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WO2025168631A1 - Antibody combination and methods for dendritic cells enrichment - Google Patents

Antibody combination and methods for dendritic cells enrichment

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Publication number
WO2025168631A1
WO2025168631A1 PCT/EP2025/052964 EP2025052964W WO2025168631A1 WO 2025168631 A1 WO2025168631 A1 WO 2025168631A1 EP 2025052964 W EP2025052964 W EP 2025052964W WO 2025168631 A1 WO2025168631 A1 WO 2025168631A1
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WO
WIPO (PCT)
Prior art keywords
antigen
antibody
binding fragment
directed against
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/052964
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French (fr)
Inventor
Pierre TONNERRE
Fanny ONODI
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Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
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Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Cite filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2025168631A1 publication Critical patent/WO2025168631A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Definitions

  • the present disclosure relates to antibody combinations and methods for enriching a human cell sample with dendritic cells.
  • the present disclosure relates to antibody combinations and methods for enriching a human cell sample with all the five dendritic cell subsets including AS-DC, pDC, cDCl, DC2 and DC3.
  • DCs Dendritic cells
  • DCs Given their frontline position in the immune defense, DCs exhibit a remarkable capacity to adapt to a spectrum of microbial and host-derived stimuli, resulting in functional plasticity and the acquisition of distinct phenotypical and functional properties, including the ability to induce specific T cell polarization programs.
  • DCs In human blood, the initial categorization of DCs includes three subsets namely, conventional dendritic cells (eDCs), including cDCl and cDC2, and plasmacytoid dendritic cells (pDCs). These three subsets share a common DC progenitor (Guilliams et al., Nat Rev Immunol. 2014).
  • eDCs conventional dendritic cells
  • pDCs plasmacytoid dendritic cells
  • AS-DC AXL+ Siglec6+ dendritic cells
  • markers similar to both pDCs and cDC2s notably the CD123 antigen and the CD11c antigen, while being specifically characterized by the expression of the AXL antigen, the Siglec6 antigen, the CD2 antigen, and the CD5 antigen markers (Villani AC et al. Science, 2017).
  • DC3 originates from a monocyte cell precursor (Liu et al., Immunity. 2023 Aug 8;56(8): 1761-1777.e6.).
  • Miltenyi pan-DC enrichment kit effectively retrieves various dendritic cell populations, it fails in capturing all DC subsets, specifically missing the newly identified DC3 subset (CDllc+ CD163+ CD14+). Similarly, the Stemcell pan-DC enrichment kit isolates different DC subsets but leads to a reduction in total dendritic cell numbers without specifically capturing the newly identified DC3 subset.
  • the symbol means a negative expression.
  • a complement receptor in the characterization of a cell, for instance CD14- DC3 cell, it means that the antigen is not expressed by the cell.
  • the CD3 antigen also known as the T-cell surface glycoprotein CD3 delta chain, is a protein complex composed of two CD3s chains, one CD3y chain, one CD35 chain, one CD3 ⁇ chain, and a T-cell receptor (a/p or y/5) heterodimer.
  • CD3s chain can be identified by the UniProt number P07766.
  • CD3y chain can be identified by the UniProt number P09693.
  • CD35 chain can be identified by the UniProt number P04234.
  • the CD5 antigen also known as the T-cell surface glycoprotein CD5, can be identified by the UniProt number P06127.
  • the CD1 lb antigen also known as the Integrin alpha-M, can be identified by the UniProt number Pl 1215.
  • the CD 14 antigen also known as the Monocyte differentiation antigen CD 14, can be identified by the UniProt number P08571.
  • the CD 15 antigen also known as Sialyl Lewis x , can be identified by the CAS number 98603-84-0.
  • the CD19 antigen also known as the B-lymphocyte antigen CD19, is a protein that can be identified by the UniProt number P15391.
  • the CD20 antigen also known as the B-lymphocyte antigen CD20, is a protein that can be identified by the UniProt number Pl 1836.
  • the CD41 antigen also known as the Integrin alpha- lib, can be identified by the UniProt number P08514.
  • the CD61 antigen also known as the Integrin beta-3, can be identified by the UniProt number P05106.
  • the CD66b antigen also known as the Carcinoembryonic antigen-related cell adhesion molecule 8 can be identified by the UniProt number P31997.
  • the CD 123 antigen also known as the interleukin-3 receptor, can be identified by the UniProt number P26951.
  • the CD141 antigen also known as the Thrombomodulin, can be identified by the UniProt number P07204.
  • the CD 163 antigen also known as the Scavenger receptor cysteine -rich type 1 protein Ml 30, can be identified by the UniProt number Q86VB7.
  • the CD235a antigen also known as the Glycophorin-A, can be identified by the UniProt number P02724.
  • the CD303 antigen also known as the C-type lectin domain family 4 member C, can be identified by the UniProt number Q8WTT0.
  • the CD304 antigen also known as the Neuropilin- 1, can be identified by the UniProt number 014786.
  • CLEC9A antigen or CD370 antigen also known as the C-type lectin domain family 9 member A, can be identified by the UniProt number Q6UXN8.
  • TLR Toll-Like Receptor
  • PAMPs pathogen-associated molecular patterns
  • TLRs Upon binding to these pathogen-associated molecular patterns (PAMPs), TLRs initiate signaling cascades that trigger immune responses from dendritic cells, including the production of cytokines, such as Interleukin-1 beta (IL-10) or Interferon gamma-induced protein 10 (IP-10).
  • IL-1 beta Interleukin-1 beta
  • IP-10 Interferon gamma-induced protein 10
  • Each dendritic cell subset express particular class of TLR; dendritic cells of the pDC subset express TLR7 (UniProt number Q9NYK1) and TLR9; dendritic cells of the DC2 and DC3 subsets express, but are not limited to, TLR3 and TLR8.
  • target cell herein means any cell that may be targeted by an antibody combination of the present disclosure.
  • a target cell also includes a cell expressing a target antigen that can be recognized by an antibody combination of the present disclosure.
