Patent Description:
PD-L1 is a <NUM> kDa type I transmembrane protein that has been speculated to play a major role in suppressing the immune system. Formation of the PD-L1 /PD-<NUM> and PD-L1 /B7. <NUM> complexes negatively regulates T-cell receptor signaling, resulting in the subsequent downregulation of T cell activation and suppression of anti-tumor immune activity.

PD-L1 regulates the immune response in the suppression of immune system responses during chronic infections, pregnancy, tissue allografts, autoimmune diseases, and cancer.

PD-L1 binds to its receptor, PD-<NUM>, found on activated T cells, B cells, monocytes and myeloid cells, to modulate activation or inhibition. PD-L1 also has an appreciable affinity for the costimulatory molecule CD80 (B7-<NUM>).

Engagement of PD-L1 with its receptor PD-<NUM> on T cells delivers a signal that inhibits TCR-mediated activation of IL-<NUM> production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3ζ. PD-L1 binding to PD-<NUM> also contributes to ligand-induced TCR down-modulation during antigen presentation to naive T cells, by inducing the up-regulation of the E3 ubiquitin ligase CBL-b.

PD-L1 is overexpressed in many cancers, including a wide variety of solid tumors and hematological malignances, such as myeloma, prostate, breast, colon, lung, melanoma, ovarian, salivary, stomach, thyroid tumors, lymphoma and bladder. PD-L1 overexpression in tumor cells may advance tumor invasion and is often associated with poor prognosis.

Furthermore, in many cancers, PD-L1 is over expressed on tumor cells and tumor-infiltrating immune cells, such as macrophages and dendritic cells.

Given the role of PD-L1 in cancer development and immune system regulation, additional tools to detect the presence of PD-L1 , for example for diagnosis and/or patient selection, are desirable.

The blockade therapy of PD-L1 target shows promising clinical benefits in many types of cancer. There is a need in the art for agents that target PD-L1 for the treatment of PD-L1 - associated conditions, such as cancer. The invention fulfills that need and provides other benefits.

Monoclonal antibodies to PD-L1 are known in the art and have been described, for example, in US Patent/Publication Nos. <CIT>, <CIT>, <CIT>, <CIT> and WO Patent/Publication Nos. <CIT>, <CIT>. <CIT>, <CIT>, <CIT>, <CIT>.

<CIT> disclsoes anti-PD-L1 antibodies, nucleic acid encoding the same, therapeutic compositions thereof, and their use enhance T-cell function to upregulate cell-mediated immune responses and for the treatment of T cell dysfunctional disorders, including infection (e.g., acute and chronic) and tumor immunity.

<NPL>, discuss A randomized, open-label phase <NUM> study of pembrolizumab in combination with lenalidomide and low-dose dexamethasone in newly diagnosed and treatment-naive multiple myeloma (MM).

The present invention relates to anti-PD-L1 antibodies and methods of using the same.

Specifically, two antibodies were derived and defined from mouse. They comprise a heavy chain variable region (VH) and/or a light chain variable region (VL) and their Complementarity Determining Region (CDR) as summarized in Table below:.

In some embodiments, the antibody is IgG1 or IgG4, and preferably IgG1λ or IgG1κ.

Antigen-binding fragments of said antibodies are also contemplated. In some embodiments, the antibody fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')<NUM>, and diabody.

in another aspect, provided herein is an isolated nucleic acid or DNA molecule that encodes any of the antibodies of the invention.

In a further aspect, provided herein is an isolated polynucleotide composition, comprising a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain of an antibody or fragment thereof according to the invention.

in another aspect, provided herein is a bispecific molecule comprising the an anti-PD-L1 antibody or antigen-binding fragment thereof according to the invention. Antibody or antigen binding portion of the invention can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand receptor) to generate at least two different the bispecific binding molecule binding sites or target molecules. Antibodies of the invention can in fact be derivatized or linked to more than one other functional molecule to generate more than two different binding sites and / or target molecule binding multispecific molecule; such multispecific molecules are also intended to be as used herein.

In some embodiments, the anti-PD-L1 antibody, or antigen-binding fragment thereof, according to the invention increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay.

