 5.63 N / A 5.22 bs24963D 4.01 E-10 5.32 9.96E- 10 10.94 hCD28 parental 1 (for bs32897 D) N / D 1.58 N / D 1.42 parental hCD28 2 (for bs24963D) N / D 2.46 1.07E- 10 2.05 H4sH Isotype Control N / C 1.09 N / C 1.11 Table 17. Tabulated ECso values ​​and maximum induction factor of IL-2 release on background signal from CD4+ T cells coincubated with OVCAR3 or PEO-1 and a constant 5 nM REGN4018 (ant¡- Muc16 x anti-CD3). Abbreviations: N / D = not determined, because the dose-response curve did not reach saturation or was bell-shaped; N / C = not calculated 159 Example 7. Bispecific anti-MUC16xCD28 antibodies enhance T-lymphocyte activation and cytotoxicity in ovarian tumor cells in the presence of TCR stimulation by anti-MUCI6xCD3 To examine whether the exemplary anti-MUC16xCD28 bispecific antibodies of the invention could enhance anti-MUCI6xCD3-mediated T-lymphocyte activation and cytotoxicity in ovarian tumor cells, FACS was used to examine tumor cell viability and phenotypic T-lymphocytes after from in vitro coculture with a dose titration of MUC16xCD3 alone or in combination with MUC16xCD28 (Figure 2A). Human peripheral blood mononuclear cells (PBMC) containing T lymphocytes were cocultured with PEO-1 ovarian cancer cells expressing endogenous levels of MUC16 (Coscia, F. et al, Nat. Commun, (2016), Aug 26; 7:12645). Two FACS-based cytotoxicity studies were performed. In the first study, FACS-based cytotoxicity was performed on MUC16+ cells in the presence of human peripheral blood mononuclear cells (PBMCs) and anti-MUCl6xCD3 in the presence or absence of anti-MUC16 x CD28 stimulation (FACS-based cytotoxicity on cells). MUC16 + PBMC + / stimulation with MUC16xCD28 (array configuration L / RQnn / Lznz / E / Yi 160 MUC16xCD28 x Mucl6xCD3)) . The second study is otherwise identical to the first study, except that Macaca fascicularis PBMCs are used instead of human PBMCs (FACS-based cytotoxicity in MUC16 cells + Macaca fascicularis PBMCs + / - stimulation with MUC16xCD28 (MUC16xCD28 x matrix configuration). MUC16xCD3)). experimental procedure In order to monitor the inactivation of MUC16+ cells in the presence of a combination of an exemplary anti-MUC16xCD3 antibody and an anti-MUCl6xCD28 antibody of the invention, cell lines endogenously expressing MUC16 (PEO1, MUC16+) were labeled with ΙμΜ of Violet cell tracer and plated overnight at 37°C. Separately, human PBMCs (New York Blood Center) or Macaca fascicularis PBMCs (Covance, Cranford NJ) were plated in RPMI medium supplemented at 1x106 cells / mL and incubated overnight at 37°C in order to enrich lymphocytes by decreasing adherent macrophages, dendritic cells, and some monocytes. The following day, target cells were coincubated with naïve and depleted human PBMC in adherent cells (4:1 effector cell / target ratio) and a serial dilution of anti-MUCl6xCD3 or bispecific based L / EQnn / Lznz / E / Yi 161 on untargeted CD3 (bsl7664D), alone or in combination with a fixed concentration (2.5 pg / mL) of an example bispecific antiMUC16xCD28 for 96 hours at 37°C. After incubation, cells were removed from cell culture plates by trypsin-EDTA dissociation buffer and analyzed by flow cytometry (FACS). For FACS analysis, cells were stained with a Far Red Cell Tracker (Invitrogen) viability tracker and directly conjugated antibodies against CD2, CD4, CD8 and CD25 (BD). Samples were processed with calibration microspheres for cell counting. For inactivation specificity assessment, target cells were sorted as Violet cell tracer positive populations. The percentage of live target cells was calculated as follows: percentage of viable cells=(R1 / R2)*100, where Rl= percentage of live target cells in the presence of antibody, and R2= percentage of live target cells in the absence of antibody. test antibody. T-lymphocyte activation was measured by the percentage of activated T-lymphocytes (CD25+) out of total CD2+ / CD4+ or CD2+ / CD8+ T-lymphocytes. Upregulation of the PD-1 marker was assessed by incubating cells with antibodies conjugated directly against CD2, CD4, CD8, CD25, and PD-1, and L / RQnn / Lznz / E / Yi 162 by reporting the percentage of PD-1+ T cells out of total T cells (CD2+). T cell count was measured by calculating the number of live CD4+ or CD8~ cells per calibration bead. Accumulated cytokine levels in the media were assayed with BD's Microsphere Cytometric Array (CBA) Thl / Th2 / Thl7 Human Cytokine Kit according to the manufacturer's protocol. Results, summary and conclusions: The anti-MUCI6xCD3 bispecific antibody was evaluated for its ability to induce naïve human T cells to inactivate PEO1 target cells expressing human MUGI6 as a single agent, or in the presence of a costimulatory anti-MUC16xCD28 antibody. The anti-MUCI6xCD3 bispecific antibody activated and directed human T cells to downregulate PEO1 cells. Furthermore, MUC16xCD3 only induced moderate T-lymphocyte inactivation of PEO-1 cancer cells, reducing their viability to -60% in a dose-dependent manner (Figure 2B and Table 18). Inactivation of target cells was observed in the presence of the anti-MUCI6xCD3 bispecific antibody and PEO1 cells were inactivated in a dose-dependent manner with ECso values ​​at the picomolar (pM) level (Figure 2B). No inactivation of target cells was observed when no anti was present. L / RQnn / Lznz / E / Yi 163 MUC16xCD3 (Figure 2B). The observed lysis in target cells was associated with upregulation of CD25+ and PD-1+ cells on CD2+ T cells, again with ECso values ​​at the picomolar level (Table 18). Anti-MUCl6xCD3 induced the release of human cytokines. The cytotoxic activity observed with anti-MUCl6xCD3 as a single agent was enhanced in the presence of the exemplary anti-MUCl6xCD28 costimulatory molecules of the present invention, bs24963D and bs32897D. The addition of the exemplary anti-MUC16xCD28 of the invention was found to increase the potency and depth of MUC16xCD3-induced cytotoxicity, resulting in a further reduction of PEO-1 cancer cell viability to less than 20% ( more than 3-fold increase in T cell inactivation) (Figure 2B). In addition, the exemplary anti-MUC16xCD28 of the invention increased the levels of MUC16xCD3-induced IFNγ release by more than 10-fold (Figure 2C). The combination of MUC16xCD28 and MUC16xCD3 expanded CD4 and CD8 T cells and increased the expression level of the activation marker CD25 (Figures 2D-E). In particular, MUC16xCD28 in combination with a bispecific against CD3 non-targeting did not induce cytotoxicity or activation of T lymphocytes (Figure 2B). ί / βοπη / ίζηζ / Ε / γι 164 In summary, costimulation increased T cell activation, PD-1 upregulation, and cytokine release compared to what was seen with MUC16xCD3 as a single agent. Tables 18 and 19 and Figures 2A-2E summarize the experimental results with human PBMCs. Table 18: Effects of anti-MUC16xCD28 on anti-MUC16xCD3 cytotoxicity in PEO1 cells in the presence of human ί / βΟΠΠ / ί7Π7 / Β / ΥΙ PBMCs PiD ECso of inactivation of PEO1[M] % min. of viability of PEO1 ECso of activation of T lymphocytes (CD8+ / CD25+) [M] % max. from PD-1 upregulation (CD4+ / PD1+) MUC16xCD3 1.27E-10 57% 2.94E-10 27.2% 1.07E-10 to 1.86E-10 to MUC16xCD3+bs24963D 5.31 E-11 9.8% 3.94E-11 65.7% 2.6E-10a 4.82E-10a MUC16xCD3+bs32897D 7.40E-11 17.7% 4.42E-11 58.4% The anti-MUCI6xCD3 bispecific antibody was also evaluated for its ability to induce untreated Macaca fascicularis T lymphocytes to inactivate target cells expressing human MUGI6 as a single agent, or in the presence of an anti-MUCI6xCD28 bispecific antibody. 165 costimulator. The same assays were performed and similar results were obtained with PBMC from Macaca fascicularis (Figures 2F-H). Figure 21 shows that the exemplary anti-MUCI6xCD28 bispecific antibody of this invention binds to cellular targets as measured by flow cytometry. These results demonstrated that the anti-MUCI 6xCD28 bispecific antibodies of the invention can potently enhance MUC16xCD3-mediated T cell activation not only through proliferation and cytokine release but also through cytotoxicity. In the selected antibody titer, the anti-MUCI6xCD3 bispecific antibody activated human T cells, but did not direct T cells to downregulate PEO1 cells (Table 19). Costimulation with an exemplary anti-MUCl6xCD28 antibody of the invention resulted in increased T cell activation, increased cytotoxic activity, and upregulation of the PD-1 marker on T cells (Table 19). L / RQnn / Lznz / E / Yi L / RQnn / Lznz / B / Yii 166 Table 19: Effects of anti-MUC16xCD28 on anti-MUC16xCD3 cytotoxicity in PEO1 cells in the presence of Macaca fascicularis PBMC PiD EC50 of PEO1 inactivation [M] % min. PEO1 viability EC50 activation of T lymphocytes (CD8+ / CD25+) [M] % max. PD-1 upregulation (CD4+ / PD1+) MUC16xCD3 2.09E-10 69% 1.59E-10 24.4% MUC16xCD3+bs24963D 1.29E-10 to 3.34E-11 18.1% 1.04E-10a 9.07E11 44.3% MUC16x CD3+bs32897D 3.92E-10 to 1.09E-10 29.7% 2.81E-10a 7.67E-11 40.3% Example 8. In vivo study of the anti-MUC16xCD28 antibody The combination of anti-CD3xMUC16 and anti-CD28xMUCl6 bispecific antibodies directed to tumor antigen enhanced tumor clearance in a mouse model. As shown in the details below, OVCAR-3 tumor growth was significantly inhibited in mice administered exemplary anti-CD3xMUCl6 and anti-CD28xMUCl6 of the invention compared to mice administered anti -CD3xMUCl6 alone or control isotype. 167 To examine whether MUC16xCD28 could enhance the antitumor efficacy of MUC16xCD3 in vivo, two different tumor models, a tumor xenogeneic ascites model and a syngeneic mouse tumor model, were used as described in details below. Tumor xenogeneic ascites model In a model of tumor xenogeneic ascites, high-grade serous carcinoma OVCAR-3 ovarian cancer cells of human origin, expressing endogenous high levels of MUC16, are implanted intraperitoneally into NSG mice pre-engrafted with human PBMCs (Crawford A, Haber L, Kelly MP, Vazzana K, Canova L, Ram P, Pawashe A, Finney J, Jalal S, Chiu D, Colleton CA, Garnova E, Makonnen S, Hickey C, Krueger P, Delfino F, Potocky T, Kuhnert J , Godin S, Retter MW, Duramad P, MacDonald D, Olson WC, Fairhurst J, Huang T, Martin J, Lin JC, Smith E, Thurston G, Kirshner JR. A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer.Science Translational Medicine 2019 Jun 19:Vol. 11, Issue 497, eaau7534). OVCAR-3 cells were engineered with luciferase reporter to track tumor growth over time using L / RQnn / Lznz / E / Yi bioluminescence in vivo (BLI). 