Bispecific antibodies against human PD-L1 and PD-L2 and methods of use thereof

Abstract

The present disclosure is directed to bispecific antibodies that bind to both PD-L1 and PD-L2 and methods of using such antibodies to treat cancer, e.g., cancers that express or overexpress PD-L1, PD-L2, or both.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 326,456, filed April 1, 2022, and U.S. Provisional Patent Application No. 63 / 378,196, filed October 3, 2022, the entire contents of both applications being incorporated herein by reference.

[0002] Sequence Listing Reference This application contains an electronically submitted Sequence Listing XML, which is incorporated herein by reference in its entirety. The Sequence Listing XML, created on March 27, 2023, is named UTFCP1514WO.xml and is approximately 30 kilobytes in size.

[0003] Field The present disclosure relates generally to the fields of medicine, oncology, and immunology, and more specifically to a human bi-antibody with high affinity for PD-L1 and PD-L2, and its use in cancer treatment. [Background technology]

[0004] background Blockade of the interaction of the T cell co-inhibitory receptor PD-1 with its ligand PD-L1 has become a mainstay of modern oncology and is now available even in the first-line setting for a subset of melanoma and lung cancer patients (Boussiotis, 2016). Numerous antibodies targeting PD-1 or PD-L1 are currently FDA approved or in clinical trials; however, agents targeting the second PD-1 ligand, PD-L2, have not been clinically investigated. PD-L2 binds PD-1 with approximately three-fold higher affinity than PD-L1 and, like PD-L1, delivers inhibitory signals that attenuate T cell function (Cheng et al., 2013; Latchman et al., 2001; Lee et al., 2016; Li et al., 2017; Youngnak et al., 2003). Historically, PD-L2 was thought to be primarily an inducible co-inhibitory molecule whose expression was restricted to the tumor stroma; however, improved PD-L2 detection reagents have revealed widespread PD-L2 expression in both the tumor microenvironment and tumor cells themselves (Baptista et al., 2016; Danilova et al., 2016; Derks et al., 2015; Dong et al., 2016; Howitt et al., 2016; Kim et al., 2015; Kim et al., 2015; Nomi et al., 2007; Obeid et al., 2016; Ohigashi et al., 2005; Roemer et al., 2016; Shi et al., 2014; Shin et al., 2015; Xu et al., 2016). Recently, PD-L2 has been shown to be an independent predictor of response to the PD-1 antibody pembrolizumab in multiple cancers ( Yearley et al., 2017 ).

[0005] Amplification of chromosomal region 9p24.l, first described in the majority of classical Hodgkin lymphomas (cHL), leads to direct upregulation of PD-L1 and PD-L2 (present there), as well as indirect induction via enhanced JAK2 activity (Roemer et al., 2016; Shi et al., 2014; Green et al., 2010; Van Roosbroeck et al., 2016). In addition to cHL, this genetic driver of high PD-L1 / PD-L2 co-expression is also found in the majority of primary mediastinal large B-cell lymphomas (PMBL), T-cell lymphomas, and a variety of histiocytic and dendritic cell malignancies. Not surprisingly, many of these cancers have been shown to respond to PD-1 blockade. More recently, 9p24.1 amplification has been demonstrated in solid tumors, e.g., triple-negative breast cancer (TNBC) ( Howitt et al., 2016 ; Barrett et al., 2015 ). Relatively high co-expression of PD-L1 and PDL2 has also been observed in numerous other cancers, including gastric cancer, melanoma, lung, head and neck, cervical, and vulvar squamous cell carcinoma, bladder cancer, and hepatocellular carcinoma, among others (Baptista et al., 2016; Danilova et al., 2016; Derks et al., 2015; Dong et al., 2016; Howitt et al., 2016; Kim et al., 2015; Nomi et al., 2007; Obeid et al., 2016; Xu et al., 2016; Yearley et al., 2017; Van Roosbroeck et al., 2016; Barrett et al., 2015; Shin et al., 2016; Inoue et al., 2016; Wang et al., 2011). In addition to expression by the tumor itself, stromal and endothelial expression of PD-L2 has also been documented for many of these tumors (Yearley et al., 2017). These findings suggest limited therapeutic potential for PD-L1 blockade in these cancers.

[0006] The PD-1 co-inhibitory receptor is expressed primarily by activated T cells and NK cells and can be targeted by antibodies that bind to it and prevent engagement by PD ligands. In contrast, PD-L1 is expressed by tumor cells and suppressive stromal populations and can be targeted by antibodies capable of cytotoxic effector function. Although the theoretical benefits of these PD-L1 antibodies with antibody-dependent cellular cytotoxicity (ADCC) capabilities can be demonstrated in vitro, there are no patient data demonstrating actual effector function in patients or improved outcomes compared to blocking-only variants (Boyerinas et al., 2015).

[0007] PD-L1 and PD-L2 share only about 40% identity because each binds to an additional receptor distinct from PD-1 (Latchman et al., 2001). PD-L1 also binds to B7-1 in an additional negative T cell regulatory interaction (Butte et al., 2007; Butte et al., 2008). In mice, PD-L2 can bind to RGMb on myeloid cells or T cells and control tolerance to inhaled antigens (Xiao et al., 2014; Nie et al., 2017). Neither the role of PD-L2 binding to RGMb in tumors nor the relevance of this interaction in humans has yet to be described. Having bispecific antibodies against PD-L1 and PD-L2 could prove to be highly advantageous from a therapeutic standpoint. Summary of the Invention

[0008] overview The present disclosure is directed to the inventors' surprising discovery of highly specific bispecific antibodies that selectively bind both PD-L1 and LD-L2 with little or no off-target binding. Starting with ADI-16415, the inventors developed a series of PD-L1 / PD-L2 dual binding antibodies, as illustrated in Table 1 below. Multiple rounds of affinity maturation of ADI-16415 resulted in ADI-37464, which exhibited high selectivity for PD-L1 and PD-L2, but also exhibited off-target binding to the insulin-like growth factor 1 (IGF-1) receptor, a transmembrane receptor that belongs to a large class of tyrosine kinase receptors. The inventors continued to modify Ab-37464 to identify novel antibodies that maintain selective binding to both PD-L1 and LD-L2, while also avoiding the off-target binding exhibited by ADI-37464. The inventors have engineered the ADI-38000 series of antibodies (e.g., Ab-38000 to Ab-38004), which are derivatives of 37464 with light chain CDR mutations designed to avoid off-target insulin receptor binding.

[0009] [Table 1]

[0010] Table 2 below summarizes the light chain CDR sequence variations of the Ab-38000 series of antibodies, as well as their characteristic off-target insulin receptor binding and affinity for PD-L1 and PD-L2.

[0011] [Table 2]

[0012] Thus, the present disclosure is based on the surprising and unexpected identification of a bispecific antibody, ADI-38002, that exhibits high affinity for both PD-L1 and LD-L2 while exhibiting little or no off-target binding. The inventors were particularly surprised that the engineered modification that distinguishes ADI-37464 from ADI-38002 is based on a single non-conservative amino acid point mutation (e.g., substitution of a glycine in ADI-37464 with an aspartic acid in ADI-38002) that would normally be expected to disrupt binding and / or reduce binding affinity.

[0013] Thus, in accordance with the present disclosure, there is provided a method for the treatment of cancer that selectively binds to both PD-L1 and PD-L2, and (i) (ii) having a heavy chain CDR sequence of TIFF2025512888000003.tif4138, and CDR3 ARDGRRMGTPSFDI (SEQ ID NO:9), and a light chain CDR sequence of CDR1 RASQDINSFLA (SEQ ID NO:10), CDR2 AASSLNS (SEQ ID NO:11), and CDR3 QKSVYFPPT (SEQ ID NO:12); TIFF2025512888000004.tif4138, and a heavy chain CDR sequence of CDR3 ARDGRRMGTPSFDI (SEQ ID NO:9), and a light chain CDR sequence of CDR1 RASQGINSFLA (SEQ ID NO:13), CDR2 AADSIQS (SEQ ID NO:14), and CDR3 QKAVYFPPT (SEQ ID NO:15); or (iii) TIFF2025512888000005.tif4138, and a heavy chain CDR sequence of CDR3 ARDGRRMGTPSFDI (SEQ ID NO:9), and a light chain CDR sequence of CDR1 RASQGINSFLA (SEQ ID NO:13), CDR2 AADSIQS (SEQ ID NO:14), and CDR3 QKSVYFPPT (SEQ ID NO:12); or (iv) or (v) having a heavy chain CDR sequence of TIFF2025512888000006.tif4139, and CDR3 ARDGRRMGTPSFDI (SEQ ID NO:9), and a light chain CDR sequence of CDR1 RASKGISSFLA (SEQ ID NO:16), CDR2 AASSLNS (SEQ ID NO:11), and CDR3 QKAVYFPPT (SEQ ID NO:15); or TIFF2025512888000007.tif4139, and CDR3 ARDGRRMGTPSFDI (SEQ ID NO:9), and light chain CDR sequences of CDR1 RASQGISSFLA (SEQ ID NO:17), CDR2 AASSLQS (SEQ ID NO:18), and CDR3 QSAVYFPPT (SEQ ID NO:19). The antibody or antibody fragment is provided having a heavy chain CDR sequence of CDR1 RASQGISSFLA (SEQ ID NO:17), CDR2 AASSLQS (SEQ ID NO:18), and CDR3 QSAVYFPPT (SEQ ID NO:19). (a) a heavy chain nucleotide sequence having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TIFF2025512888000008.tif164146; and (b) a light chain nucleotide sequence having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TIFF2025512888000009.tif77146 It can be encoded by a variable sequence having the following structure:

[0014] The antibody or antibody fragment may be a heavy chain having an amino acid sequence having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TIFF2025512888000010.tif57146; and a light chain having an amino acid sequence having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TIFF2025512888000011.tif24145; may include.

[0015] Also provided is a method of treating cancer in a subject, comprising contacting a PD-L1 positive, PD-L2 positive, or PD-L1 positive PDL2 positive cancer cell in the subject with the antibody. The PD-L1 positive, PD-L2 positive, or PD-L1 positive PDL2 positive cancer cell may be a solid tumor cell, such as a lung cancer cell, a brain cancer cell, a head and neck cancer cell, a breast cancer cell, a skin cancer cell, a liver cancer cell, a pancreatic cancer cell, a gastric cancer cell, a colon cancer cell, a rectal cancer cell, a uterine cancer cell, a cervical cancer cell, an ovarian cancer cell, a testicular cancer cell, a skin cancer cell, an esophageal cancer cell, a lymphoma cell, a renal cell cancer cell, or a leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma.

[0016] The method may further include contacting the PD-L1 positive, PD-L2 positive, or PD-L1 positive PDL2 positive cancer cells with a second anti-cancer agent or treatment, such as chemotherapy, radiation therapy, immunotherapy, hormone therapy, or toxin therapy. The second anti-cancer agent or treatment may inhibit the function of PD-L1 or PD-L2 in the cells. The second anti-cancer agent or treatment may be given simultaneously with the first agent, or may be given before and / or after the first agent. The PD-L1 positive or PD-L2 positive cancer cells may be metastatic cancer cells, multidrug resistant cancer cells, or recurrent cancer cells.

[0017] The antibody fragment may be a recombinant scFv (single chain variable fragment) antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric antibody, a humanized antibody, or an IgG. The antibody may be a human antibody, a murine antibody, an IgG, a humanized antibody, or a humanized IgG. The antibody or antibody fragment may further comprise a label, for example, a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody or antibody fragment may further comprise an antitumor drug linked to the antibody or antibody fragment, for example, by a photolabile linker or an enzymatically cleavable linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme. The antibody or antibody fragment may be conjugated to a nanoparticle or a liposome.

[0018] In another embodiment, a method of treating cancer in a subject is provided, the method comprising: (i) administering to a subject a peptide having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9; and light chain CDR sequences of CDR1 SEQ ID NO:10, CDR2 SEQ ID NO:11, and CDR3 SEQ ID NO:12; (ii) administering to a subject a peptide having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:14, and CDR3 SEQ ID NO:15; or (iii) administering to a subject a peptide having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:15, or (iv) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:16, CDR2 SEQ ID NO:11, and CDR3 SEQ ID NO:15; or (v) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:17, CDR2 SEQ ID NO:18, and CDR3 SEQ ID NO:19 to the subject. The antibody fragment may be a recombinant scFv (single chain variable fragment) antibody, a Fab fragment, a F(ab'h fragment, or an Fv fragment. The antibody may be an IgG. The antibody may be a chimeric or humanized antibody. The delivering step may include administration of the antibody or antibody fragment, or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

[0019] The antibody or antibody fragment may be encoded by the light and heavy chain variable sequences shown in SEQ ID NO:3 and SEQ ID NO:5, or may be encoded by light and heavy chain variable sequences having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to sequences derived from SEQ ID NO:3 and SEQ ID NO:5. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to sequences derived from SEQ ID NO:4 and SEQ ID NO:6, or may comprise light and heavy chain variable sequences having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to sequences derived from SEQ ID NO:4 and SEQ ID NO:6, or may comprise light and heavy chain variable sequences having at least, or about, 95%, 96%, 97%, 98%, 99% or 100% identity to sequences derived from SEQ ID NO:4 and SEQ ID NO:6.

