Method for detecting antibody-dependent cellular phagocytosis

Abstract

Methods are provided for increasing antibody-dependent cellular phagocytosis (ADCP) activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes. In some embodiments, the phagocytes are cultured in the presence of target cells. Also provided are methods for assaying ADCP activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes, and detecting the ADCP activity of the phagocytes in the sample. In some embodiments, the phagocytes are cultured in the presence of target cells.

Description

[Technical Field]

[0001] The general concept of the present invention relates to the field of methods for detecting antibody-dependent cellular phagocytosis (ADCP), which are useful for quantifying ADCP activity of antibodies, and in particular, are useful for quantifying ADCP activity of therapeutic antibodies useful in the treatment of cancer.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 379,599, filed October 14, 2022, and U.S. Provisional Application No. 63 / 379,612, filed October 14, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0003] Several therapeutic antibodies have been developed and approved for use in cancer treatment (Mossner et al., 2010; Shuptrine, Surana, & Weiner, 2012; Weiner, Murray, & Shuptrine, 2012; Weiner, Surana, & Wang, 2010). These therapeutic antibodies can directly affect tumor growth by interfering with receptor signaling, blocking downstream signaling, and inducing apoptosis. They can also indirectly affect tumor growth by activating complement-dependent cytotoxicity and affecting immune cell effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (Herter et al., 2014; Weiner et al., 2010). Unlike ADCC, which relies on effector cells to secrete molecules that activate apoptotic cell death, ADCP relies on macrophages to directly destroy target cells by phagocytosis. These macrophages in particular are poised to become impressive effectors of cancer immunotherapy.

[0004] Macrophages are present in tissues throughout the body (Geissmann et al., 2010), and there are specialized tissue-specific macrophage populations, such as Kupffer cells in the liver, microglia in the brain, osteoclasts in bone, and alveolar macrophages in the lungs. Importantly, macrophages are known to infiltrate solid tumors, thereby gaining access to tumor cells. These macrophages are important for the efficacy of many antibodies. In particular, in vivo and in vitro studies have demonstrated that ADCP is an important and potent mechanism of action (MOA) for the treatment of different cancers with different therapeutic antigens, such as the treatment of multiple myeloma (MM) with the anti-CD38 antibody daratumumab (Khagi & Mark, 2014; Overdijk et al., 2015) and the treatment of B-cell malignancies with the anti-CD20 antibody rituximab (Oflazoglu & Audoly, 2010), both of which have been shown to have important ADCP components. ADCP of solid tumors has also been demonstrated in vitro using anti-HER-2 / neu antibodies in breast cancer cells (Watanabe et al., 1999) and anti-epidermal growth factor receptor (EGFR) antibodies in colon cancer cells (Weiskopf et al., 2013). Additionally, comparability studies have shown the importance of differences in ADCP for different therapeutic antibodies that bind to the same target, such as the anti-CD20 antibodies ofatumumab, obinutuzumab, and rituximab (Rafiq et al., 2013).

[0005] The process of ADCP is essentially a two-step mechanism involving (1) binding to target cells (e.g., tumor cells or suitable target cells mimicking a patient's tumor) and (2) binding of macrophages or other phagocytes, such as monocytes, neutrophils, or dendritic cells, to the fragment crystallizable (Fc) domain of an antibody via Fc receptors on the phagocyte, followed by phagocytosis of the target cell. These Fc receptors that mediate ADCP on phagocytes include FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a), although many studies suggest that FcγRIIa is the primary FcγR receptor involved in the induction of ADCP by macrophages (Richards et al., 2008; Weiskopf & Weissman, 2015).

[0006] In response to the recognition and appreciation of the role of ADCP as a critical MOA in effective cancer treatment, regulatory authorities now require data on the effect of ADCP on antibody-mediated cytotoxicity for the approval of therapeutic antibodies. Additionally, ADCP assays can also provide insight into the safety of therapeutic antibodies at different stages, such as the manufacturing or storage of therapeutic products. As a result, drug developers and researchers are rapidly adopting ADCP assays as a critical step during the development and manufacturing of therapeutic antibodies. Unfortunately, however, prototype ADCP assays using primary macrophages from human donors and target cell phagocytosis as an endpoint can be very challenging, time-consuming, and subject to variability, making them unsuitable for routine use. In particular, for primary macrophages generated in vitro from peripheral blood mononuclear cell (PBMC) preparations from human donors, a long differentiation time exists, and donor-to-donor differences can add considerable variability to the assay. Furthermore, the signal-to-noise ratio may not be optimal for distinguishing ADCP activity from nonspecific background. Summary of the Invention

[0007] A method is provided for increasing antibody-dependent cellular phagocytosis (ADCP) activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes.

[0008] In some embodiments, the low % CO2 is about 0.1% CO2 to about 1% CO2. In some embodiments, the low % CO2 is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% CO2. In further embodiments, the low % CO2 is about 1% CO2.

[0009] In some embodiments, the period of time sufficient to increase phagocyte ADCP activity is between 3 and 15 days, and in further embodiments, the period of time sufficient to increase phagocyte ADCP activity is between 3 and 7 days.

[0010] In some embodiments, the method further comprises determining ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

[0011] In some embodiments, the phagocytes are J774A.1 cells or donor cells. In further embodiments, the donor cells are human macrophage cells.

[0012] In some embodiments, the phagocytes are cultured in the presence of target cells. In further embodiments, the target cells are added to the phagocyte culture after about 1 to about 14 days. In still further embodiments, the target cells are added to the phagocyte culture after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In certain embodiments, the target cells are added to the phagocyte culture after about 7 days. In further embodiments, the target cells are Daudi cells, B cells, leukemia cells, or lymphoma cells. In still further embodiments, the target cells are Daudi cells.

[0013] In some embodiments, the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0014] In some embodiments, the expression of a regulator of ADCP is increased. In further embodiments, the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0015] In some embodiments, the expression of a regulator of ADCP is decreased. In further embodiments, the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0016] In some embodiments, the target cell is contacted with an antibody or fragment thereof.

[0017] In some embodiments, the sample is incubated in a humidified chamber or incubator.