  • a target cell may be a T cell, a B cell, a NK cell, a monocyte, a red blood cell and/or a platelet.
  • the term “enrichment” or “enriching” or equivalents, in the context of enriching a human cell sample with dendritic cells, refers to the isolation of dendritic cells from the cells presents in the human cell sample. It thus means that the percentage of the dendritic cells relative to the total number of cells in a human cell sample has increased in said sample. At the same time this means that the number of non-dendritic cells present in the human cell sample has decreased in said human cell sample.
  • These non-dendritic cells which are depleted from the human cell sample may for example include T cells, B cells, NK cells, platelet, red blood cells, basophils, neutrophils and/or monocytes.
  • the dendritic cell enrichment method of the present disclosure may lead to an enrichment of all the five dendritic cell subsets, specifically the AS-DC subset, the pDC subset, the cDCl subset, the DC2 subset and the DC3 subset.
  • the dendritic cell enrichment method of the present disclosure may lead to an enrichment of dendritic cells from 30% to 90% of the total cells within a given human cell sample.
  • the dendritic cell enrichment method of the present disclosure may lead to an enrichment of the DC3 subset, expressing at least the CD 14 antigen, from 5% to 15% of the total cells within a given human cell sample.
  • the dendritic cell enrichment method of the present disclosure may lead to a human cell sample depleted in CD88-positive (CD88+) cells and CD89-positive (CD89+) cells. In some embodiments, the dendritic cell enrichment method of the present disclosure may lead to a human cell sample depleted in HLA-DR-positive (HLA-DR+) myeloid cells other than dendritic cells.
  • human refers to the species Homo sapiens, encompassing individuals belonging to the biological classification of humans.
  • “human” specifically denotes characteristics, biological components, or attributes unique to, or derived from, the human species. This may include but is not limited to, cells, tissues, proteins, or any other biological materials originating from or characteristic of humans.
  • magnetic beads refers to solid nanoparticles typically composed of a magnetic core, often made from materials like iron oxide or magnetite, and coated with a surface layer that enables the binding or immobilization of specific target molecules such as proteins other biomolecules. Magnetic beads, in combination with antibodies, can be used for separation, purification, and isolation of cells from biological samples using magnetic forces.
  • a magnetic bead may consist of MojoSortTM Human anti-APC Nanobeads purchased from BioLegend.
  • a magnetic bead may have a diameter from about 20 nanometer (nm) to about 800 nm.
  • a magnetic bead may have a diameter of about 20 nm, about 50 nm, about 75 nm, about 100 nm, about 115 nm, about 130 nm, about 145 nm, about 160 nm or about 800 nm.
  • a magnetic bead that may be used in the present disclosure may have a diameter of about 130 nm.
  • the dendritic cell enrichment method according to the present disclosure is carried out on a cell sample from an individual, including dendritic cells and non-dendritic cells.
  • an individual is a human.
  • a cell sample may be a human cell sample.
  • a human cell sample may be a whole blood sample.
  • a whole blood sample may be obtained from an individual who may be healthy or sick.
  • a whole blood sample may comprise a plurality of blood cells comprising the major types of blood cells including platelets, red blood cells and white blood cells.
  • red blood cells also known as erythrocyte, refers to a type of non-nucleated cells found in the bloodstream and characterized by its biconcave disc shape. Red blood cell’s primary function is to deliver dioxygen (O2) to the body tissues and to carry carbon dioxide (CO2) to the lungs for exhalation/elimination. Red blood cells express the surface membrane antigen CD235a, also called Glycophorins A protein.
  • CD235a also called Glycophorins A protein.
  • blood platelets or “platelets”, also known as thrombocytes, are small, disc-shaped non-nucleated cell fragments derived from megakaryocytes in the bone marrow. These cell fragments play a critical role in hemostasis and blood clotting mechanisms by adhering to damaged blood vessel walls, aggregating to form a plug at the site of injury, and releasing various factors essential for coagulation cascade activation, ultimately aiding in the cessation of bleeding.
  • Blood platelets express the CD41, also known as Integrin allb (GPIIb), and/or the CD61 surface membrane antigen, also known as integrin 03 (GPIIIa).
  • the CD41/CD61 complex is required for platelet aggregation and clotting.
  • white blood cells also known as leukocytes, are a group of nucleated cells found in the bloodstream and lymphatic system of an individual. These white blood cells form a critical component of the human’s immune defense mechanism, functioning primarily to combat infections, diseases, and foreign substances by various means such as phagocytosis, antibody production, and cellular immune responses.
  • White blood cells encompass several subtypes, including, but not limited to, neutrophils, lymphocytes (T cells, B cells, and NK cells), monocytes, dendritic cells, eosinophils, and basophils, each playing distinct roles in immune surveillance, response, and regulation within the human body.
  • a human cell sample may be obtained from peripheral circulation, buffy coat, bone marrow and/or cord blood from an individual.
  • a human cell sample may be obtained from peripheral circulation from an individual.
  • a human cell sample may be an isolated fraction of a whole blood sample comprising or consisting essentially of a plurality of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Isolated PBMC sample may be derived from a whole blood sample by separated peripheral blood mononuclear cells from the other blood cells.
  • isolated PBMCs comprise or consist essentially of peripheral blood cells having one round nucleus such as T cells, B cells, monocytes, Natural Killer (NK) cells, and dendritic cells.
  • Non-nucleated blood cells may include red blood cells and platelets whereas multi-lobed nuclei blood cells may include neutrophils, basophils, and eosinophils.
  • an isolated PBMC sample may be obtained by density gradient centrifugation, in particular with the Ficoll-Paque method.
  • an isolated PBMC sample may contain residual non-nucleated blood cells or multi-lobed nuclei blood cells that have not been depleted after the initial cell separation process.
  • a human cell sample to be used in methods as disclosed herein may be an isolated PBMCs sample.