168 experimental procedure Experiments were performed as described in (Crawford A, Haber L, Kelly MP, Vazzana K, Canova L, Ram P, Pawashe A, Finney J, Jalal S, Chiu D, Colleton CA, Garnova E, Makonnen S, Hickey C , Krueger P, Delfino F, Potocky T, Kuhnert J, Godin S, Retter MW, Duramad P, MacDonald D, Olson WC, Fairhurst J, Huang T, Martin J, Lin JC, Smith E, Thurston G, Kirshner JR. A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer.Science Translational Medicine June 19, 2019:Vol. 11, Number 497, eaau7534) . Briefly, mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin (Perkin Elmer), suspended in PBS. Ten minutes later, BLI images of mice were performed under isoflurane anesthesia using the Xenogen IVIS system (Perkin Elmer). Image acquisition was performed with the field of view at D, subject height 1.5 cm, and medium clustering level for an exposure time of 0.5 min. BLI signals were extracted using Living Image software (Xenogen; Alameda, CA). Regions of interest were drawn around each tumor mass, and photon intensities were recorded as p / s / cm2 / sr (photons per second per square centimeter per steradian). The mice that did not receive cells L / RQnn / Lznz / E / Yii 169 OVCAR-3 / Luc served as the reference read for BLI activity. These control mice (N=3) without tumors were imaged each day and the lower limit of detection (LOD) was calculated as the average BLI reading in all tumor-free mice imaged. Eight to ten (8-10) week old NSG (NOD SCID gamma chain knockout) mice (Jackson Laboratory, MD) were injected with 5x106 human PBMC (ReachBio, Seattle, WA). Ten to fourteen (10-14) days later, the mice were bled via the tail vein to determine human T cell engraftment. Within two weeks of PBMC transfer, 2x106 ascites cells of the OVCAR-3 / Luc cell line, previously passaged in vivo, were administered intraperitoneally (IP) within two weeks (day 0). Mice were checked for T cell engraftment by flow cytometry and then assigned to groups by BLI to ensure similar tumor burden. Four days after tumor implantation, mice were divided into groups of 5 animals each with a median BLI of 1.49xl05 or 3.03xl05p / s / cm2 / sr for the two studies. Mice were treated with the indicated bispecific or control antibodies on days 5 and L / Rann / Lznz / E / Yii 170 8. Mice were administered anti-MUCI6xCD3 or a CD3 binding control with or without exemplary anti-MUCI6xCD28 (bs24963D) of the invention twice by intravenous (IV) injection. Images were obtained several times throughout the study to track tumor growth. Blood serum cytokine levels were also obtained at the indicated time point. At the indicated time points, blood was collected by submandibular puncture into microtainer serum tubes (BD 365967). Cytokine levels were assayed with the V-plex Human Prolnflammatory-10 Plex kit according to the manufacturer's instructions (Meso Scale Diagnostics, Rockville, MA). All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Regeneren Pharmaceuticals Institutional Animal Care and Use Committee. A total of 2 studies with 5 mice per group were completed. Results, summary and conclusions For studies of xenogeneic tumors, two models were used. For the first xenogeneic model, NSG mice were injected intraperitoneally (IP) with previously in vivo passaged OVCAR3 / Luc cells (day 0) thirteen days later. L / RQnn / Lznz / E / Yi 171 of the graft with the human PBMCs. Mice were treated IV on days 5 and 8. Mice received 12.5 pg antiMUC16xCD3 or 12.5 pg CD3-binding control (hIgG4p_pvA isotype). Some of the mice were also administered the exemplary anti-MUC16xCD28 of the invention (bs24963D) at 100 pg. Tumor burden was assessed by BLI on days 4, 8, 12, 15, 20, and 25 after tumor implantation. No apparent reduced BLI tumors were observed when the example bs24963D was administered without anti-MUCI6xCD3. In contrast, while treatment with 12.5 pg antiMUC16xCD3 significantly reduced overt BLI tumors, the exemplary anti-MUCI6xCD28 of the invention significantly improved efficacy over anti-MUCI6xCD3 alone (Tables 20-22). Table 20 summarizes the level of bioluminescence on day 4 after tumor implantation in the first xenogeneic model of 0VCAR-3 / Luc. L / RQnn / Lznz / E / Yi 172 Table 20: OVCAR-3 / Luc model. Bioluminescence level on day L / Rann / Lznz / E / Yii after tumor implantation Antibody (pg) Mean radiance [p / s / cm22 / sr] 4 days after implantation (median ± SEM) CD3 binding control (12.5) 1.51X105 ± 2.81xl04 Anti-MUCl6xCD3 (12.5) 1.5xl05 ± 1.05xl04 CD3 binding control (12.5) + anti-MUC16xCD28 (100) 1.53xl05 + 2.OlxlO4 Anti-MUC16xCD3 (12.5) + anti-MUCl6xCD28 (100) 1.27x1o5 ± 2.29xl04 Table 21 summarizes the level of bioluminescence at day 25 after tumor implantation in the first xenogeneic model of OVCAR-3 / Luc. Table 21: OVCAR-3 / Luc model. Bioluminescence level on day 25 after tumor implantation Antibody (pg) Average radiance [p / s / cm22 / sr] 25 days after implantation (median ± SEM) CD3 binding control (12.5) 7.71x10® + 1.07x10® Anti-MUCI6xCD3 (12.5) 7.44xl03 + 3.llxlO3 CD3 binding control (12.5) + anti-MUC16xCD28 (100) 6.04x10® ± 8.32xl05 Anti-MUC16xCD3 (12.5) + anti-MUCI6xCD28 (100) 1.31X103 ± 3.05x1o1 Table 22 summarizes the ratio of change in BLI between day 4 and day after tumor implantation in the first xenogeneic model of OVCAR-3 / Luc. Table 22: OVCAR-3 / Luc model. Ratio of change in BLI between day 4 and day 25 after tumor implantation Antibody (pg) Mean Brightness Change Ratio [p / s / cm22 / sr] from day 4 to day 25 post-implantation (mean ± SD) CD3 binding control (12.5) 50.72 ± 29.67 Anti-MUC16xCD3 (12.5 ) -0.94 ± 0.05 CD3 binding control (12.5) + antiMUC16xCD28 (100) 35.38 ± 8.18 Anti-MUC16xCD3 (12.5) + anti-MUCI6xCD28 (100) -0.99 ± 0.00 173 For the second xenogeneic model, NSG mice were injected with pre-passaged OVCAR-3 / Luc cells (day 0) eleven days after engraftment with the human PBMCs. Mice were treated IV with 0.5 mg / kg anti-MUC16xCD3 or given 0.5 mg / kg CD3-binding control on days 5 and 8. Tumor burden was assessed by BLI on days 4, 8, 11, 14 , 21, 28 and 34. Some of the mice were also administered the exemplary anti-MUCl6xCD28 of the invention (bs24963D) at 0.2 mg / kg, 1 mg / kg or 5 mg / kg. The example bs24963D did not decrease tumor burden when administered without anti-MUCI6xCD3. In contrast, while treatment with 0.5 mg / kq anti-MUCl6xCD3 significantly reduced overt BLI tumors, the exemplary antiMUC16xCD28 improved efficacy over antiMUCl 6xCD3 alone (Tables 23-25 ​​and Figure 4A). Table 23 summarizes the level of bioluminescence on day 4 after tumor implantation in the second xenogeneic model of OVCAR-3 / Luc. L / RQnn / Lznz / E / Yi Table 23: OVCAR-3 / Luc model. Bioluminescence level the day after tumor implantation L / Rann / Lznz / E / Yi Antibody (mg / kg) Mean radiance [p / s / cm22 / sr] 4 days after implantation (median ¿ SEM) CD3 binding control (0.5) 3.65XÍ05 + 5.50xl04 Anti-MUC16xCD3 (0.5) 3.76xl05 ± 2.40xl04 CD3 binding control (0.5) + anti-MUC16xCD28 (5) 2.71X105 ± 2.65xl04 Anti-MUC16xCD3 (0.5) + anti-MUC16xCD28 (5) 3.18xl05 ± 4.45xl04 Anti-MUC16xCD3 (0.5) + anti-MUC1 6xCD28 (1) 3.07xl05 + 4.37X104 Anti-MUC16xCD3 (0.5) + anti-MUC16xCD28 (0.2) 2.86X105 ± 4.95xl04 Table 24 summarizes the level of bioluminescence at day 25 after tumor implantation in the second xenogeneic model of OVCAR-3 / Luc. Table 24: OVCAR-3 / Luc model. Bioluminescence level at day 34 after tumor implantation Antibody (mg / kg) Mean radiance [p / s / cm22 / sr] 34 days after implantation (median ± SEM) CD3 binding control (0.5) 1.79xl07 ± 2.17xl06 Anti-MUC16xCD3 (0.5) 9.60xl04 + 4.55X104 Anti-MUC16xCD3 (0.5) + antiMUC16xCD28 (5) 2.34xl07 ± 1.12xl06 Anti-MUC16xCD3 (0.5) + antiMUC16xCD28 (5) 2.45xl03 + 4.49xl03 Anti-MUC16xCD3 (0.5) + antiMUC16 xCD28 (1) 1.62xl03 ± 2.32xl03 Anti-MUC16xCD3 (0.5) + anti-MUC16xCD28 (0.2) 1.29xl03 ± 4.77xl04 Table 25 summarizes the ratio of change in BL1 between day 4 and day 34 after tumor implantation in the second xenogeneic model of OVCAR-3 / Luc. Table 25: OVCAR-3 / Luc model. Ratio of change in BLI between day 4 and day after tumor implantation Antibody (mg / kg) Ratio of change in mean radiance [p / s / cm22 / sr] from day 4 to day 34 after implantation (mean + SD) CD3 binding control (0.5) 51.35 1 27.59 Anti-MUC16xCD3 (0.5) -0.64 + 0.31 Anti-Muc16xcd3 (0.5) + Anti-Muc16xCD28 (5) 64.62 + 36.38 ANTI-MUC16XCD3 (0.5) + ANTI-MUC16XCD28 (5) -0.97 1 0.04 ANTI-MUC16XCD3 (0.5) (1) -0.99 ± 0.02 Anti-MUC16xCD3 (0.5) + anti-MUC16xCD28 (0.2) -1.00 + 0.00 175 Other results from the second xenogeneic model with different doses are shown in Figure 3A. Mice treated with MUC16xCD3 at 2.5 pg on day 5 and 8 after tumor implantation had significantly reduced tumor burden compared to mice treated with a CD3-binding control antibody (EGFRvIIIxCD3), but did not completely kill tumor cells. OVCAR-3 / Luc (Figure 3A). The combination of MUC16xCD3 at 2.5 pg with MUC16xCD28 at 100 pg further inhibited tumor growth with longer lasting rejection of tumor cells over time (Figure 3Aj_. In the same experiment, serum cytokine levels were also obtained Figure 3B shows cytokine levels (pg / mL) in mice treated with different antibodies and / or combinations of antibodies Figure 3C shows tumor burden and correlation with serum CA-125 levels on day 26. To assess the ability of bispecifics against CD28 and CD3 to promote tumor inactivation in vivo, the well-established xenogeneic intraperitoneal ovarian OVCAR-3 tumor model was used. In this model, tumor cells are introduced into immunodeficient mice that are reconstituted with human PBMC. Like other ovarian cancer cell lines, OVCAR-3 cells express MUC16. Before the L / RQnn / Lznz / E / Yi 176 implantation, OVCAR-3 cells were engineered with a luciferase reporter to allow in vivo monitoring of tumor growth over time using bioluminescence (BLI). Implanted OVCAR-3 tumors grew incessantly in mice treated with the EGFRvIIIxCD3 bispecific, a control CD3 bispecific that did not bind to these cells, and in mice treated only with the MUC16xCD28 bispecific (Figure 3A). Although the bispecific against MUC16xCD3 alone demonstrated significant antitumor activity, it did not completely eliminate OVCAR-3 tumors (Figure 3A) whereas the addition of the bispecific against MUC16xCD28 to the bispecific against MUC16xCD3 enhanced the antitumor effect in vivo (Figure 3A) over MUC16xCD3 alone. Consistent with the enhanced antitumor activity, the combination of both bispecifics also increased circulating cytokine secretion (Figure 3B). Bispecifics against MUC16 bind to the remaining nub of MUC16 (the cell surface remnant after CA-125 cleavage and release) on the ovarian cancer cell surface after proteolytic cleavage has released the prognostic biomarker of