[0018] A method is provided for assaying ADCP activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO2 atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes, and detecting the ADCP activity of the phagocytes in the sample.

[0019] In some embodiments, the low % CO2 is about 0.1% CO2 to about 1% CO2. In some embodiments, the low % CO2 is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% CO2.

[0020] In some embodiments, low % CO2 is 1% CO2.

[0021] In some embodiments, the period of time sufficient to increase phagocyte ADCP activity is between 3 and 15 days, and in further embodiments, the period of time sufficient to increase phagocyte ADCP activity is between 3 and 7 days.

[0022] In some embodiments, the method further comprises determining ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

[0023] In some embodiments, the phagocytes are J774A.1 cells or donor cells. In further embodiments, the donor cells are human macrophage cells.

[0024] In some embodiments, the phagocytes are cultured in the presence of target cells, hi further embodiments, the target cells are added to the phagocyte culture after about 3 to about 10 days, e.g., after about 7 days.

[0025] In some embodiments, the target cells are labeled with a fluorescent dye.

[0026] In some embodiments, the phagocytes are J774A.1 cells or donor cells. In further embodiments, the donor cells are human macrophage cells.

[0027] In some embodiments, the target cell is a Daudi cell, a B cell, a leukemia cell, or a lymphoma cell. In yet further embodiments, the target cell is a Daudi cell.

[0028] In some embodiments, the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0029] In some embodiments, the expression of a regulator of ADCP is increased. In further embodiments, the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0030] In some embodiments, the expression of a regulator of ADCP is decreased. In further embodiments, the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0031] In some embodiments, the target cell is contacted with an antibody or fragment thereof.

[0032] In some embodiments, the sample is incubated in a humidified chamber or incubator. [Brief explanation of the drawings]

[0033] [Figure 1A] The ADCP assay was evaluated by flow cytometry using human donor macrophages cultured in 5% CO2. Figure 1A shows representative flow cytometry quadrat data. Figure 1B shows dose-response curves (0.03 to 2000 ng / mL) for daratumumab (black circle) or rituximab (black triangle). [Figure 1B] The ADCP assay was evaluated by flow cytometry using human donor macrophages cultured in 5% CO2. Figure 1A shows representative flow cytometry quadrat data. Figure 1B shows dose-response curves (0.03 to 2000 ng / mL) for daratumumab (black circle) or rituximab (black triangle). [Figure 2A] Figure 2 shows the ADCP assay evaluated by flow cytometry using J774A.1 cells cultured in 5% CO2. Figure 2A shows representative flow cytometry quadrat data. Figure 2B shows dose-response curves (0.03 to 2000 ng / mL) for daratumumab (black circle) or rituximab (black triangle). [Figure 2B]Figure 2 shows the ADCP assay evaluated by flow cytometry using J774A.1 cells cultured in 5% CO2. Figure 2A shows representative flow cytometry quadrat data. Figure 2B shows dose-response curves (0.03 to 2000 ng / mL) for daratumumab (black circle) or rituximab (black triangle). [Figure 3A] Micrographs of J774A.1 cells cultured for 2 days in 5% CO2 or 5 days in 1% CO2 are shown. Figure 3A: Brightfield (200x magnification) of J774A.1 cells cultured for 2 days in 5% CO2. Figure 3B: Phase contrast (200x magnification) of J774A.1 cells cultured for 5 days in 1% CO2. Arrows indicate typical morphological changes in J774A.1 cells cultured in 1% CO2. [Figure 3B] Micrographs of J774A.1 cells cultured for 2 days in 5% CO2 or 5 days in 1% CO2 are shown. Figure 3A: Brightfield (200x magnification) of J774A.1 cells cultured for 2 days in 5% CO2. Figure 3B: Phase contrast (200x magnification) of J774A.1 cells cultured for 5 days in 1% CO2. Arrows indicate typical morphological changes in J774A.1 cells cultured in 1% CO2. [Figure 4A] Figure 4A shows the ADCP assay evaluated by flow cytometry with J774A.1 cells cultured for 2 days in 5% CO2 or for 3 or 7 days in 1% CO2. Figure 4A: Representative flow cytometry quadrat data. Figure 4B: Dose-response curves for daratumumab (0-1000 ng / mL, in duplicate): 2 days at 5% CO2 (filled circles), 3 days at 1% CO2 (filled triangles), and 7 days at 1% CO2 (filled squares). [Figure 4B] Figure 4A shows the ADCP assay evaluated by flow cytometry with J774A.1 cells cultured for 2 days in 5% CO2 or for 3 or 7 days in 1% CO2. Figure 4A: Representative flow cytometry quadrat data. Figure 4B: Dose-response curves for daratumumab (0-1000 ng / mL, in duplicate): 2 days at 5% CO2 (filled circles), 3 days at 1% CO2 (filled triangles), and 7 days at 1% CO2 (filled squares). [Figure 5] ADCP assay in 96-well plate format with J774A.1 cells cultured for 3 days in 5% CO2 or 7, 11, or 15 days in 1% CO2. Dose-response curves for daratumumab (0-5000 ng / mL, in duplicate): 3 days at 5% CO2 (closed circles), 7 days at 1% CO2 (closed triangles), 11 days at 1% CO2 (closed squares); 15 days at 1% CO2 (open circles). [Figure 6] ADCP assay in 96-well plate format with J774A.1 cells cultured for 5 days in 1% CO2. Dose response for daratumumab or rituximab (0-1000 ng / mL): daratumumab (black circle), rituximab (black triangle). [Figure 7] ADCP assay in 96-well plate format with J774A.1 cells cultured for 5 days in 1% CO2. Dose response of freshly thawed daratumumab (0-1000 ng / mL) or stored at 37°C for 3 or 6 months: freshly thawed (filled circle), 3 months at 37°C (filled triangle), and 6 months at 37°C (filled square). [Figure 8A] Figure 8A shows that differential gene expression was observed between J774A.1 cells cultured in 1% CO2 and J774A.1 cells grown in 5% CO2. Figure 8A: Representative PCA between group A (1% CO2) and group B (5% CO2). Figure 8B: Graphical summary of IPA showed upregulated immune system pathways in J774A.1 cells grown in 1% CO2. In addition, corresponding upstream regulators, such as cytokines and transcription factors, were also activated. In the figure, HMOX1, TRAF3, and HR are downregulated genes. The remaining genes shown in the figure were upregulated. Figure 8C: Clustering of gene expression related to macrophage cell morphology, characteristics, and phagocytosis. [Figure 8B]Figure 8A shows that differential gene expression was observed between J774A.1 cells cultured in 1% CO2 and J774A.1 cells grown in 5% CO2. Figure 8A: Representative PCA between group A (1% CO2) and group B (5% CO2). Figure 8B: Graphical summary of IPA showed upregulated immune system pathways in J774A.1 cells grown in 1% CO2. In addition, corresponding upstream regulators, such as cytokines and transcription factors, were also activated. In the figure, HMOX1, TRAF3, and HR are downregulated genes. The remaining genes shown in the figure were upregulated. Figure 8C: Clustering of gene expression related to macrophage cell morphology, characteristics, and phagocytosis. [Figure 8C] Figure 8A shows that differential gene expression was observed between J774A.1 cells cultured in 1% CO2 and J774A.1 cells grown in 5% CO2. Figure 8A: Representative PCA between group A (1% CO2) and group B (5% CO2). Figure 8B: Graphical summary of IPA showed upregulated immune system pathways in J774A.1 cells grown in 1% CO2. In addition, corresponding upstream regulators, such as cytokines and transcription factors, were also activated. In the figure, HMOX1, TRAF3, and HR are downregulated genes. The remaining genes shown in the figure were upregulated. Figure 8C: Clustering of gene expression related to macrophage cell morphology, characteristics, and phagocytosis. DETAILED DESCRIPTION OF THE INVENTION