[0020] Monoclonal antibodies are also provided, wherein the antibody or antibody fragment has (i) heavy chain CDR sequences SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and light chain CDR sequences SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; (ii) heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:14, and CDR3 SEQ ID NO:15; or (iii) heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:14, and CDR3 SEQ ID NO:16. or (iv) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:16, CDR2 SEQ ID NO:11, and CDR3 SEQ ID NO:15, or (v) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:17, CDR2 SEQ ID NO:18, and CDR3 SEQ ID NO:19. The antibody fragment may be a recombinant scFv (single chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. The antibody may be a chimeric antibody, a humanized antibody, or an IgG.

[0021] Nucleic acids encoding antibodies or antibody fragments having light and heavy chain variable sequences as shown in SEQ ID NO:3 and SEQ ID NO:5, or nucleic acids encoding light and heavy chain variable sequences having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to sequences derived from SEQ ID NO:3 and SEQ ID NO:5, are also provided. Nucleic acids encoding antibodies or antibody fragments having light and heavy chain variable sequences according to sequences derived from SEQ ID NO:4 and SEQ ID NO:6, or nucleic acids encoding antibodies or antibody fragments comprising light and heavy chain variable sequences having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to sequences derived from SEQ ID NO:4 and SEQ ID NO:6, are also provided.

[0022] In yet another embodiment, a hybridoma or engineered cell expressing an antibody or antibody fragment is provided, wherein the antibody or antibody fragment has heavy chain CDR sequences SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and light chain CDR sequences SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; (ii) heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:14, and CDR3 SEQ ID NO:15; or (iii) heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:13, CDR2 SEQ ID NO:14, and CDR3 SEQ ID NO: or (iv) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:16, CDR2 SEQ ID NO:11, and CDR3 SEQ ID NO:15; or (v) having heavy chain CDR sequences of CDR1 SEQ ID NO:7, CDR2 SEQ ID NO:8, and CDR3 SEQ ID NO:9, and light chain CDR sequences of CDR1 SEQ ID NO:17, CDR2 SEQ ID NO:18, and CDR3 SEQ ID NO:19. The antibody fragment may be a recombinant scFv (single chain variable fragment) antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. The antibody may be a chimeric antibody, a humanized antibody, or an IgG.

[0023] Further embodiments include cancer vaccines comprising one or more antibodies or antibody fragments characterized by the heavy and light chain CDR sequences described above. At least one antibody fragment may be a recombinant scFv (single chain variable fragment) antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. At least one antibody may be a chimeric antibody or an IgG. At least one antibody or antibody fragment may be encoded by the light and heavy chain variable sequences described herein, or may be encoded by the light and heavy chain variable sequences having at least or about 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences described above. At least one antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences set forth above, or may comprise light and heavy chain variable sequences having at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth above.

[0024] In another embodiment, a method is provided for detecting cells expressing PD-L1 or PD-L2 in a subject, the method comprising contacting a sample from the subject with an antibody or antibody fragment characterized by the heavy and light chain CDR sequences described above, and detecting cells expressing PD-L1 or PD-L2 in the sample by binding of the antibody or antibody fragment to cells in the sample. The sample may be a body fluid or tissue sample. The cell may be a cancer cell, such as a lymphoma cell, a breast cancer cell, or a renal cell carcinoma cell. The cell may be a cell associated with immunosuppression. The cell associated with immunosuppression may be a non-cancerous cell in the tumor microenvironment, such as a stromal cell or an endothelial cell. The detection may include ELISA, RIA, or Western blot. The method may further comprise performing the method a second time and determining a change in antigen level compared to the first assay. The antibody or antibody fragment may be encoded by the light and heavy chain variable sequences set forth above or have at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth above. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences set forth above or have at least, or about, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth above.

[0025] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0026] As used herein, "essentially free" with respect to a particular component means that the particular component is not intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts.The total amount of the particular component resulting from unintentional incorporation of the composition is preferably less than 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0027] As used in this specification and the claims, "a" or "an" can mean one or more. As used in this specification and the claims, when used in conjunction with the word "comprising," the word "a" or "an" can mean one or more. As used in this specification and the claims, "another" or "further" can mean at least a second or more.

[0028] As used in this specification and the claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among study subjects.

[0029] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain particular embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figure 3 shows the affinity measurements of the 38000 series by Octet. The affinity of each of the 38000 series low / non-insulin receptor binding members to the indicated species PD-L1 and PD-L2 was measured by Octet. Affinities to human monovalent PD-L1 and PD-L2 were also measured by Biacore. [Diagram 2] Figures 2A-B show treatment of CT26 parental and CT26-PD-L2 tumors with 38000 series PD-L1 / PD-L2 bispecific antibodies. Figure 2A shows parental CT26 tumors were implanted into BALB / C mice and treated with the indicated antibody at 10 mg / kg on days 3, 6, and 9. Figure 2B shows CT26-PDL2 tumors were implanted into BALB / C mice and treated with the indicated antibody at 10 mg / kg on days 3, 6, and 9. Tumor size was measured with a caliper with an endpoint of 1000 mm3. [Diagram 3] Shown is a second experiment of treatment of CT26 parental tumors with 38000 series PD-L1 / PD-L2 bispecific antibodies. Parental CT26 tumors were implanted into BALB / C mice and treated with the indicated antibodies at 10 mg / kg on days 3, 6, and 9. Tumor size was measured with a caliper with an endpoint of 1000 mm3. [Figure 4] 1 shows treatment of BFTC-909 renal cell carcinoma in humanized mice. BFTC-909 tumor xenografts were implanted into huNOG-ExL CD34+ stem cell humanized mice and treated with the indicated antibody at 10 mg / kg twice weekly after tumors reached 50 mm3. [Diagram 5]Figure 3 shows that 38002 PD-L1 / PD-L2 antibody enhances IFNγ secretion in human peripheral blood mixed lymphocyte reaction. In vitro generated dendritic cells from one donor (donor 1) were co-cultured with T cells from two other donors (donors 2 or 3) and IFNγ secretion was measured by ELISA after 72 hours of co-culture. [Figure 6] Figure 3 shows that 38002 and 38004 do not bind to human insulin receptor at physiological concentrations. CHO cells expressing high levels of insulin receptor were stained with the indicated PD-L1 / PD-L2 antibodies and a secondary anti-human PE conjugate. The amount of binding was measured by flow cytometry on a BD LSRII. [Figure 7] The complete heavy chain variable region sequence of ADI-38002 (SEQ ID NOs:20 and 21) is shown. [Figure 8] The complete light chain variable region sequence of ADI-38002 (SEQ ID NOs:22 and 23) is shown. [Figure 9] The human IgG1 (GASDIE) constant region sequences of ADI-38002 (SEQ ID NOs:24 and 25) are shown. [Figure 10] Figure 1 shows histogram plots showing PE-labeled isotype, anti-human INSR, anti-human PD-L1, and anti-human PD-L2 staining in the CHO-INSR cells used in the study. Gates were set on the isotype control samples. PE expression is displayed on the x-axis. [Figure 11] Shown are results for INSR+CHO cells incubated with serial dilutions of 38002 (circles), 37464 (squares), or control antibody (open circles). Fluorescence measured by flow cytometry. Mean values ​​± SDM (n=2) are displayed for each antibody at each concentration. A dose range of 33.333 to 0.00511 nM was tested on INSR+CHO cells. [Figure 12A] A depiction of an antibody-dependent cellular cytotoxicity reporter bioassay (ADCC, Promega, 2020) is shown. [Figure 12B]Shown is the ADCC (Antibody-Dependent Cellular Cytotoxicity Reporter Bioassay (Promega, 2020)) activity of 38002 and 37464 against CHO-INSR cells. Concentrations of 38002, 37464, or IgG isotype are displayed on the x-axis and luminescence activity is displayed on the y-axis. For each antibody at each concentration, the mean ± SDM (n=2) is displayed. A dose range of 22.22-0.0302 nM was tested against INSR+CHO cells. [Figure 13A] A depiction of the antibody-dependent cellular phagocytosis reporter bioassay (ADCP, Promega, 2016) is shown. [Figure 13B] ADCP activity of 38002 and 37464 against CHO-INSR cells is shown. Concentrations of 38002, 37464, or IgG isotype are displayed on the x-axis and luminescence activity is displayed on the y-axis. Mean values ​​± SD (n=2) are displayed for each antibody at each concentration. A dose range of 22.22 - 0.0302 nM was tested against INSR+CHO cells. [Figure 14]Figure 14A-E show that selected bispecific antibodies can engage Fcy receptors and induce antibody-dependent cellular cytotoxicity of NK cells. (Figure 14A) Calcein-labeled U2940 target cells were incubated with the indicated mouse IgG2a bispecific antibodies and co-cultured with NK cells isolated from mouse spleens. The percentage of cell lysis was measured using calcein release of samples and controls. Mean values ​​± SEM (n=2) are displayed for each antibody at each concentration. (Figure 14B-C) Chinese hamster ovary (CHO), PD-L1+ CHO cells and PD-L2+ CHO cells (1:1 mixture of the two cell lines) were incubated with bispecific antibodies at the concentrations indicated on the x-axis and co-cultured with NFAT-RE luciferase reporter Jurkat T cells (FcgRIIIa for ADCC, FcgRIIa-H for ADCP). Higher signals indicate efficient ADCC (Figure 14B) or ADCP (Figure 14C) activation pathways. Mean values ​​± SEM (n = 3) are displayed for each antibody at each concentration. (Figures 14D-E) Mouse melanoma cancer cells B16F10 ectopically expressing PD-L2 were incubated with bispecific antibodies at the concentrations indicated on the x-axis and co-cultured with NFAT-RE luciferase reporter Jurkat T cells (FcgRIIIa for ADCC and FcgRIIa-H for ADCP). Higher signals indicate efficient ADCC (Figure 14D) or ADCP (Figure 14E) activation pathways. Mean values ​​± SEM (n = 3) are displayed for each antibody at each concentration. Nonlinear regression was used to generate best-fit curves in GraphPad Prism. [Figure 15]Figures 15A-H show that selected bispecific antibodies delay tumor growth and increase survival in B16 melanoma by increasing T cell activation and decreasing myelosuppression. Treatment with mouse IgG1 anti-PD-1 (clone RMP1-14) and mouse IgG2a bispecific antibodies was injected on days 3, 6, 9, and 12. (Figures 15A-B) C57Bl / 6 mice were subcutaneously injected with 50,000 PD-L2-overexpressing B16 cells. Mice were monitored to determine tumor growth (Figure 15A) and survival (Figure 15B). Mean values ​​± SEM (n=20) are displayed. (Figures 15C-H) C57Bl / 6 mice were subcutaneously injected with 100,000 PD-L2-overexpressing B16 cells and treated as indicated in (Figure 15A). On D14, lymph nodes (FIGS. 15C-E) and tumors (FIGS. 15F-H) were collected and analyzed by flow cytometry. Individual and mean values ​​are presented. [Figure 16] Figures 16A-B show that antibody 37464 exhibits therapeutic advantage over 27869 and 27907. (Figure 16A) Balb / c mice were subcutaneously injected with 100,000 CT26 cells and treated with mouse IgG1 anti-PD-1 (clone RMP1-14) or mouse IgG2a bispecific antibody on days 3, 6, 9, and 12. Mice were monitored to determine tumor growth. Mean values ​​± SEM (n=10) are displayed. (Figure 16B) Serial dilutions of BiPDL antibody or isotype control antibody were preincubated with CHO / PDL-1 cells combined with human recombinant B7.1, followed by secondary staining with PE-labeled anti-human B7.1 antibody. Binding of the labeled PE-labeled anti-human B7.1 antibody was an indication of PD-L1 on the cell surface freely available for binding to B7.1. Nonlinear regression was used to generate best-fit curves in GraphPad Prism. [Figure 17]Figures 17A-I show the selection of optimized bispecific antibodies without off-target binding. (Figures 17A-B) Vectors encoding all hits from library screening, as well as control vectors encoding EGFR (transfection and negative control) and CD20 (positive control), were rearranged / re-expressed in two replicates. After cell fixation, cells were probed with (Figure 17A) 5 μg / mL 37464 or (Figure 17B) 20 μg / mL 38002. Parallel slides were probed with 1 μg / mL rituximab biosimilar (positive control) or no test molecule (AF647 anti-hIgG Fc secondary antibody only). Specific interactions of the test antibodies are shown in blue (low intensity) and green (low / medium intensity and above). Interactions that are nonspecific but have a large intensity difference between the test and control treatments are shown in purple; stronger nonspecific interactions are shown in black; positive control interactions are shown in orange. Spots of INSR (long and short isoforms) and INSR+IGF1R cells are highlighted in red. (Figure 17C) INSR+CHO cells were incubated with serial dilutions of 38002 (red), 37464 (blue), or control antibody (black). Fluorescence was measured by flow cytometry. Mean values ​​± SDM (n=2) are displayed for each antibody at each concentration. A dose range of 33.333 to 0.00511 nM was tested on INSR+CHO cells. (Figure 17D-E) Chinese hamster ovary (CHO) cells expressing PD-L1 (D) or PD-L2 (Figure 17E) were incubated with bispecific antibodies at the concentrations indicated on the x-axis and co-cultured with NFAT luciferase reporter Jurkat T cells. Higher signals indicate more efficient blockade of the PD-1 pathway. (Figure 17F-G) Chinese hamster ovary (CHO), PD-L1+ CHO cells and PD-L2+ CHO cells (1:1 mixture of the two cell lines) were incubated with bispecific antibodies at the concentrations indicated on the x-axis and co-cultured with NFAT-RE luciferase reporter Jurkat T cells (FcgRIIIa for ADCC, FcgRIIa-H for ADCP).Higher signals indicate efficient ADCC (Figure 17F) or ADCP (Figure 17G) activation pathways. Mean values ​​± SEM (n = 3) are displayed for each antibody at each concentration. (Figure 17H-I) Mouse melanoma cancer cells B16F10 ectopically expressing PD-L2 were incubated with bispecific antibodies at the concentrations indicated on the x-axis and co-cultured with NFAT-RE luciferase reporter Jurkat T cells (FcgRIIIa for ADCC and FcgRIIa-H for ADCP). Higher signals indicate efficient ADCC (Figure 17H) or ADCP (Figure 17I) activation pathways. Nonlinear regression was used to generate best-fit curves in GraphPad Prism. [Figure 18] Figures 18A-B show the characterization of bispecific antibody 38002, which has similar affinity but does not bind INSR. (Figure 18A) Table showing the affinity of bispecific antibody 38002 for monomeric PD-L1 and PD-L2. KD was measured on the Octet® platform and is shown in molar concentration. (Figure 18B) Titration curves of 38002 binding to CD274 (PD-L1), PDCD1LG2 (PD-L2), INSR long isoform, and INSR short isoform expressed in live HEK293 cells. [Figure 19]Figure 19A-H shows that bispecific antibody 38002 increases survival in B16 melanoma by increasing T cell activation and decreasing myelosuppression. (Figure 19A) C57BL / 6 mice were implanted with PD-L2-expressing B16F10 cells. Mice received intraperitoneal treatment with anti-mouse PD-1 (clone RMP1-14), mouse IgG2a anti-PD-L1 antibody, mouse IgG2a anti-PD-L2 antibody, a 1:1 mixture of mouse IgG2a anti-PD-L1 and anti-PD-L2 antibodies, or human IgG1-GASDIE 38002 bispecific antibody on days 3, 6, 9, and 12. Mean tumor volume ± SEM (mm3) is plotted against days after tumor inoculation. (Figure 19B-H) C57Bl / 6 mice were subcutaneously injected with 100,000 PD-L2-overexpressing B16 cells. Treatment with mouse IgG1 anti-PD-1 (clone RMP1-14) and mouse IgG2a 38002 was injected on days 3, 6, 9, and 12, and tumors were harvested and analyzed by flow cytometry on D14. Individual and mean values ​​are shown. [Figure 20] Figure 20A-D shows that C57BL / 6 mice were injected with 500,000 B16-PD-L2 cells. On days 3, 6, 9, and 12, 20 mg / kg of human IgG1-GASDIE 38002 bispecific antibody was given via IP. On day 18, mice were euthanized and lymph nodes were collected and stained with PE-labeled antibodies against CD11b, Gr-1, arginase-1, and FOXP3, or isotope controls, and analyzed by flow cytometry. [Figure 21]Figures 21A-B show that treatment of nu / nu mice bearing MDA-MB-231 tumors with dual IMGS-001 inhibits tumor growth and reduces infiltration of CD11b+ cells. (Figure 21A) Mean tumor volume ± SEM (mm3) is plotted on the y-axis against days after tumor challenge on the x-axis. Unpaired t-test was used to compare groups. *p<0.05; **p<0.01; ***p<0.001. (Figure 21B) Immunohistochemistry with anti-CD11b in untreated tumors and tumors treated with dual IMGS-001 (magnification 200x). Histograms show the mean ± SD of the percentage of CD11b+ cells detected in whole slide tumor tissues. Unpaired t-test was used to compare groups. p=0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Description of Exemplary Embodiments The inventors have previously generated monoclonal antibodies with binding specificity for both human PD-L1 and PD-L2 proteins. These antibodies have been shown to bind to both PD-L1 and PD-L2, and therefore provide an opportunity to block the binding of PD-L1, PD-L2, or PD-L1 and PD-L2, to PD-1, for example, by blocking the interaction of PD-L1 / PD-L2 with PD-1 and the interaction of PD-L1 with B7-1. They may also be used to deliver therapeutic payloads to cancer cells expressing PD-L1, PD-L2, or PD-L1 and PD-L2.