ovarian cancer CA-125 (I. Mylonas et ai., Immunohistochemical expression of the tumor marker CA-125 in normal, hyperplastic and malignant L / RQnn / Lznz / E / Yi 177 endometrial tissue. Anticancer Res 23, 1075-1080 (2003)), but do not bind to soluble CA-125 (Figures 9A and 9B). To determine whether the bispecific against MUC16xCD28 perturbed the ability to use CA-125 as a biomarker of ovarian tumor burden, CA-125 levels were measured in the mice. CA-125 levels correlated with tumor burden regardless of treatment. The lowest levels of CA-125 were observed in the mice treated with the bispecific combination (Figure 3C) as previously demonstrated for the bispecific against MUC16xCD3. Syngeneic mouse model experimental procedure Syngeneic studies were carried out in mice engineered to express human CD3 and a portion of human MUC16 for MC38 studies using VelociGene® technology, as previously described (Valenzuela et al., (2003) Nat. Biotechnol Jun;21(6):652-9), (Crawford A, Haber L, Kelly MP, Vazzana K, Canova L, Ram P, Pawashe A, Finney J, Jalal S, Chiu D, Colleton CA, Garnova E , Makonnen S, Hickey C, Krueger P, Delfino F, Potocky T, Kuhnert J, Godin S, Retter MW, Duramad P, MacDonald D, Olson WC, Fairhurst J, Huang T, Martin J, Lin JC, Smith E, Thurston G, Kirshner JR A Mucin 16 bispecific T L / RQnn / Lznz / E / Yi 178 cell-engaging antibody for the treatment of ovarian cancer. Science Translational Medicine June 19, 2019:Vol. 11, Number 497, eaau7534). Mice expressing human CD3, human CD28 and a portion of human MUGI6 were used for ID8-VEGF studies. For humanization of CD3, a targeting vector was engineered that replaced the extracellular portions of the mouse CD3 (γδε) genes with the corresponding human region of the genes. For humanization of CD28, a targeting vector was engineered that replaced the extracellular portions of the mouse CD28 gene with the corresponding human region of the gene. For MUC16, mouse SEA 13-17 repeats were replaced with human SEA 1216 repeats. For each humanized mouse, correct gene targeting in F1H4 embryonic stem (ES) cell clones (C57BL / 6 χ 129 hybrid) was identified by an allele loss assay as described previously (Poueymirou et al. (2007), Nat. Biotechnol. Jan; 25(1):91-9). Target ES cells were injected into 8-cell stage Swiss Webster embryos to produce complete F0 generation heterozygous mice for breeding with C57BL / 6N mice (Taconic, Rensselaer, NY) to obtain homozygosity. Mice expressing the human extracellular portion of CD3 L / RQnn / Lznz / E / Yii 179 (γδε), the human extracellular portion of CD28 and a portion of human MUC16 were then crossed for homozygosity (referred to as hCD3 / hMucl6 or hCD3 / hCD28 / hMUCl6 humanized mice). To examine the efficacy in an immunocompetent model, a knock-in mouse was generated. The T lymphocytes of this mouse express human CD3 and instead of murine MUGI6, a chimeric molecule is expressed that contains a portion of human MUGI6 where the exemplary bispecific antibody of the invention binds. Consequently, the anti-MUCl6xCD3 molecule can be used in this study. To investigate whether the addition of a bispecific molecule directed against CD28 can improve efficacy in these mice, a surrogate bispecific antibody was also generated. The surrogate antibody recognized human MUC16 but not murine CD28 to examine the effects of CD28 costimulation and is sometimes referred to as antiMUC16xmCD28. For the syngeneic tumor model, the MC38 cell line engineered to express a portion of human MUGI6 was used. Mice were implanted with MC38 / huMUC16 cells subcutaneously (SC) and treated with 0.01 mg / kg anti-MUCl 6xCD3 on the day of implantation, twice weekly until day 21. Treatment with 0.01 mg / kg L / RQnn / Lznz / E / Yi of anti-MUCl6xCD3 resulted in antitumor efficacy 180 significant and the addition of MUC16xmCD28 ameliorated this effect. (See Figures 6A, 6B, 6C and 6D). Implantation and measurement of syngeneic tumors Mice expressing human CD3 and a murine-human MUC16 chimera at the corresponding mouse loci were implanted with IxlO6 MC38 / huMUC16 cells subcutaneously. Mice were administered anti-MUCl6xCD3 or an isotype control intraperitoneally (IP) with or without a surrogate bispecific antibody that recognizes human MUC16 and mouse CD28 twice weekly throughout the study until day 21. treatment began on the day of implantation. Tumor growth was measured with calipers twice a week. Mice were euthanized 50 days after tumor implantation. Calculation of growth and inhibition of syngeneic tumors To determine tumor volume using an external caliper, the largest longitudinal diameter (length) and the largest transverse diameter (width) were determined. Tumor volume was calculated based on caliper measurements using the formula: Volume = (length x width2) / 2. Tumor growth was monitored over time using ί / βοπη / ίζηζ / Ε / γι X and Y diameter caliper measurements. 181 underwent euthanasia when the size of the tumor was greater than 2000 mm3. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney t-test. Results Tumor sizes in the MC38 / huMUC16 model with different treatments are summarized in Table 26. Table 26: Model MC38 / huMUC16. Tumor size (mm3) on day 21 L / Rann / Lznz / E / Yii Antibody (pg) Tumor size (mm3) (mean ± SEM) Isotype control (0.5) 1191 ±424 Anti-MUC16xCD3 (0.01) 639.5 ± 186.8 Anti-MUC16xmCD28 (0.5) 648.5 ± 129.7 Anti-MUC16xCD3 (0.01) + anti-MUCl 6xmCD28 (0.5) 167.3 ±71.9 It was evaluated whether the exemplary anti-MUC16xCD28 bispecific antibodies of the invention could enhance the antitumor efficacy of MUC16xCD3 in a syngeneic mouse model in mice with a fully intact immune system. Mice were engineered to express human CD3 and human MUGI6 instead of mouse genes using Velocigene technology (Crawford A, Haber L, Kelly MP, Vazzana K, Canova L, Ram P, Pawashe A, Finney J, Jalal S, Chiu D, Colleton CA, Garnova E, Makonnen S, Hickey C, 182 Krueger P, Delfino F, Potocky T, Kuhnert J, Godin S, Retter MW, Duramad P, MacDonald D, Olson WC, Fairhurst J, Huang T, Martin J, Lin JC, Smith E, Thurston G, Kirshner JR. A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer. Science Translational Medicine June 19, 2019:Vol. 11, Number 497, eaau7534). The MC38 colon carcinoma cell line was engineered to express human MUC16 (pLVX.EFla.MUC16, MC38 / hMUC16) and implanted subcutaneously. Mice were given by intraperitoneal injection 2x per week from the day of implantation (day 0) isotype control (Iso Ctrl), 0.01 mg / kg MUC16xCD3, 0.5 mg / kg MUC16xmCD28, or a combination. Tumor growth was monitored over time (Figure 6A). Monotherapy with MUC16xCD3 or MUC16xCD28 significantly inhibited tumor growth. Tumor growth was furthermore significantly inhibited by co-treatment with MUC16xCD3 and MUC16xCD28 (Table 26). In the same experiment, serum cytokine levels were also obtained. Figure 6B shows cytokine levels in mice treated with different antibodies and / or combinations of antibodies. Suitable humanized MC38 / hMUC16 mice received implanted tumor cells and were treated with control, ί / βοπη / ίζηζ / Ε / γι bispecific against individual CD3 or CD28 or combinations (Figures 6A, 6C and 6D). In the MUC16 tumor model, the combination of the bispecifics against CD3 and CD28 provided the best antitumor responses (Figure 6A), as was also observed in cytokine production assays (Figures 6C and 6D). To investigate whether the addition of targeting of the major MUC16xCD28 can improve efficacy in a syngeneic model, mice expressing human CD3 instead of murine MUC16, human CD28 instead of murine CD28, and a chimeric molecule containing a portion of murine CD28 were used. Human MUGI6 where the exemplary bispecific antibody of the invention is fixed. The ID8-VEGF cell line was engineered to express human MUC16 (ID8-VEGF / hMUCI6) and implanted intraperitoneally. Mice were administered 1 mg / kg of EGFRvIIIxCD3 or MUC16xCD3 alone or in combination with MUC16xCD28 on days 3, 6, and 10 after tumor implantation. Tumor growth was controlled by weight gain (Figure 5). MUC16xCD3 inhibited tumor growth and the combination with MUC16xCD28 further delayed tumor growth. Notably, unlike previous in vitro and in vivo assays in which bispecifics against CD28 had L / RQnn / Lznz / B / Yi With very limited single-agent activity (see above), the bispecifics against CD28 in this syngeneic MC38 / MUC16 model had more remarkable single-agent activity. This suggested that signal 1 was already being activated to some degree on these MC38 models. Consistent with this, it has previously been shown that MC38 tumor cells express high levels of endogenous reactivated retroviral proteins such as pl5E, and that C57BL6 mice can generate endogenous T cells that recognize and respond to this neoepitope (J. C. Yang, D. Perry -Lalley, The envelope protein of an endogenous murine retrovirus is a tumor-associated T-cell antigen for multiple murine tumors. J Immunother 23, 177-183 (2000); H. J. Zeh, 3rd, D. Perry-Lalley, Μ. E. Dudley , S. A. Rosenberg, J. C. Yang, High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J Immunol 162, 989-994 (1999)). In fact, it was confirmed that in the MC38 models of this invention, intratumoral T cells responsive to this pl5E neoantigen could be easily detected (data not shown). Thus, bispecifics against CD28 in this syngeneic MUC16 tumor model may enhance TCR / CD3-dependent endogenous T cell responses, which can then be further increased by providing additional signal activation. L / Rann / Lznz / E / Yii 185 by means of a bispecific antibody against CD3. It has long been appreciated that activation of T cells through the TCR complex (signal 1) can be markedly enhanced by costimulatory signals such as those mediated when the T cell receptor CD28 binds to its liqands (CD80 / B7. 