[0034] While the general inventive concept is susceptible to embodiment in many forms, specific embodiments thereof are shown in the drawings and described in detail herein, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the general inventive concept. Accordingly, the general inventive concept is not intended to be limited to the specific embodiments shown herein.

[0035] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0036] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the object of the article. By way of example, "a cell" means one cell or more than one cell.

[0037] As used herein, "about," when referring to measurable values ​​such as amounts and temporal durations, is meant to encompass variations of ±5%, preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for practicing the disclosed methods.

[0038] As used herein, "antibody-dependent cellular phagocytosis" (ADCP) refers to the mechanism of elimination of antibody-bound target cells by internalization by phagocytes, e.g., macrophages generated in vitro from peripheral blood mononuclear cell (PBMC) preparations derived from human donors or other species (e.g., monkeys, mice, etc.), and also macrophage cell lines, e.g., J774A.1 cells (ATCC® TIB67™) and other macrophage-like phagocyte lines known to those skilled in the art. In a particularly preferred embodiment, the phagocytes are J774A.1 cells.

[0039] The phagocytic "signal" can be measured, for example, by using microscopic imaging techniques, flow cytometry, or a plate reader to track target cells, e.g., target cells labeled with a fluorescent tag or dye known to those skilled in the art. In a particularly preferred embodiment, target cells are labeled with the fluorescent dye pHrodo-Red (Invitrogen), and the phagocytic signal is measured by flow cytometry (Aziz, Yang, & Wang, 2013) or using a plate reader, e.g., the SpectraMax® Paradigm® Multi-Mode Microplate Reader (Molecular Devices). The phagocytic signal can also be corrected for background, e.g., by subtracting a control without antibody in an ADCP assay.

[0040] As used herein, a "target cell" can be a tumor cell (e.g., a B-cell leukemia cell, a lymphoma cell, or a multiple myeloma cell) derived from a cancer patient, or a tumor cell line, such as Daudi cells, Ramos cells, Raji cells, and other tumor cell lines known to those skilled in the art. In a particularly preferred embodiment, the target cell is a Daudi cell (ATCC® CCL-213™).

[0041] The phrase "percent (%) CO2" as used herein means percent (v / v) CO2.

[0042] The phrase "percent (%) CO2 atmosphere" as used herein means an atmosphere having the stated percent (v / v) CO2.

[0043] The terms "antibody" and "antibodies," as used herein, are intended to have broad meanings and include immunoglobulin molecules, including polyclonal antibodies, monoclonal antibodies, including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, and single-chain antibodies.

[0044] Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0045] The term "antibody fragment" refers to a portion of an immunoglobulin molecule having a heavy chain and / or light chain antigen-binding site, for example, heavy chain complementarity determining regions (HCDRs) 1, 2, and 3, light chain complementarity determining regions (LCDRs) 1, 2, and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of one antibody arm; and a domain antibody (dAb) fragment consisting of the VH domain. Although VH and VL domains can be engineered and linked together via synthetic linkers to form a variety of single-chain antibody designs, the VH / VL domains pair intramolecularly, or, when the VH and VL domains are expressed as separate single-chain antibody constructs, pair intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or diabody. These are described, for example, in WO 1998 / 44001, WO 1988 / 01649, WO 1994 / 13804, and WO 1992 / 01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0046] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD38 is substantially free of antibodies that specifically bind to antigens other than human CD38). However, an isolated antibody that specifically binds human CD38 may have cross-reactivity to other antigens, such as orthologs of human CD38, such as Macaca fascicularis (cynomolgus monkey) CD38. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0047] A "humanized antibody" refers to an antibody in which the antigen-binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Because humanized antibodies may contain substitutions within the framework regions, such frameworks may not be exact copies of expressed human immunoglobulin or germline gene sequences.

[0048] A "human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen-binding site are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody includes a heavy or light chain variable region "derived" from sequences of human origin when the variable region of the antibody is obtained from a system that uses human germline immunoglobulins or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage and transgenic non-human animals, such as mice, carrying human immunoglobulin loci described herein. A "human antibody" may contain amino acid differences when compared to human germline or rearranged immunoglobulin sequences, due, for example, to naturally occurring somatic mutations or the introduction of intentional substitutions in the framework or antigen-binding site. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to the amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0049] The isolated humanized antibody can be synthetic. Human antibodies are derived from human immunoglobulin sequences, but can be generated using systems such as phage display that incorporate synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve the properties of the antibody, resulting in an antibody that does not naturally occur within the in vivo human antibody germline repertoire.