In some embodiments, the anti-PD-L1 antibody, or antigen-binding fragment thereof, according to the invention kills cancer in a cytotoxic T lymphocyte (CTL) assay.

In some embodiments, the anti-PD-L1 antibody, or antigen-binding fragment thereof, according to the invention kills myeloma cancer cells and prolongs survive rates in NSG mice.

In one aspect of the invention, the antibody or antigen-binding fragment thereof according of the invention is for use in a method of treating cancer, preferably myeloma.

in some particular embodiments, the PD-L1 antibody is combined with the chemotherapeutic drug lenalidomide. This combination kills myeloma cells in SCID myeloma mice and increases its survive rate.

The invention further provides the use of an antibody or antigen-binding fragment according of the invention for the in vitro detection of PD-L1, or as a diagnostic agent in vitro.

Anti-PD-L1 mouse monoclonal antibodies were generated by hybridoma techniques. Briefly, The DNA sequence encoding the human PD-L1 was expressed with the Fc region of mouse IgG1 at the C-terminus in human 293T cells. Balb/c mice were immunized with the purified PD-L1 antigen emulsified with complete Freund's adjuvant followed by boosting a series of PD-L1 antigen emulsified with incomplete Freund's adjuvant. The antibody-expressing fusioned cells were screened by Enzyme-Linked ImmunoabSorbant Assay (ELISA) using the coated PD-L1 antigen. All ELISA positive clones producing the antibody with the highest specificity were further selected. Two anti-PD-L1 monoclonal antibodies named as Q106 and Q107 among them were finally chosen and further produced using in serum-free medium by in vitro cell culture method, and subsequently purified by Protein A affinity chromatography.

Screen of anti-PD-L1 antibodies against recombinant human PD-L1 was measured by indirect ELISA assay. Ninety-six well Falcon <NUM> polyvinylchloride microtiter plates (Becton Dickinson Inc, Oxnard, CA) were coated with <NUM>µL of recombinant human PD-L1-Fc at <NUM> overnight. The plate was washed three times in PBST (PBS with <NUM>% Tween-<NUM>), and then blocked with PBS containing <NUM>% BSA to prevent nonspecific binding. <NUM>µL of hybridoma supernatant was added to each well and incubated at room temperature for <NUM> hours. Wells were washed three times with PBST and <NUM>µL of HPR-conjugated goat anti-mouse secondary antibody (Biolegend, cat#<NUM>) was added, and further incubated for <NUM> hours at room temperature. After washing, o-Phenylenediamine dihydrochloride (OPD) peroxidase substrate was added to each well and plates were incubated for <NUM> minutes at room temperature. Reactions were stopped using <NUM> N H<NUM>SO<NUM> and the optical density read by an ELISA plate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of <NUM>. The wells filled with anti-PD-L1 serum were served as the positive control. <NUM> positive clones were totally obtained, and continously passaged by limited dilution of cloning. The step was repeated three times for got a stable single of hybridoma. Results showed that two positive clones Q106 and Q107 with the highest titers can stably secrete monoclonal antibodies against human PD-L1.

This example shows the specificity for the anti-PD-L1 antibody of the invention for human PD-L1. In addition, it shows the affinity of two antibodies Q106 and Q107 for human PD-L1 expressed at the cell membrane on 293T-transfected cells (<FIG>). Human PD-L1 were stably transfected into 293T cells. Cells were harvested and plated at <NUM>,<NUM> cells per well in a <NUM>-well plate for binding assay. The PD-L1 antibodies Q106, Q107 or Isotype antibody control were titrated starting at 10µg/ml, in a serial of three-fold dilutions and bound to cells in 50µl volumes for <NUM> minutes on ice. Cells were washed and then detected with anti-mouse IgG PE (BD Biosciences) at <NUM>µg/ml for <NUM> minutes on ice.

All samples were run on a MiltenyiBiotech MACSQuant and Mean Fluorescence Intensity of PD-L1 binding data as a function of anti-PD-L1 antibody concentration was analyzed using FlowJo® software provided by Tree Star. EC<NUM> values (antibodies concentration associated with an half-maximal binding) were calculated using Kaleidagraph. These values are summarized below in Table <NUM>:
Flow cytometry analysis also shows that the specificity of PD-L1 antibodies Q106 and Q107 in wildtype and PD-L1 knockdown mantle cell lymphoma cell line, Granta519 (<FIG>), and multiple myeloma cell line U266 (<FIG>).