1 and CD86 / B7.2) on target cells (signal 2) (J. H. Esensten, Y. A. Helou, G. Chopra, A. Weiss, J. A. Bluestone, CD28 Costimulation:From Mechanism to Therapy. Immunity 44, 973-988 (2016 )) . According to the data reported herein, the potential of CD28 costimulation to enhance T cell antitumor activity was first demonstrated by studies in which B7 ligands were overexpressed on tumor cells (R. H. Schwartz, Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7 / BB1 in interleukin-2 production and immunotherapy, Cell 71, 1065-1068 (1992), L. Chen et al., Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 71, 10931102 (1992)), which showed increased rejection by T lymphocytes of such B7-expressing tumors. This potential inspired efforts to evaluate CD28-activating antibodies in human trials. Tragically, the 2006 trial of such an antibody (TGN1412) had potentially life-threatening complications. L / RQnn / Lznz / E / Yi 186 fatal in the six human volunteers (G. Suntharalingam et al., Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med 355, 1018-1028 (2006)), due to multi-organ failure resulting from a massive cytokine release syndrome (CRS). This catastrophe led to the cessation of any further testing of CD28-activating antibodies in humans. Bispecific antibodies against CD28 that would not directly activate CD28, unless clustered on tumor cell surfaces, offered the potential to promote costimulation only at the tumor site, without the systemic toxicity of conventional CD28-activating antibodies. Initial versions of such CD28 bispecifics were proposed and evaluated in the 1990s (C. Renner et al., Cure of xenografted human tumors by bispecific monoclonal antibodies and human T cells. Science 264, 833-835 (1994); G Jung et al., Local immunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and resting autologous lymphocytes: evidence for in situ t-cell activation and therapeutic efficacy.Int J Cancer 91, 225-230 (2001);M. Brandl, L. Grosse-Hovest, E. Holler, H. J. Kolb, G. Jung, Bispecific antibody fragments with CD20 X CD28 specificity allow effective autologous and allogeneic T L / RQnn / Lznz / E / Yi 187 cell activation against malignant cells in peripheral blood and bone marrow cultures from patients with B-cell lineage leukemia and lymphoma. Exp Hematol 27, 1264-1270 (1999)); however, the initial technology available at the time required chemical cross-linking or hybrid / hybridoma fusions to create the proposed biotherapeutic agents, and resulted in suboptimal reagents that had profound activity by themselves regardless of their clustering in tumor cells (which resembles conventional CD28 antibodies, presumably due to the non-specific aggregation of these bispecifics). In addition, these early approaches also required prior activation of T lymphocytes in vitro, in order to observe any antitumor activity in vivo. Taken together, the catastrophic clinical results with the CD28-activating antibody TGN1412, as well as the limitations of these early CD28 bispecific approaches, discouraged further exploration of these approaches. Described herein is a novel class of costimulatory bispecific antibodies against CD28 that can markedly and safely promote antitumor activity by providing a costimulatory signal 2 . These bispecifics against CD28 have limited activity per se. L / RQnn / Lznz / E / Yi themselves (in the absence of signal 1), but can markedly enhance antitumor activity in the vicinity of signal 1, as can be provided when these CD28 bispecifics are combined with the emerging class of CD3 bispecifics (or if these CD28 bispecifics are used in settings where endogenous populations of tumor-specific T cells already exist). The generation, testing, and success of this new CD28 bispecific approach depended on (1) the use of a novel bispecific platform that was initially developed to produce CD3 bispecifics and has recently been both technologically validated (E. J. Smith et al. , A novel, native-format bispecific antibody triggering Tcell killing of B cells is robustly active in mouse tumor models and cynomolgus monkeys. Sel Rep 5, 17943 (2015)) and clinically (A. Crawford et al., REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer.Proceedings of the American Association for Cancer Research Annual Meeting 2018, (2018)) (Clinicaltrials.gov: NCT02290951, Clinicaltrials.gov: NCT03564340) for these CD3 bispecifics, and then it was adapted to efficiently produce bispecifics against CD28 that show minimal activity in the absence of a 1-specific signal; (2) the development of L / RQnn / Lznz / E / Yi 189 multiple genetically humanized xenogeneic and syngeneic animal tumor models (D. M. Valenzuela et al., Highthroughput engineering of the mouse genome coupled with highresolution expression analysis. Nat Biotechnol 21, 652-659 (2003); W. T. Poueymirou et al., F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.Nat Biotechnol 25, 91-99 (2007)) to assess these CD28 bispecifics by themselves and in combination with CD3 bispecifics; and (3) along with a much deeper understanding of cytokine release syndrome and its clinical development (A. Shimabukuro-Vornhagen et al., Cytokine release syndrome. J Immunother Cancer 6, 56 (2018); D. W. Lee et al. , Current concepts in the diagnosis and management of cytokine release syndrome. Blood 124, 188195 (2014); C. L. Bonifant, H. J. Jackson, R. J. Brentjens, K. J. Curran, Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 3, 16011 ( 2016)), validation of a monkey model in which any potential toxicity of these bispecifics against CD28 could be compared with that of conventional CD28-activating antibodies. The generation and evaluation of costimulatory bispecific antibodies against TSAxCD28 directed against a TSA for ovarian cancer (MUC16xCD28) is described herein. HE L / RQnn / Lznz / E / Yi 190 demonstrated that, in the absence of signal 1, these bispecifics against CD28 have minimal activity, in vitro or in vivo. However, these CD28 bispecifics can be combined with CD3 bispecifics to form artificial immune synapses containing the tumor antigens as well as the TCR and CD28 complexes. In addition, when combined with appropriate CD3 bispecifics in vitro, these CD28 bispecifics can effectively and specifically promote T cell activation and tumor cell inactivation in an antigen-dependent manner. In addition, these CD28 bispecifics also effectively enhance the antitumor activity of CD3 bispecifics in vivo, in a tumor antigen-specific manner, in xenogeneic and syngeneic tumor models; in such models, bispecifics against CD28 have minimal activity as a single agent unless tumor-specific T cells are already present, and in such settings appear to enhance this specific activity in a tumor antigen-dependent manner. Furthermore, co-treatment with TSAxCD28 and TSAxCD3 significantly leads to expansion of an intratumoral activated / memory T cell phenotype in vivo. Lastly, toxicology studies in humanized immunocompetent mice L / RQnn / Lznz / E / Yi 191 genetically, as well as in Macaca fascicularis, demonstrate that these bispecifics exhibit limited activity and no toxicity as individual agents, in direct comparison with conventional CD28-activating antibodies. Characterization of human-specific clinical candidates in the field of immuno-oncology is often limited to testing in xenogeneic tumor models with engrafted human immune cells. Although these xenogeneic models (such as the OVCAR3 model used) can be very useful, they do have limitations. Mice used in such xenogeneic models do not express the human tumor target in their normal tissues, which precludes evaluation of the test agent in the setting of normal tissue expression of the target. In fact, if a target is also normally expressed at high levels in normal tissues, this could limit antitumor efficacy by diverting the test agent from the tumor and could lead to toxicity in these normal tissues; none of this could be assessed in a xenogeneic model. A further limitation might involve the activity of engrafted human peripheral blood mononuclear cells (PBMC) that are transferred to a mouse. L / RQnn / Lznz / E / Yii immunocompromised, which could differ from that of T cells 192 normal host found in an immunocompetent system. To overcome these limitations and provide better models for evaluating specific clinical candidates for humans, double and triple genetically humanized mice were created. In these models, tumor antigens were genetically humanized to allow their normal expression in the appropriate host tissues (for MUC16), and CD3 and / or CD28 components were genetically humanized to allow immunocompetent host cells to respond to specific clinical candidates. for humans. In these genetically humanized immunocompetent syngeneic animal models, it was found that, as in xenogeneic animal models, CD28 bispecies for the tumor target MUC16 enhanced the antitumor activity of their appropriate CD3 bispecies. The similar enhancement of the antitumor efficacy of the different bispecifics against TSAxCD28 (eg, MUC16 and PSMA (data not shown)) in multiple preclinical models suggests that this therapeutic modality is robust and not limited to a specific tumor model, and could have broader utility as a novel class of combination target for immunotherapy. In general, the findings highlight that bispecies against L / RQnn / Lznz / E / Yi 193 TSAxCD28 may act synergistically with TSAxCD3 bispecifics and may provide a biologic solution that could markedly improve the efficacy of well-studied TSAxCD3 bispecifics in a reasonably safe and well-tolerated manner, warranting testing in human trials. TSAxCD3 bispecifics represent a promising emerging class of immunotherapy, but further optimization of antitumor activity will surely be required in many cases. Just as CAR-T approaches have used chimeric receptors that artificially activate both signal 1 and signal 2 to enhance their antitumor activity (E. A. Zhukovsky, R. J. Morse, Μ. V. Maus, Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection. Curr Opin Immunol 40, 24-35 (2016); S. L. Maude et al., Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 378, 439-448 (2018) ), shown now the possible benefit of combining specific against CD3 (providing signal 1) with bispecific against CD28 (providing signal 2) to improve antitumor activity. In addition to the practical benefits that such an approach could have over CAR-T treatments, since it does not require preparation L / RQnn / Lznz / E / Yi 194 laborious cellular therapy that must be individually tailored to each patient, nor does it require the depletion of patients' lymphocytes preemptively through toxic chemotherapy for them to accept this cellular therapy often associated with adverse effects (A. Shimabukuro- Vornhagen et al., Cytokine release syndrome. J Immunother Cancer 6, 56 (2018); C. H. June, R. S. O'Connor, O. U. Kawalekar, S. Ghassemi, M. C. Milone, CAR T cell immunotherapy for human cancer. Science 359, 1361- 1365 (2018)), the bispecific approach according to the invention offers the potential for greater efficacy, as well as greater safety and specificity of action. That is, it is possible to take advantage of combinatorial targeting, by combining a bispecific against CD3 for one antigen with a bispecific against CD28, specific for a second antigen (the increase in efficacy will only occur in tumor cells that express both antigens). to thus concentrate inactivation, by