  • a human cell sample to be used in methods as disclosed herein may be an isolated PBMCs sample derived from a whole blood sample.
  • An isolated PBMC sample may comprises T cells, B cells, NK cells, monocytes, and/or dendritic cells.
  • an isolated PBMC sample may contain a low concentration of residual granulocytes (basophils, neutrophils, eosinophils), red blood cells and/or platelets that have not been depleted after the initial cell separation process from whole blood cell.
  • Granulocytes refer to a cell subset of white blood cells characterized by the presence of cytoplasmic granules. This category includes neutrophils, eosinophils, and basophils, each playing distinct roles in the immune response. [00118] Generally, during the isolation of PBMC through separation techniques such as ficoll gradient, the majority of granulocytes, including neutrophils, basophils, and eosinophils, are eliminated. However, a minor fraction of neutrophils and basophils from the granulocyte family may persist despite these methods.
  • Neutrophils can be characterized by the expression of at least the surface membrane CD15 antigen and/or the CD66b antigen.
  • Eosinophils can be characterized by the expression of at least the surface membrane antigen CCR3 (also known as CD193) and the absence of the CD16 antigen.
  • CCR3 also known as CD193
  • Basophils can be characterized by the expression of at least the CD88 surface membrane antigen, also named C5AR1.
  • T cells or blood T cells refer to a subset of lymphocytes derived from the thymus gland and circulating in the bloodstream, playing a pivotal role in adaptive immunity. These cells express T cell receptors (TCRs) on their surface and are involved in recognizing specific antigens presented by major histocompatibility complex (MHC) molecules. T cells encompass various subsets, including cytotoxic T cells, helper T cells, and regulatory T cells. Furthermore, T cells also encompass a heterogeneous group of cells called Natural killer T (NKT) cells, that share properties of both T cells and natural killer cells. These different subsets can be discriminated based on selective phenotypic extracellular markers, in combination with intracellular transcription factor and/or cytokine staining.
  • TCT Natural killer T
  • All the various T cell subsets, including NKT cells, can be characterized by commonly expressing at least the CD3 surface membrane antigen and/or the TCRa/p surface membrane antigen and the TCRy/6 surface membrane antigen.
  • NKT cells can also be characterized by the expression of at least the CD56 surface membrane antigen, also named NCAM, shared with NK cells.
  • B cells or blood B cells refer to a subset of lymphocytes originating from the bone marrow and circulating in peripheral blood, characterized by the presence of surface B-cell receptors (BCRs) crucial for antigen recognition.
  • B cells play a pivotal role in adaptive immunity by differentiating into plasma cells, producing antibodies, and facilitating immunological memory, thereby contributing significantly to the body's defense against pathogens and foreign substances.
  • B cells can be characterized by the expression of at least the surface membrane CD 19 antigen and/or the CD20 antigen. While the CD20 antigen is acquired during late stages of B -cell lymphogenesis and is then lost upon differentiation into plasma cells, CD 19 expression covers the entire spectrum of early B-cell genesis and maturation.
  • Natural Killer Cells also called NK cells or large granular lymphocytes (LGL), refer to a subset of cytotoxic lymphocytes belonging to the innate immune system and represent about 5% to 20% of all circulating lymphocytes in humans.
  • NK cells originate, differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the blood circulation.
  • NK cells are characterized by their ability to recognize and eliminate virally infected cells, tumor cells, and cells displaying stress-induced surface markers, without prior sensitization or the need for specific antigens.
  • NK cells exert their cytotoxic functions through the release of cytolytic granules containing perforin and granzymes, inducing apoptosis in target cells. Additionally, NK cells modulate immune responses by secreting cytokines and chemokines, thereby contributing to the regulation of adaptive immune reactions.
  • NK cell subsets can be characterized by commonly expressing at least the surface membrane CD56 antigen and/or the CD16a antigen (FcyRIIIa) and/or the CD57 antigen.
  • monocytes were classified following three subsets based on expression of the CD14 antigen and/or the CD16 antigen: classical monocytes expressing the CD 14 surface membrane antigen and lack the expression of the CD 16 marker; intermediate monocytes expressing both the CD 14 and CD 16 surface membrane antigens; and nonclassical monocytes expressing the CD 16 surface membrane antigen and lack the expression of the CD 14 antigen.
  • the expression of the CD 14 antigen is shared with the DC3 subset.
  • All the three monocyte subsets also express the CD88 surface membrane antigen and/or the CD89 surface membrane antigen.
  • the present disclosure aims at enriching a cell sample with all the identified dendritic cell subsets.
  • All the dendritic cell subsets can commonly be characterized by the expression, on their surface, of at least the CD45 and HLA-DR antigens. These antigens are shared with other white blood cells such as, for instance, monocytes. To distinguish and isolate dendritic cells from other white blood cells, specific markers are used for further characterization of dendritic cells.
  • Axl+ Siglec6+ dendritic cells or AS-DCs or pre dendritic cells can be characterized by the expression, on its surface, of at least the Axl antigen.
  • pDCs can be characterized by the absence of expression of the CDllc antigen, the AXL antigen and/or the CD88 antigen.
  • pDCs can have the capacity to secrete interferon alpha and can have the capacity to secrete interferon gamma-induced protein 10 (IP- 10).
  • Conventional dendritic cells 1 or eDC 1 s can be characterized by the expression, on its surface, of at least one antigen among the CD141 antigen and the CLEC9A antigen, and optionally at least the XCR1 antigen.
  • cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, the XCR1 antigen, and the CLEC9A antigen.
  • cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, and optionally at least the CLEC9A antigen, and/or the CDllc antigen.
  • cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, and by the absence of expression of the CDlc antigen, the AXL antigen and the CD88 antigen.
  • cDCls can have the capacity to secrete interleukin- 1 beta (IL- 10).
  • the conventional dendritic cell subset comprises two sub-populations which are dendritic cells 2 and dendritic cells 3.
