WO2024079711A1 - Procédé de détection de phagocytose cellulaire dépendante des anticorps - Google Patents

Procédé de détection de phagocytose cellulaire dépendante des anticorps Download PDF

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WO2024079711A1
WO2024079711A1 PCT/IB2023/060349 IB2023060349W WO2024079711A1 WO 2024079711 A1 WO2024079711 A1 WO 2024079711A1 IB 2023060349 W IB2023060349 W IB 2023060349W WO 2024079711 A1 WO2024079711 A1 WO 2024079711A1
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cells
cell
adcp
phagocytic
antibody
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PCT/IB2023/060349
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Chi Kwong So
Tammy SODEN
Wen-I TSOU
Diana STAFA
Wenyu Li
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Janssen Research & Development, Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages

Definitions

  • the general inventive concepts relate to the field of methods of detecting antibodydependent cellular phagocytosis (ADCP) that are useful for quantifying the ADCP activity of antibodies, and in particular for quantifying the ADCP activity of therapeutic antibodies useful for the treatment of cancer.
  • ADCP antibodydependent cellular phagocytosis
  • a number of therapeutic antibodies have been developed and approved for use in the treatment of cancer (Mossner et al., 2010; Shuptrine, Surana, & Weiner, 2012; Weiner, Murray, & Shuptrine, 2012; Weiner, Surana, & Wang, 2010).
  • These therapeutic antibodies can affect tumor growth directly by interfering with receptor signaling, by blocking receptor downstream signaling, and by inducing apoptosis, but they can also affect tumor growth indirectly 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).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • ADCP Unlike ADCC, which depends on effector cells to secrete molecules that activate apoptosis-based cell death, ADCP depends on macrophages to directly destroy target cells by phagocytosis. These macrophages in particular are poised to be tremendous effectors of cancer immunotherapy.
  • Macrophages reside in tissues throughout the body (Geissmann et al., 2010) and specialized tissue-specific macrophage populations exist, e.g., Kupffer cells in the liver, microglia in the brain, osteoclasts in bone, and alveolar macrophages in the lungs. And importantly, macrophages are known to infiltrate solid tumors thus gaining proximity to the tumor cells. These macrophages are crucial to the efficacy of many antibodies.
  • ADCP is an important and potent mechanism of action (MO A) for the treatment of different cancers with different therapeutic antibodies, e.g., treatment of multiple myeloma (MM) with Daratumumab, an anti-CD38 antibody (Khagi & Mark, 2014; Overdijk et al., 2015), and treatment of B-cell malignancies with Rituximab, an anti-CD20 antibody (Oflazoglu & Audoly, 2010), have both been shown to have an important ADCP component.
  • MO A mechanism of action
  • ADCP of solid tumors has also been demonstrated in vitro using anti-HER- 2/neu antibodies with breast cancer cells (Watanabe et al., 1999) and using anti-epidermal growth factor receptor (EGFR) antibodies with colon cancer cells (Weiskopf et al., 2013).
  • EGFR anti-epidermal growth factor receptor
  • comparability studies have shown the importance of differences in ADCP for different therapeutic antibodies that bind the same target, e.g., anti-CD20 antibodies Ofatumumab, Obinutuzumab, and Rituximab (Rafiq et al., 2013).
  • the process of ADCP is essentially a two-step mechanism involving (1) binding of an antibody to a target cell, e.g., a tumor cell or a suitable target cell mimicking a patient’s tumor; and (2) macrophages or other phagocytic cells such as monocytes, neutrophils or dendritic cells binding to the fragment crystallizable (Fc) domain of the antibody through Fc receptors on the phagocytic cells with subsequent phagocytosis of the target cell.
  • a target cell e.g., a tumor cell or a suitable target cell mimicking a patient’s tumor
  • macrophages or other phagocytic cells such as monocytes, neutrophils or dendritic cells binding to the fragment crystallizable (Fc) domain of the antibody through Fc receptors on the phagocytic cells with subsequent phagocytosis of the target cell.