T lymphocytes, only of tumor cells that express both antigens, while limiting off-target toxicity in normal tissues that express only one of the antigens. Taken together, the data described herein demonstrate that the combination of CD28-based bispecies with bispecies L / EQnn / Lznz / E / Yi 195 based on CD3 can provide ready-to-use, well-tolerated biologic solutions with markedly enhanced and synergistic antitumor activity. Initial testing of this possibility in human trials will take place this year. Example 9. MUC16xCD28 alone or in concomitant treatment does not induce systemic T cell activation compared to CD28 superagonist in Macaca fascicularis The exemplary MUC16xCD28 antibodies of the invention enhance T cell activation against MUC16xCD3 in Macaca fascicularis (Figures 2F-2H). To determine the safety and tolerability of the exemplary anti-MUCI6xCD28 bispecific antibodies of the invention alone or in combination with anti-MUCI6xCD3, a single dose toxicity study was performed in Macaca fascicularis. Male or female Macaca fascicularis were assigned to treatment groups as indicated in Table 27. The study in Macaca fascicularis was carried out according to the IACUC guidelines. Male Macaca fascicularis (3 animals / group) received a single dose of each test article by intravenous infusion over approximately 30 minutes (the conjoint treatment was administered as a separate infusion for a total of 1 hour). The evaluation of the L / Rann / Lznz / E / Yii toxicity was based on clinical observations, qualitative food consumption, body weight, neurological examinations, vital signs (body temperature, heart rate, pulse oximetry, and respiratory rate), and clinical and anatomical pathology. Blood and tissue samples were collected for cytokine analysis, immunophenotyping analysis, histopathology, and toxicokinetic evaluation. CRP levels were analyzed on a Roche Modular P 800 system. Cytokines were measured by Meso Scale Diagnostics (MSD, Rockville, MD). For peripheral blood flow cytometry, blood was collected in potassium EDTA tubes, lysed, stained with anti-CD3, anti-Ki67 and anti-ICOS (BD Biosciences) and analyzed with FACS Canto II. Animals received a single dose of each test article by intravenous infusion over approximately 30 minutes (the conjoint treatment was administered as a separate infusion for a total of 1 hour). Toxicity assessment was based on clinical observations, qualitative food consumption, body weight, neurological examinations, vital signs (body temperature, heart rate, pulse oximetry, and respiratory rate), and clinical and anatomical pathology. Blood samples were collected for cytokine analysis, L / RQnn / Lznz / E / Yi 197 FACS immunophenotyping and toxicokinetic evaluation. No significant cytokine release, T cell margination, or T cell activation marker upregulation was observed after administration of a single dose of exemplary anti-MUC16xCD28 of the invention at 1 or 10 mg / kg, MUC16xCD3 at 1 or 10 mg / kg or joint treatments. Table 27 summarizes different readings including absolute numbers of T lymphocytes, marker of T lymphocyte activation (ki67), CRP and cytokine levels in serum from blood obtained at the indicated time point from individual animals. In addition, these findings were validated by dry and wet coating human T cell proliferation assays, which demonstrated that anchoring of MUC16xCD28 to assay plates by either a dry coating or a wet coating method does not induce lymphocyte activation. T in the absence of CD3 stimulation in contrast to a CD28 superagonist antibody (Figure 7). In fact, it was found that the exemplary anti-MUCI6xCD28 bispecific antibodies of the invention, as well as the parental bivalent anti-CD28 antibodies, failed to induce human T cell proliferation compared to the CD28 superagonist antibody. In general, the single dose exploratory toxicology study L / RQnn / Lznz / B / Yi 198 in monkeys and in vitro human T-cell based assays suggest that the exemplary anti-MUCl6xCD28 antibodies of the invention are safe and well tolerated. L / RQnn / Lznz / E / Yi 199 Table 27: Summary of the study of toxicity in Macaca, fascicularis Mole cule Description Dose (mg / kg ) Animal No. 1 Day 1 Clinical Observ ations Any Obs. CRP (mg / d L) Plasma cytokine at 5 h post-dose (pg / mL) (E3 / pL) Ki67+ T cell (E3 / pL) Days 2-4 Pretest 5 h Pretest 72 h test 24 h IL -6 IL -8 IL 10 IF Ng TNF -a IL -2 IL -4 REGN 4018 anti- Mucl6 x CD3 (h!gG4) 1 1501 - - 2.28 1.67 0.11 0.1 0 13.8 10 2 3 BL Q BLQ 7 BL Q 1502 - - 3.12 1.71 0.25 0.2 9 7.9 24 2 4 BL Q 4 BL Q BL Q 1503 - - 3.84 1.58 0.21 0.1 7 6 5 2 BL Q BL Q BLQ 3 BL Q bs24 963D antiMu cl6 x CD28 (h!gG4) 1 2501 - - 3.07 2.40 0.13 0.2 0 0.4 3 3 BL Q BL Q 4 3 BL Q 2502 - - 1.97 2.73 0.10 0.1 5 0.1 4 3 3 BL Q 4 4 BL Q 2503 - - 1.64 3.0 5 0.10 0.1 9 0.2 7 3 4 46 6 4 BL Q REGN 4018 + bs24 963D anti- Mucló x CD3 -H anti- Mucl6 x CD28 1 + 1 3501 - - 2.89 1.98 0.19 0.1 0 13.3 24 3 4 47 4 13 BL Q 3502 - - 1.62 1.18 0.10 0.0 6 13.3 22 3 BL Q 80 5 7 BL Q 3503 - - 1.80 1.37 0.10 0.0 9 9.8 7 BL Q BL Q BL Q 4 9 BL Q REGN 4018 antiMucl6 x CD3 (h!gG4) 10 450 1 - - 2.48 0.89 0.13 0.1 2 14.2 11 4 4 28 4 3 BL Q 4502 - - 1.16 0.52 0.10 0.1 2 7.6 7 4 4 31 BLQ 6 BL Q 4503 - - 3.75 1.01 0.23 0.2 1 2.5 5 4 4 38 4 4 BL Q bs24 963D antiMucl6 x CD28 (h!gG4) 10 9501 - - 1.86 2.91 0.09 0.1 7 0.1 4 4 4 BL Q 5 3 BL Q 9502 - - 0.57 0.96 0.04 0.0 7 0.2 9 4 4 BL Q BLQ 4 BL Q 95 03 - - 1. 49 2.98 0.18 0.1 9 0.5 7 4 5 BL Q 4 3 BL Q REGN 4018 + bs24 963D anti- Mucl6 x CD3 + anti- Mucl6 x CD28 10 + 10 6501 - - 3.58 0.75 0.21 0.0 9 14.3 31 5 3 BL Q BLQ 7 BL Q 6502 - - 3.98 1.29 0.31 0.2 9 14.6 73 5 3 BL Q BLQ 38 BL Q 6503 - - 2.01 0.7 9 0.17 0.1 0 5.3 7 3 3 BL Q 4 BL Q BL Q REGN 4018 + bs24 963D anti- Mu cl6 x CD3 + anti - Mucl6 x CD28 1 + 10 5501 - - 1.70 1.37 0.14 0.2 3 14.2 6 4 4 36 BLQ 6 BL Q 5502 - - 3.11 3.24 0.18 0.1 7 5.5 7 5 4 BL Q BLQ 4 BL Q 5503 - - 2 .38 1.85 0.20 0.1 9 6.2 31 4 4 BL Q BLQ 2 BL Q REGN 4018 + bs24 963D anti- Mucl6 x CD3 + Mucl6 x CD28 1 + 1, repeated dose 8501 - - 3.36 1.04 0.26 0.0 5 14.4 12 4 4 BL Q 3 11 BL Q 850 2 - - 2.49 2.09 0.14 0.0 6 11.7 9 4 5 BL Q 5 7 BL Q 8503 - - 5.93 4.73 0.31 0.1 5 14.6 25 4 4 BL Q 4 4 BL Q BLQ: below the quantification limit LLOQ (lower limit of quantification): IFN-g = 37 pg / mL; TNF-α = 3 pg / mL; IL-2 = 2.4 pg / mL; IL-6 = 2 pg / mL; IL-8 = 1.7 pg / mL; IL-4 = 1.8 pg / mL; IL-10 = 3 pg / mL L / Rann / Lznz / E / Yi 200 Blood samples were collected for cytokine analysis, flow cytometry, and immunophenotyping. While CD28-SA administered to monkeys induced significant cytokine release, lymphocyte margination, and T cell activation, it was notable that no cytokine release, T cell margination, or T cell activation was observed after MUC16xCD28 administration ( Figures 8A-8C and Table 27). Overall, these preliminary observations suggest that TSAxCD28 bispecifics are well tolerated in primates and do not induce cytokine release or T cell activation as seen with CD28-SA (data not shown). It should be noted that previous studies with CD28-SA in monkeys failed to predict the profound cytokine release and T cell activation observed in humans (Tegenaro AG, www.circare.org / foia5 / tgnl412investigatorbrochure.pdf), and this was attributed to decreased CD28 expression in monkeys (D. Eastwood et al., Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells. Br J Pharmacol 161, 512-526 (2010)). Although tolerability studies in Macaca fascicularis might not be predictive of CRS in humans, the strong signals seen with CD28-SA in monkeys suggest that this is ί / βοπη / ίζηζ / Ε / γι 201 lost in Tegenaro et al. simply because they did not examine the first appropriate time points at which these responses can be robustly observed. Example 10: Binding of bs24963D (Ab against MUC16 X CD28, also called REGN5668) and REGN4018 (against MUC16 X CD3) to cell lines expressing human MUC16 or Macaca fascicularis, to primary cells of human PBMC and Macaca fascicularis and a cell line of T lymphocytes Materials and Methods: Summary of Experimental Procedures Flow cytometry analysis was used to determine the binding of bs24963D to human ovarian cancer cell lines (OVCAR-3 and PEO1) endogenously expressing human MUGI 6, and of bs24963D and REGN4018 to mouse ID8 cells modified by engineered to express human or Macaca fascicularis MUC16, 3T3 cells engineered to express human MUC16, human and Macaca fascicularis T cells, and the reporter T cell line engineered. Briefly, 1x105 cells / well were incubated for 30 min at 4°C with a serial dilution of antibodies including bs24963D, REGN4018 and control antibodies (IgG4p~PVA non-binding control mAb, bispecific antibody of L / fiann / Lznz / E / Yii 202 unbridged control against CD28, or parental controls against CD28 or CD3). Antibody dilutions ranged from 12.2 pM to 200 nM for human and Macaca fascicularis primary T cells and engineered indicator T cells, while 8.1 pM to 133 nM were chosen for MUC16+ target cells. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS followed by detection with a phycoerythrin (PE)-labeled anti-human IgG (MUGI6+ cells) or Alexa-labeled anti-human IgG antibody. 