[0050] The term "recombinant antibody," as used herein, includes antibodies isolated from animals transgenic or transchromosomal for human immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody, antibodies isolated from recombinant combinatorial antibody libraries, as well as antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences, or all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies generated in vitro using Fab arm exchange (e.g., bispecific antibodies).

[0051] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition, displaying a single binding specificity and affinity for a particular epitope, or, in the case of bispecific monoclonal antibodies, dual binding specificities for two distinct epitopes.

[0052] The term "epitope," as used herein, refers to the portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In discontinuous epitopes, amino acids in different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule.

[0053] "Variant," as used herein, refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide in one or more modifications, such as, for example, a substitution, insertion, or deletion.

[0054] "Synergy," "synergistic," or "synergistic" means more than the additive effect expected from a combination.

[0055] As used herein, the term "in combination with" means that two or more therapeutic agents may be used together in a mixture, simultaneously as individual agents, or sequentially in any order as individual agents.

[0056] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder, such as the progression or spread of a tumor or tumor cells. Beneficial or desired clinical results include alleviation of symptoms, lessening of the extent of the disease, a stable (i.e., not worsening) disease state, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging the survival of a subject as compared to expected survival if the subject were not receiving treatment. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease, or those in whom the condition or disease is to be prevented.

[0057] "Inhibiting growth" (e.g., when referring to cells such as tumor cells) refers to a measurable decrease in cell growth in vitro or in vivo when contacted with a therapeutic agent or combination of therapeutic agents or drugs, compared to the growth of the same cells grown under appropriate control conditions known to those of skill in the art. Inhibition of cell growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Inhibition of cell growth can occur by various mechanisms, for example, by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, necrosis, or inhibition of cell proliferation.

[0058] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Examples of indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health, a decrease in tumor burden, a cessation or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other locations in the body.

[0059] Daratumumab (HUMAX®-CD38), an exemplary antibody used in the ADCP assay in the Examples, is a human monoclonal antibody that binds to human CD38; see, e.g., U.S. Pat. No. 7,829,673 and (de Weers et al., 2011). Daratumumab has been shown to have positive effects on multiple myeloma. Rituximab (Rituxan®), another exemplary antibody used in the ADCP assay in the Examples, is a chimeric anti-CD20 antibody that targets the CD20 protein, which is expressed in more than 95% of B-cell lymphomas. Monoclonal antibody therapy with rituximab has been shown to be an effective treatment for B-cell lymphomas, such as non-Hodgkin's lymphoma (see, e.g., U.S. Pat. No. 8,557,244).

[0060] In some embodiments of any of the compositions or methods described herein, ranges are intended to include every integer or fraction or value within the range.

[0061] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" and / or "consisting essentially of" such features.

[0062] Methods for increasing ADCP activity A method is provided for increasing antibody-dependent cellular phagocytosis (ADCP) activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes.

[0063] In some embodiments, the low % CO2 is between about 0.1% CO2 and about 1% CO2. In further embodiments, the low % CO2 is about 1% CO2. In some embodiments, the low % CO2 is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% CO2.

[0064] In some embodiments, the low % CO2 atmosphere is maintained by CO2 injected into a humidified chamber or incubator.

[0065] In some embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 15 days, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3, 4, 5, 6, or 7 days.

[0066] In some embodiments, the method further comprises determining ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

[0067] In some embodiments, the phagocytes are J774A.1 cells or donor cells. J774A.1 cells (ATCC® TIB67™) are a largely adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). Donor cells can be obtained from a human subject. In further embodiments, donor cells are human macrophage cells. In some embodiments, human macrophage cells are derived from exudates, e.g., peritoneal exudates (US8975040).

[0068] In some embodiments, the phagocytes are cultured in the presence of target cells. In further embodiments, the target cells are added to the phagocyte culture after about 1 to about 14 days. In still further embodiments, the target cells are added to the phagocyte culture after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In certain embodiments, the target cells are added to the phagocyte culture after about 7 days. In further embodiments, the target cells are Daudi cells, B cells, leukemia cells, or lymphoma cells. In still further embodiments, the target cells are Daudi cells. Daudi cells (ATCC® CCL213™) are a well-characterized B-lymphoblastoid cell line derived from a 16-year-old Black male with Burkitt's lymphoma.

[0069] In some embodiments, the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0070] In some embodiments, the expression of a regulator of ADCP is increased. In further embodiments, the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0071] In some embodiments, the expression of a regulator of ADCP is decreased. In further embodiments, the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0072] In some embodiments, the target cell is contacted with an antibody or fragment thereof.

[0073] In some embodiments, the sample is incubated in a humidified chamber or incubator.

[0074] Assaying ADCP activity Also provided is a method for assaying ADCP activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO2 atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes, and detecting the ADCP activity of the phagocytes in the sample.

[0075] In some embodiments, the low % CO2 is about 0.1% CO2 to about 1% CO2. In some embodiments, the low % CO2 is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% CO2.

[0076] In some embodiments, low % CO2 is 1% CO2.

[0077] In some embodiments, the low % CO2 atmosphere is maintained by CO2 injected into a humidified chamber or incubator.

[0078] In some embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 15 days, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3, 4, 5, 6, or 7 days.

[0079] In some embodiments, the method further comprises determining ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

[0080] In some embodiments, the phagocytes are J774A.1 cells or donor cells. J774A.1 cells (ATCC® TIB67™) are a largely adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) and are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). Donor cells can be obtained from a human subject. In further embodiments, donor cells are human macrophage cells.

[0081] In some embodiments, the phagocytes are cultured in the presence of target cells, hi further embodiments, the target cells are added to the phagocyte culture after about 3 to about 10 days, e.g., after about 7 days.

[0082] In some embodiments, the target cells are labeled with a fluorescent dye.

[0083] In some embodiments, the phagocytes are J774A.1 cells or donor cells. J774A.1 cells (ATCC® TIB67™) are a largely adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) and are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). Donor cells can be obtained from a human subject. In further embodiments, donor cells are human macrophage cells.