  • FcyRIIa CD32a
  • FcyRI CD64
  • FcyRIIIa CD16a
  • ADCP assays can also provide insight into the stability of therapeutic antibodies at different stages of production or storage of the therapeutic product.
  • drug developers and researchers are rapidly adopting ADCP assays as a key step during development and production of the therapeutic antibodies.
  • the prototypical ADCP assay using primary macrophages derived from human donors and target cell phagocytosis as an end-point can be a very challenging and time-consuming with variability that makes it unsuitable as a routine assay.
  • PBMC peripheral blood mononuclear cell
  • ADCP antibody-dependent cellular phagocytosis
  • the low % CO2 is from 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.
  • the period of time sufficient to increase ADCP activity of the phagocytic cells is 3 to 15 days. In further embodiments, the period of time sufficient to increase ADCP activity of the phagocytic cell is 3 to 7 days.
  • the method further comprises determining the ADCP activity of the phagocytic cell using target cells labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • the donor cell is a human macrophage cell.
  • the phagocytic cells are cultured in the presence of target cells.
  • target cells are added to the phagocytic cell culture after about 1 to about 14 days.
  • target cells are added to the phagocytic cell culture after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
  • target cells are added to the phagocytic cell culture after about 7 days.
  • the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.
  • the target cells are Daudi cells.
  • the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • a regulator of ADCP is increased.
  • the regulator is ATF4, FOXO3, IL IB, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof
  • expression of a regulator of ADCP is decreased.
  • the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS or combinations thereof.
  • the target cells are contacted with an antibody or fragment thereof.
  • the sample is cultured in a humidified chamber or incubator.
  • a method for assaying ADCP activity of a phagocytic cell in a sample comprising: culturing the phagocytic cell in a low percent (%) CO2 atmosphere for a period of time sufficient to increase ADCP activity of the phagocytic cell; and detecting ADCP activity of the phagocytic cell in the sample.
  • the low % CO2 is from 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.
  • the low % CO2 is 1% CO2.
  • the period of time sufficient to increase ADCP activity of the phagocytic cells is 3 to 15 days. In further embodiments, the period of time sufficient to increase ADCP activity of the phagocytic cell is 3 to 7 days.
  • the method further comprises determining the ADCP activity of the phacobytic cell using target cells labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • the donor cell is a human macrophage cell.
  • the phagocytic cells are cultured in the presence of target cells.
  • target cells are added to the phagocytic cell culture after about 3 to about 10 days, for example after about 7 days.
  • the target cells are labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • the donor cell is a human macrophage cell.
  • the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells. In yet further embodiments, the target cells are Daudi cells.
  • the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • a regulator of ADCP is increased.
  • the regulator is ATF4, FOXO3, IL IB, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof.
  • expression of a regulator of ADCP is decreased.
  • the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS or combinations thereof.
  • the target cells are contacted with an antibody or fragment thereof.
  • the sample is cultured in a humidified chamber or incubator.
  • FIGs. 1A-1B illustrate ADCP assay assessed by flow cytometry with human donor macrophages cultured in 5% CO2.
  • FIG. 1A Representative flow cytometry quadrats data.
  • FIG. IB Dose response curves (0.03 to 2000 ng/mL) for Daratumumab, filled circle (•), or Rituximab, filled triangle (A).
  • FIGs. 2A-2B illustrate ADCP assay assessed by flow cytometry with J774A.1 cells cultured in 5% CO2.
  • FIG. 2A Representative flow cytometry quadrats data.
  • FIG. 2B Dose response curve (0.03 to 2000 ng/mL) for Daratumumab, filled circle (•); or Rituximab, filled triangle (A).
  • FIGs. 3 A-3B show photomicrographs for J774A.1 cells cultured in 5% CO2 for 2 days or 1% CO2 for 5 days.
  • FIG. 3A 200X bright field of J774A.1 cells cultured in 5% CO2 for 2 days.
  • FIG. 3B 200X phase-contrast of J774A.1 cells cultured in 1% CO2 for 5 days. Arrows indicate representative morphology change of J774A.1 cells cultured in 1% CO2.