[0032] Recently, the inventors engineered bispecific PD-L1 / PD-L2 antibodies to avoid significant binding to the human insulin receptor while retaining PD-L1 / PD-L2 affinity. This was achieved by modifying the light chain CDR amino acids. It was surprisingly found that the ability of the antibodies to mediate a curative response as a monotherapy, superior to PD-1 blockade, was not affected by reduced off-target effects. The inventors surprisingly identified a new subclass of immune checkpoint antibodies that have the additional property of checkpoint cytoreduction, thus enabling the disruption of the stromal barrier to T cell infiltration of tumors, as well as direct tumor cytoreduction and anti-metastatic activity. This surprising and unexpected result is of particular relevance for "cold" cancers that are hyporesponsive (<5%) to existing PD-1 antibodies, due to the elimination and suppression of effector T cell responses.

[0033] Furthermore, the inventors then used clinically proven mutagenesis to engineer the Fc portion of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of multiple tumor cell lines in vitro. Mice injected with immune excluded (B16-PDL-2) and immune infiltrated (CT26, CT26-PD-L2, MC38) tumors showed greater than 50% survival when treated with anti-PD-L1 / PD-L2 bispecific antibodies, whereas treatment with mouse anti-PD1 showed little or no survival benefit in cold tumors. Ex vivo cell analysis of tumor and stromal components from mice treated with bispecific antibodies resulted in increased T cell activation and reduced myeloid cell compartments. Finally, treatment of nu / nu mice bearing human triple-negative breast cancer (MDA-MB-231) with dual PD-L1 / PD-L2 antibodies significantly inhibited tumor growth and reduced CD11b + reduced cellular infiltration, whereas clinically proven anti-PD-L1 had very limited effect.

[0034] These findings and other aspects of the disclosure are described in further detail below.

[0035] I.PD-L1 A. Structure Programmed cell death ligand 1 (PD-L1) is a protein encoded by the CD274 gene. PD-L1 is a 40 kDa type 1 transmembrane protein that may play a key role in immune suppression in a variety of events, such as pregnancy, allogeneic tissue transplantation, autoimmune diseases, cancer, and other disease states. The human PD-L1 protein is encoded by the amino acid sequence shown below: TIFF2025512888000012.tif29145.

[0036] B. Function PD-L1 is a ligand for its receptor PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to PD-1 modulates the activation or inhibition of T cells and B cells and transmits inhibitory signals that reduce the proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. Binding of PD-L1 to PD-1 also induces apoptosis. This reduction in CD8+ T cells and CD4+ helper T cells is thought to help PD-L1-expressing cancer cells escape from antitumor immunity (Dong et al., 2002). Upregulation of PD-L1 has been associated with escape from the host immune system and is thought to be responsible for increased tumor malignancy (Thompson et al., 2004). Due to the role of PD-L1 in evading antitumor immunity, PD-L1 is an attractive target for therapeutic intervention.

[0037] II.PD-L2 A. Structure Programmed cell death ligand 2 (PD-L2) is a protein encoded by the CD273 gene. PD-L2 is a 31 kDa protein that may play a key role in immune suppression in a variety of events, such as pregnancy, allogeneic tissue transplantation, autoimmune diseases, cancer, and other disease states. The human PD-L2 protein is encoded by the amino acid sequence shown below: TIFF2025512888000013.tif24145.

[0038] PD-L2 is initially produced containing a signal peptide corresponding to amino acids 1-19 of SEQ ID NO:2, which is then removed to yield the mature protein. The mature PD-L2 protein, corresponding to amino acids 20-273 of SEQ ID NO:2, is composed of an Ig-like V domain, an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic tail.

[0039] B. Function PD-L2 is the ligand for its receptor PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. Binding of PD-L2 to PD-1 initiates an immunological cascade that impairs T cell proliferation, cytokine production, cytolytic function, and survival. PD-1 transmits inhibitory signals that reduce proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. PD-L2 has also been shown to be an independent predictor of response to the PD-1 antibody pembrolizumab in multiple cancers (Yearley et al., 2017).

[0040] III. Monoclonal Antibodies and Their Production A. General Method Antibodies to PD-L1 and PD-L2 can be made by standard methods well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). Methods for generating monoclonal antibodies (mAbs) generally begin similarly to the preparation of polyclonal antibodies. The first step in both of these methods is the immunization of a suitable host, or the identification of a subject who has immunity due to past natural infection. As is well known in the art, a given composition for immunization can vary in immunogenicity. Thus, it is often necessary to boost the host immune system, as accomplished by coupling the immunogen, which is a peptide or polypeptide, to a carrier. Exemplary preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means of conjugating polypeptides with carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine.As is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants.Exemplary preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response containing killed Mycobacterium tuberculosis bacteria), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0041] The amount of immunogen composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen and the animal used for immunization. A variety of routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal) can be used to administer the immunogen. The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. A second booster injection may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate monoclonal antibodies.

[0042] After immunization, somatic cells that can produce antibodies, specifically B lymphocytes (B cells), are selected for use in mAb generation protocols. These cells can be obtained from biopsied spleen or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of immortal myeloma cells, generally of the same species as the immunized animal, or with human or human / mouse chimeric cells. The myeloma cell line suitable for use in the fusion method of hybridoma production preferably does not produce antibodies, has high fusion efficiency, and has enzyme deficiencies that prevent it from growing in certain selective media that support the growth of only the desired fused cells (hybridomas).

[0043] As known to those skilled in the art, any of a number of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, when the animal to be immunized is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 41, Sp210-Ag14, PO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul can be used; when the animal to be immunized is a rat, R210.RCY3, Y3-AG 1.2.3, IR983F, and 4B210 can be used; U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful in connection with human cell fusion. One specific mouse myeloma cell is the NS-1 myeloma cell line (also called P3-NS-1-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that can be used is the 8-azaguanine-resistant mouse myeloma SP2 / 0 non-producing cell line. More recently, additional fusion partner lines have been described for use with human B cells, such as KR12 (ATCC CRL-8658); K6H6 / B5 (ATCC CRL-1823), SHM-D33 (ATCC CRL-1668), and HMMA2.5 (Posner et al., 1987). The antibodies in this disclosure were generated using the SP2 / 0 / mIL-6 cell line, which is an IL-6-secreting derivative of the SP2 / 0 line.

[0044] The method for generating hybrids of antibody-producing spleen cells or lymph node cells and myeloma cells generally involves mixing somatic cells and myeloma cells in a ratio of 2:1, but the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. A fusion method using Sendai virus is described by Kohler and Milstein (1975; 1976), and a method using polyethylene glycol (PEG), e.g., 37% (v / v) PEG, is described by Gefter et al. (1977). The use of electrically induced fusion is also suitable (Goding, pp. 71-74, 1986).

[0045] The fusion method generally involves approximately 1 × 10 -6 From 1×10 -8 Viable hybrids are produced at a low frequency of 10-20%. However, this is not a problem because viable fused hybrids are differentiated from the injected parent cells (specifically the injected myeloma cells, which usually continue to divide indefinitely) by culturing in selective medium. Selective medium generally contains an agent that blocks de novo synthesis of nucleotides in tissue culture medium. Exemplary preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine. When the source of B cells is a human B cell line transformed by Epstein-Barr virus (EBV), ouabain is added to eliminate EBV-transformed lines that have not fused with myeloma.

[0046] The preferred selection medium is HAT or HAT with ouabain. In HAT medium, only cells that can function in the nucleotide salvage pathway can survive. Myeloma cells are defective in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. B cells can function in this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in selection medium are hybrids formed from myeloma cells and B cells. When the source of the B cells used for fusion is a line of B cells transformed by EBY, ouabain is also used for drug selection of the hybrids, because the B cells transformed by EBV are sensitive to drug killing, whereas the myeloma partner used is selected to be resistant to ouabain.

[0047] Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing the cells by single clone dilution in microtiter plates and then testing individual clonal supernatants (after about 2-3 weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc.

[0048] The selected hybridomas can then be serially diluted or single-cell sorted by flow cytometry sorting, cloned into individual antibody-producing cell lines, and then the clones can be propagated indefinitely to provide mAbs. Cell lines can be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often intraperitoneally) into an animal (e.g., a mouse). Optionally, the animal is primed with a hydrocarbon, specifically an oil, such as pristane (tetramethylpentadecane), prior to injection. In this way, when human hybridomas are used, it is optimal to inject into an immunodeficient mouse, e.g., a SCID mouse, to prevent tumor rejection. In the injected animal, a tumor develops that secretes the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, e.g., serum or ascites, can then be utilized to provide high concentrations of mAbs. Individual cell lines can also be cultured in vitro, where the mAbs are naturally secreted into the culture medium and can be easily obtained therefrom in high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulin in the cell supernatant. These cell lines can be adapted for growth in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulin.