647 (CD28 + cells). Near-infrared reactive live / dead cell dye was added to human and Macaca fascicularis T-lymphocytes. Wells containing no antibody or containing only a secondary antibody were used as controls. After incubation with MUC16+ cells or the cell line J.RT3.T3.5 / NF-KB-Luc / lG4AB / hCD8ap / hCD28, cells were washed, resuspended in 200 pL of FACS buffer (cold PBS containing containing 1% filtered FBS and 1 mM EDTA) and analyzed by flow cytometry on a BD FACS Canto II. L / RQnn / Lznz / E / Yi 203 After incubation with human or Macaca fascicularis T cells, cells were washed and stained with a cocktail of anti-CD2, anti-CD16, anti-CD4 and anti-CD8 in FACS buffer for 20 min at 4°C. After washing, cells were resuspended in FACS buffer, sorted as live / CD2+ / CD4+ / CD16~ or live / CD2+ / CD8+ / CD16~ and analyzed by flow cytometry on a BD LSRFortessa-X20. For ECso determinations, the measured MFI values ​​were analyzed with a four-parameter logistic equation on a 9-point response curve with GraphPad Prism. The fold increase in maximum MFI was determined by taking the ratio of the highest detected MFI to the MFI of wells containing only the secondary antibody. Flow cytometry was also used to determine the binding of bs24963D and a commercial anti-PD-L1 antibody to MUC16+ human pancreatic cancer cells, SW1990 and SW1990 / hPD-L1 cells. Briefly, 2xl05 cells were incubated with 5 pL (66.7 nM) of bs24963D, anti-PD-L1 (2.5 pL) or AlexaFluor647-conjugated non-binding control antibody (bs24963D) or APC (anti-PD-L1) and incubated on ice. for 30 minutes. Cells were washed once with staining buffer, L / EQnn / Lznz / E / Yi 204 were centrifuged and washed with D-PBS. Cells were stained with 100 pL of a 1:1000 dilution of LIVE / DEAD Fixable violet viability dye and incubated for 15 minutes at room temperature. Cells were washed 3 times in staining buffer and resuspended in 100 µL of staining buffer and 1:1 Cytofix solution and analyzed by flow cytometry with the Cytoflex cytometer. The coefficient of binding on viability was calculated by dividing the MFI of the antibody of interest by the MFI of viability alone. Materials and methods NF-kB Luciferase Reporter Bioassay The ability of bs24963D to enhance TCR-mediated signaling was assessed in a reporter assay based on antigen presenting cells / engineered T lymphocytes as shown in Figure 10. TCRs recognize specific MHC / peptide complexes and activate lymphocytes. T through numerous transcription factors such as activator protein 1 (AP-1), nuclear factor of activated T lymphocytes (NFAT) or nuclear factor kappa-light chain enhancer of activated B lymphocytes (NF -κΒ) (Goldrath, 1999; Nature 402:255-62) (Shapiro, 1998; J. Immunology; 161(12):6455-8). The answer L / EQnn / Lznz / E / Yi 205 of T cells is further refined through the coupling of costimulatory receptors, such as CD28, which in turn is activated by its endogenous ligands, CD80 or CD86, and subsequently potentiates cellular signals, such as pathways controlled by the transcription factor NF-κΒ, after TCR activation. In this assay, engineered T cells are activated directly through the 1G4 TCR (IG4AB), which recognizes the NY-ESO-1 157-165 peptide (NYESOlp) in complex with the human MHC class I molecule, HLA-A2, and hp2M displayed in genetically engineered 3T3 antigen-presenting cells (Robbins, 2008; J. of Immunology; 180(9): 6116-31). TCR activation leads to luciferase production, which is driven by the transcription factor NF-κΒ in engineered reporter T cells. CD8 facilitates TCR / MHC interaction and promotes T cell activation by recruiting lymphocyte-specific protein tyrosine kinase (Lck) to the TCR / CD3 complex, thereby enhancing TCR signaling through phosphorylation of TCR motifs. tyrosine-activatable intracellular immunoreceptors (ITAM) (Colé, 2012; Immunology; 137(2):139-48) (Guirado, 2002; Biochem. Biophys. Res. Comm. 291(3):574-81). L / RQnn / Lznz / E / Yi 206 Two-fold serial dilutions of bs24963D, non-bridging control (non-anti-TAAxCD28 bispecific antibody) or non-binding control (39 pM at 10 nM) were added in duplicate to 5xl04 engineered reporter T cells (J.RT3.T3 .5 / NF-kB-Luc / lG4AB / hCD8ap / hCD28) per well in the presence of 1.5xl04 antigen-presenting cells that were either MUC16~ (3T3 / hp2M / HLA-A2 / NYES01p) or MUC16+ (3T3 / hp2M / HLAA2 / NYESOlp / hMUCl6). Antibody dilutions and bioassay were performed in complete medium (RPMI supplemented with 10% FBS and a cocktail of penicillin, streptomycin, and L-glutamine). Wells containing no antibodies were included as additional controls and used to calculate the fold increase in activity and EC50 values. Plates were incubated at 37 °C and 5% CO2 for 5 hours and then ONE-Glo luciferase substrate (100 pL) was added to each well. Luciferase activity was recorded as a luminescence signal with the ENVISION plate reader expressed as relative light units (RLU). The detected RLU values ​​were analyzed using a 4-parameter logistic equation on a 10-point response curve with GraphPad Prism. The maximum activation signal was determined as the mean of maximum RLU response detected within the range of L / RQnn / Lznz / E / Yi 207 antibody concentration evaluated. The fold increase in activity was calculated as the ratio of the mean of the highest RLU value recorded within the tested antibody concentration range over the mean of the RLU value recorded in the absence of antibody. T cell activation assays for T cell proliferation and IL-2 release The ability of bs24963D to mediate IL2 release and T cell proliferation in the presence of a constant concentration of REGN4018 (assessed in OVCAR-3 and PEO1 human ovarian cancer cell lines) or in the presence of a constant concentration of cemiplimab ( evaluated in human pancreatic cancer cell lines [SW1990 and SW1990 / hPD-L1]) was determined by T cell activation assays with spiked human primary T cells from 3 or 2 donors, respectively. Isolation of human primary T cells Human PBMC were isolated from leukocyte concentrates from 4 healthy donors. For donors 555014 and 555109, PBMC were isolated from peripheral blood by density gradient centrifugation. Briefly, 15 mL of Ficoll Plaque Plus was added to 50 mL conical tubes and subsequently 30 mL of blood diluted 1:1 was layered with PBS containing L / RQnn / Lznz / E / Yii 208 contained 2% FBS on top. After a 30 minute centrifugation at 400 x g, without the brake, the mononuclear cell layer was transferred to a fresh tube, diluted 5x with BBS containing 2% FBS and spun for 8 minutes at 300 x g. For donors 555131 and 555129, peripheral blood PBMCs were isolated from a healthy donor using the EasySep Direct Human PBMC Isolation Kit from Stem Cell Technologies and according to the manufacturer's protocol. Isolated PBMCs were frozen in FBS containing 10% DMSO. For the isolation of CD3+ T cells, frozen vials of PBMC were thawed in a 37°C water bath and diluted in stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA and 0.01 mM β-mercaptoethanol [BME]) containing 50 U / mL of Benzonase® nuclease. Cells were centrifuged at 1200 rpm for 10 minutes, resuspended in EasySep buffer and isolated with StemCell Technologies EasySep T cell isolation kit according to the manufacturer's protocol. T cell activation assay with human OVCAR-3, PEO1, SW1990, SW1990 / hPD-L1 cells and human primary T cells CD3+ T cells, which were resuspended in ί / βοπη / ίζπζ / Ε / γι 209 stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA and 0.01 mM BME), were plated in 96-well round bottom plates at a concentration of 1x105 cells / well. OVCAR3, PEO1, SW1990 or SW1990 / hPD-Ll cells were treated with 25 pg / mL (OVCAR-3), 10 pg / mL (PEO1) or 30 pg / mL (SW1990 and SW1990 / hPD-Ll) mitomycin C to stop proliferation. After incubation for 1 hour at 37°C, 5% CO2, mitomycin C-treated cells were washed 3 times with D-PBS containing 2% FBS, followed by a final resuspension in stimulation medium. OVCAR-3, PEO1, SW1990 and SW1990 / hPD-II cells were added to wells containing CD3+ T cells at a final concentration of IxlO4, 2.5xl04 cells or 5xl04 cells, respectively, for OVCAR-3, PEO1 and both SW1990 cells. A constant concentration of REGN4018 or non-bridged control bispecific antibody against CD3 (5 nM), or cemiplimab or non-binding IgG4ps control (20 nM), was added to wells containing OVCAR-3, PEO1, SW1990, or SW1990 / hPD cells. -ll. Subsequently, bs24963D antibodies, non-anti-TAAxCD28 control or non-fixing control, were titered from 7.6 pM to 500 nM in a 1:4 serial dilution and added to the wells. The end point of the 10-point concentration curve did not contain antibodies and was used to calculate the L / RQnn / Lznz / E / Yi 210 times increase in activity. After incubating the plates for 72 (OVCAR-3 and PEO1) or 96 (SW1990 and SW1990 / hPD-Ll) hours at 37°C, 5% CO2, 50 pL of medium supernatant was collected to measure IL release. -2 before treatment with [methyl-3H]-thymidine to quantify proliferation. For IL-2 release, 5 pL (for assays using OVCAR-3 and PEO1 cells) or 20 pL (for assays using SW1990 and SW1990 / hPD-L1 cells) of supernatant was assayed with the IL-2 AlphaLISA kit. 2 human according to the manufacturer's protocol. IL-2 measurements were acquired on the Perkin Elmer Envision Multi-Label Plate Reader and reported as relative fluorescence units (RFU). For proliferation assays, 50 pL of [methyl-3H]-thymidine diluted to 2 mCi / mL in stimulation medium was added to the wells and the plates were incubated for 6 hours (for assays using OVCAR-3 and PEO1 cells). ) or 16 hours (for assays using SW1990 and SW1990 / hPD-L1 cells). [Methyl-3H]-thymidine will be incorporated in higher amounts in dividing cells. After incubation, cells were harvested on filter plates and prepared for measurement on the TopCount NXT microplate luminescence and scintillation counter instrument. L / RQnn / Lznz / E / Yi 211 All serial dilutions were assessed in triplicate for IL-2 release and proliferation. Antibody EC50 values ​​were determined from a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software. Peak levels of IL-2 release and proliferation are given as the mean of maximal response detected within the dose range evaluated. The fold increase in maximal IL-2 release or bs24963D-mediated T cell proliferation relative to maximal IL-2 release or proliferation mediated by no antibody was calculated. The ability of bs24963D to activate T cells was evaluated in an assay where signal 1 is provided by stimulatory antigen-presenting cells. This assay used J.RT3.T3.5 reporter T cells engineered to express human CD8, Human CD28, a TCR described in the literature (1G4) that recognizes a peptide NY-ESO-1 (NYESOlp) in complex with HLA-A2, and a luciferase reporter of NF-κΒ. Stimulatory antigen-presenting cells providing signal 1 were 3T3 cells engineered to express HLA-A2, Ββ2Μ and NYESOlp with or without human MUGI6 (hMUC16). A non-bridged control bispecific antibody was tested against CD28 (not antiL / EQnn / Lznz / E / Yi 212 TAAxCD28) and a non-binding control mAb (IgG4p~PVA) in parallel with bs24963D. NF-κΒ signaling was measured with a luminescent reagent to detect luciferase reporter activity. The results are summarized in Table 28. In this test system, bs24963D mediated a concentration-dependent increase in NF-κΒ signaling on reporter T cells in the presence of MUC16+ antigen-presenting cells; no activity was observed with cells lacking MUC16 expression (Figures 11A and 11B). No increase in NF-κΒ signaling was observed with the non-bridged control bispecific antibody against CD28. Table 28: Summary of NF-kB luciferase activation mediated by bs24963D ί / βοπη / ίζηζ / Ε / γι Antibody Antigen-presenting cells (+ / - MUC16) 3Τ3 / Ηβ2Μ / ΗΙΛA2 / NYESOlp / hMUCl6 3T3 / hp2M / HLA-A2 / NYESOlp ECso (M) max RLU a Increase in times ECso (M) RLU max a Increase in times bs24963D 2.88x10' 10 254,300 2.11 ND 65,920 1.00 No antiTAAxCD28 ND 133,220 1.24 ND 64,740 1.02 aMax RLU is the mean of the highest RLU value observed within the tested antibody concentration