  • FIGs. 4A-4B show ADCP assay assessed by flow cytometry with J774A.1 cells cultured in 5% CO2 for 2 days, or 1% CO2, for 3 days or 7 days.
  • FIG. 4A Representative flow cytometry quadrats data.
  • FIG. 4B Dose response curves for Daratumumab (0 to 1000 ng/mL, in duplicate): 5% CO2 for 2 days, filled circle (•); 1% CO2 for 3 days, filled triangle (A); 1% CO2 for 7 days, filled square ( ⁇ ).
  • FIG. 5 shows ADCP assay in 96-well plate format with J774A.1 cells cultured in 5% CO2 for 3 days, or 1% CO2, for 7 days, 11 days, or 15 days.
  • Dose response for Daratumumab (0 to 5000 ng/mL, in duplicate): 5% CO2 for 3 days, filled circle (•); 1% CO2 for 7 days, filled triangle (A); 1% CO2 for 11 days, filled square ( ⁇ ); 1% CO2 for 15 days, open circle (o).
  • FIG. 6 shows ADCP assay in 96-well plate format with J774A.1 cells cultured in 1% CO2 for 5 days.
  • Dose response for Daratumumab or Rituximab (0 to 1000 ng/mL): Daratumumab, filled circle (•); Rituximab, filled triangle (A).
  • FIG. 7 shows ADCP assay in 96-well plate format with J774A.1 cells cultured in 1% CO2 for 5 days.
  • Dose response for Daratumumab (0 to 1000 ng/mL) that was freshly thawed or stored at 37°C for 3 months or 6 months: freshly thawed, filled circle (•); 37°C for 3 months, filled triangle (A); 37°C for 6 months, filled square ( ⁇ ).
  • FIGs. 8A, 8B and 8C show that a different gene expression is observed between J774A.1 cells cultured in 1% CO2 vs J774A.1 cells grown in 5% CO2.
  • FIG. 8A Representative PCA between Group A (1% CO2) and Group B (5% CO2).
  • FIG. 8B IPA Graphical Summary illustrated upregulated immune system pathways in J774A.1 cells in 1% CO2. In addition, the corresponding upstream regulators such as cytokine and transcription factors were also activated. In the figure, HM0X1, TRAF3 and HR were downregulated genes. The rest of the genes shown in the Figure were upregulated.
  • FIG. 8C Clustering of gene expression related to macrophage cell morphology, characteristics, and phagocytosis.
  • a cell means one cell or more than one cell.
  • ‘About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 5%, preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • ADCP antibody-dependent cellular phagocytosis
  • phagocytic cells e.g., macrophages generated in vitro from peripheral blood mononuclear cell (PBMC) preparations derived from human donors or other species (e.g., monkey and mouse, etc.) and also macrophage cell lines, e.g., J774A.1 cells (ATCC® TIB67TM) and other macrophage- like phagocytic cell lines known by those skilled in the art.
  • PBMC peripheral blood mononuclear cell
  • macrophage cell lines e.g., J774A.1 cells (ATCC® TIB67TM) and other macrophage- like phagocytic cell lines known by those skilled in the art.
  • the phagocytic cells are J774A.1 cells.
  • the phagocytosis “signal” can be measured, for example, by using microscopy imaging techniques, flow cytometry, or a plate reader for tracking target cells, e.g., target cells labeled with fluorescent tags or dyes known by those skilled in the art.
  • target cells e.g., target cells labeled with fluorescent tags or dyes known by those skilled in the art.
  • the target cells are labeled with the fluorescent dye pHrodo-Red (Invitrogen) and the phagocytosis signal is measured by flow cytometry (Aziz, Yang, & Wang, 2013) or with a plate reader, e.g., SpectraMax® Paradigm® Multi-Mode Microplate Reader (Molecular Devices).
  • the phagocytosis signal can also be corrected by subtracting background, e.g., control with no antibody added in the ADCP assay.
  • target cells can be tumor cells derived from cancer patients (e.g., B cell leukaemia cells, lymphoma cells, multiple myeloma cells), or tumor cell lines such as, e.g., Daudi cells, Ramos cells, Raji cells, and other tumor cell lines known by those skilled in the art.