[0049] Monoclonal antibodies produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure may be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure may be synthesized using an automated peptide synthesizer.

[0050] It is also contemplated that molecular cloning approach can be used to generate monoclonal antibodies. For this purpose, RNA can be isolated from hybridoma strains, antibody genes can be obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phagemid library can be prepared from the RNA isolated from cell strains, and phagemids expressing suitable antibodies can be selected by panning with viral antigens. The advantage of this approach compared to traditional hybridoma technology is that approximately 104 times more antibodies can be produced and screened at one time, and the combination of H and L chains can lead to new specificities, further increasing the probability of finding suitable antibodies.

[0051] For example, yeast-based antibody libraries can be rationally designed and antibodies can be selected and / or isolated from such yeast-based antibody display libraries as disclosed in WO2012 / 009568; WO2009 / 036379; WO2010 / 105256; WO2003 / 074679; U.S. Patent No. 8,691,730; and U.S. Patent No. 9,354,228. Antibodies can be expressed and purified as full-length IgG from any desired cell type disclosed above.

[0052] Other U.S. patents that teach the production of antibodies useful in this disclosure, each of which is incorporated herein by reference, include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using combinatorial approaches; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0053] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure may be defined primarily by their binding specificity, i.e., binding to PD-L1 and PD-L2. One of skill in the art may determine whether such an antibody falls within the scope of the present invention by assessing the binding specificity / affinity of a given antibody using techniques well known to those of skill in the art. In one aspect, monoclonal antibodies are provided having CDRs derived from the heavy and light chains described above. Such antibodies may be produced by the clones described in the Examples section below using the methods described herein.

[0054] In a second aspect, the antibodies may be defined by variable sequences that include additional "framework" regions. These are provided, for example, in SEQ ID NO:3 / SEQ ID NO:4 and SEQ ID NO:5 / SEQ ID NO:6, which encode or represent complete variable regions. Furthermore, the antibody sequences may optionally differ from these sequences using methods described in more detail below. For example, the nucleic acid sequences may differ from those described above in the following ways: (a) the variable region is separated from the light and heavy chain constant domains; (b) the nucleic acid differs from the above without affecting the residues encoded thereby; (c) the nucleic acid differs from the above by a predetermined percentage, e.g., at least or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; (d) the nucleic acid is capable of being isolated under high stringency conditions, e.g., low salt and / or high temperature conditions, e.g., about 0.02M to about 0.15M. (e) differs from said one by a predetermined percentage of amino acids, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, or (f) differs from said one by allowing for conservative substitutions (described below), each of which applies to said nucleic acid sequences and said amino acid sequences.

[0055] C. Manipulation of Antibody Sequences In various embodiments, one may choose to engineer the sequence of an identified antibody for a variety of reasons, such as improving expression, improving cross-reactivity, or reducing off-target binding. Below is a review of relevant techniques for antibody engineering.

[0056] Hybridomas can be cultured, then the cells can be lysed and total RNA can be extracted. Random hexamers can be used with RT to generate cDNA copies of the RNA, then PCR can be performed using a multiplex mixture of PCR primers that are expected to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies collected from hybridoma supernatants and purified by FPLC using protein G columns.

[0057] Recombinant full-length IgG antibodies can be produced by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfecting into 293 Freestyle cells or CHO cells, and harvesting and purifying the antibody from the 293 or CHO cell supernatant.

[0058] The rapid availability of antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to shorten the duration of process development programs. Lonza has developed a general method for the rapid production of small amounts (up to 50 g) of antibodies in CHO cells using pooled transfectants grown in CD ACF medium. Although slightly slower than true transient systems, advantages include higher product concentration and the use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools expressing model antibodies in disposable bioreactors: Harvest antibody concentrations of 2 g / L were achieved within 9 weeks of transfection in disposable bag bioreactor cultures (5 L working volume) operated in fed-batch mode.

[0059] Antibodies and antibody libraries that can select and / or isolate such antibodies can be rationally designed and synthesized, for example, by Adimab® technology, disclosed in WO2012 / 009568; WO2009 / 036379; WO2010 / 105256; WO2003 / 074679; U.S. Patent No. 8,691,730; and U.S. Patent No. 9,354,228. This antibody synthesis method requires inserting the nucleotide sequence encoding the desired or designed antibody into a vector for ectopic expression. The desired antibody can then be expressed as a full-chain IgG molecule and purified.

[0060] Antibody molecules include, for example, fragments produced by proteolytic cleavage of mAbs (e.g., F(ab'), F(ab'h), or single chain immunoglobulins which can be produced, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form "chimeric" binding molecules. Importantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

[0061] In related embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody that contains the same CDR sequence as that of the disclosed antibody (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, it may be desirable to perform modifications, for example, the introduction of conservative changes to the antibody molecule. In performing such changes, the hydropathic index of amino acids may be considered. The importance of the amino acid hydropathic index to confer interactive biological functions to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0062] It is also understood in the art that substitution of similar amino acids can be effectively performed based on hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, discloses that the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Pat. No. 4,554,101, amino acid residues are assigned the following hydrophilicity values: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic non-ionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2); the amino acids most commonly found were phenylalanine (+0.2), and threonine (-0.4); the sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); the hydrophobic nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), praline (-0.5±1), alanine (-0.5), and glycine (0); the hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0063] It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity to generate a biologically or immunologically modified protein. In such changes, amino acid substitutions with hydrophilicity values ​​within ±2 are preferred, particularly within ±1, and more particularly within ±0.5 are preferred.

[0064] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various characteristics noted above are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0065] The present disclosure also contemplates isotype modification. By modifying the Fe region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve binding valency.

[0066] The modified antibody may be produced by any technique known to those of skill in the art, such as expression by standard molecular biology techniques or chemical synthesis of the polypeptide. Methods of recombinant expression are set forth elsewhere herein.

[0067] D. Single chain antibody Single chain variable fragments (scFv) are fusions of the variable regions of immunoglobulin heavy and light chains linked together by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification generally leaves the specificity unchanged. These molecules were historically created to facilitate phage display, where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFvs may be created directly from subcloned heavy and light chains derived from hybridomas. Single chain variable fragments lack the constant Fc region found in complete antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, since protein L interacts with the variable region of the kappa light chain.

[0068] Flexible linkers are generally composed of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues may also function. Tang et al. (1996) used phage display as a means to rapidly select specialized linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which genes for the variable domains of the heavy and light chains were linked by segments encoding 18-amino acid polypeptides of variable composition. The scFv repertoire (approximately 5×106 different members) was displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected variants showed a significant increase in binding activity while retaining considerable sequence diversity. Subsequent screening of 1054 individual variants yielded catalytically active scFvs that were efficiently produced in a soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VHC terminus as the only common feature of the selected tethers, as well as abundant arginines and pralines in other positions.

[0069] The recombinant antibody of the present disclosure may also comprise a sequence or portion that allows receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow multimerization with J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chain may be modified with an agent that allows the combination of two antibodies, such as biotin / avidin.

[0070] In another embodiment, a single chain antibody can be produced by joining the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced in separate cells, purified, and then appropriately linked together (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via a suitable chemical bridge).

[0071] Cross-linking reagents, such as stabilizers and coagulants, are used to form molecular bridges that connect the functional groups of two different molecules.However, it is contemplated that the heteromeric complexes that are composed of dimers or multimers of the same analogue or different analogues can be created.To link two different compounds stepwise, heterobifunctional cross-linking agents can be used that eliminate unnecessary homopolymer formation.

[0072] Exemplary heterobifunctional crosslinkers contain two reactive groups; one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogens, etc.). The crosslinker can react with a lysine residue of one protein (e.g., a selected antibody or fragment) through the primary amine reactive group, and the crosslinker, already tethered to the first protein, reacts with a cysteine ​​residue (free sulfhydryl group) of the other protein (e.g., a selected agent) through the thiol reactive group.

[0073] It is preferable to use a crosslinker that has reasonable blood stability.Many types of disulfide bond-containing linkers are known that can be successfully used to conjugate targeting agent and therapeutic / preventive agent.Containing sterically hindered disulfide bond linkers may prove to provide higher stability in vivo and prevent targeting peptide from being released before reaching the site of action.Therefore, these linkers are one group of linking agents.

[0074] Another cross-linking reagent is SMPT, a bifunctional cross-linker that contains a "sterically hindered" disulfide bond with adjacent benzene rings and methyl groups. It is believed that the steric hindrance of the disulfide bond serves to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent dissociation of the conjugate before the attached agent is delivered to the target site.

[0075] SMPT cross-linking reagents, like many other known cross-linking reagents, offer the ability to cross-link functional groups, such as the SH of cysteines or primary amines (e.g., the ε-amino group of lysine). Another possible type of cross-linker includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide (upon photolysis) reacts nonselectively with any amino acid residue.

[0076] In addition to hindered crosslinkers, unhindered linkers can also be used according to the present specification. Other useful crosslinkers that do not contain or are not expected to generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment includes the use of flexible linkers.

[0077] US Patent No. 4,680,338 describes a bifunctional linker useful for making conjugates of ligands with amine-containing polymers and / or proteins, specifically for forming conjugates of antibodies with chelators, drugs, enzymes, detectable labels, etc. US Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest can be directly attached to the linker, the cleavage of which results in the release of the active agent. Particular uses include the addition of free amino groups or free sulfhydryl groups to proteins, such as antibodies, or drugs.

[0078] US Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide components to create fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length, contain at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by praline, and are characterized by higher stability and reduced aggregation. US Patent No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separation techniques.

[0079] E. Purification In certain embodiments, the antibody of the present disclosure may be purified. The term "purified" as used herein refers to a composition in which a protein is purified to any degree relative to its naturally available state and can be isolated from other components. Thus, a purified protein also refers to a protein that is separated from the environment in which it may naturally occur. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, for example, making up about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0080] Protein purification techniques are well known to those skilled in the art. These techniques include, at one level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods that are particularly suitable for preparing pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or precipitation by heat denaturation, followed by centrifugation; gel filtration chromatography, reverse phase chromatography, hydroxylapatite chromatography, and affinity chromatography; and combinations of such techniques and other techniques.

[0081] In purifying the antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide.As is generally known in the art, it is considered possible to change the order of performing various purification steps or omit certain steps to still result in a suitable method for preparing substantially purified protein or peptide.

[0082] Typically, complete antibodies are fractionated using an agent that binds to the Fe moiety of the antibody (i.e., protein A). Alternatively, antigens can be used to simultaneously purify and select suitable antibodies. Such methods often use a selection agent bound to a support, e.g., a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., washed off), and the antibody is released by applying conditions (salt, heat, etc.).

[0083] In view of the present disclosure, various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the purity. The actual units used to express the amount of activity will of course depend on the particular assay technique chosen to track purification, and whether the expressed protein or peptide exhibits detectable activity.

[0084] It is known that the mobility of polypeptides can vary, sometimes significantly, under different conditions of SDS / PAGE (Capaldi et al., 1977). It is therefore understood that the apparent molecular weight of purified or partially purified expression products can vary under different electrophoretic conditions.

[0085] IV. Pharmaceutical Formulations and Cancer Treatment A. Cancer Cancer results from the proliferation of a clonal population of cells derived from a tissue. The development of cancer, called carcinogenesis, can be modeled and characterized in many ways. The link between cancer development and inflammation has been understood for a long time. The inflammatory response is involved in the host defense against microbial infection and also drives tissue repair and regeneration. Considerable evidence points to a relationship between inflammation and cancer development risk, namely that chronic inflammation can lead to metaplasia.

[0086] Cancer cells to which the methods of the present disclosure may be applied generally include any cells that express PD-L1, PD-L2, or PD-L1 and PD-L2, and more specifically, overexpress PD-L1, PD-L2, or PD-L1 and PD-L2. Suitable cancer cells may be cells of breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, blood cancer (e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. Furthermore, the methods of the present disclosure may be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers may be recurrent, metastatic, and / or multidrug resistant, and the methods of the present disclosure may be applied to such cancers, specifically to render them resectable, to prolong or reinduce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multidrug resistant cancers. At the cellular level, this may translate as the death of cancer cells, the inhibition of cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0087] B. Formulation and Administration The present disclosure provides a pharmaceutical composition comprising a bispecific antibody (DSPDL) against PD-L1 and PD-L2. In a specific embodiment, the term "pharmaceutical acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more specifically, in humans. The term "carrier" refers to a diluent, excipient, or vehicle for administration of a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, and the like.

[0088] The compositions may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0089] The antibodies of the present disclosure may include classical pharmaceutical preparations. The administration of these compositions according to the present disclosure is via any common route, so long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal, or topical. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are usually administered as pharma- ceutically acceptable compositions as described above. Of particular interest are direct intratumoral administration, tumor perfusion, or local or regional administration to tumors, for example, local or regional vascular or lymphatic systems, or administration to resected tumor beds.