[0084] In some embodiments, the target cell is a Daudi cell, a B cell, a leukemia cell, or a lymphoma cell. In yet further embodiments, the target cell is a Daudi cell. Daudi cells (ATCC® CCL213™) are a well-characterized B-lymphoblastoid cell line derived from a 16-year-old Black male with Burkitt's lymphoma.

[0085] In some embodiments, the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0086] In some embodiments, the expression of a regulator of ADCP is increased. In further embodiments, the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0087] In some embodiments, the expression of a regulator of ADCP is decreased. In further embodiments, the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0088] In some embodiments, the target cell is contacted with an antibody or fragment thereof.

[0089] In some embodiments, the sample is incubated in a humidified chamber or incubator.

[0090] Assaying ADCP activity A method is provided for assaying ADCP activity of phagocytes in a sample, the method comprising culturing the phagocytes in a low percent (%) CO2 atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes, and detecting the ADCP activity of the phagocytes in the sample.

[0091] In some embodiments, the low % CO2 is about 0.1% CO2 to about 1% CO2. In some embodiments, the low % CO2 is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% CO2.

[0092] In some embodiments, low % CO2 is 1% CO2.

[0093] In some embodiments, the low % CO2 atmosphere is maintained by CO2 injected into a humidified chamber or incubator.

[0094] In some embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 15 days, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity in phagocytes is 3, 4, 5, 6, or 7 days.

[0095] In some embodiments, the method further comprises determining ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

[0096] In some embodiments, the phagocytes are J774A.1 cells or donor cells. J774A.1 cells (ATCC® TIB67™) are a largely adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) and are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). Donor cells can be obtained from a human subject. In further embodiments, donor cells are human macrophage cells.

[0097] In some embodiments, the phagocytes are cultured in the presence of target cells, hi further embodiments, the target cells are added to the phagocyte culture after about 3 to about 10 days, e.g., after about 7 days.

[0098] In some embodiments, the target cells are labeled with a fluorescent dye.

[0099] In some embodiments, the phagocytes are J774A.1 cells or donor cells. J774A.1 cells (ATCC® TIB67™) are a largely adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) and are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). Donor cells can be obtained from a human subject. In further embodiments, donor cells are human macrophage cells.

[0100] In some embodiments, the target cell is a Daudi cell, a B cell, a leukemia cell, or a lymphoma cell. In yet further embodiments, the target cell is a Daudi cell. Daudi cells (ATCC® CCL213™) are a well-characterized B-lymphoblastoid cell line derived from a 16-year-old Black male with Burkitt's lymphoma.

[0101] In some embodiments, the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0102] In some embodiments, the expression of a regulator of ADCP is increased. In further embodiments, the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0103] In some embodiments, the expression of a regulator of ADCP is decreased. In further embodiments, the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0104] In some embodiments, the target cell is contacted with an antibody or fragment thereof.

[0105] In some embodiments, the sample is incubated in a humidified chamber or incubator. [Example]

[0106] Materials and Methods Human donor macrophages Human donor macrophages were prepared by standard methods known to those skilled in the art. Briefly, frozen PBMCs were enriched with a human monocyte enrichment cocktail (STEMCELL Technology® 19058) without CD16 depletion. Monocytes were cultured in X-VIVO10 medium (Lonza® 04-380Q) supplemented with 10% fetal bovine serum (FBS) and 25 ng / mL M-CSF at 37°C and 5% CO2. On days 3-4, 50% of the medium was replaced with fresh X-VIVO10 + 10% FBS. IFN-γ (50 ng / mL) was added on day 6, and cells were harvested for ADCP assay on day 7.

[0107] J774A.1 cells J774A.1 cells (ATCC® TIB67™) are a predominantly adherent monocyte / macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) and are active in antibody-dependent phagocytosis (Ralph & Nakoinz, 1975). J774A.1 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS to a final concentration of 10%. Subcultures were prepared by cell dissociation and scraping with the cell dissociation reagent Accutase (eBioscience, 00-4555-56). For parental J774A.1, cells were cultured at 37°C in 5% CO2. In low CO2 conditions, as described in the figures and examples, J774A.1 cells were grown at 8 x 10 6 Cells / 40 mL were seeded and incubated in low CO2 for the indicated periods.

[0108] Daudi cells Daudi cells (ATCC® CCL213™) are a well-characterized B-lymphoblastoid cell line derived from a 16-year-old Black male with Burkitt's lymphoma. Daudi cells were cultured in suspension in RPMI-1640 medium (ATCC 30-2001) supplemented with FBS to a final concentration of 10% at 37°C and 5% CO. Fresh medium was added every 2-3 days (depending on cell density) until the day of the ADCP assay.

[0109] Cell labeling Target cells (e.g., Daudi cells) were washed twice with phosphate buffered saline (PBS) and diluted to 10 6 25 μL of 1 mg / mL pHrodo-Red (stock solution in DMSO) was added per 50 ml of cell suspension (i.e., final concentration of 500 ng / mL and 10 6 After 30 min of incubation at 37°C, cells were washed twice with PBS and resuspended in DMEM medium at 5 × 10 cells / mL before use in the ADCP assay. 6 Cells were resuspended at 1000 cells / mL. pHrodo-Red SE was from Invitrogen (Waltham, MA, P36600).

[0110] Macrophages (e.g., human donor macrophages and J774A.1 cells) were stained with Alexa Fluor® 488-labeled rat anti-mouse CD11b antibody [M1 / 70] (Biolegend, San Diego, CA). Macrophages were lifted from culture flasks with Accutase at 37°C for 30 minutes, washed twice with PBS, and 2.5 μL / mL of anti-mouse CD11b antibody was added for 20 minutes at 4°C. This stain provided uniform surface staining of macrophages and was also useful for distinguishing the cell surface of cells during flow cytometry.

[0111] Flow cytometry For flow cytometry, data were acquired on a FACScan Flow Cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Flowjo, LLC, Ashland, OR).