  • Dendritic cells 2 or DC2s can be characterized by the expression, on its surface, of at least one antigen among the CDlc and CD11c antigens, and optionally at least one antigen among the CD172a and CDllb antigens, and by the absence of the expression of the CD163 antigen.
  • DC2s can be characterized by the expression, on its surface, of at least one antigen among the CDlc and CD11c antigens, and optionally at least one antigen among the CD172a and CD1 lb antigens, and by the absence of expression of the CD163 antigen.
  • DC2s can be further characterized by the absence of expression of the AXL antigen and/or the CD88 antigen.
  • DC2s can have the capacity to secrete interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
  • IL- 10 interleukin- 1 beta
  • IP- 10 interferon gamma-induced protein 10
  • Dendritic cells 3 or DC3s can be characterized by the expression, on its surface, of at least both the CD163 and CD11c antigens, and optionally at least one of the antigens among the CDlc antigen, the CD172a antigen, and the CD14 antigen.
  • DC3s can be characterized by the expression, on its surface, of at least the CD163 antigen, the CDllc antigen and the CDlc antigen, and optionally at least one antigen among the CD172a antigen and the CD14 antigen.
  • DC3s can be further characterized by the absence of expression of the AXL antigen and/or the CD88 antigen.
  • DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CD163 antigen and the CDlc antigen; and by the absence of expression of the AXL antigen and the CD88 antigen.
  • DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CD 163 antigen, the CDlc antigen, and the CD 14 antigen. [00169] In some embodiments, DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CDlc antigen and the CD163 antigen, and by the absence of expression of the CD 14 antigen.
  • DC3s can have the capacity to secrete interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
  • IL- 10 interleukin- 1 beta
  • IP- 10 interferon gamma-induced protein 10
  • a human cell sample as described above, may be exposed to an antibody combination specifically designated for enriching the human cell sample with dendritic cells.
  • an antibody combination as described herein as a negative selection technique allows to prepare a human cell sample which is enriched in all the five dendritic cell subsets as mentioned herein.
  • an object of the present disclosure relates to an antibody combination comprising a plurality of antibodies or fragments thereof directed against non-dendritic cell antigens.
  • the antibody combination as described herein is comprised in a composition.
  • the expression “directed against” refers to the ability of an antibody or antigen-binding fragment thereof to detectably bind an epitope present on a specific antigen, while having relatively no or little detectable reactivity with different antigen.
  • the antibody or antigen-binding fragment thereof forms a complex with the specific antigen that is relatively stable under suitable conditions.
  • antibody encompasses an “antigen-binding fragment”.
  • the antibodies described herein may be isolated antibodies.
  • the antibodies may be monoclonal antibodies.
  • the antibodies may be labelled with a detectable molecule and/or conjugated to magnetic beads.
  • a human cell sample as described herein may contain a low concentration of residual red blood cells and platelets that have not been depleted after the initial cell separation process from whole blood cell. Therefore, the antibody composition as describe herein may further comprise antibodies or fragments thereof directed against antigens specific of platelets and/or red blood cells.
  • an antibody combination disclosed herein, for enriching a human cell sample with dendritic cells may further comprise antibodies of groups (vii) and (viii) defined as:
  • Antibodies or an antigen-binding fragments thereof of group (i) described herein may specifically bind to an antigen present at the surface of T cells, including the subgroup of NKT cells.
  • an antibody or an antigen-binding fragment thereof of group (i) may consist of an antibody or an antigen-binding fragment thereof directed against the CD3 antigen and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/5 antigen.
  • an antibody or an antigen-binding fragment thereof of group (i) may consist of an antibody or an antigen-binding fragment thereof directed against the CD3 antigen.
  • Antibodies or an antigen-binding fragments thereof from group (i), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions.
  • this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
  • an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1.5 pg, or about 2
  • an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 pg to about 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of about 0,05 pg, or about 0,1 pg, or about 0,19 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of 0,192 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (i) may consist of an anti-human CD3 Antibody purchased from BioLegend.
  • Antibodies or an antigen-binding fragments thereof of group (i) described herein may specifically bind to an antigen present at the surface of monocytes and basophils.
  • the use of an antibody specific to both the CD88 antigen and the CD89 antigen allows for targeting and binding monocytes and basophils without binding to, and thus depleting, the DC3 subset.
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen.
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and an antibody or an antigen-binding fragment thereof directed against the CD89 antigen.
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD89 antigen.
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen.
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and formulated in the antibody combination in a concentration ranging from about 0,001
  • an antibody or an antigen-binding fragment thereof of group (ii) may consist of an anti-human CD88 Antibody purchased from BioLegend.
  • Antibodies or an antigen-binding fragments thereof of group (iii) described herein may specifically bind to an antigen present at the surface of NK cells and NKT cells.
  • an antibody or an antigen-binding fragment thereof of group (iii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD56 antigen.
  • an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of about 0,001pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001
  • an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of about 0,05 pg, or about 0,1 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,33 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of 0,336 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iii) may consist of an anti-human CD56 Antibody purchased from BioLegend.
  • Antibodies or an antigen-binding fragments thereof of group (iv) described herein may specifically bind to an antigen present at the surface of NK cells and monocytes, such as intermediate monocytes and non-conventional monocytes.
  • an antibody or an antigen-binding fragment thereof of group (iv) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen.
  • Antibodies or an antigen-binding fragments thereof directed against the CD 16 antigen can be directed against both the CD 16a antigen and the CD 16b antigen.
  • an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen can be an antibody or an antigen-binding fragment thereof directed against the CD 16a antigen and/or the CD 16b antigen.
  • An antibody or an antigen-binding fragment thereof from group (iv), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions.
  • this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
  • an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl0 6 peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration of about 0,001 pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,01 pg to about 3 pg, or from about 0,02 pg to about 2 pg, or from about 0,02 pg to about 1 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration of 0,675 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (iv) may consist of an anti-human CD 16 Antibody purchased from BioLegend.
  • Antibodies or an antigen-binding fragments thereof of group (v) described herein may specifically bind to an antigen present at the surface of B cells.