[0090] The active compound may be administered parenterally or intraperitoneally.The solution of the active compound as free base or pharmacologically acceptable salt may be prepared in water mixed appropriately with surfactant, for example, hydroxypropylcellulose.Dispersion may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and oil.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0091] C. Combination Therapy In accordance with the present disclosure, it is also contemplated that the bispecific antibodies to PD-L1 and PD-L2 (bi-PDL) described herein may be used in conjunction with chemotherapy or radiation therapy interventions, or other treatments. In particular, combining bispecific antibodies to PD-L1 and PD-L2 with other therapies that target different aspects of PD-L1 or PD-L2 function, such as peptides and small molecules that target the cytoplasmic domain of PD-L1 or PD-L2, may also prove effective.

[0092] To use the methods and compositions of this disclosure to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells, a "target" cell is generally contacted with a bispecific anti-PD-L1 anti-PD-L2 antibody according to the present disclosure and at least one other agent. These compositions are provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may include contacting the cell with a bispecific anti-PD-L1 anti-PD-L2 antibody according to the present disclosure and the other agent or factor simultaneously. This may be accomplished by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by simultaneously contacting the cell with two separate compositions or formulations, one composition including a bispecific anti-PD-L1 anti-PD-L2 antibody according to the present disclosure and the other including the other agent.

[0093] Alternatively, the bispecific anti-PD-L1-anti-PD-L2 antibody therapy may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the bispecific anti-PD-L1-anti-PDL2 antibody are applied separately to the cells, it is generally ensured that no significant period of time elapses between each delivery, so that the agents and expression constructs can still exert their advantageously combined effect on the cells. In such cases, it is contemplated to contact the cells with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, with a delay time of no more than about 12 hours being most preferred. However, in some circumstances, it may be desirable to significantly extend the duration of treatment, ranging from a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0094] It is also contemplated that multiple administrations of either the bispecific anti-PD-L1 anti-PD-L2 antibody or other agents may be desired. Various combinations may be used, such as those exemplified below, where a bispecific anti-PD-L1 anti-PD-L2 antibody treatment according to the present disclosure is "A" and the other treatment is "B": TIFF2025512888000014.tif17128.

[0095] The administration of the therapeutic agent of the present invention to patients follows the general protocol for administration of the specific second-line treatment, taking into account the toxicity of antibody treatment, if any. It is expected that the treatment cycle will be repeated as necessary. It is also contemplated that various standard therapies and surgical interventions may be applied in combination with the described cancer treatment.

[0096] Those skilled in the art can refer to "Remington's Pharmaceutical Sciences" 15th Edition, Chapter 33, specifically pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations must meet the standards of sterility, pyrogenicity, general safety, and purity required by the standards of FDA Office of Biologics.

[0097] 1.Chemotherapy Cancer treatments also include a variety of combination therapies with both chemical and radiation-based treatments. Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, temazolomide (aqueous form of DTIC), or any analog or derivative variants thereof. The combination of chemotherapy and biological therapy is known as biochemotherapy. The present invention contemplates any chemotherapeutic agent that may be used or known in the art to treat or prevent cancer.

[0098] 2. Radiation therapy Other widely used agents that cause DNA damage include y-rays, commonly known as X-rays, and / or the direct delivery of radioisotopes to tumor cells. Other types of DNA damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells.

[0099] The terms "contact" and "exposure," when applied to a cell, are used herein to describe the process by which a therapeutic substance and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with a target cell. Both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing, to effect cell death or stasis.

[0100] 3.Immunotherapy Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may function alone as an effector of therapy or may recruit other cells that actually effect cell death. The antibody may be conjugated with a drug or toxin (chemotherapeutic drug, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function solely as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity combined with inhibition or reduction of Fortilin, provides therapeutic benefit in the treatment of cancer.

[0101] Immunotherapy can also be used as part of combination therapy. The general approach of combination therapy is described below. In one aspect of immunotherapy, the tumor cells must bear some marker that can be targeted, i.e., not present on the majority of other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. Another aspect of immunotherapy is the anti-cancer effect due to immune stimulating effects. There are also immune stimulating molecules, such as cytokines, such as IL-2, IL-4, IL-12, GM-CSP, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FL T3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery, in combination with tumor suppressors, such as mda-7, has been shown to enhance antitumor effects (Ju et al., 2000).

[0102] As mentioned above, examples of immunotherapies currently under investigation or in use include immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Patent No. 5,801,005; U.S. Patent No. 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferons, as well as IL-1, GM-CSP, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent No. 5,830,880 and U.S. Patent No. 5,846,945), and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has antitumor activity and is approved for use in the treatment of malignant tumors (Dillman, 1999). Combination cancer treatment with Herceptin and chemotherapy has been shown to be more effective than either treatment individually. Therefore, it is contemplated that one or more anticancer treatments may be used with the tumor-associated HLA-restricted peptide treatment described herein.

[0103] In adoptive immunotherapy, the patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated by lymphokines, e.g., IL-2, or transduced with genes for tumor necrosis, and re-administered (Rosenberg et al., 1988; 1989). To achieve this, an immunologically effective amount of activated lymphocytes is administered to an animal or human patient in combination with an antigenic peptide composition incorporating an adjuvant, as described herein. The activated lymphocytes are most preferably the patient's own cells, previously isolated from blood or tumor samples and activated (or "expanded") in vitro. This type of immunotherapy has caused regression in some cases of melanoma and renal cancer, but the percentage of responders is small compared to non-responders.

[0104] There are many different approaches for passive immunotherapy of cancer, which can be broadly classified as follows: injection of antibodies alone, injection of antibodies coupled to toxins or chemotherapeutic agents, injection of antibodies coupled to radioisotopes, injection of anti-idiotypic antibodies, and finally, purging of tumor cells in the bone marrow.

[0105] Human monoclonal antibodies are used in passive immunotherapy because they cause little or no side effects in patients. However, their application is somewhat limited by their rarity and, so far, they have only been administered intralesionally. Human monoclonal antibodies against ganglioside antigens have been administered intralesionally to patients suffering from recurrent cutaneous melanoma (Irie & Morton, 1986). After daily or weekly intralesional injections, regression was observed in 6 out of 10 patients. In another study, moderate success was achieved from intralesional injections of two human monoclonal antibodies (Irie et al., 1989). Possible therapeutic antibodies include anti-TNF, anti-CD25, anti-CD3, anti-CD20, CTLA-4-IG, and anti-CD28.

[0106] It may be advantageous to administer multiple monoclonal antibodies against two different antigens, or even antibodies with multiple antigen specificities. Treatment protocols may also include administration of lymphokines or other immune enhancing agents, such as those described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in more detail elsewhere herein.

[0107] 4. Gene Therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or at the same time as the tumor-associated HLA-restricted peptide is administered. Delivering a vector encoding a tumor-associated HLA-restricted peptide together with a second vector encoding one of the following gene products has a combined anti-hyperproliferative effect on target tissue. Alternatively, a single vector encoding both genes may be used. A variety of proteins are encompassed by the present invention, some of which are described below. The various genes that can be targeted for any type of gene therapy combined with the present invention are well known to those skilled in the art and may include any gene involved in cancer.

[0108] Inducer of cell proliferation. Proteins that induce cell proliferation are further classified into various categories according to their functions. What all of these proteins have in common is their ability to control cell proliferation. For example, one of the PDGFs, the sis oncogene, is a secreted growth factor. Oncogenes rarely arise from genes that code for growth factors, and at present, sis is the only known naturally occurring oncogenic growth factor. In one embodiment of the present invention, it is contemplated that antisense mRNA against a particular inducer of cell proliferation is used to prevent the expression of the inducer of cell proliferation.

[0109] The proteins PMS, ErbA, ErbB, and neu are growth factor receptors. Mutations in these receptors result in a loss of controllable function. For example, a point mutation affecting the transmembrane domain of the Neu receptor protein results in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. The altered oncogenic ErbA receptor is thought to compete with the endogenous thyroid hormone receptor, causing unregulated growth.

[0110] The largest class of oncogenes includes signal transduction proteins (e.g., Src, Abl, and Ras). The protein Src is a cytoplasmic protein-tyrosine kinase that in some cases undergoes proto-oncogene to oncogene conversion via a mutation at tyrosine residue 527. In contrast, the proto-oncogene to oncogene conversion of the GTPase protein ras occurs, as an example, by a valine to glycine mutation at amino acid 12 in the sequence, which reduces ras GTPase activity. The proteins Jun, Fos, and Myc are proteins that exert direct effects on nuclear function as transcription factors.

[0111] Inhibitors of cell proliferation. Tumor suppressor oncogenes function to inhibit excessive cell proliferation. Inactivation of these genes destroys their inhibitory activity, resulting in uncontrolled proliferation. The most common tumor suppressors are Rb, p53, p21, and p16. Other genes that may be used according to the invention include APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, C-CAM, MMACl / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / p16 fusions, and p21 / p27 fusions.

[0112] Regulators of programmed cell death. Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintenance of homeostasis in adult tissues, and suppression of carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like proteases have been shown to be important regulators and effectors of apoptosis in other systems. Bcl-2 proteins, discovered in association with follicular lymphoma, play prominent roles in regulating apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein is now recognized as a member of a family of related proteins that can be classified as cell death agonists or cell death antagonists.

[0113] After its discovery, Bcl-2 was shown to act to suppress cell death induced by a variety of stimuli. It is now clear that there is a family of Bcl-2 cell death control proteins that share structural and sequence homology. These various family members have been shown to possess functions similar to Bcl-2 (e.g., BclxL, Bclw, Bcls, Mcl-1, Al, Bfl-1) or to oppose Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0114] 5.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0115] Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue. Tumor resection refers to the physical removal of at least a portion of the tumor. Surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery) in addition to tumor resection. It is further contemplated that the present invention may be used in conjunction with the removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0116] A cavity may be formed in the body by the removal of a part of cancerous cells, tissue, or entire tumor. Treatment may be accomplished by perfusion, direct injection, or local application of the area with additional anti-cancer treatment. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be at varying dosages.

[0117] V. Antibody Conjugates The antibody can be linked to at least one agent to form an antibody conjugate. It is common to link, covalently bind or complex at least one desired molecule or moiety to increase the effectiveness of the antibody molecule as a diagnostic or therapeutic agent. Such a molecule or moiety can be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as immunosuppression / anti-inflammatory. Non-limiting examples of such molecules are given above. Such molecules are optionally attached via a cleavable linker designed to allow the molecule to be released at or near the target site.

[0118] In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands such as biotin.

[0119] Antibody conjugates are generally preferred for use as diagnostic agents.Antibody diagnostic agents are generally divided into two classes: those for use in in vitro diagnosis, such as in various immunoassays, and those for use in in vivo diagnostic protocols, generally known as "antibody-directed imaging".Many suitable imaging agents are known in the art, and methods for attaching them to antibodies are also known (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509).The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, substances detectable by NMR, and X-ray imaging agents.

[0120] In the case of paramagnetic ions, examples include ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0121] In the case of radioisotopes for therapeutic and / or diagnostic applications, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, Iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, Technetium 99m , and / or yttrium 90 is often preferred for use in certain embodiments, with technetium 99m and / or indium 111 Radiolabeled monoclonal antibodies can be said to be often preferred because they are low energy and suitable for long-distance detection. Radiolabeled monoclonal antibodies can be made according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies can be labeled with technetium-99m by a ligand exchange process, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium onto a Sephadex column, and applying the antibody to the column. Alternatively, direct labeling techniques can be used, for example, by incubating pertechnetate with a reducing agent, such as SNCh, with a buffer solution, such as sodium potassium phthalate solution, and with the antibody. Intermediate functional groups often used to bind radioisotopes to antibodies and which exist as metal ions are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0122] Among the fluorescent labels contemplated for use as conjugates are Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPYFL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0123] Another type of antibody conjugate contemplated is linked to an enzyme (enzyme tag) that produces a colored product when contacted with a secondary binding ligand and / or a chromogenic substrate, and is primarily intended for in vitro use. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin compounds, avidin compounds, and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0124] Yet another known method of site-specific attachment of molecules to antibodies involves the reaction of antibodies with hapten-based affinity labels. In essence, hapten-based affinity labels react with amino acids in the antigen-binding site, thereby destroying this site and preventing specific antigen reaction. However, this may not be advantageous, as it may result in loss of antigen binding by the antibody conjugate.

[0125] Molecules containing azide groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). Specifically, 2- and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binding agents.

[0126] Several methods for attachment or conjugation of antibodies to conjugate moieties are known in the art. Some attachment methods include, for example, the use of metal chelate complexes with organic chelating agents attached to antibodies, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3a-6a-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may be reacted with enzymes in the presence of coupling agents, such as glutaraldehyde or periodic acid. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, with a detectable imaging moiety attached to the antibody using a linker, such as methyl-p-hydroxybenzimidate or N-succinimidyl 1-3-(4-hydroxyphenyl)propionate.

[0127] In other embodiments, immunoglobulins are derivatized by selectively introducing sulfhydryl groups into the Fe region of the immunoglobulin using reaction conditions that do not alter the binding site of the antibody. Antibody conjugates made according to this methodology have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to the carbohydrate group of the Fe region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to generate diagnostically and therapeutically promising antibodies that are currently undergoing clinical evaluation.