[0112] Flow cytometry and microplate reader readout in 96-well plate format For the ADCP assay performed in a 96-well plate (Corning, 3799) for flow cytometry, serial dilutions of daratumumab or rituximab were added to each well at 2.5 × 10 4 The cells were pre-incubated with 1 × 10 pHrodo-red labeled Daudi cells at room temperature for 15 minutes. 5 100 of human macrophages or J774A.1 cells were added and mixed. The plates were centrifuged at 40 x g for 1 minute with minimal acceleration and deceleration and incubated in a 37°C incubator with 5% or 1% CO2 for 3 or 24 hours. The cells were then washed once with DPBS and detached with Accutase for further CD11b staining and flow cytometry. For a microplate reader, serially diluted daratumumab or rituximab were added at 1.25 x 100 per well (Corning, 3904). 4 The cells were pre-incubated with 5 × 10 pHrodo-red labeled Daudi cells at room temperature for 15 minutes. 4 Human macrophages or J774A.1 cells were added and mixed. The plates were centrifuged at 40 x g for 1 minute with minimal acceleration and deceleration and incubated for 2.5 hours in a 37°C incubator with 1% CO2. Data were acquired using a SpectraMax® Paradigm® Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at Ex / Em 560 / 600 nm in well scan mode. Results were plotted after subtracting the signal from wells without antibody (subtracting the signal from the background / control).

[0113] Example 1: ADCP by flow cytometry using human donor macrophages Human donor macrophages (1 × 10 5 ) in a 96-well plate with pHrodo-red-labeled Daudi cells (2.5 × 10 4 ) and titrated monoclonal antibodies (daratumumab or rituximab, 0.03–2000 ng / mL) for 3 hours. Cells were detached from the wells and stained with anti-CD11b-A488 antibody. ADCP was assessed by flow cytometry and analyzed using FlowJo software. Dose-response curves were generated by plotting the percentage of cells in the Q2 gating for pHrodo-red Daudi cells phagocytosed by human donor macrophages (Figures 1A–1B).

[0114] The results confirmed that daratumumab and rituximab could induce ADCP of tumor cells in human macrophages. ADCP flow cytometry analysis showed that Daudi cells were opsonized by human macrophages in the presence of either daratumumab or rituximab. In addition, a dose-dependent response curve for daratumumab and rituximab was observed in human macrophages that phagocytosed pHrodo-labeled Daudi cells.

[0115] Example 2: ADCP by flow cytometry using J774A.1 cells cultured in 5% CO J774A.1 cells (1 × 10) maintained in 5% CO 5 ) in a 96-well plate with pHrodo-red-labeled Daudi cells (2.5 × 10 4) and titrated monoclonal antibodies (daratumumab or rituximab, 0.03–2000 ng / mL) for 24 hours. Cells were detached from the wells and stained with anti-CD11b-A488 antibody. ADCP was assessed by flow cytometry and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). A dose-response curve was generated by plotting the percentage of cells in the Q2 gating for pHrodo-red Daudi cells engulfed by J774A.1 cells (Figures 2A–2B).

[0116] The results confirmed that daratumumab and rituximab can induce ADCP of tumor cells in the murine macrophage cell line J774A.1. ADCP flow cytometry analysis showed that Daudi cells were opsonized by J774A.1 in the presence of either daratumumab or rituximab. The percentage of phagocytosis from the J774A.1 cell line was lower than that from human macrophages, suggesting that the J774A.1 cell line has lower ADCP activity. A less dynamic dose-dependent response curve of daratumumab and rituximab was observed in J774A.1 cells that phagocytosed pHrodo-labeled Daudi cells.

[0117] Example 3: Morphological changes of J774A.1 cells cultured in 1% CO J774A.1 cells were subcultured for 2 days in a 5% CO2 humidified chamber or for 5 days in a 1% CO2 humidified chamber. Photographs were taken under a microscope at 200x bright field magnification or 200x phase contrast magnification (Figures 3A-3B).

[0118] Bright field microcopy showed morphological changes in J744A.1 cells after 5 days of culture in 1% CO. Furthermore, J744A.1 cells with macrophage morphology were more spread than round J744A.1 cells when they were cultured in 1% CO.

[0119] Example 4: Increase in ADCP of J774A.1 cells cultured in 1% CO J774A.1 cells were cultured in a 5% CO2 humidified chamber for 2 days, or in a 1% CO2 humidified chamber for 3 or 7 days. J774A.1 cells (1 x 10 5 ) in a 96-well plate with pHrodo-red-labeled Daudi cells (2.5 × 10 4 ) and titrated monoclonal antibody (daratumumab, 0-1000 ng / mL) for 24 hours. Cells were detached from the wells and stained with anti-CD11b-A488 antibody. ADCP was assessed by flow cytometry and analyzed using FlowJo software. A dose-response curve was generated by plotting the percentage of cells in the Q2 gating for pHrodo-red Daudi cells engulfed by J774A.1 cells (Figures 4A-4B).

[0120] The data showed that 1% CO2 cell culture conditions enhanced ADCP activity in J744A.1 cells. ADCP results were compared for 2 days in 5% CO2, 3 days in 1% CO2, and 1% CO2, respectively. 2 Comparisons were made between J744A.1 cells cultured in 1% CO2 for 7 days. ADCP flow cytometry analysis showed that, in the presence of daratumumab, Daudi cells were opsonized by J744A.1 at a higher level in 1% CO2 culture conditions than in 5% CO2 culture conditions. The percent phagocytosis from J774A.1 cells cultured in 1% CO2 was higher than that from J774A.1 cells cultured in 5% CO2. A favorable dynamic daratumumab dose-dependent response curve was observed in J774A.1 cells cultured in 1% CO2.

[0121] Example 5: Effect of time on ADCP of J774A.1 cells cultured in 1% CO J774A.1 cells were cultured in a 5% CO2 humidified chamber for 3 days, or in a 1% CO2 humidified chamber for 7, 11, or 15 days. J774A.1 cells (5 x 10 4) were cultured in 96-well plates containing titrated monoclonal antibody (daratumumab, 0–5000 ng / mL) on pHrodo-red-labeled Daudi cells (1.25 × 10 4 ) for 2 hours. ADCP was assessed by a microplate reader and the results were plotted after subtraction of the no-antibody control (Figure 5).