Affinity purified both Q106 and Q107 antibodies in Example <NUM> were conjugated to the fluorochromes PE (Invitrogen). Human myeloma, breast cancer and prostate cancer cell lines were maintained in RPMI-<NUM> medium (Fisher Scientific, Herndon, VA) supplemented with <NUM>% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Cells were centrifuged and washed with PBS, then seprately stained with PE-conjugated Q106 and Q107 antibodies, incubated for <NUM> on ice, and washed <NUM> times before analysis. Flow cytometry data were collected with a FACSCantoll (Becton Dickinson) and analyzed with FlowJo V. <NUM> software (TreeStar). Both Q106 and Q107 PD-L1 antibodies can bind to the cellular PD-L1 protein of Human myeloma cell lines (ARH-<NUM> and U266), breast cancer cell lines (MB-<NUM> and MCF-<NUM>), and prostate cancer cell lines (PC-<NUM> and LN3) in FACS assays (<FIG>).

Allogeneic CD3+, CD4+ and CD8+ T cells were purified from PBMCs from healthy donors using magnetic cell sorting (Miltenyi Biotec). CD3+ T cells were labeled with <NUM>(<NUM>)-carboxyfluorescein diacetate succinimidyl ester (CFSE; <NUM> □M; Invitrogen) for <NUM> minutes at <NUM>. After washing, T cells (<NUM> × <NUM><NUM>/<NUM>µL/well) were seeded into <NUM>-well U-bottomed tissue culture plates (Corning Glassworks) and cocultured with irradiated MM cell line ARH-<NUM> at <NUM> for <NUM>-<NUM> days in <NUM>% CO<NUM> in Aim-V medium supplemented with <NUM>% pooled human serum (T-cell medium). Flow cytometry analysis was used to detect dilution of CFSE. Anti-PD-L1 antibodies Q106 and Q107 can significantly increase CD3+ (<FIG>), CD4+ (<FIG>) and CD8+ (<FIG>) T cell proliferations in CFSE dilution assay.

Generation of tumor-reactive, alloantigen-specific cytotoxic T lymphocyte lines.

Allogeneic CD3+ T cells were cocultured in T-cell medium with irradiated ARH-<NUM>. After <NUM> days of coculture, CD3+ T cells were harvested and restimulated with newly irradiated above described tumor cells. The cultures were fed with fresh T cell medium containing recombinant IL-<NUM> (<NUM> IU/ml), IL-<NUM> (<NUM> ng/ml), and IL-<NUM> (<NUM> ng/ml) (R&D Systems). The frequencies of CD3+CD8+ T cells were monitored every week by flow cytometry. After at least <NUM> repeated cycles of in vitro restimulation, cytotoxic T lymphocyte (CTL)-cell line was generated, and named as CTL-ARH-<NUM>. The CTL-cell line was expanded in T-cell medium containing recombinant IL-<NUM> (<NUM> IU/ml), IL-<NUM> (<NUM> ng/ml), and IL-<NUM> (<NUM> ng/ml) for <NUM> weeks and subjected to functional tests.

The standard <NUM>-hour <NUM>Cr-release assay was performed to measure cytolytic activity of the T-cell line with target cells including ARH-<NUM>, U266, ARP-<NUM>, K562, B cells, PBMCs and primary tumor cells isolated from multiple myeloma patients. To determine whether the cytolytic activity was restricted by Major Histocompatibility Complex (MHC) class I or II molecules, target cells were pretreated with <NUM>µg/ml antibodies against HLA-ABC (Serotec Ltd), HLA-DR (Immunotech), or control IgG (eBioscience).