[0128] VI. Immunodetection Methods Additionally, in further embodiments, there are immunodetection methods for binding, purifying, removing, quantifying, and otherwise generally detecting PD-L1 or PD-L2 and their associated antigens. Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays, and Western blots, to name a few. In particular, competitive assays for the detection and quantification of PD-L1 and PD-L2 antibodies are also provided. Steps of various useful immunodetection methods are described in the scientific literature, e.g., Doolittle and BenZeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, immunobinding methods include obtaining a sample and, optionally, contacting the sample with a first antibody under conditions effective to allow the formation of an immune complex according to the embodiments described herein.

[0129] Contacting a selected biological sample with an antibody under conditions effective and for a time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture for a time sufficient for the antibody to form immune complexes, i.e., bind, with any PD-L1 and PD-L2 present. After this time, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove non-specifically bound antibody species, allowing only specifically bound antibodies within the primary immune complexes to be detected.

[0130] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches.These methods are generally based on the detection of any of the following labels or markers, for example: radioactive, fluorescent, biological, and enzymatic tags.Patents relating to the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.Of course, as known in the art, additional advantages can be found by using secondary binding ligands, for example, second antibodies and / or biotin / avidin ligand binding arrangements.

[0131] The antibody used in detection may itself be linked to a detectable label, in which case it is possible to determine the amount of primary immune complexes in the composition simply by detecting this label. Alternatively, the first antibody bound in the primary immune complexes can be detected by a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody, and therefore may be called a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand or secondary antibody under conditions effective to allow the formation of secondary immune complexes for a sufficient time. The secondary immune complexes are then washed to remove non-specifically bound labeled secondary antibodies or secondary ligands, and the label remaining in the secondary immune complexes is then detected.

[0132] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, e.g., an antibody, having binding affinity for the antibody is used to form secondary immune complexes as described above. After washing, the secondary immune complexes are again contacted with a third binding ligand or antibody having binding affinity for the second antibody under effective conditions and for a sufficient time to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label that allows the detection of the tertiary immune complexes thus formed. This system can provide signal amplification if desired.

[0133] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin attached to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first stage antibody. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in sequential solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, where each step adds an additional biotin site to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second stage antibody against biotin. This second stage antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. By appropriate amplification, macroscopically visible conjugates can be made.

[0134] Another known method of immunodetection utilizes immuno-PCR (polymerase chain reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple incubations of streptavidin with biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed away with a low pH or high salt buffer that releases the antibody. The resulting washing solution is then used to perform a PCR reaction with suitable primers, along with appropriate controls. At least theoretically, the enormous amplification power and specificity of PCR can be used to detect a single antigen molecule.

[0135] A. ELISA Immunoassays are, in the simplest sense, binding assays. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it is easily understood that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. can also be used.

[0136] In one exemplary ELISA, the antibodies of the disclosure are immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing PD-L1 and / or PD-L2 is then added to the well. After binding and washing away non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by the addition of another bispecific antibody to PD-L1 and PD-L2 linked to a detectable label, or an anti-PD-L1 or anti-PD-L2 antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by the addition of a second bispecific antibody to PD-L1 and PD-2, or an anti-PD-L1 or anti-PD-L2 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label.

[0137] In another exemplary ELISA, samples suspected of containing PD-L1 and / or PD-L2 antigens are immobilized onto a well surface and then contacted with the bispecific anti-PD-L1 anti-PD-L2 antibody. After binding and washing away non-specifically bound immune complexes, the bound bispecific anti-PD-L1 anti-PD-L2 antibody is detected. Immune complexes may be detected directly if the primary bispecific anti-PD-L1 anti-PDL2 antibody is linked to a detectable label. Again, immune complexes may be detected using a second antibody that has binding affinity for the first bispecific anti-PD-L1 anti-PD-L2 antibody, where the second antibody is linked to a detectable label.

[0138] Regardless of the format used, ELISAs share certain features, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0139] In coating a plate with either antigen or antibody, the wells of the plate are generally incubated overnight or for a specified period of time with a solution of the antigen or antibody. The wells of the plate are then washed to remove incompletely adsorbed material. The remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum to the surface.

[0140] In ELISA, it is probably more common to use secondary or tertiary detection means than direct methods. Thus, after binding proteins or antibodies to the wells, coating with a non-reactive material to reduce background, and washing to remove unbound materials, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in combination with a labeled tertiary antibody or third binding ligand.

[0141] By "under conditions effective to allow the formation of immune complexes (antigen / antibody)" is meant that the conditions preferably include dilution of the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0142] "Appropriate" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 hour to about 2-4 hours, preferably at a temperature of about 25°C to 27°C, or may be overnight at about 4°C.

[0143] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of immune complexes, even if only in trace amounts, can be determined.

[0144] To provide detection means, the second or third antibody is associated with a label to allow detection.Preferably, this is an enzyme that produces color when incubated with a suitable chromogenic substrate.Thus, it is desirable to contact or incubate the first and second immune complexes with the antibody conjugated with urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase for a period and under conditions favorable for further immune complex formation (e.g., incubation in PBS-containing solution, e.g., PBS-Tween, at room temperature for 2 hours).

[0145] After incubation with the labeled antibody, followed by washing to remove unbound material, the amount of label is quantified, for example, by incubation with a chromogenic substrate, e.g., urea or bromocresol purple or 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) or H202 if the enzyme label is peroxidase. Quantitation is then accomplished by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0146] B. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Gel electrophoresis is used to separate native or denatured proteins according to polypeptide length (denaturing conditions) or the three-dimensional structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) where they are probed (detected) using an antibody specific for the target protein.

[0147] Samples may be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (if the sample volume is large), a homogenizer (if the volume is small), or by sonication. Cells may also be disrupted by one of the mechanical methods mentioned above. However, it should be noted that bacterial, viral, or environmental samples may be the source of proteins, and thus Western blotting is not limited to cell studies only. A combination of detergents, salts, and buffers may be used to aid in cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0148] The proteins of a sample are separated using gel electrophoresis. Protein separation can be by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful format for determining proteins. It is also possible to use two-dimensional (2D) gels, which spread the proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (pH with a neutral net charge) in the first dimension and according to molecular weight in the second dimension.

[0149] To make the proteins available for antibody detection, they are transferred from within the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers is placed on top of it. The entire stack is placed in a buffer solution, which moves along the paper by capillary action, carrying the proteins with it. Another method for transferring proteins, called electroblotting, uses an electric current to attract the proteins from the gel to a PVDF or nitrocellulose membrane. The proteins move from within the gel to the membrane while maintaining the organization they had in the gel. As a result of this blotting process, the proteins are exposed in a thin surface layer for detection (see below). Both types of membranes are chosen for their nonspecific protein binding properties (i.e., they bind all proteins equally). Protein binding is based on hydrophobic interactions and on charged interactions between the membrane and the proteins. Nitrocellulose membranes are less expensive than PVDF, but are much more fragile and do not stand up well to repeated probing. The uniformity and overall effectiveness of the transfer of proteins from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. After transfer, proteins are detected using a labeled primary antibody or an unlabeled primary antibody followed by indirect detection using labeled Protein A or a labeled secondary antibody that binds to the Fe region of the primary antibody.

[0150] C. Immunohistochemistry The antibodies may be used with both fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). Methods for preparing tissue blocks from these granular specimens have been used successfully in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0151] Briefly, frozen sections may be prepared by rehydrating 50 ng of frozen "ground" tissue in a small plastic capsule in phosphate buffered saline (PBS) at room temperature; pelleting the particles by centrifugation; resuspending in viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; flash freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen tissue cylinder; securing the tissue cylinder to a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used for cutting serial sections.

[0152] Permanent sections may be prepared by a similar method including rehydrating a 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours of fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; chilling in ice water to harden the agar; removing the tissue / agar block from the tube; immersing and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0153] D. Immunodetection Kit Moreover, in a further embodiment, there is an immunodetection kit for use with the immunodetection methods described above. The immunodetection kit thus comprises, in suitable container means, a first bispecific antibody that binds to PD-L1 antigen and / or PD-L2 antigen, and optionally an immunodetection reagent.

[0154] In certain embodiments, the bispecific antibody against PD-L1 and PD-L2 can be pre-bound to a solid support, such as a column matrix and / or a well of a microtiter plate. The immunodetection reagent of the kit can take any of a variety of forms, including a detectable label associated with or linked to a given antibody. Detectable labels associated with or attached to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0155] Further suitable immunodetection reagents for use in the kits of the present invention include bicomponent reagents comprising a second antibody having binding affinity for the first antibody together with a third antibody having binding affinity for the second antibody, where the third antibody is linked to a detectable label. As mentioned above, numerous exemplary labels are known in the art, and all such labels may be used in connection with the embodiments described herein.

[0156] The kit may further comprise appropriately aliquoted compositions of PD-L1 antigen and PD-L2 antigen, which may be labeled or unlabeled, that may be used to prepare a standard curve for the detection assay. The kit may contain the antibody-label conjugate in fully conjugated form, in the form of an intermediate, or as separate moieties that are conjugated by the user of the kit. The components of the kit may be packaged in either aqueous media or in lyophilized form.

[0157] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably, suitably aliquoted. The kits will also include means for containing the antibody, antigen, and other reagent containers in seal for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held. EXAMPLES

[0158] VII. Examples The following examples are included to illustrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore may be considered to constitute preferred modes for its practice. However, in light of this disclosure, it should be understood by those skilled in the art that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0159] Example 1 – Materials and Methods Antibody characterization. Antibody candidates generated from yeast surface display screening were tested for their ability to bind PD-L1 or PD-L2. To generate affinity KDs for human PD-L1 and PD-L2, candidate monoclonal Abs were loaded onto an Anti-Human Fc Capture (AHC) biosensor at 100 nM (15 μg / mL) and tested for association and dissociation with human PD-L1 or PD-L2 proteins in a dilution series ranging from 30 to 0.37 nM. Binding and release of the analyte (PD-L1 or PD-L2) was recorded in real time by the Octet instrument and then used to calculate the KD, Kon, and Kdis; results are derived from 2:1 global fit modeling by reference well subtraction. To generate affinity KDs for mouse PD-L1 and PD-L2, candidate antibodies were covalently immobilized at 100 nM (15 μg / mL) on activated amine-reactive second generation (AR2G) biosensors (quenched by 1 M ethanolamine pH 8.5 after protein loading) and tested for association and dissociation with mouse PD-L1 or PD-L2 proteins in a dilution series from 300 to 1 nM. Analyte binding and release were recorded in real time by the Octet instrument and then used to calculate KD, Kon, and Kdis; results are derived from 2:1 global fit modeling by reference well subtraction.

[0160] Dual PD-L1 / PD-L2 specific antibody activity in mixed lymphocyte reaction. CD14+ monocytes were isolated from peripheral blood mononuclear cells using CD14 microbeads. Cells were seeded at 1 million / mL and stimulated with IL-4 and GM-CSF in 10% FCS / RPMI / P / S cell culture medium. To differentiate the cells into immature dendritic cells (IDCs), they were cultured for 7 days and various concentrations of PD-L1 antibody, both PD-L1 and PD-L2 antibodies, DiPDL antibody, or commercial antibodies were added. IDCs were then used to stimulate CD4+ T cells at a CD4:IDC ratio of 10:1. IFNγ was assayed by ELISA according to the protocol provided by R&D systems.

[0161] Antibody activity against syngeneic mouse colorectal cancer. 1×10 5 CT26 or CT26-PDL2 colorectal cancer cells are implanted subcutaneously into the right flank of female BALB / c mice. On days 3, 6, 9, 12, and 15, mice receive PD-L1 / PD-L2 antibody (250ug), control antibody (e.g., anti-PD-1), or vehicle intraperitoneally. Tumor growth is tracked by caliper measurement, and mice are scored as no longer surviving when they die or tumor volume exceeds 1000mm3.

[0162] Antibody activity against BFTC909 renal cell carcinoma in humanized mice. 5 × 10 6 BFTC909 renal cell carcinoma cells will be implanted subcutaneously. Tumors >50 mm 3 Upon reaching tumor mass, mice receive PD-L1 / PD-L2 antibody (250 μg), control antibody (e.g., anti-PD-1), or vehicle intraperitoneally twice weekly for three weeks. Tumor growth is tracked by caliper measurement and tumors are then annihilated when mice die or tumor volumes reach 1000 mm. 3 When the number of days between 0 and 15 is exceeded, the mouse is scored as no longer viable.

[0163] Measurement of human insulin receptor binding. 2×10 6CHO-K cells or CHO-K-human insulin receptor (CHO-INSR) cells are placed into wells of a 96-well round-bottom tissue culture plate and pelleted by centrifugation at 2000 RPM for 2 minutes. The pellet is resuspended in 100μl of flow cytometry staining buffer containing a range of PD-L1 / PD-L2 bispecific antibodies at a concentration range (typically 0.1, 1, 10μg / mL) and incubated at 4°C for 30 minutes to 1 hour. The cells are then pelleted and resuspended with a 1:300 dilution of anti-human IgG PE secondary antibody. After a further 30 minutes of incubation, the cells are finally pelleted and resuspended in 100μl of flow cytometry buffer. The resuspended cells are run on a BD LSR II flow cytometer to assess the intensity of PE fluorescence in CHO-INSR cells compared to CHO-K cells to assess the amount of binding to the human insulin receptor.