  • the target cells are Daudi cells (ATCC® CCL-213TM).
  • antibody and “antibodies” as used herein are meant in a broad sense 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.
  • Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAl , IgA2 , IgGl , IgG2 , IgG3 and IgG4 .
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
  • antibody fragments refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL).
  • HCDR heavy chain complementarity determining regions
  • LCDR light chain complementarity determining regions
  • VH heavy chain variable region
  • VL light chain variable region
  • Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain.
  • Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting of the VH and CHI domains
  • a Fv fragment consisting of the VL and VH domains of a single arm of an antibody
  • dAb domain antibody
  • VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. WO 1998/44001, WO1988/01649, WO1994/13804, and W01992/01047.
  • scFv single chain Fv
  • diabody diabody
  • 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 specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than human CD38).
  • An isolated antibody that specifically binds CD38 can have cross-reactivity to other antigens, such as orthologs of human CD38, such sMacaca fascicularis (cynomolgus monkey) CD38.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • Humanized antibody refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
  • Human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites 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 comprises heavy or light chain variable regions that are "derived from” sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin 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 as described herein.
  • a human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites.
  • 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 an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.
  • Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.
  • recombinant antibody includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.
  • epitope means a portion of an antigen to which an antibody specifically binds.
  • Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope can be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3 -dimensional space through the folding of the protein molecule.
  • Variant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.
  • combination with means that two or more therapeutics can be used together in a mixture, concurrently as single agents or sequentially as single agents in any order.
  • treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of tumor or tumor cells.
  • beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if a subject was not receiving treatment.
  • Inhibits growth refers to a measurable decrease in the cell growth in vitro or in vivo when contacted with a therapeutic or a combination of therapeutics or drugs when compared to the growth of the same cells grown in appropriate control conditions well known to the skilled in the art. Inhibition of growth of a cell 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 a variety of mechanisms, for example by antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, necrosis, or by inhibition of cell proliferation.
  • ADCC antibodydependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • apoptosis necrosis
  • necrosis or by inhibition of cell proliferation.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual.
  • Exemplary indicators of an effective therapeutic or combination of therapeutics include, for example, improved well-being of the patient, reduction of a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.
  • An exemplary antibody used in the ADCP assays in the examples is a human monoclonal antibody that binds 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 against multiple myeloma.
  • Another exemplary antibody used in the ADCP assays in the examples, Rituximab (Rituxan®) is a chimeric anti-CD20 antibody targeting the CD20 protein which is expressed on over 95% of B cell lymphomas.
  • a range is intended to comprise every integer or fraction or value within the range.
  • Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features.
  • ADCP antibody-dependent cellular phagocytosis
  • the low % CO2 is from about 0.1 % CO2 to 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.
  • the low % CO2 atmosphere is maintained by CO2 being infused into a humidified chamber or incubator.
  • the period of time sufficient to increase ADCP activity of the phagocytic cells is 3 to 15 days, for example 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 of the phagocytic cell is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity of the phagocytic cell is 3, 4, 5, 6, or 7 days.
  • the method further comprises determining the ADCP activity of the phagocytic cell using target cells labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis ((Ralph & Nakoinz, 1975).
  • Donor cells may be obtained from a human subject.
  • the donor cell is a human macrophage cell.
  • the human macrophage cell is derived from an effusion fluid, for example a peritoneal exudate (US8975040).
  • the phagocytic cells are cultured in the presence of target cells.
  • target cells are added to the phagocytic cell culture after about 1 to about 14 days.
  • target cells are added to the phagocytic cell culture after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
  • target cells are added to the phagocytic cell culture after about 7 days.
  • the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.
  • the target cells are Daudi cells.
  • Daudi cells (ATCC® CCL213TM) are a well characterized B lymphoblast cell line derived from a 16-year-old black male with Burkitt's lymphoma.
  • the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • a regulator of ADCP is increased.
  • the regulator is ATF4, FOXO3, IL IB, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof.
  • expression of a regulator of ADCP is decreased.
  • the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS or combinations thereof.
  • the target cells are contacted with an antibody or fragment thereof.