ARH-<NUM>-reactive CTL-cell line from HLA-A*<NUM>+ healthy blood donors wa generated as described above. As shown in <FIG>, the percentages of tumor-reactive CD8+ T cells in CTL line were increased while applied Q106 antibody in CFSE dilution assay. Next, the cytolytic activity of ARH-<NUM> CTL cell line was examined. The data showed that the CTL cells not only killed the stimulatory ARH-<NUM> cell lines, but also killed HLA-A*<NUM>+U266 and primary MM cells (patients <NUM> and <NUM>). No killing was observed on HLA-A*<NUM>-ARP-<NUM>, ARK and primary MM cells (patient <NUM> and <NUM>) or K562 cells (<FIG>) at all, indicating that NK cells were not responsible for the killing. Moreover, purified normal allogeneic (to the T cells) PBMCs and B cells from a HLA-A*<NUM>+ donor and a MM patient were used as target cells to demonstrate whether the CTL cells were cytolytic to normal cells. As shown in <FIG>, no killing was observed against normal B cells or PBMCs, although the T cells were alloantigen-specific.

More importantly, when MM passaged cell lines or MM primary cells were pre-incubated with anti-PD-L1 antibodies, Q106 and Q107, respectively, these cells became more sensitive to the killing (<FIG>; P< <NUM> to P<<NUM>).

ADCC was measured by <NUM>-Chromium (<NUM>-Cr) release assays. In ADCC assay, purified PBMCs from normal volunteers was used as effector cells. Target cells (<NUM>×<NUM><NUM>) were incubated with <NUM> µCi of <NUM>-Cr for <NUM> at <NUM> with gentle resuspension of pellet at <NUM> intervals. After washing, cells were plated at <NUM>,<NUM> cells/well in <NUM>-well U-bottom plate with a different concentration of PBMCs. This is followed by the addition of antibody solution in a final concentration ranging from <NUM> to <NUM> µg/ml. Both anti-PD-L1 antibodies, Q106 and Q107 and mouse IgG1 (BioLegend) were used as tested groups and isotype control. Cells are then incubated for <NUM> at <NUM>, and released <NUM>-Cr was analyzed using a Gamma Counter. Spontaneous release was determined from target cells without the addition of antibody and PBMCs, and maximum release was determined from target cells with <NUM>% Triton X-<NUM> without the addition of antibody and PBMCs. Percent cytotoxicity was calculated as [(counts in sample - spontaneous release)/(maximum counts - spontaneous release)] ×<NUM>%. All experiments were performed in triplicates. Data showed that ADCC induced by anti-PD-L1 antibodies, Q106 and Q107 killed hematological tumor cells (<FIG>) and solid cancer cells(<FIG>).

CDC was measured by <NUM>-Chromium (<NUM>-Cr) release assays as demonstrated in Example <NUM>. In CDC assay, guinea pig serum (Sigma-Aldrich) was used as complement source. Target cells (<NUM>×<NUM><NUM>) were incubated with <NUM>µCi of 51Cr for <NUM> at <NUM> with gentle resuspension of pellet at <NUM> intervals. After washing, cells were plated at <NUM>,<NUM> cells/well in <NUM>-well U-bottom plate with different concentration of guinea pig serum. This is followed by the addition of antibody solution, in a final concentration ranging from <NUM> to <NUM>µg/ml. Both Q106 and Q107 PD-L1 antibodies and mouse IgG1 (BioLegend) were used as tested group and isotype control. Cells are then incubated for <NUM> at <NUM>, and released <NUM>-Cr was analyzed using a Gamma Counter. Spontaneous release was determined from target cells without the addition of antibody and guinea pig serum, and maximum release was determined from target cells with <NUM>% Triton X-<NUM> without the addition of antibody and guinea pig serum. Percent cytotoxicity was calculated as [(counts in sample - spontaneous release)/(maximum counts - spontaneous release)] ×<NUM>%. All experiments were performed in triplicate. The data showed that CDC induced by anti-PD-L1 antibodies, Q106 and Q107 killed both hematological tumor cells(<FIG>) and solid cancer cells(<FIG>).

It is now apparent that many tumors exploit expression of PD-<NUM> ligands as a means to attenuate anti-tumor T cells responses. Several human cancers have been characterized to express elevated levels of PD-L1 on both tumors and tumor-infiltrating leukocytes and this elevated PD-L1 expression is often associated with a worse prognosis. Mouse tumor models demonstrate similar increases in PD-L1 expression within tumors and demonstrate a role for the PD-<NUM> /PD-L1 pathway in inhibiting tumor immunity.