[0164] Example 2 - Results Generation of PD-L1 / PD-L2 bispecific antibodies without insulin receptor cross-reactivity. PD-L1 and PD-L2 heavy and light chains from several rounds of Adimab® selection generated the high affinity PD-L1 / PD-L2 bispecific antibody ADI-37464. This antibody, despite its potent antitumor activity, was also found to have high affinity for the human insulin receptor (INSR) making it unsuitable for clinical use. Figure 1 shows that five new clones (38000-38004) were generated by targeted point mutations in the light chain complementarity determining regions (CDRs) with the goal of eliminating insulin receptor binding while preserving high PD-L1 / PD-L2 dual affinity. Surprisingly, such modifications resulted in little discernible difference in binding affinity between the parent clones and the modified antibodies. Three of these derivatives (38002, 38003, 38004) showed significantly reduced INSR binding along with retained PD-L1 / PD-L2 dual affinity and were carried forward for further studies.

[0165] Affinity measurements of 38000 series PD-L1 / PD-L2 antibodies. Using Octet and Biocore, the affinities of the 38000 series antibodies to PD-L1 and PD-L2 were measured relative to the parent antibody ADI-37464 (Figure 2). By all measures, 38002 demonstrated binding affinities to PD-L1 and PD-L2 that were not significantly different from 37464.

[0166] Treatment of CT26 parental and CT26-PD-L2 tumors with 38000 series PD-L1 / PD-L2 bispecific antibodies. In the parental CT26 syngeneic model of colorectal cancer, 38002 showed comparable therapeutic activity to the parental antibody 37464 (Figure 3A). In the high PD-L2 overexpressing CT26-PD-L2 model, 38002 showed comparable tumor growth inhibition to the parental 37464 clone (Figure 3B). Both 38002 and 37464 were able to cure CT26-PD-L2 animals, with 37464 curing a greater proportion, although the difference between 38002 and 37464 in this experiment was not statistically significant. In the second CT26 parental study, 38002 demonstrated greater tumor growth inhibition than PD-1 blockade, and was again statistically comparable to the parental PD-L1 / PD-L2 bispecific antibody 37464 (Figure 4).

[0167] Treatment of BFTC-909 renal cell carcinoma in humanized mice. In treating human BFTC909 renal cell carcinoma tumors in bone marrow stem cell humanized huNOG-ExL mice, 38002 demonstrated the highest reduction in tumor growth compared to Keytruda or another PD-L1 / PD-L2 bispecific clone 27907 over 10 days of treatment (Figure 5).

[0168] Human Mixed Lymphocyte Reaction (MLR). In paired mixed lymphocyte reactions from two separate donors, the addition of PD-L1 / PD-L2 bispecific antibody 38002 significantly enhanced interferon-gamma release to similar or greater levels compared to PD-L1 / PD-L2 bispecific antibody 27907 (Figure 5).

[0169] Measurement of off-target insulin receptor binding. The 37464 antibody and all 38000 antibodies showed high binding to PD-L1 CHO cells, PD-L2 CHO cells, and dual PD-L1 and PD-L2 CHO cells by flow cytometry, while 37464 also showed significant binding to CHO cells overexpressing INSR at 1 μg / mL. In contrast, the two derived clones 38002 and 38004 show no detectable binding to INSR at 1 μg / mL, as shown in Figure 6. The lack of INSR binding at concentrations achievable in vivo is a key feature that makes the 38000 series of PD-L1 / PD-L2 antibodies suitable for therapeutic use in patients, unlike the conventional 37464 antibody.

[0170] Example 3 - Analysis of binding to insulin receptor on CHO cells, ADCC activity, and ADCP activity of 38002 compared to 37464 Expression of INSR, PD-L1, and PD-L2 in CHO-INSR cells. The cells used in this example, Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-INSR), were obtained from ATCC (CHO INSR 1284-CRL-3307|ATCC). The cells were first characterized for expression of INSR, PD-L1, and PD-L2 by flow cytometry. The engineered CHO cells showed high expression of INSR (>96%) and no expression of PD-L1 or PD-L2, as expected (Figure 10). INSR+CHO cells were incubated with PE-tagged antibodies against INSR, PD-L1, and PD-L2 as a control. Fluorescence was measured by flow cytometry.

[0171] Assessment of binding to INSR on CHO cells. The ability of 38002 and 37464 antibodies to bind live cells expressing human INSR was assessed by using flow cytometry. INSR-CHO cells were incubated with serial dilutions of 38002, 37464, or isotype control antibody, followed by washing four times and incubation with goat anti-human IgG (H+L) secondary antibody, Alexa Fluor 555. Fluorescence was measured by flow cytometry. The data show that 37464, but not 38002, binds to INSR+ cells at high concentrations when compared to the isotype control (Figure 11).

[0172] ADCC activity. To test the ADCC ability of 38002 and 37464 against INSR-expressing target cells, we used a reporter cell assay with Jurkat cells engineered to express the FcγRIIIa receptor, which contains an NFAT response element driving luciferase expression (Figure 12A). The 38002 and 37464 antibodies were incubated with INSR-CHO target cells, which express INSR but not PD-L1 or PD-L2. Antibody 38002 showed no activity against INSR-expressing target cells, whereas antibody 37464 did (Figure 12B).

[0173] ADCP activity. To test the ADCP activity of 38002, a reporter cell assay was used with Jurkat cells engineered to express the FcγRIIa receptor, which contains an NFAT response element driving luciferase expression (FIG. 13A). Antibodies 38002, 37464, and a control isotype were tested against target CHO cells stably expressing INSR. The data showed activity against INSR+ cells at a concentration of 37464, but not 38002 (FIG. 13B).

[0174] This example further illustrates the surprising lack of INSR binding of 38002 at concentrations achievable in vivo, which is a key feature of the 38000 series of PD-L1 / PD-L2 antibodies, distinguishing them from the previously generated 37464 antibody and making them suitable for therapeutic use in patients.

[0175] Example 4 –Evaluation of the binding profiles of 38002, 38003, and 38004 using human plasma membrane protein cell arrays Cell microarrays were used to screen for specific off-target binding interactions of three human IgG1 antibodies: 38002, 38003, and 38004 (collectively the "Test Antibodies") that target human CD274 (PD-L1) and PDCD1LG2 (PD-L2).

[0176] Examination of the binding levels of each test antibody with fixed untransfected HEK293 cells and cells overexpressing CD274 (PD-L1) and PDCD1LG2 (PD-L2) indicated that 20 μg / mL of each test antibody was an appropriate screening concentration. Therefore, test antibody 38002 at 20 μg / mL was screened for binding against fixed human HEK293 cells individually expressing 5861 full-length human plasma membrane proteins and cell surface-tethered human secreted proteins plus an additional 371 human heterodimers. This revealed a total of 37 library hits.

[0177] Each library hit was re-expressed along with two control receptors and re-tested by treatment with 20 μg / mL of each test antibody or control, and this was performed in both fixed and live cells.

[0178] Test antibody 38002 showed specific interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) in fixed and live cell microarrays. Additional interactions with CST1, CST4, and FBLN1 were observed in fixed cell microarrays, and interactions with CST1, CST4, INSR isoform long, INSR isoform short, and INSR+IGF1R heterodimer in live cell microarrays. Interactions with primary targets and INSR isoform long and isoform short were further investigated in flow cytometry dose response studies. EC for interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) 50 The EC50 values ​​for the interaction of INSR with the long and short isoforms were found to be 144.9 and 87.5 μg / mL, respectively. Thus, the EC50 values ​​for the interaction of INSR with the long and short isoforms were found to be 0.03 μg / mL and 0.1 μg / mL, respectively. 50 The EC values ​​are 4830-fold and 1449-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively. 50 These values ​​are 2930- and 875-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively. These data suggest that the secondary interaction of 38002 with INSR is not biologically relevant.

[0179] Test antibody 38003 was not screened against the entire protein library, but against hits generated by test antibody 38002, and showed specific interactions with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) on both fixed and live cell microarrays. Additional interactions with CST4 and FBLN1 were observed on fixed cell microarrays, and additional interactions with CST1, CST4, and INSR isoform short were observed on live cell microarrays. Similarly, test antibody 38004 was screened against hits generated by test antibody 38002, and showed specific interactions with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) on both fixed and live cell microarrays. Additional interactions with CST1 and CST4 were observed only on live cell microarrays.

[0180] Additional flow cytometry validation studies. Human HEK293 cells were transfected with expression vectors encoding ZsGreen1 alone, or ZsGreen1 plus CD274 (PD-L1), PDCD1LG2 (PD-L2), INSR isoform long, INSR isoform short, or CD20 (assay control). Live cell transfectants were incubated with a dose range (0-300 μg / mL) of 38002, 1 μg / mL of rituximab biosimilar (assay control), or assay buffer alone, as indicated. Cells were washed and incubated with the same AF647 anti-human IgG Fc detection antibody used in the cell microarray screen. Cells were washed again and analyzed by flow cytometry using an Accuri flow cytometer (BD). 7AAD live / dead dye was used to exclude dead cells from the analysis, and ZsGreen+ (transfected) cells were selected for analysis.

[0181] Confirmation / specificity screening (fixed cells). In a subsequent confirmation / specificity screening, all 37 library hits, INSR+IGFR, as well as two control receptors (CD20 and EGFR) were overexpressed in HEK293 cells. As expected, the rituximab biosimilar showed moderate / high strength interaction with overexpressed CD20, validating the incubation conditions and detection system.

[0182] Test antibody 38002 showed specific interactions with primary targets CD274 (PD-L1) (high intensity) and PDCD1LG2 (PD-L2) (high intensity). Test antibody 38002 showed additional interactions with CST1 (very low and low intensity), CST4 (low / moderate intensity), and FBLN1 (low / moderate intensity).

[0183] Test antibody 38003 showed specific interactions with primary targets CD274 (PD-L1) (high intensity) and PDCD1LG2 (PD-L2) (high intensity) when screened against library hits generated by test antibody 38002. Test antibody 38003 also showed additional interactions with CST4 (high and medium / high intensity) and FBLN1 (medium / high intensity).

[0184] When screened against library hits generated by test antibody 38002, test antibody 38004 showed specific interactions with primary targets CD274 (PD-L1) (high strength) and PDCD1LG2 (PD-L2) (high strength).

[0185] Confirmation / specificity screening (live cells). In confirmation / specificity screening performed on live cells without cell fixation, test antibody 38002 showed specific interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), as well as additional interactions with INSR (isoform long and isoform short), CST1, CST4, and INSR_IGF1R. The interaction of 38002 with FBLN1, which was low / moderate in fixed cell microarrays, was very low (non-significant) in live cell microarrays.

[0186] The test antibody 38003 showed specific interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), as well as additional interactions with INSR (isoform short), CST1, and CST4.

[0187] The test antibody 38004 showed specific interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), as well as additional interactions with CST1 and CST4.

[0188] Flow cytometry follow-up findings (live cells). To further investigate some of the identified specific interactions of test antibody 38002, CD274 (PD-L1), PDCD1LG2 (PD-L2), INSR isoform long, INSR isoform short, or CD20 were overexpressed in HEK293 cells. Live cell transfectants, including cells transfected with ZsGreen1 alone, were incubated with a dose range (0-300 μg / mL) of 38002, 1 μg / mL of rituximab biosimilar (assay control), or assay buffer alone. The interactions were then investigated by flow cytometry.

[0189] Test antibody 38002 demonstrated binding to its primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) and demonstrated EC 50The EC values ​​were 0.03 μg / mL and 0.1 μg / mL, respectively. Binding to the long and short isoforms of INSR was also observed, with EC 50 The EC50 values ​​for the long isoforms were 144.9 μg / mL and 87.5 μg / mL, respectively. Thus, the EC50 values ​​for the long isoforms were 4830-fold and 1449-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively. The EC50 values ​​for the short isoforms were 2930-fold and 875-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively.

[0190] Conclusion: Test antibody 38002 showed specific interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) in fixed and live cell microarrays. Additional interactions with CST1, CST4, and FBLN1 were observed in fixed cell microarrays, and additional interactions with CST1, CST4, INSR isoform long, INSR isoform short, and INSR+IGF1R heterodimer were observed in live cell microarrays. When the interactions with the primary targets and INSR isoform long and isoform short were further investigated in flow cytometry dose response studies, the EC50 values ​​for interaction with primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2) were found to be 0.03 μg / mL and 0.1 μg / mL, respectively, while the EC50 values ​​for interaction with INSR isoform long and isoform short were found to be 0.02 μg / mL and 0.1 μg / mL, respectively. 50 The EC values ​​were found to be 144.9 μg / mL and 87.5 μg / mL, respectively. Thus, the EC 50 The EC values ​​are 4830-fold and 1449-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively. 50These values ​​are 2930- and 875-fold greater than the primary targets CD274 (PD-L1) and PDCD1LG2 (PD-L2), respectively. These data suggest that the secondary interaction of 38002 with INSR is not biologically relevant.