  • the sample is cultured in a humidified chamber or incubator.
  • Also provided is a method for assaying ADCP activity of a phagocytic cell in a sample comprising: culturing the phagocytic cell in a low percent (%) CO2 atmosphere for a period of time sufficient to increase ADCP activity of the phagocytic cell; and detecting ADCP activity of the phagocytic cell in the sample.
  • the low % CO2 is from 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.
  • the low % CO2 is 1% CO2.
  • the low % CO2 atmosphere is maintained by CO2 being infused into a humidified chamber or incubator.
  • the period of time sufficient to increase ADCP activity of the phagocytic cells is 3 to 15 days, for example 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 of the phagocytic cell is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity of the phagocytic cell is 3, 4, 5, 6, or 7 days.
  • the method further comprises determining the ADCP activity of the phacobytic cell using target cells labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis (Ralph & Nakoinz, 1975).
  • Donor cells may be obtained from a human subject.
  • the donor cell is a human macrophage cell.
  • the phagocytic cells are cultured in the presence of target cells.
  • target cells are added to the phagocytic cell culture after about 3 to about 10 days, for example after about 7 days.
  • the target cells are labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis (Ralph & Nakoinz, 1975).
  • Donor cells may be obtained from a human subject.
  • the donor cell is a human macrophage cell.
  • the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells. In yet further embodiments, the target cells are Daudi cells. Daudi cells (ATCC® CCL213TM) are a well characterized B lymphoblast cell line derived from a 16-year- old black male with Burkitt's lymphoma.
  • the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • a regulator of ADCP is increased.
  • the regulator is ATF4, FOXO3, IL IB, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof
  • expression of a regulator of ADCP is decreased.
  • the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS or combinations thereof.
  • the target cells are contacted with an antibody or fragment thereof.
  • the sample is cultured in a humidified chamber or incubator.
  • a method for assaying ADCP activity of a phagocytic cell in a sample comprising: culturing the phagocytic cell in a low percent (%) CO2 atmosphere for a period of time sufficient to increase ADCP activity of the phagocytic cell; and detecting ADCP activity of the phagocytic cell in the sample.
  • the low % CO2 is from 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.
  • the low % CO2 is 1% CO2.
  • the low % CO2 atmosphere is maintained by CO2 being infused into a humidified chamber or incubator.
  • the period of time sufficient to increase ADCP activity of the phagocytic cells is 3 to 15 days, for example 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 of the phagocytic cell is 3 to 7 days. In yet further embodiments, the period of time sufficient to increase ADCP activity of the phagocytic cell is 3, 4, 5, 6, or 7 days. [0102] In some embodiments, the method further comprises determining the ADCP activity of the phacobytic cell using target cells labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis (Ralph & Nakoinz, 1975).
  • Donor cells may be obtained from a human subject.
  • the donor cell is a human macrophage cell.
  • the phagocytic cells are cultured in the presence of target cells.
  • target cells are added to the phagocytic cell culture after about 3 to about 10 days, for example after about 7 days.
  • the target cells are labeled with a fluorescent dye.
  • the phagocytic cell is a J774A.1 cell or a donor cell.
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis (Ralph & Nakoinz, 1975).
  • Donor cells may be obtained from a human subject.
  • the donor cell is a human macrophage cell.
  • the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.
  • the target cells are Daudi cells.
  • Daudi cells ATCC® CCL213TM are a well characterized B lymphoblast cell line derived from a 16-year- old black male with Burkitt's lymphoma.
  • the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • expression of a regulator of ADCP is increased.
  • the regulator is ATF4, FOXO3, IL IB, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof.
  • expression of a regulator of ADCP is decreased.
  • the regulator is TP53, TNF, TGFB1, STAT6, MYD88, HRAS or combinations thereof.
  • the target cells are contacted with an antibody or fragment thereof.
  • the sample is cultured in a humidified chamber or incubator.
  • Human donor macrophages were prepared by standard methods as known by those skilled in the art. In brief, frozen PBMC were enriched by Human monocyte enrichment cocktail without CD16 depletion (STEMCELL Technology® 19058). 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. At day 3-4, 50% medium were replaced with fresh X- VIVO10 + 10% FBS. At day 6, IFN-y (50ng/mL) were added and cells were harvested for ADCP assay at day 7.