[0122] The results showed that prolonging the incubation time of J774A.1 cells in 1% CO2 did not enhance ADCP activity. The daratumumab dose-dependent response curve with the lowest fluorescence signal-to-noise ratio was obtained using J744A.1 cells in 5% CO2. The daratumumab dose-dependent response curve with the highest fluorescence signal-to-noise ratio was obtained using J744A.1 cells incubated in 1% CO2 for 7 days. Long-term incubation (i.e., days 11 and 15) in 1% CO2 did not increase the fluorescence signal-to-noise ratio or the dynamics of the curve.

[0123] Example 6: Dose-dependent ADCP with daratumumab or rituximab J774A.1 cells were cultured in a 1% CO2 humidified chamber for 5 days. Then, J774A.1 cells (5 × 10 4 ) were cultured on pHrodo-red-labeled Daudi cells (1.25 × 10 ) in a 96-well plate containing titrated antibodies (daratumumab or rituximab, 0–1000 ng / mL). 4 ) for 2.5 hours. ADCP was assessed by a microplate reader and the results were plotted after subtraction of the no-antibody control (Figure 6).

[0124] The results showed that both daratumumab and rituximab could induce ADCP of tumor cells in the murine macrophage cell line J774A.1 cultured at 1% CO. A dose-dependent response curve of daratumumab and rituximab was observed in J774A.1 cells that phagocytosed pHrodo-labeled Daudi cells, indicating ADCP activity.

[0125] Example 7: Stability of Daratumumab with ADCP J774A.1 cells were cultured in a 1% CO2 humidified chamber for 5 days. Then, J774A.1 cells (5 × 10 4 ) were cultured in 96-well plates containing pHrodo-red labeled Daudi cells (1.25 × 10 cells) containing titrated daratumumab (0–1000 ng / mL) that had been freshly thawed or stored at 37°C for 3 or 6 months. 4 ) for 2.5 hours. ADCP was assessed by a microplate reader, and the results were plotted after subtraction of the no-antibody control (Figure 7).

[0126] Data demonstrated stability characteristics of the ADCP assay using J744A.1 cells cultured in 1% CO2. The dose-response curves from daratumumab stability samples stored at 37°C for 3 and 6 months were shifted to the right compared to freshly thawed samples. Results indicated that these heat-exposed samples had lower potency (i.e., less than 100%).

[0127] Example 8: RNA-Seq analysis Two groups of J774A.1 cells were incubated at different CO2 concentrations (1% vs. 5%). J774A.1 cells (1 × 10 6 and 4 x 10 6J744A.1 cells (1000 cells / mL) were cultured in either a 5% or 1% CO2 humidified chamber for 4 days. RNA from the corresponding culture conditions was extracted for RNA-Seq analysis (Figures 8A, 8B, and 8C). Principal component analysis (PCA) showed that the gene expression profiles of J744A.1 cells cultured at 1% CO2 and 5% CO2 were significantly different. Ingenuity Pathways Analysis (IPA) analysis revealed that several key upstream regulators of immune system pathways, such as several cytokines (i.e., TNF, IL6) and transcription factors (i.e., ATF4, STAT3), were upregulated in J744A.1 cells cultured at 1% CO2. Activation of these upstream regulators may contribute to J744A.1 cells acquiring a more macrophage-like morphology, which exhibits enhanced phagocytic activity.

[0128] conclusion As described above, ADCP activity was surprisingly and unexpectedly increased for J774A.1 cells cultured in 1% CO. The increased ADCP activity provided an improved ADCP assay with an increased signal relative to background. The improved ADCP assay is suitable for determining the ADCP activity of antibodies, for comparing the ADCP activity of different antibodies or different antibody preparations, and for measuring antibody stability, such as required for quality control during antibody manufacturing or storage.

[0129] Embodiment The following exemplary embodiments further describe optional aspects of the technology of the present disclosure and are part of the detailed description. Although these exemplary embodiments are described in a format substantially similar to claims (each with a numerical designation followed by a capital letter), they are not technical claims of this application. The following exemplary embodiments are referenced to each other in a dependent relationship as "embodiments" instead of "claims."

[0130] 1A. A method for increasing antibody-dependent cellular phagocytosis (ADCP) activity of phagocytes in a sample, comprising culturing the phagocytes in a low percent (%) CO2 atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes.

[0131] 2A. The method of embodiment 1A, wherein the low % CO2 is about 0.1% CO2 to about 1% CO2.

[0132] 3A. The method of embodiment 2A, wherein the low % CO2 is about 1% CO2.

[0133] 4A. The method of any one of embodiments 1A-3A, wherein the period of time sufficient to increase ADCP activity of phagocytes is 3 to 15 days.

[0134] 5A. The method of any one of embodiments 1A-4A, wherein the period of time sufficient to increase ADCP activity of phagocytes is 3 to 7 days.

[0135] 6A. The method of any one of embodiments 1A-5A, further comprising determining ADCP activity of phagocytes using target cells labeled with a fluorescent dye.

[0136] 7A. The method of any one of embodiments 1A-6A, wherein the phagocytes are J774A.1 cells or donor cells.

[0137] 8A. The method of any one of embodiments 1A-7A, wherein the phagocytes are cultured in the presence of target cells.

[0138] 9A. The method of embodiment 8A, wherein target cells are added to the phagocyte culture after about 7 days.

[0139] 10A. The method of any one of embodiments 8A-9A, wherein the target cell is a Daudi cell, a B cell, a leukemia cell, or a lymphoma cell.

[0140] 11A. The method of any one of embodiments 6A to 10A, wherein the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0141] 12A. The method of any one of embodiments 1A to 11A, wherein the expression of a regulator of ADCP is increased.

[0142] 13A. The method of embodiment 12A, wherein the modulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0143] 14A. The method of any one of embodiments 1A to 11A, wherein the expression of a regulator of ADCP is reduced.

[0144] 15A. The method of embodiment 14A, wherein the modulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0145] 16A. The method of any one of embodiments 8A to 15A, wherein the target cells are contacted with an antibody or fragment thereof.

[0146] 17A. The method of any one of embodiments 1A to 16A, wherein the sample is cultured in a humidified chamber or incubator.

[0147] 18A. A method for assaying ADCP activity of phagocytes in a sample, comprising culturing phagocytes in a low percent (%) CO2 atmosphere for a period of time sufficient to increase the ADCP activity of the phagocytes, and detecting the ADCP activity of the phagocytes in the sample.