range (39 pM to 10 nM). bThe fold increase in peak RLU mediated by bs24963D or no anti-TAAxCD28 relative to peak RLU mediated by no antibody was calculated. Abbreviations: ND, not determined because no concentration-dependent increase in luciferase activity was observed 213 Example 11. Assessment of IL-2 release and proliferation of human primary T lymphocytes mediated by bs24963D (anti-MUC16 X anti-CD28) in the presence or absence of REGN4018 (anti-MUC16 X anti-CD3) or cemiplimab (a antagonist antibody against PD-1) The ability of bs24963D to activate primary human T cells, as determined by IL-2 release and T cell proliferation, was assessed in the presence of 2 different MUC16+ human ovarian cancer cell lines (OVCAR-3 and PEO1). As these cells do not provide sufficient signal 1 of an allogeneic response, a fixed concentration of REGN4018 (a bispecific antibody against MUC16 X CD3) was included to provide signal 1. Results for OVCAR-3 and PEO1 cells are summarized in Table 29 for IL-2 release and Table 30 for proliferation. The ability of bs24963D to activate primary human T cells, as determined by IL-2 release and T cell proliferation, was assessed in the presence of a human pancreatic cancer cell line MUC16+(SW1990) and SW1990 engineered to overexpressing human PD-L1 (SW1990 / hPD-L1). Both cell lines provide an allogeneic response that is sufficient for L / RQnn / Lznz / E / Yi 214 serve as signal 1. In addition, the ability of fixed concentrations of cemiplimab (20 nM) to enhance the effects of bs24963D was also evaluated. Results for SW1990 and SW1990 / hPD-II cells are summarized in Table 31 for IL-2 release and Table 32 for proliferation. Ability of bs24963D (REGN5668) to enhance IL-2 release and proliferation of human primary T cells in the presence or absence of REGN4018 with OVCAR-3 and PE 01 target cells When incubated with OVCAR-3 and PEO1 cancer cells, bs24963D mediated the concentration-dependent enhancement of IL-2 release (Figure 12) and proliferation of (Figure 13) human T cells only in the presence of REGN4018. Control non-bridged bispecific antibodies against CD3 and CD28 did not enhance IL-2 release in the presence or absence of REGN4018. In this assay, 5 nM REGN4018 alone did not increase IL-2 release, but did show a modest enhancement of T-cell proliferation relative to the non-binding control. L / RQnn / Lznz / B / Yi 215 Table 29: Summary of bs24963D-mediated enhancement of IL-2 release from human primary T cells in the presence or absence of REGN4018 with OVCAR-3 and PEO1 target cells L / Rann / Lznz / E / Yi Target cell line Donor Ab at fixed concentration (5 nM) Antibody tested at variable concentrations (7.6 pM at 500 nM) IL-2 release from primary human T cells EC5o (M) IL-2 max (RFU) IL-2 fold increaseb (IL -2) OVCAR- 3 Donor 555014 REGN4018 bs24963D 7 . Ο7χ1Ο-10 46,931 20.01 Not anti-TAAxCD28 NC 4,741 1.89 Not antiTAAxCD3 bs24963D NO 1, 361 2.75 Not anti-TAAxCD28 NC 3, 033 6.62 Donor 555109 REGN4018 bs24 963D 1.22 xl0~9 36,725 32.10 No anti-TAAxCD28 NC 1, 638 1.38 No antiTAAxCD3 bs24 963D ND 893 1.82 No anti-TAAxCD28 NC 1,403 2.84 Donor 555131 REGN4018 bs24 963D 5.90 xlO-10 46,209 13.08 No anti-TAAxCD28 NC 4, 443 1.33 No antiTA AxCD3 bs24 963D NC 1,814 2.71 No anti-TAAxCD28 NC 3, 136 5.95 PEO1 Donor 555014 REGN4018 bs24 963D 1.95x10-' 31024 30.30 Not anti-TAAxCD28 NC 2312 2.16 Not antiTAAxCD3 bs24 963D NC 2490 5.56 Not anti-TAAxCD28 NC 2776 6.12 Donor 555 109 REGN4018 bs24 963D 3.10x10-' 16,421 20.03 No anti-TAAxCD28 NA 897 1.33 Not antiTAAxCD3 bs24 963D NC 2, 039 4.36 Not anti-TAAxCD28 NC 1, 360 2.35 Donor 555014 REGN4018 bs24 963D 2.42 xlO-' 29,217 25.70 Not anti-TAAxCD28 NC 2, 175 1.8 2 No antiTAAXCD3 bs24963D NC 2,911 5.77 No anti-TAAxCD28 NC 2,443 4.42 aMaximum IL-2 concentration is the mean of the highest IL-2 concentration value recorded within the tested antibody concentration range. bThe fold increase in bs24963D-mediated maximal IL-2 release, in the presence or absence of REGN4018, relative to no antibody-mediated maximal IL-2 release was calculated. Abbreviations: NO, not calculated because the data does not fit a 4-parameter logistic equation; ND, not determined because a concentration-dependent increase in IL-2 release was not observed. 216 Table 30: Summary of bs24963D-mediated enhancement of human primary T cell proliferation in the presence or L / Rann / Lznz / E / Yi absence of REGN4018 with OVCAR-3 and PEO1 target cells Target cell line Donor Ab at fixed concentration (5 nM) Antibody evaluated at variable concentrations (7.6 pM at 500 nM) EC5o T-lymphocyte proliferation (M) Max proliferation (CPM) Fold increaseb (proliferation) OVCAR- 3 Donor 555014 REGN4018 bs24963D 8 . 89X1Q-11 13,968 2.03 Not antiTAAxCD28 NC 11,260 1.59 Not antiTAAxCD3 bs24963D NC 336 1.38 Not antiTAAxCD28 NC 579 1.60 Donor 555109 REGN4018 bs24963D 9.45 xlO-11 14,818 3.18 Not antiTAAxCD28 4.61 xlO-8 8,141 1.66 Not antiTAAxCDS bs24963D ND 416 1.37 Not antiTAAxCD28 ND 475 1.33 Donor 555131 REGN4018 bs24963D 2.47 xlO-11 13,607 1.66 Not antiTAAxCD28 NC 10,154 1.16 Not antiTAAxCD3 bs24963D NC 622 1.82 Not antiTAAxCD28 NC 562 1.68 PEDI Donor 5550 14 REGN4018 bs24963D 1.73 xlO-10 9,605 3.27 Not antiTAAxCD28 NC 6, 953 2.26 Not antiTAAxCD3 bs24963D NC 603 2.58 Not antiTAAxCD28 NC 551 3.09 Donor 555109 REGN4018 bs24963D 4.04 xlO-10 10,304 8.42 Not antiTAAxCD28 NC 4,888 4.62 Not antiT7\AxCD3 bs24963D NC 733 2.84 Not antiTAAxCD28 NC 419 1.72 Donor 555014 REGN4018 bs24963D 2.22 xlO-10 10,335 3.69 Non-antiTAAxCD28 4.22 xlO-8 5,631 2.06 No antiTAAxCD3 bs24963D NC 835 4.44 No antiTAAxCD28 NC 523 2.29 aMaximum proliferation is the mean of the highest CPM value recorded within the tested antibody concentration range. bThe fold increase in bs24963D-mediated maximal T cell proliferation, in the presence or absence of REGN4018, relative to maximal proliferation in the absence of bs24963D or non-anti-TAAxCD28 control, was calculated. Abbreviations: NO, not calculated because data do not fit a 4-parameter logistic equation, ND, not determined because no concentration-dependent increase in proliferation was observed 217 Ability of bs24963D (REGN5668) to enhance IL-2 release and proliferation of human primary T cells in the presence or absence of cemiplimab with SW1990 and SW1990 / hPD-L1 target cells When incubated with MUGI6+ SW1990 and SW1990 / hPD-L1 human pancreatic cancer cells, bs24963D mediated the concentration-dependent enhancement of IL-2 release (Figure 14) and proliferation of (Figure 15) human T cells in presence and absence of cemiplimab. Overexpression of human PD-L1 in SW1990 cells suppressed IL-2 and T cell proliferation in the presence of bs24963D and these were modestly increased with the addition of cemiplimab. At high concentrations, the control non-bridged bispecific antibody against CD28 mediated some IL-2 release in the presence of SW1990 and SW1990 / hPD-L1 cells. In the absence of bs24963D, cemiplimab did not increase IL-2 release or T cell proliferation relative to the control non-bridged bispecific antibody against CD28. Table 31: Summary of bs24963D-mediated enhancement of IL-2 release from human primary T cells in the presence or absence of cemiplimab with SW1990 and SW1990 / hPD-Ll ί / βΟΠΠ / ί7Π7 / Β / ΥΙ target cells Target cell line Donor Ab at fixed concentration (20 nM) Antibody tested at variable concentrations (7.6 pM at 500 nM) IL-2 release from human primary T cells ECso (M) IL-2 max (RFU) IL-2 fold increaseb (IL -2) SW1990 Donor 555109 cemiplimab bs24963D 2.98x10'9 3,773 3.31 No anti-TAAxCD28 ND 1,415 1.98 Control of lgG4p bs24963D 2.31x10'9 2,922 4.83 No anti-TAAxCD28 ND 944 1.31 Donor 555129 cemiplimab bs24963D 8.60x10'10 4,833 3.39 No anti -TAAxCD28 NC 2,927 2.28 lgG4p control bs24963D 1.21x10 9 3,589 2.89 No anti-TAAxCD28 NC 2,027 1.51 SW1990 / hPD- L1 Donor 555109 cemiplimab bs24963D 1.01x10'9 1,692 2.73 No anti-TAAxCD28 NC 1,102 1.60 lgG4p control bs24963D 2.55x10' 9 1,053 1.70 No anti-TAAxCD28 NA 616 1.23 Donor 555129 cemiplimab bs24963D 1.41x10'9 3,391 2.33 No anti-TAAxCD28 NC 1,977 1.57 lgG4p control bs24963D 2.22x109 2,161 2.14 No anti-TAAxCD28 NA 1,459 1.49 L / RQnn / Lznz / E / YiaThe maximum IL-2 concentration is the mean of the highest IL-2 concentration value recorded within the tested antibody concentration range. bThe fold increase in bs24963D-mediated maximal IL-2 release, in the presence or absence of cemiplimab, relative to no antibody-mediated maximal IL-2 release was calculated. Abbreviations: NC, not calculated because the data does not fit a 4-parameter logistic equation; ND, not determined because a concentration-dependent increase in IL-2 release was not observed. 219 Table 32: Summary of bs24963D-mediated enhancement of human primary T cell proliferation in the presence or absence of cemiplimab with SW1990 and SW1990 / hPDL1 target cells L / EQnn / Lznz / E / Yi 220 Target cell line Donor Ab at fixed concentration (20 nM) Antibody evaluated at variable concentrations (7.6 pM at 500 nM) Proliferation of T lymphocytes EC50 (M) Max proliferation (CPM) Increase in timesb (proliferation) SW1990 Donor 555109 cemiplimab bs24963D 1.68x10- 557 4.52 No antrTAAxCD28 ND 161 1.41 IgG4p control bs24963D 2.86x10- 421 4.36 No antiTAAxCD28 ND 123 1.46 Donor 555129 cemiplimab bs24963D 4.59x10“ 10 545 2.47 No antrTAAxCD28 ND 279 1.66 IgG4p control bs24963D 4.83x10“ 10 569 2.17 No antiTAAxCD28 NA 353 1.46 SW1990 / H PD-L1 Donor 555109 cemiplimab bs24963D 1.40x10- 279 4.70 No antrTAAxCD28 NA 151 1.98 IgG4p control bs24963D 1.54 x10“ 9 140 2.35 No antiTAAxCD28 ND 84.0 1.42 Donor 555129 cemiplimab bs24963D 2.53x10- 601 3.20 No antrTAAxCD28 NC 222 1.73 IgG4p control bs24963D 1.66x10“ 333 2.74 No antrTAAxCD28 ND 146 1.62 aMaximum proliferation is the mean of the highest CPM value recorded within the tested antibody concentration range. bThe fold increase in bs24963D-mediated maximal T cell proliferation, in the presence or absence of cemiplimab, relative to maximal proliferation in the absence of bs24963D or non-anti-TAAxCD28 control, was calculated. Abbreviations: NC, not calculated because the data does not fit a 4-parameter logistic equation; ND, not determined because no concentration-dependent increase in proliferation was observed l i «αηη / Lznz / E / YiA 221 The scope of the present invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention, in addition to those described herein, will be apparent to the mid-level trade person from the above description and accompanying figures. Such modifications are intended to be within the scope of the L / fiann / Lznz / E / Yii appended claims.