  • an antibody or an antigen-binding fragment thereof of group (v) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen.
  • Antibodies or an antigen-binding fragments thereof from group (v), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
  • an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about
  • an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 pg to about 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group may be formulated in the antibody combination in a concentration of about 0,01 pg, or about 0,05 pg, or about 0,1 pg, or about 0,12 pg, or about 0,15 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration of 0,1 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • Antibodies or an antigen-binding fragments thereof of group (vii) described herein may specifically bind to an antigen present at the surface of platelets.
  • an antibody or an antigen-binding fragment thereof of group (vii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen.
  • Antibodies or an antigen-binding fragments thereof from group (vii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
  • an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of about 0,001pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about
  • an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of about 0,01 pg, or about 0,012 pg, or about 0,013 pg, or about 0,014 pg, or about 0,015 pg, or about 0,016 pg, or about 0,017 pg, or about 0,018 pg, or about 0,019 pg, or about 0,02 pg, or about 0,03 pg, or about 0,04 pg, or about 0,05 pg, or about 0,06 pg, or about 0,07 pg, or about 0,08 pg, or about 0,09 pg, or about 0,1 pg, or about 0,2 pg, or about 0,25 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of 0,0125pg per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (vii) may consist of an anti-human CD41 Antibody purchased from BioLegend.
  • Antibodies or an antigen-binding fragments thereof of group (viii) described herein may specifically bind to an antigen present at the surface of red blood cells.
  • an antibody or an antigen-binding fragment thereof of group (viii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
  • Antibodies or an antigen-binding fragments thereof from group (viii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions.
  • this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
  • an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50x10 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6
  • an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,1 pg to about 3 pg, or from about 0,2 pg to about 2 pg, or from about 0,2 pg to about 1 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, or about 0,55 pg, or about 0,6 pg, or about 0,65 pg, or about 0,67 pg, or about 0,7 pg, or about 0,75 pg, or about 0,8 pg, or about 0,85 pg, or about 0,9 pg, or about 0,95 pg, or about 1 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of 0,5 pg, per about 50xl0 6 cells, in particular peripheral blood mononuclear cells.
  • an antibody or an antigen-binding fragment thereof of group (viii) may consist of an anti-human CD235a Antibody purchased from BioLegend.
  • an antibody combination disclosed herein may comprise or consist of:
  • an antibody combination disclosed herein may comprise or consist of:
  • an antibody combination disclosed herein may comprise or consist of:
  • an antibody combination disclosed herein may comprise or consist of:
  • a detectable molecule may be, but is not limited to, biotin or a fluorochrome.
  • the human cell sample may be incubated in suitable conditions for the binding of each antibody or its fragment thereof to antigens at the surface of the cells present in the human cell sample.
  • the human cell sample may be incubated in the dark at about 4°C for about 10 minutes.
  • the antibody combination as described herein is reacted with a human cell sample as described herein in a single step. Specifically, the antibody combination is introduced at once in the human cell sample in order to react the cells with the antibodies that are comprised in the antibody combination.
  • DC3s expressing at least both the CD163 antigen and the CDllc antigen, and optionally at least one antigen among the CDlc antigen, the CD172a antigen, and the CD14 antigen.
  • the method of the present disclosure may lead to an enrichment of cDCls from about 0.4% to about 3% of the total cells within a given human cell sample.
  • the method of the present disclosure may lead to an enrichment of DC2s from about 2.5% to about 6%, or from about 2.5% to about 5.5% of the total cells within a given human cell sample.
  • the method of the present disclosure may lead to an enrichment of DC3s from about 14% to about 25% of the total cells within a given human cell sample.
  • the human cell sample enriched in dendritic cells may include at least one conventional dendritic cell expressing at least the antigens CDllc, CD 163 and CD 14.
  • the method of the present disclosure may lead to an enrichment of DC3s expressing at least the antigens CDllc, CD163 and CD14 from about 5% to about 15% of the total cells within a given human cell sample.
  • the human cell sample enriched in dendritic cells may include at least one conventional dendritic cell expressing the CDllc and CD163 antigens and lacking the expression of the CD 14 antigen.
  • the human cell sample enriched in dendritic cells may include the least one conventional dendritic cell secreting interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
  • IL- 10 interleukin- 1 beta
  • IP- 10 interferon gamma-induced protein 10
  • the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include non-dendritic cells.
  • the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include CD88-positive (CD88+) cells and/or CD89-positive (CD89+) cells.
  • the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include HLA-DR-positive (HLA-DR+) myeloid cells other than dendritic cells.
  • the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include basophils, neutrophils and monocytes.
  • the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include T cells, B cells, NK cells, platelet, red blood cells, basophils, neutrophils and/or monocytes.
  • the present disclosure also provides uses of an antibody combination as disclosed herein in a method for preparing a human cell sample enriched in dendritic cells as described herein.
  • the uses are performed in vitro.
  • the present disclosure also relates to the specific use of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, in a method for preparing a human cell sample enriched in dendritic cells.
  • the antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally the antibody or an antigenbinding fragment thereof directed against the CD89 antigen as described herein may be used in a method for preparing a human cell sample enriched in dendritic cells as described herein.
  • the human cell sample enriched in dendritic cells, using an antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, may include at least one conventional dendritic cell expressing at least the antigens CDllc, CD163 and CD14.
  • the human cell sample enriched in dendritic cells may include the least one conventional dendritic cell secretes interleukin-1 beta (IL-10) and/or interferon gamma-induced protein 10 (IP-10).
  • IL-10 interleukin-1 beta
  • IP-10 interferon gamma-induced protein 10
  • Embodiments of the methods for preparing a human cell sample enriched in dendritic cells as disclosed herein are applicable to the present uses.
  • the present disclosure also provides a kit-of-part useful for enriching a human cell sample in dendritic cells.
  • a kit-of-part useful for enriching a human cell sample in dendritic cells.