Here we present an experiment demonstrating the impact of blocking PD-L1 on multiple myeloma U266 of growth in NSG mice (<FIG> and <FIG>). These cells express PD-L1, but not PD-L2 on their cell surface as assessed by Flow Cytometry. Mice were inoculated intravenously with <NUM> million U266 cells on Day <NUM>. On Day <NUM> (when tumors were seen in luminous image), <NUM> mice/group were treated with <NUM>/kg of PD-L1 antibody(Q106) for the 3x/week duration of the study. In the study, mouse IgG was set up as control. Blockade of PD-L1 in late intervention is highly effective as a single agent therapy at preventing tumor growth. In contrast, control IgG showed no evidence of inhibiting tumor growth. These results demonstrate the unique role of the PD-<NUM>/PD-L1 axis in suppression of the anti-tumor immune response and support the potential for the treatment of human cancers with the antibody that blocks the PD-L1 interaction with PD-<NUM> and B7.

U266 NSG tumor model: Methodically, on Day <NUM>, <NUM> of mice were inoculated intraveneously with <NUM> million of U266-luciferase cell in <NUM> microliters of PBS. Take image in IVIS imaging system every week after tumor inoculation. About <NUM>-<NUM> weeks later, <NUM> of <NUM> mice with similar-sized tumors were recruited into one of <NUM> treatment groups as listed below. The tumors were measured by taking image every week. Mice not recruited into below treatment groups, due to dissimilar tumor volume were euthanized:.

Shown are bioluminescence images (<FIG>), tumor burdens (<FIG>) and survival (<FIG>) of mice received a different treatments. Representative results from two independent experiments performed are shown. Error bars = SEM. *P< <NUM>, compared with mouse IgG control.

On Day <NUM>, <NUM> of SCID mice were inoculated subcutaneously with <NUM> million of U266-luciferase myeloma cells in <NUM> microliters of PBS plus matrigel. Mice are allowed to grow tumors. Mice are weighed and measured <NUM> x/week until Day <NUM> (when the tumor volume is between <NUM>-<NUM> mm3). On Day <NUM>, following tumor measurement, mice are recruited into <NUM> of the <NUM> treatment groups below. Mice not recruited into below treatment groups, due to dissimilar tumor volume are euthanized.

Tumors are measured, luminous image taken and mice weighed 2X/week. Animals exhibiting weight loss of ><NUM>% will be weighed daily and euthanized if they lose ><NUM>% body weight. Mice will be euthanized when tumor volumes exceed <NUM>,<NUM> mm3, or after <NUM> months if tumors do not form.

This study showed that combination immunotherapy of anti-PD-L1 antibody, Q106 with lenalidomide blockade was more effective than treatment with the PD-L1 antibody or lenalidomide chemotherapy alone(<FIG>).

Claim 1:
An anti-PD-L1 antibody, or antigen binding fragment, which comprises
a) a heavy chain variable region (HCVR) having sets of complementarity determining regions (CDRs) consisting of: HCDR1 of SEQ ID NO: <NUM>; HCDR2 of SEQ ID NO: <NUM>; and HCDR3 of SEQ ID NO: <NUM>;
and a light chain variable region (LCVR) having sets of complementarity determining regions (CDRs) consisting of: LCDR1 of SEQ ID NO: <NUM>; LCDR2 of SEQ ID NO: <NUM>; and LCDR3 of SEQ ID NO: <NUM>; or
or
b) a heavy chain variable region (HCVR) having sets of complementarity determining regions (CDRs) consisting of: HCDR1 of SEQ ID NO: <NUM>; HCDR2 of SEQ ID NO: <NUM>; and HCDR3of SEQ ID NO: <NUM>,
and a light chain variable region (LCVR) having sets of complementarity determining regions (CDRs) consisting of: LCDR1 of SEQ ID NO: <NUM>; LCDR2 of SEQ ID NO: <NUM>; and LCDR3 of SEQ ID NO: <NUM>.