[0148] 19A. The method of embodiment 18A, wherein the low % CO2 is about 0.1% CO2 to about 1% CO2.

[0149] 20A. The method of embodiment 19A, wherein the low % CO2 is about 1% CO2.

[0150] 21A. The method of any one of embodiments 18A to 20A, wherein the period of time sufficient to increase ADCP activity of phagocytes is 3 to 15 days.

[0151] 22A. The method of any one of embodiments 18A to 21A, wherein the period of time sufficient to increase ADCP activity of phagocytes is 3 to 7 days.

[0152] 23A. The method of any one of embodiments 18A to 22A, further comprising determining ADCP activity of phagocytes using target cells labeled with a fluorescent dye.

[0153] 24A. The method of any one of embodiments 18A to 23A, wherein the phagocytes are J774A.1 cells or donor cells.

[0154] 25A. The method of any one of embodiments 18A to 24A, wherein the phagocytes are cultured in the presence of target cells.

[0155] 26A. The method of embodiment 25A, wherein target cells are added to the phagocyte culture after about 7 days.

[0156] 27A. The method of any one of embodiments 25A-26A, wherein the target cells are labeled with a fluorescent dye.

[0157] 28A. The method of any one of embodiments 18A to 27A, wherein the phagocytes are J774A.1 cells or donor cells.

[0158] 29A. The method of any one of embodiments 25A to 28A, wherein the target cell is a Daudi cell, a B cell, a leukemia cell, or a lymphoma cell.

[0159] 30A. The method of any one of embodiments 23A to 29A, wherein the fluorescent dye is pHrodo-Red, pHAb, or AcidiFluor.

[0160] 31A. The method of any one of embodiments 18A to 30A, wherein the expression of a regulator of ADCP is increased.

[0161] 32A. The method of embodiment 31A, wherein the modulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

[0162] 33A. The method of any one of embodiments 18A to 32A, wherein the expression of a regulator of ADCP is reduced.

[0163] 34A. The method of embodiment 33A, wherein the modulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

[0164] 35A. The method of any one of embodiments 25A to 34A, wherein the target cells are contacted with an antibody or fragment thereof.

[0165] 36A. The method of any one of embodiments 18A to 35A, wherein the sample is cultured in a humidified chamber or incubator.

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[0167] All publications and patents mentioned herein are incorporated herein by reference. Various modifications and variations of the subject matter described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention that will be apparent to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. 1. A method for increasing antibody-dependent cellular phagocytosis (ADCP) activity of phagocytes in a sample, comprising: Low percent (%) CO ２ and culturing said phagocytes in an atmosphere for a period of time sufficient to increase ADCP activity of said phagocytes.

2. The low % CO ２ However, approximately 0.1% CO ２ ~Approximately 1% CO ２ The method of claim 1, wherein

3. The low % CO ２ However, about 1% CO ２ The method of claim 2, wherein

4. The method according to any one of claims 1 to 3, wherein the period sufficient to increase the ADCP activity of the phagocytes is 3 to 15 days.

5. The method of any one of claims 1 to 4, further comprising determining the ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

6. The method of any one of claims 1 to 5, wherein the phagocytes are J774A.1 cells or donor cells.

7. The method of any one of claims 1 to 6, wherein the phagocytes are cultured in the presence of target cells.

8. 8. The method of claim 7, wherein target cells are added to the phagocyte culture after about 7 days.

9. The method of any one of claims 7 to 8, wherein the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.

10. The method according to any one of claims 5 to 9, wherein the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.

11. The method according to any one of claims 1 to 10, wherein the expression of a regulator of ADCP is increased.

12. 12. The method of claim 11, wherein the modulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

13. The method of any one of claims 1 to 10, wherein the expression of a regulator of ADCP is reduced.

14. 14. The method of claim 13, wherein the modulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

15. The method of any one of claims 7 to 14, wherein the target cells are contacted with an antibody or a fragment thereof.

16. The method of any one of claims 1 to 15, wherein the sample is incubated in a humidified chamber or incubator.

17. 1. A method for assaying ADCP activity of phagocytes in a sample, comprising: Low percent (%) CO ２ culturing the phagocytes in an atmosphere for a period of time sufficient to increase ADCP activity in the phagocytes; detecting ADCP activity of said phagocytes in said sample.

18. The low % CO ２ However, approximately 0.1% CO ２ ~Approximately 1% CO ２ 18. The method of claim 17, wherein:

19. The low % CO ２ However, about 1% CO ２ 19. The method of claim 18, wherein:

20. The method of any one of claims 17 to 19, wherein the period of time sufficient to increase the ADCP activity of the phagocyte is 3 to 15 days.

21. The method of any one of claims 17 to 20, further comprising determining the ADCP activity of the phagocytes using target cells labeled with a fluorescent dye.

22. 22. The method of any one of claims 17 to 21, wherein the phagocytes are J774A.1 cells or donor cells.

23. The method of any one of claims 17 to 22, wherein the phagocytes are cultured in the presence of target cells.

24. 24. The method of claim 23, wherein the target cells are added to the phagocyte culture after about 7 days.

25. The method of any one of claims 23 to 24, wherein the target cells are labeled with a fluorescent dye.

26. 26. The method of any one of claims 17 to 25, wherein the phagocytes are J774A.1 cells or donor cells.

27. The method of any one of claims 23 to 26, wherein the target cell is a Daudi cell, a B cell, a leukemia cell or a lymphoma cell.

28. The method according to any one of claims 21 to 27, wherein the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.

29. The method of any one of claims 17 to 28, wherein the expression of a regulator of ADCP is increased.

30. 30. The method of claim 29, wherein the modulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or a combination thereof.

31. The method of any one of claims 17 to 30, wherein the expression of a regulator of ADCP is reduced.

32. 32. The method of claim 31, wherein the modulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS, or a combination thereof.

33. The method of any one of claims 23 to 32, wherein the target cells are contacted with an antibody or fragment thereof.

34. The method of any one of claims 17 to 33, wherein the sample is incubated in a humidified chamber or incubator.

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