  • Such a kit may be suitable to implement the uses and methods disclosed herein.
  • a kit-of-part may include a plurality of antibodies of groups (i), (ii), (iii), (iv), (v) and (vi) as described herein.
  • kit-of-part may further include a plurality of antibodies of groups (vii) and (viii) as described herein.
  • a kit-of-part may include:
  • a kit-of-part may include:
  • the antibodies may be packaged individually or in combination.
  • PBMCs Human Peripheral blood mononuclear cells
  • PBMC sample for counting, it is added 500 mF of cold DPBS IX (GIBCO® ThermoFishcer Ref: #14190094), to top up the PBMC sample tube.
  • the obtained solution is mixed thoroughly and set aside an aliquot for counting.
  • the total PBMCs is counted using an optical microscope (Malassez chamber) during centrifugation of the PBMCs sample tube at 200 x g for 10 minutes at 4°C. After centrifugation, the supernatant is gently discarded and the PBMC pellet is resuspended at a concentration of 50xl0 6 cells/ml with a cold solution of PBS+.
  • the APC-conjugated antibody combination is prepared for a sample comprising 50xl0 6 PBMC by mixing, in a separate tube, the antibodies as described in Table 1.
  • both DC2 and DC3 subsets were capable of secreting IL- 10 (in response to R848) or IP-10 (in response to Poly IC) ( Figure 8A, 8B), although DC3s secreted higher levels of these two molecules than DC2s.
  • the DC2 subset differed from DC3 subset by their capability to secrete IL-10 in response to PolylC ( Figure 8A), suggesting functional differences between those DC2 and DC3 subsets depending on the type of stimulation.
  • the lab-made method of the disclosure allows enriching biological cell samples with all dendritic cell subsets without causing a significant loss in total cell numbers.
  • PBMC of healthy donors were either left untouched or Pan-DC enriched using the lab-made method with the antibody combinations comprising either an anti-CD88 antibody alone (20pl for 50.106 PBMC), anti-CD89 antibody alone (20pl for 50.10 6 PBMC), or a combination of both the two anti-CD89 and anti-CD88 antibodies (lOpl of each for 50.106 PBMC) to deplete monocytes.
  • dendritic cells are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti- CD88 and anti-CD89 antibodies.
  • the lab-made method is more efficient for enriching a human cell sample with all the dendritic cell subsets with the use of an antibody combination comprising anti-CD88 antibodies without anti-CD89 antibodies.

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Abstract

The present application relates to a novel antibody combination, for enriching a human cell sample with all the five dendritic cell subsets including AS-DC, pDC, cDC1, DC2 and DC3, comprising antibodies or antigen-binding fragments thereof directed against the CD3 antigen, and/or the TCRα/β antigen, and/or the TCRγ/δ antigen; (ii) antibodies or antigen-binding fragments thereof directed against the CD88 antigen and, optionally the CD89 antigen; (iii) antibodies or antigen-binding fragments thereof directed against the CD56 antigen; (iv) antibodies or antigen- binding fragments thereof directed against the CD16 antigen; (v) antibodies or antigen-binding fragments thereof directed against the CD19 antigen, and/or the CD20 antigen; and (vi) antibodies or antigen-binding fragments thereof directed against the CD15 antigen, and/or the CD66b antigen.

Description

[TITLE]
ANTIBODY COMBINATION AND METHODS FOR DENDRITIC CELLS ENRICHMENT
[TECHNICAL FIELD]
[0001] The present disclosure relates to antibody combinations and methods for enriching a human cell sample with dendritic cells. In particular, the present disclosure relates to antibody combinations and methods for enriching a human cell sample with all the five dendritic cell subsets including AS-DC, pDC, cDCl, DC2 and DC3.
[BACKGROUND]
[0002] Dendritic cells (DCs) play critical roles as innate immune sentinels and expert antigen-presenting cells. Recent advancements in omics technologies have revealed a diverse array of distinct DC subsets characterized by particular phenotypes and functional capabilities.
[0003] The utilization of advanced techniques enabling analyses at the single -cell level has provided invaluable insights into the diversity of dendritic cell subpopulations across various anatomical compartments under both healthy and pathological conditions. The classification of DC subsets, based on ontogeny, anatomical localization, phenotypic markers, and functional specialization, has significantly contributed to the understanding at a steady state.
[0004] Given their frontline position in the immune defense, DCs exhibit a remarkable capacity to adapt to a spectrum of microbial and host-derived stimuli, resulting in functional plasticity and the acquisition of distinct phenotypical and functional properties, including the ability to induce specific T cell polarization programs.
[0005] In human blood, the initial categorization of DCs includes three subsets namely, conventional dendritic cells (eDCs), including cDCl and cDC2, and plasmacytoid dendritic cells (pDCs). These three subsets share a common DC progenitor (Guilliams et al., Nat Rev Immunol. 2014).
[0006] Further investigations through single-cell RNA-sequencing have revealed a rare circulating DC subset termed AS-DC (AXL+ Siglec6+ dendritic cells), exhibiting markers similar to both pDCs and cDC2s, notably the CD123 antigen and the CD11c antigen, while being specifically characterized by the expression of the AXL antigen, the Siglec6 antigen, the CD2 antigen, and the CD5 antigen markers (Villani AC et al. Science, 2017).
[0007] Furthermore, recent studies have elucidated heterogeneity within the cDC2 population, distinguishing two sub-populations which are CD11C+ CD 163- cells (DC2s) and CDllc+ CD163+ CD14+/-, leading to a new subset named DC3. DC3 originates from a monocyte cell precursor (Liu et al., Immunity. 2023 Aug 8;56(8): 1761-1777.e6.).
[0008] Examining the shared and distinct functionalities of these diverse DC subsets has driven the need for efficient methods to enrich and isolate these rare DC fractions from biological cell samples, becoming pivotal tools for immunologists. Several companies have introduced commercial kits, like Miltenyi and Stemcell, for the enrichment of pan-DC subsets from mouse or human samples.