[0191] Example 5 BiPDL antibodies with effector function mediate efficient ADCC against tumor cells expressing PD-L1 and PD-L2. We evaluated the effector function of BiPDL antibodies by using primary mouse NK cells and calcein-AM labeled U9240 PMBL target cells. Percent specific lysis showed that mouse IgG2a 21680 and 21661 could efficiently mediate ADCC against U2940 (Figure 14A). To investigate the effector function of BiPDL antibodies for ADCC and ADCP, we used Promega's reporter bioassays for ADCC (Jurkat / FcyRIIa) and ADCP (Jurkat / FcyRIIIa). The data show that 27869, 27907, and 37464 could induce much stronger ADCC and ADCP signaling when compared with avelumab or control isotype.

[0192] BiPDL inhibits tumor growth and increases survival in the "cold" tumor model B16-PD-L2 melanoma by increasing T cell activation and reducing bone marrow suppression. To evaluate the efficacy of BiPDL antibodies in the "cold" tumor context, we used B16F10 melanoma cells overexpressing mouse PD-L2. Mice were treated with control anti-mouse PD-1 clone RMP1-14, a combination of mouse IgG2a anti-PD-L1 and anti-PD-L2 antibodies, or BiPDL antibodies 27869 and 37464. As previously observed, B16 tumor growth was not affected by anti-PD1 treatment. However, treatment with BiPDL antibodies could reduce tumor growth (Figure 15A) and increase mouse survival (Figure 15B), suggesting an immunomodulatory function of BiPDL antibodies in the tumor microenvironment. A slightly higher efficacy was observed for 27869.

[0193] To determine the mechanism behind this therapeutic response, we analyzed immune cell populations and their activation status in tumors and lymph nodes. Lymph nodes of mice treated with BiPDL antibody showed higher Ki67 fluorescence by CD4 (Figure 15C) and CD8 (Figure 15D) T cells than mice treated with anti-PD1, anti-PD-L1, or anti-PD-L2 antibodies. Mice treated with BiPDL showed significantly higher Ki67 fluorescence by CD8 T cells in lymph nodes than other treatment conditions. + The proportion of dendritic cells was also shown (Figure 15E). Lower percentages of intratumoral macrophages (Figure 15F), granulocytic myeloid-derived suppressor cells (MDSCs, Figure 15G), and monocytic MDSCs (Figure 15H) expressed the immunosuppressive marker arginase in 37464-treated mice compared to anti-PD1-treated mice.

[0194] 27869 and 37464 behaved similarly in vitro, with 27869 showing minimal therapeutic benefit in the B16 model. However, in mice implanted with colon cancer cell line CT26 (considered a hot tumor mouse model), treatment with 37464 resulted in a more dramatic reduction in tumor growth than 27869 (Figure 16A). To further explore these differences, we hypothesized that 27869 and 37464 may have different effects on PD-L1-B7.1 interactions that provide costimulatory function for T cells. Therefore, we designed a combination bioassay by using PE-labeled human recombinant B7.1 in live CHO / PD-L1 cells analyzed by flow cytometry. These data show that the binding of PE-labeled human recombinant B7.1 is blocked when CHO / PD-L1 cells are pretreated with 37464, but not when preincubated with 27869 or a control antibody. These highly significant functional and structural results demonstrate that BIPDL is a very rare antibody with triple blocking (blocking PD-L1, PD-L2, and B7.1) along with enhanced effector functions ADCC and ADCP. Overall, these results indicate that the 37464 lineage is a better lead than the 27869 lineage.

[0195] Off-target profiling of the BiPDL antibody and further variable region engineering / selection rounds eliminated off-target binding and preserved triple blocking and effector function. Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy, and toxicity of therapeutic antibodies. Here, the inventors performed a primary screen and did not reveal significant off-target binding of the BiPDL antibody, but further human plasma membrane protein cell arrays revealed that 37464 exhibited binding to the insulin receptor INSR (Figure 17A). To eliminate off-target binding with INSR while retaining therapeutic affinity for PD-L1 and PD-L2, two targeted point mutations were performed in the light chain of 37464, in the variable regions CDRL1 and CDRL3. K for PD-L1 was eliminated while eliminating off-target binding with INSR. d =7.62×10 -9 , and K for PD-L2. d =1.9×10 -9 The inventors obtained a new engineered antibody, designated 38002, that preserves affinity for PD-L1 and PD-L2 (monomeric human ligands, FIG. 18A), as measured by

[0196] To confirm the elimination of 38002 binding to INSR, BiPDL antibody was incubated with INSR-expressing cells at different concentrations. 37464 showed binding to INSR at concentrations as low as 1 nM, whereas 38002 binding to INSR showed no significant binding at 33 nM and remained minimal at 100 nM (Figure 17C) for both short and long isoforms of the protein (Figure 18B). Furthermore, flow cytometry staining confirmed that 38002 preserved high binding activity to PD-L1 and PD-L2, and the retained PD-1 blocking activity in Promega bioassays with its ligands remained comparable to anti-PD-1 Keytruda (Figure 17D-E). Furthermore, 38002 retained effector functions ADCC and ADCP only when cells expressed PD ligands (Figure 17F-G), but not when expressing INSR or control (Figure 17F).

[0197] The novel antibody increases cytotoxic cell infiltration and decreases suppressive cell infiltration in B16-PD-L2 melanoma. To confirm that the novel antibody 38002 maintains the in vivo efficacy previously observed, mice were implanted with B16 melanoma and treated with mouse IgG2a anti-PD-1, anti-PD-L1 and / or anti-PD-L2, or 38002. In this model, PD-1 blockade does not improve survival, but 38002 increases survival more efficiently than other treatments, with a 50% survival rate observed. When tumors were analyzed ex vivo by flow cytometry, a non-significant increase in the proportion of NK cells in the infiltrated tumor immune was observed (Figure 19B). Interestingly, after treatment with 38002, there was a significant increase in the percentage of CD8 T cells (Figure 19C), as well as their cytotoxic function, exemplified by the expression of granzyme B (Figure 19D) and perforin (Figure 19E). In association with this cytotoxic response, the inventors also observed a decrease in the proportion of immunosuppressive cells, including granulocytic (FIG. 19F) and monocytic MDSCs (FIG. 19G) as well as macrophages (FIG. 19H).

[0198] Novel antibody inhibits tumor growth in human MDA-MB-231 breast cancer associated with myeloid cell depletion. To evaluate the effect of IMGS001 on MDA-MB-231 growth in vivo in a T cell-free microenvironment, 1 × 10 6 MDA-MB-231 cells were challenged. Starting 3 days after challenge, mice were treated ip with IMGS-001, avelumab, or PBS as a control every 3 days for a total of 7 doses. Tumors were measured twice weekly. Mice receiving IMGS-001 or avelumab had slower tumor growth compared to the control group treated with PBS (Figure 21A). On days 14, 17, and 19, mice treated with IMGS-001 had significantly less tumor volume compared to the control group, whereas mice treated with avelumab had significantly lower tumor volume compared to the control only on day 17. On day 19, some of the mice were sacrificed and tumors were collected for microscopic examination. Immunohistochemical analysis showed a uniformly distributed infiltration of numerous CD11b-positive cells in untreated tumors, which was significantly and consistently reduced by about one-third in tumors derived from mice treated with IMGS-001.

[0199] It will be readily understood that the components of the various embodiments of the present invention, as generally described and illustrated in the figures herein, could be arranged and designed in a wide variety of different configurations. Thus, the detailed description of the embodiments of the present invention, as depicted in the accompanying figures, is not intended to limit the scope of the invention as claimed, but rather represents only selected embodiments of the invention.

[0200] The features, structures, or characteristics of the invention described herein may be combined in any suitable manner in one or more embodiments. For example, reference herein to "certain embodiments," "some embodiments," or similar words means that the specific features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the invention. Thus, the appearance herein of "certain embodiments," "some embodiments," "other embodiments," or similar words does not necessarily refer to the same group of embodiments, but rather the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0201] It should be noted that references to features, advantages, or similar terms herein do not imply that all of the features and advantages that may be realized by the present invention should or will be present in any single embodiment of the present invention. Rather, terms referring to features and advantages are understood to mean that the specific features, advantages, or characteristics described in connection with an embodiment are included in at least one embodiment of the present invention. Thus, descriptions of features and advantages and similar terms herein may, but do not necessarily, refer to the same embodiment.

[0202] Furthermore, the described features, advantages, and characteristics of the invention may be combined in any suitable manner in one or more embodiments.Those skilled in the relevant art will recognize that the invention may be practiced without one or more of the specific features or advantages of a particular embodiment.In other cases, additional features and advantages that are not present in all embodiments of the invention may be recognized in a particular embodiment.

[0203] Those skilled in the art will readily appreciate that the invention described above may be practiced with steps in a different order and / or hardware elements in a different configuration than those disclosed. Thus, while the invention has been described based on these preferred embodiments, it will be apparent to those skilled in the art that certain modifications, variations, and alternative constructions will become apparent that are within the spirit and scope of the invention. Accordingly, reference should be made to the appended claims in order to determine the metes and bounds of the invention.

[0204] VIII. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025512888000015.tif191146TIFF2025512888000016.tif224138TIFF2025512888000017.tif224119TIFF2025512888000018.tif170144

Claims

1. An antibody or its antigen-binding fragment that binds to PD-L1 and PD-L2, The heavy chain CDR1 amino acid sequence shown in SEQ ID NO:7, the heavy chain CDR2 amino acid sequence shown in SEQ ID NO:8, and the heavy chain CDR3 amino acid sequence shown in SEQ ID NO:9, as well as the light chain CDR1 amino acid sequence shown in SEQ ID NO:10, the light chain CDR2 amino acid sequence shown in SEQ ID NO:11, and the light chain CDR3 amino acid sequence shown in SEQ ID NO:

12. The antibody or its antigen-binding fragment, including the antibody.

2. The antibody or antigen-binding fragment according to claim 1, comprising a light chain sequence with SEQ ID NO:6 and a heavy chain sequence with SEQ ID NO:

4.

3. The antibody or antigen-binding fragment according to claim 1, comprising a light chain sequence having at least 90% identity with SEQ ID NO:6 and a heavy chain sequence having at least 90% identity with SEQ ID NO:

4.

4. The antibody or antigen-binding fragment according to claim 1, comprising a light chain sequence having at least 95% identity with SEQ ID NO:6 and a heavy chain sequence having at least 95% identity with SEQ ID NO:

4.

5. The antibody or antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment.

6. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a chimeric antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is IgG.

8. The antibody or antigen-binding fragment according to claim 1, further comprising a cell-permeable peptide and / or being an intrabody.

9. The antibody according to claim 1, which is a humanized antibody, or an antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody comprises a human IgG1 Fc region.

11. The antibody or antigen-binding fragment according to claim 10, wherein the human IgG1 Fc region comprises an amino acid sequence having G236A, S239D, and I332E amino acid substitutions.

12. The antibody or antigen-binding fragment according to claim 10, wherein the human IgG1 Fc region comprises the amino acid sequence shown in SEQ ID NO:

25.

13. An antibody or antigen-binding fragment according to any one of claims 1 to 9, which exhibits reduced binding to the human insulin-like growth factor 1 receptor (IGF1R) compared to an antibody containing the CDR region of ADI-37464.

14. 1 × 10 for PD-L1 -8 M or less K D An antibody or antigen-binding fragment according to any one of claims 1 to 9, having the following characteristics.

15. 1 × 10 for PD-L2 -8 M or less K D An antibody or antigen-binding fragment according to any one of claims 1 to 9, having the following characteristics.

16. A pharmaceutical composition for treating cancer in a subject having cancer, comprising the antibody or antigen-binding fragment according to any one of claims 1 to 9.

17. The cancer is: (a) Solid tumors; (b) recurrent cancer, metastatic cancer, and / or multidrug-resistant cancer; and / or (c) Cancers selected from the group consisting of lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, lymphoma, kidney cancer, renal cell carcinoma, bone cancer, nerve tissue cancer, melanoma, prostate cancer, vaginal cancer, bladder cancer, acute myeloid leukemia, chronic myeloid leukemia, and multiple myeloma. The pharmaceutical composition according to claim 16.

18. A hybridoma or engineered cell expressing the antibody or antigen-binding fragment according to any one of claims 1 to 9.

19. A polynucleotide encoding the antibody or its antigen-binding fragment according to any one of claims 1 to 9.

20. A vaccine formulation comprising one or more antibodies or antigen-binding fragments according to any one of claims 1 to 9.

21. A conjugate comprising an antibody according to any one of claims 1 to 9 or an antigen-binding fragment thereof, which is bound to an active substance via a linker.

22. The conjugate according to claim 21, wherein the active substance comprises a drug.

23. The conjugate according to claim 21, wherein the active substance comprises a reporter molecule.

24. A method for detecting cells expressing PD-L1 or PD-L2 in a target, (a) a step of contacting a sample derived from the subject with an antibody or antigen-binding fragment according to any one of claims 1 to 9; and (b) A step of detecting cells expressing PD-L1 or PD-L2 in the sample by binding the antibody or its antigen-binding fragment to cells in the sample. The method, including the method.

25. The method according to claim 24, wherein the sample is a body fluid or tissue sample.

26. The method according to claim 24, wherein detection comprises ELISA, RIA, or Western blot.

27. The method according to claim 24, wherein the cells are cancer cells or cells associated with immunosuppression.

28. A pharmaceutical composition for treating immunosuppression in the tumor microenvironment or increasing the immune response against a tumor or cancer, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 9.

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