  • FBS fetal bovine serum
  • J774A.1 cells are a mostly adherent monocyte/macrophage cell line derived from mice (Ralph, Moore, & Nilsson, 1976) that are active in antibody dependent phagocytosis (Ralph & Nakoinz, 1975). J774A.1 cells were cultured in cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with FBS to a final concentration of 10%. Subcultures are prepared by cell dissociation reagent, Accutase (eBioscience. 00-4555-56) and scraping. For parental J774A.1 , cells are cultured in 5% CO2 at 37°C. For low CO2 is noted in the Figures and Examples, J774A.1 cells were seeded at 8x10 6 cell / 0mL in a T150 flask and incubated in low CO2 for the period of time indicated. Daudi cells
  • Daudi cells are a well characterized B lymphoblast cell line derived from a 16-y ear-old black male with Burkitt's lymphoma.
  • the Daudi cells were cultured in suspension in RPMI-1640 medium (ATCC 30-2001), with FBS added to a final concentration of 10%, at 37°C and 5% CO2. Fresh medium was added every 2 to 3 days (depending on cell density) until the day of the ADCP assay.
  • Target cells e.g., Daudi cells
  • PBS phosphate buffered saline
  • 25pl of 1 mg/ml pHrodo-Red stock solution in DMSO
  • pHrodo-Red SE was from Invitrogen (Waltham, MA; P36600).
  • Macrophages e.g., human donor macrophages and J774A.1 cells
  • a rat anti-Mouse CD1 lb antibody [MI/70] labelled with Alexa Fluor® 488 (Biolegend, San Diego, CA).
  • the macrophages were lifted from a culture flask with Accutase for 30 min at 37°C and washed two times with PBS and 2.5 pl/mlof the anti-Mouse CD1 lb antibody were added for 20 min at 4°C. This staining provided a homogenous surface staining of macrophages and was also useful to distinguish cell surfaces of cells during flow cytometry.
  • ADCP assay done in 96-well plates (Corning, 3799) for flow cytometry serial diluted Daratumumab or Rituximab were pre-incubated with 2.5 x 10 4 of pHrodo-red labeled Daudi cells in each well for 15 minutes at room temperature. Then, lx 10 5 human macrophages or J774A.1 cells were added and mixed. The plate were centrifuge at 40xg for 1 minute with minimal acceleration and deceleration and incubated in 37°C incubator with 5% or 1% CO2 for 3 or 24 hours. The cells then wash once with DPBS and detached with Accutase for further CD1 lb staining and flow cytometry.
  • serial diluted Daratumumab or rituximab were pre- incubated with 1.25 x 10 4 of pHrodo-red labeled Daudi cells in each well (Corning, 3904) for 15 minutes at room temperature. Then, 5x 10 4 human macrophages or J774A.1 cells were added and mixed. The plate were centrifuge at 40xg for 1 minute with minimal acceleration and deceleration and incubated in 37°C incubator with 1% CO2 for 2.5 hours. Data was acquired using a SpectraMax® Paradigm® Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at Ex/Em 560/600 nm with well scan mode. Results were plotted after the subtraction of the signal from wells with no antibody added (subtracting signal from background/control).
  • Example 1 ADCP by flow cytometry with human donor macrophages
  • Human donor macrophages (IxlO 5 ) were incubated with pHrodo-red labeled Daudi cells (2.5xl0 4 ) and titrated monoclonal antibodies (Daratumumab or Rituximab, 0.03 to 2000 ng/mL) for 3 hours in a 96- well plate. Cells were detached from the wells and stained with an antiCD 1 lb-A488 antibody. ADCP was assessed by flow cytometry and analyzed by FlowJo software and dose response curves were generated by plotting percentage for cells of Q2 gating for pHrodo-red Daudi cells engulfed by human donor macrophages (FIGs. 1 A-1B).