[0009] While the Miltenyi pan-DC enrichment kit effectively retrieves various dendritic cell populations, it fails in capturing all DC subsets, specifically missing the newly identified DC3 subset (CDllc+ CD163+ CD14+). Similarly, the Stemcell pan-DC enrichment kit isolates different DC subsets but leads to a reduction in total dendritic cell numbers without specifically capturing the newly identified DC3 subset.
[0010] Consequently, it remains a need to provide solutions to enrich and recover all the different dendritic cell subsets from a biological cell sample.
[0011] There is a need for solutions that efficiently enrich a biological cell sample with all dendritic cell subsets without causing a significant loss in total cell numbers.
[0012] There is also a need for antibody combinations that can selectively isolate non- dendritic cells from a biological cell sample without targeting dendritic cell subsets.
[0013] There is notably a need for solutions to enrich a biological cell sample with the newly identify DC3 subset.
[0014] There is also a need for antibodies that can selectively isolate non-dendritic cells from a biological cell sample without targeting the newly identify DC3 subset.
[0015] The present disclosure has for goal to satisfy all or part of those needs.
[SUMMARY]
[0016] According to one of its objects, the present disclosure relates to an antibody combination, for enriching a human cell sample with dendritic cells, comprising :
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD 88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
[0017] According to another of its objects, the present disclosure relates to an antibody combination comprised in a composition, for enriching a human cell sample with dendritic cells, the antibody combination comprising:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD 88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
[0018] In some embodiments, the antibody combination as disclosed herein may further comprise (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen; and optionally (viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
[0019] As shown in the example section, the use of an antibody combination as described herein in a negative selection technique allow to prepare a human cell sample which is enriched in all the five dendritic cell subsets, specifically the Axl-i- Siglec6+ dendritic cell subset, the plasmacytoid dendritic cell subset, the conventional dendritic cell 1 subset, the dendritic cell 2 subset and the newly identified dendritic cell 3 subset as defined herein.
[0020] In some particular embodiments, the antibody combination as disclosed herein may comprise (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen; (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen; (iv) an antibody or an antigen-binding fragment thereof directed against the CD16 antigen; (v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen; and (vi) an antibody or an antigen-binding fragment thereof directed against the CD15 antigen.
[0021] In some particular embodiments, the antibody combination as disclosed herein may comprise (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen; (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen; (iv) an antibody or an antigen-binding fragment thereof directed against the CD16 antigen; (v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen; (vi) an antibody or an antigen-binding fragment thereof directed against the CD15 antigen; (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen; and (viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
[0022] In some embodiments, each antibody or its antigen-binding fragment thereof may be independently formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells.
[0023] In some embodiments, the human cell sample may consist of a whole blood sample or a peripheral blood mononuclear cell (PBMC) fraction thereof. [0024] According to another of its objects, the present disclosure relates to an in vitro use of an antibody combination as disclosed herein in a method for preparing a human cell sample enriched in dendritic cells.
[0025] According to another of its objects, the present disclosure relates to an in vitro method for preparing a human cell sample enriched in dendritic cells comprising at least the steps of:
(a) reacting a human cell sample with the antibody combination according to any one of claims 1 to 5, under conditions suitable for the binding of each antibody or its antigenbinding fragment thereof to the cells expressing at their surface one or more antigens against which the respective antibodies are directed;
(b) removing the cells bound by the antibodies from the human cell sample; and
(c) recovering a human cell sample which is enriched in dendritic cells.
[0026] In some embodiments, the human cell sample may be in a cell-culture medium for human cells suitable for isolating the dendritic cells in the human cell sample.
[0027] In some embodiments, the cell-culture medium may comprise dulbecco's phosphate-buffered saline (DPBS) and ethylenediaminetetraacetic acid (EDTA).
[0028] In some embodiments, the cell-culture medium may comprise dulbecco's phosphate-buffered saline (DPBS); ethylenediamine tetraacetic acid (EDTA); and bovine serum albumin (BAS) and/or heat inactivated human serum.
[0029] In some embodiments, each antibody or its antigen-binding fragment thereof comprised in the combination may be labelled with a detectable molecule and/or may be conjugated to magnetic beads.
[0030] In some embodiments, the human cell sample enriched in dendritic cells may include at least Axl-i- Siglec6+ dendritic cells (AS-DCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells 1 (cDCls), dendritic cells 2 (DC2s) and/or dendritic cells 3 (DC3s).
[0031] In some embodiments, AS-DCs express at least the Axl antigen; pDCs express at least one antigen among the CD 123 antigen and the CD303 antigen, and optionally the CD304 antigen; cDCls express at least one antigen among the CD141 and CLEC9A antigens, and optionally at least the XCR1 antigen; DC2s express at least one antigen among the CDlc and CDllc antigens, and optionally at least one antigen among the CD172a and CDllb antigens, and lack the expression of the CD163 antigen; and DC3s express at least both the CD163 and CDllc antigens, and optionally at least one antigen among the CDlc antigen, the CD172a antigen, and the CD 14 antigen. [0032] In some embodiments, the human cell sample enriched in dendritic cells may include at least dendritic cells, in particular DC3s, expressing the CDllc antigen, the CD163 antigen and the CD 14 antigen; and optionally the said dendritic cells further may secrete interleukin-1 beta (IL-10) and/or interferon gamma-induced protein 10 (IP-10).
[0033] In some embodiments, the human cell sample consists of a whole blood sample or a peripheral blood mononuclear cell (PBMC) fraction thereof.
[0034] According to another of its objects, the present disclosure relates to a kit-of- part useful for enriching a human cell sample in dendritic cells, including:
(A) (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/0 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD 88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen,
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen,
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen,
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen, and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen; and
(B) instructions for preparing a human cell sample enriched in dendritic cells.
[0035] According to another of its objects, the present disclosure relates to the use of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen, in a method for preparing a human cell sample enriched in dendritic cells.