Claims

1. An isolated bispecific antigen-binding molecule comprising: a) a first antigen-binding domain (DI) that binds to human CD28 with a Kd of less than about 2x10~7M as measured by surface plasmon resonance at 37°C; and b) a second antigen-binding domain (D2) that binds specifically to a human mucin 16 membrane antigen (MUGI6) on a target tumor cell, with a KD of less than about 10-9 M as measured by surface plasmon resonance at 37°C.

2. The bispecific antigen-binding molecule isolated according to claim 1, wherein the bispecific antigen-binding molecule binds to the surface of human T lymphocytes with an ECsO of less than about 10-5 M as measured by an in vitro FACS binding assay.

3. The bispecific antigen-binding molecule isolated according to claim 1, wherein the bispecific antigen-binding molecule binds to the surface of Macaca fascicularis T lymphocytes with an ECso of less than about 6xlCu6 M as measured by an L / EQnn / Lznz / E / Yi 223 FACS binding assay in vitro.

4. The bispecific antigen-binding molecule isolated according to claim 1, wherein the bispecific antigen-binding molecule is bound to the surface of MUC16-expressing cell lines with an ECso of less than about 10~9 M as measured by an in vitro FACS binding assay.

5. The bispecific antigen-binding molecule isolated according to claim 1, wherein the bispecific antigen-binding molecule demonstrates a costimulatory effect when used together with a bispecific anti-MUCl6xCD3 antibody and is evaluated in target cells expressing MUC16.

6. The bispecific antigen-binding molecule isolated according to claim 5, wherein the costimulatory effect is shown by one or more of the following: (a) the ability to activate and direct human T lymphocytes to inactivate a target cell expressing MUC16; (b) the ability to upregulate PD-1 on T lymphocytes; (c) the ability to increase the release of IFN gamma cytokines from PBMCs; (d) the ability to decrease tumor cells; (f) the ability to increase tumor clearance; and / or (g) the lack of induction of systemic T lymphocyte activation.

7. The bispecific antigen-binding molecule isolated according to claim 6, wherein the costimulatory effect is further shown by (g) measuring IL-2 cytokine production with a functional primary CD4+ T lymphocyte / APC assay.

8. The bispecific antigen-binding molecule isolated according to any of claims 1-7, wherein the target tumor cell is an ovarian cancer cell.

9. The bispecific antigen-binding molecule isolated according to any of claims 1-8, wherein the first antigen-binding domain (DB) comprises: a) three heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and 42; and b) three light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence selected from the group L / RQnn / Lznz / E / Yi 225 consisting of SEQ ID NO: 10 and 34.

10. The bispecific antigen-binding molecule isolated according to claim 9, comprising: a) an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 44, an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 46, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and 48.

11. The bispecific antigen-binding molecule isolated according to claim 10, comprising: a) an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38, and an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

12. The bispecific antigen-binding molecule isolated according to claim 9, wherein the first antigen-binding domain comprises: a) a set of HCVR CDRs (HCDR1, HCDR2, HCDR3), wherein the set comprises amino acid sequences selected from the group consisting of SEQ ID NO: 20, 22, 24 L / EQnn / Lznz / E / Yi 226 and 44, 46, 48, and a set of LCVR CDRs (LCDR1, LCDR2, LCDR3), wherein the set comprises amino acid sequences selected from the group consisting of SEQ ID NO: 12, 14, 16 and 36, 38, 40.

13. The bispecific antigen-binding molecule isolated according to claim 9, wherein the first antigen-binding domain comprises an HCVR / LCVR pair comprising amino acid sequences selected from the group consisting of SEQ ID NO: 18 / 10; and 42 / 34.

14. The bispecific antigen-binding molecule isolated according to any of claim 113, wherein the second antigen-binding domain comprises: a) three HCDRs contained within an HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 26; and b) three LCDRs contained within an LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34.

15. The bispecific antigen-binding molecule isolated according to claim 14, wherein the second antigen-binding domain comprises: a) an HCDR1 comprising an amino acid sequence L / RQnn / Lznz / B / Yi 227 selected from the group consisting of SEQ ID NO: 4 and 28; b) an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 30; and c) an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 32.

16. The bispecific antigen-binding molecule isolated according to claim 15, wherein the second antigen-binding domain comprises: a) an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38, and an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

17. The bispecific antigen-binding molecule isolated according to claim 16, wherein the second antigen-binding domain comprises: a) a set of HCVR CDRs (HCDR1, HCDR2, HCDR3), wherein the set comprises amino acid sequences selected from the group consisting of SEQ ID NO: 4, 6, 8 and 28, 30, 32; and a set of LCVR CDRs (LCDR1, LCDR2, LCDR3), wherein the set comprises amino acid sequences selected from the group consisting of SEQ ID NO: 12, 14, 16 L / EQnn / Lznz / E / Yi 228 and 36, 38, 40.

18. The bispecific antigen-binding molecule isolated according to any of claims 1 to 17, comprising: a) a first antigen-binding domain comprising HCVR CDRs comprising amino acid sequences of SEQ ID NO: 20, 22, 24, and LCVR CDRs comprising amino acid sequences of SEQ ID NO: 12, 14, 16; and b) a second antigen-binding domain comprising HCVR CDRs comprising amino acid sequences of SEQ ID NO: 4, 6, 8, and LCVR CDRs comprising amino acid sequences of SEQ ID NO: 12, 14, 16.

19. The bispecific antigen-binding molecule isolated according to any of claims 1 to 17, comprising: a) a first antigen-binding domain comprising HCDRs comprising the amino acid sequences of SEQ ID NO: 44, 46, 48, and LCDRs comprising the amino acid sequences of SEQ ID NO: 36, 38, 40; and b) a second antigen-binding domain comprising HCDRs comprising the amino acid sequences of SEQ ID NO: 28, 30, 32, and LCDRs comprising the amino acid sequences of SEQ ID NO: 36, 38, 40.

20. The bispecific antigen-binding molecule isolated according to any of claims 1 to 17, comprising: a) a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 18 / 10; and b) a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 2 / 10.

21. The bispecific antigen-binding molecule isolated according to any of claims 1 to 17, wherein a) the first antigen-binding domain comprises an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 42 / 34; and b) the second antigen-binding domain comprises an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 26 / 34.

22. An isolated bispecific antigen-binding molecule that competes for binding to MUC16, or binds to the same epitope on MUC16 as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain having an HCVR / LCVR pair that L / RQnn / Lznz / E / Yi 230 comprises the amino acid sequences of SEQ ID NO: 18 / 10 or 42 / 34 and a second antigen-binding domain having an HCVR / LCVR pair comprising the amino acid sequences of either SEQ ID NO: 2 / 10 or 26 / 34.

23. An isolated bispecific antigen-binding molecule that competes for binding to human CD28, or binds to the same epitope on human CD28 as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain having an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 18 / 10 or 42 / 34 and a second antigen-binding domain having an HCVR / LCVR pair comprising the amino acid sequences of either SEQ ID NO: 2 / 10 or 26 / 34.

24. A pharmaceutical composition comprising the bispecific antigen-binding molecule according to any of claims 1 to 23, and a pharmaceutically acceptable carrier or diluent.

25. A nucleic acid comprising a nucleotide sequence encoding a bispecific antibody according to any of claims 1 to 23.

26. An expression vector comprising the nucleic acid L / RQnn / Lznz / E / Yi according to claim 25. 231 27. A host cell comprising the expression vector according to claim 26.

28. A method for inhibiting the growth of an ovarian cell tumor in a subject, comprising administering to the subject an isolated bispecific antibody according to any of claims 1 to 23 or a pharmaceutical composition according to claim 26.

29. The method according to claim 28, further comprising administering a second therapeutic agent.

30. The method according to claim 29, wherein the second therapeutic agent comprises an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, a checkpoint inhibitor, or combinations thereof.

31. A method for treating a patient suffering from ovarian cancer, or other cellular neoplasm expressing MUC16, comprising administering to the subject a bispecific antibody isolated according to any of claims 1 to 23 or a pharmaceutical composition according to claim 24.

32. The method according to claim 31, further comprising administering a second therapeutic agent.

33. The method according to claim 32, in L / RQnn / Lznz / E / Yi 232 wherein the second therapeutic agent comprises an antitumor agent, radiotherapy, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, a checkpoint inhibitor or combinations thereof.

34. The method according to any of claims 29 or 32, wherein the second therapeutic agent is a different bispecific antibody comprising a first antigen-binding domain that binds to the same tumor target antigen and a second antigen-binding domain that binds to CD3 on T lymphocytes.

35. The bispecific antigen-binding molecule isolated according to any of claims 123, wherein the antigen-binding molecule induces T-lymphocyte-mediated cytotoxicity of human ovarian cells.

36. A bispecific antigen-binding molecule comprising a first antigen-binding domain that binds specifically to human CD28, and a second antigen-binding domain that binds specifically to human MUGI6.

37. The bispecific antigen-binding molecule according to claim 36, wherein the antigen-binding molecule binds to human T lymphocytes expressing CD28 with an EC50 value of between 1xlO-12 M to 10xlO-6 ί / βΟΠΠ / ί7Π7 / Β / YI 233 M.

38. The bispecific antigen-binding molecule according to claim 37, wherein the antigen-binding molecule binds to human T lymphocytes expressing CD28 with an ECso value of between 1x10-9 M to 10x10~6 M.

39. The bispecific antigen-binding molecule according to any of claims 36 to 38, wherein the antigen-binding molecule binds to human cells expressing human CD28 and Macaca fascicularis cells expressing Macaca fascicularis CD28.

40. The bispecific antigen-binding molecule according to any of claims 36 to 38, wherein the antigen-binding molecule induces cytokine release and upregulation of CD25 in human whole blood.

41. The bispecific antigen-binding molecule according to any of claims 36 to 38, wherein the antigen-binding molecule induces T-lymphocyte-mediated cytotoxicity of human ovarian cells.

42. The bispecific antigen-binding molecule according to any of claims 36-41, wherein the first antigen-binding domain that binds specifically to human CD28 comprises the heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and 42, and the light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34.

43. The bispecific antigen-binding molecule according to any of claims 36-42, wherein the second antigen-binding domain that binds specifically to human MUGI 6 comprises the heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising SEQ ID NO: 2 and 26, and the light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34.

44. The bispecific antigen-binding molecule L / RQnn / Lznz / E / Yi 235 according to any one of claims 36-42, wherein the first antigen-binding domain that binds specifically to human CD28 comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 44; wherein HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 46;wherein HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and 48, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38 and wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.; 45. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the second antigen-binding domain that binds specifically to human MUC16 comprises three heavy chain complementarity-determining regions L / RQnn / Lznz / B / Yi 236 (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 28; wherein HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 30;wherein HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 32, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38, and wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

46. ​​The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the first antigen-binding domain that binds specifically to human CD28 comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), and wherein the second antigen-binding domain that binds specifically to human MUGI6 comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3); wherein the first antigen-binding domain comprises an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 44;wherein HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 46; wherein HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and 48, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38 and wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40;and wherein the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 28, wherein HCDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 30, wherein HCDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 32, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38, and wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

47. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein comprising three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 44; wherein HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 46;wherein HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and 48, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 36, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38 and L / EQnn / Lznz / E / Yi 239, wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

48. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and 42, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34.

49. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the second antigen-binding domain competes for binding to human MUC16 with a reference antigen-binding protein comprising three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence selected from the group consisting of SEQ IDs 4 and 28, wherein HCDR2 comprises the amino acid sequence L / RQnn / Lznz / E / Yi 240 selected from the group consisting of SEQ IDs 6 and 30, wherein HCDR3 comprises the amino acid sequence selected from the group consisting of SEQ IDs 8 and 32, and wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ IDs 12 and 36,wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 38 and wherein LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 40.

50. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the second antigen-binding domain competes for binding to human MUC16 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 26, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34.

51. The bispecific antigen-binding molecule according to any of claims 36 to 42, wherein the first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein L / EQnn / Lznz / E / Yi 241 comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ IDs 18 and 42, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ IDs 10 and 34, and wherein the second antigen-binding domain competes for binding to human MUC16 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ IDs 2 and 26.and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 34., 52. A pharmaceutical composition comprising a bispecific antigen-binding molecule according to any of claims 36 to 51 and a pharmaceutically acceptable carrier or diluent.

53. A method for treating a type of ovarian cancer in a subject, wherein the method comprises administering to the subject the pharmaceutical composition according to claim 52. L / RQnn / Lznz / E / Yi 54. A method according to any of claims 28-34 and 53, wherein the bispecific antigen-binding molecule is administered in a fixed dose.