  • Example 2 ADCP by flow cytometry with J774A.1 cells cultured in 5% CO2
  • J774A.1 cells (IxlO 5 ) that had been maintained in 5% CO2, were incubated with pHrodo- red labeled Daudi cells (2.5xl0 4 ) and titrated monoclonal antibodies (Daratumumab or Rituximab, 0.03 to 2000 ng/mL) for 24 hours in a 96-well plate. Cells were detached from the wells and stained with an anti-CDl lb-A488 antibody.
  • ADCP was assessed by flow cytometry analyzed by FlowJo software (FlowJo, LLC, Ashland, OR) and dose response curves were generated by plotting percentage for cells of Q2 gating for pHrodo-red Daudi cells engulfed by J774A.1 cells (FIGs. 2A-2B).
  • Example 3 Morphology change for J774A.1 cells cultured in 1% CO2
  • J774A.1 cells were sub-cultured in 5% CO2 humidified chamber for 2 days or in 1% CO2 humidified chamber for 5 days. Photos were taken by microscopy, 200X bright field or 200X phase-contrast in (FIGs. 3A-3B ).
  • Example 4 Increased ADCP for J774A.1 cells cultured in 1% CO2
  • J774A.1 cells were cultured in a 5% CO2 humidified chamber for 2 days or cultured in a 1% CO2 humidified chamber for 3 days or 7 days.
  • the J774A.1 cells (IxlO 5 ) were incubated with pHrodo-red labeled Daudi cells (2.5xl0 4 ) and titrated monoclonal antibody (Daratumumab, 0 to 1000 ng/mL) for 24 hours in a 96-well plate. Cells were detached from the wells and stained with an anti-CDl lb-A488 antibody.
  • ADCP was assessed by flow cytometry analyzed by FlowJo software and dose response curves were generated by plotting percentage for cells of Q2 gating for pHrodo-red Daudi cells engulfed by J774A.1 cells (FIGs. 4A-4B).
  • ADCP results were compared between J744A.1 cells that were cultured in 5% CO2 for 2 days, 1% CO2 for 3 days, and 1% CO 2 for 7 days, respectively.
  • ADCP flow cytometry analysis showed that Dauid cells, in the presence of daratumumab, were opsonized by J744A.1 in a higher level in 1% CO2 than 5% CO2 culturing conditions.
  • the % engulfment from J774A.1 cells that were cultured in 1% CO2 was higher than J774A.1 cells that were cultured in 5% CO2.
  • a good dynamic daratumumab dose dependent response curves were observed with J774A.1 cells that were cultured in 1% CO2.
  • Example 5 Effect of time on ADCP for J774A.1 cells cultured in 1% CO2
  • J774A.1 cells were cultured in 5% CO2 humidified chamber for 3 days or 1% CO2 humidified chamber for 7 days, 11 days, or 15 days. J774A.1 cells (5x10 4 ) were then incubated with pHrodo-red labeled Daudi cells (1.25xl0 4 ) for 2 hours in a 96-well plate with titrated monoclonal antibody (Daratumumab, 0 to 5000 ng/mL). ADCP was assessed by microplate reader and the results were plotted after subtraction of the no antibody control (FIG. 5).
  • J774A.1 cells were cultured in 1% CO2 humidified chamber for 5 days. J774A.1 cells (5x10 4 ) were then incubated with pHrodo-red labeled Daudi cells (1.25xl0 4 ) for 2.5 hours in a 96-well plate with titrated antibodies (Daratumumab or Rituximab, 0 to 1000 ng/mL). ADCP was assessed by microplate reader and the results were plotted after subtraction of the no antibody control (FIG. 6).
  • J774A.1 cells were cultured in 1% CO2 humidified chamber for 5 days. J774A.1 cells (5x10 4 ) were then incubated with pHrodo-red labeled Daudi cells (1.25xl0 4 ) for 2.5 hours in a 96- well plate with titrated Daratumumab (0 to 1000 ng/mL) that was freshly thawed or stored at 37°C for 3 months or 6 months. ADCP as assessed by microplate reader and the results were plotted after subtraction of the no antibody control (FIG. 7).
  • J774A.1 cells Two groups of J774A.1 cells were incubated with different CO2 concentrations (1% vs. 5%). J774A.1 cells, 1 X 10 6 and 4 X 10 6 cells/mL were cultured in either 5% or 1% CO2 humidified chamber for 4 days, respectively. RNA from the corresponding culture conditions was extracted for RNA-Seq Analysis (FIGs. 8A, 8B, and 8C). PCA (Principal components analysis) demonstrated that gene expression profiles between J744A.1 cells cultured in 1% CO2 vs 5% CO2 were significantly different.
  • IPA Ingenuity Pathways Analysis
  • ADCP activity surprisingly and unpredictably increased for J774A.1 cells cultured in 1% CO2.
  • the increased ADCP activity provided an improved ADCP assay with increased signal compared to background.
  • the improved ADCP assay method is suitable for determining ADCP activity of an antibody, for comparing the ADCP activity of different antibodies or different antibody preparations, and for measuring the stability of an antibody as is required for quality control during production or storage of an antibody.
  • a method for increasing antibody-dependent cellular phagocytosis (ADCP) activity of a phagocytic cell in a sample comprising: culturing the phagocytic cell in a low percent (%) CO2 atmosphere for a period of time sufficient to increase ADCP activity of the phagocytic cell.
  • ADCP antibody-dependent cellular phagocytosis
  • 10A The method of any one of embodiments 8A-9A, wherein the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.
  • 11A The method of any one of embodiments 6A-10A, wherein the fluorescent dye is pHrodo-Red, pHAb or AcidiFluor.
  • 13A The method of embodiment 12A, wherein the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof.
  • the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLI1, SP3, or combinations thereof.
  • a method for assaying ADCP activity of a phagocytic cell in a sample comprising: culturing the phagocytic cell in a low percent (%) CO2 atmosphere for a period of time sufficient to increase ADCP activity of the phagocytic cell; and detecting ADCP activity of the phagocytic cell in the sample.
  • 29A The method of any one of embodiments 25A-28A, wherein the target cells are Daudi cells, B cells, leukemia cells or lymphoma cells.
  • 31 A The method of any one of embodiments 18A-30A, wherein expression of a regulator of ADCP is increased.
  • 32A The method of embodiment 31A, wherein the regulator is ATF4, FOXO3, IL1B, IL6, VEGFA, HGF, EGF, CHD1, SELP, TIMP3, DACH1, STAT3, GLU, SP3, or combinations thereof.
  • 35A The method of any one of embodiments 25A-34A, wherein the target cells are contacted with an antibody or fragment thereof.
  • 36A The method of any one of embodiments 18A-35A, wherein the sample is cultured in a humidified chamber or incubator.
  • Antibody-mediated phagocytosis contributes to the antitumor activity of the therapeutic antibody Daratumumab in lymphoma and multiple myeloma.
  • ADCP antibody dependent cellular phagocytosis
  • ADCC antibody dependent cellular cytotoxicity

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Abstract

L'invention concerne un procédé visant à augmenter l'activité de phagocytose cellulaire dépendante des anticorps (ADCP) d'une cellule phagocytaire dans un échantillon consistant à : mettre en culture la cellule phagocytaire dans une atmosphère à faible pourcentage (%) de CO2 pendant une période de temps suffisante pour augmenter l'activité de l'ADCP de la cellule phagocytaire. Dans certains modes de réalisation, les cellules phagocytaires sont cultivées en présence de cellules cibles. L'invention concerne également un procédé de dosage de l'activité de l'ADCP d'une cellule phagocytaire dans un échantillon consistant à : mettre en culture la cellule phagocytaire dans une atmosphère à faible pourcentage (%) de CO2 pendant une période de temps suffisante pour augmenter l'activité de l'ADCP de la cellule phagocytaire ; et détecter l'activité de l'ADCP de la cellule phagocytaire dans l'échantillon. Dans certains modes de réalisation, les cellules phagocytaires sont cultivées en présence de cellules cibles.
PCT/IB2023/060349 2022-10-14 2023-10-13 Procédé de détection de phagocytose cellulaire dépendante des anticorps WO2024079711A1 (fr)

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