[0036] In some embodiments, it may be further used an antibody or an antigen-binding fragment thereof directed against the CD89 antigen with an antibody or an antigen-binding fragment thereof directed against the CD88 antigen, in a method for preparing a human cell sample enriched in dendritic cells. [0037] In some embodiments, the human cell sample enriched in dendritic cells may include at least dendritic cells, in particular DC3s, expressing the CDllc antigen, the CD163 antigen and the CD 14 antigen, and optionally the said dendritic cells may secrete interleukin- 1 beta (IL-1P) and/or interferon gamma-induced protein 10 (IP-10).
[DESCRIPTION OF THE FIGURES]
[0038] FIGURE 1 patterns to the number of dendritic cells within untouched Peripheral blood mononuclear cells (PBMCs) samples or after dendritic cell enrichment. FIGURE 1A depicts flow-cytometry dot plots illustrating the percentages of dendritic cells in PBMCs samples based on the expression of class II major histocompatibility complex (HLA-DR) and the expression of Lineage (Lin) markers, including CD3 (specific for T cell), CD19 (specific for B cells), CD20 (specific for B cells), CD56 (specific for NK cells) and CD88 (specific for monocytes and basophils). From left to right box: (a) Dendritic cell percentage within an untouched PBMCs sample, (b) Dendritic cell percentage within a PBMCs sample after enrichment with the lab-made method, (c) Dendritic cell percentage within a PBMCs sample after enrichment with the Miltenyi kit (Ref: 130-100-777), and (d) Dendritic cell percentage within a PBMCs sample after enrichment with the StemCell kit (Ref: #19251). FIGURE IB depicts a scatter plot illustrating the percentage of dendritic cells among live cells within each PBMCs samples. Ordinate: Percentage of dendritic cells for 50.106PBMC processed. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method, (c) PBMCs sample after enrichment with the Miltenyi kit, and (d) PBMCs sample after enrichment with the StemCell kit. FIGURE 1C depicts a scatter plot illustrating the absolute dendritic cell numbers of all the dendritic cells in each PBMCs samples. Ordinate: Absolute number of dendritic cells for 50.106PBMC processed. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method, (c) PBMCs sample after enrichment with the Miltenyi kit, and (d) PBMCs sample after enrichment with the StemCell kit. Data are representative of n=5 experiments. **, P < 0.01; ***, P < 0.005; Repeated measures one-way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate the Standard Error of Mean (SEM).
[0039] FIGURE 2 patterns to the percentage number of AS-DCs within each PBMC samples. FIGURE 2A depicts flow-cytometry dot plots illustrating the AS-DC subset in each PBMCs samples based on the expression of CDllc and the expression of AXL. FIGURE 2B depicts a scatter plot illustrating the percentage of cells of the AS-DC subset in each PBMC samples. Ordinate: Percentage of AS-DCs among live PBMCs. Abscissa 2A and 2B : From left to right: (a) untouched PBMC sample, (b) PBMC sample after enrichment with the lab-made method, (c) PBMC sample after enrichment with the Miltenyi kit, and (d) PBMC sample after enrichment with the StemCell kit. ***, P < 0.005. Repeated measures one-way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate the SEM.
[0040] FIGURE 3 patterns to the percentage number of pDCs within each PBMC samples. FIGURE 3A depicts flow-cytometry dot plots illustrating the pDC subset in each PBMCs samples based on the expression of CD11c and the expression of CD123. FIGURE 3B depicts a scatter plot illustrating the percentage of cells of the pDC subset in each PBMC samples. Ordinate: Percentage of pDCs among live PBMCs. Abscissa 3 A and 3B: From left to right: (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method, (c) PBMCs sample after enrichment with the Miltenyi kit, and (d) PBMCs sample after enrichment with the StemCell kit. ***, P < 0.005. Repeated measures one-way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate the SEM.
[0041] FIGURE 4 patterns to the percentage number of cDCls within each PBMC samples. FIGURE 4A depicts a flow-cytometry dot plot illustrating the cDCl subset in each PBMCs samples based on the expression of CDlc and the expression of CD141. FIGURE 4B depicts a scatter plot illustrating the percentage of cells of the cDCl subset in each PBMCs samples. Ordinate: Percentage of cDCls among live PBMCs. Abscissa 4A and 4B : From left to right: (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method, (c) PBMCs sample after enrichment with the Miltenyi kit, and (d) PBMCs sample after enrichment with the StemCell kit. ***, P < 0.005. Repeated measures one-way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate.
[0042] FIGURE 5 patterns to the percentage number of DC2s and DC3s within each PBMC samples. FIGURE 5A depicts flow-cytometry dot plots illustrating the DC2 and DC3 subsets in each PBMCs samples based on the expression of CD163 and the expression of CD14. FIGURE 5B depicts a scatter plot illustrating the percentage of cells of the DC2 subset in each PBMCs samples. Ordinate: Percentage of DC2s among live PBMCs. FIGURE 5C depicts a scatter plot illustrating the percentage of cells of the DC3 subset in each PBMCs samples. Ordinate: Percentage of DC3s among live PBMCs. FIGURE 5D depicts a scatter plot illustrating the percentage of cells of the CD14+ DC3 subset in each PBMCs samples. Ordinate: Percentage of CD14+ DC3s among live PBMCs. Abscissa 5B to 5D: From left to right: (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method, (c) PBMCs sample after enrichment with the Miltenyi kit, and (d) PBMCs sample after enrichment with the StemCell kit. ns, not significant; **, P < 0.01; ***, P < 0.005. Repeated measures one- way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate the SEM.
[0043] FIGURE 6 depicts flow-cytometry dot plots illustrating the dendritic cell expression of CD86 and CD80 after 24h of culture with (from left to right plot) (a) culture medium without stimulation, (b) medium supplemented with Poly IC (at 25pg/ml, Invivogen, ref: tlrl-pic), (c) medium supplemented with R848 (Ipg/ml, Invivogen, ref: tlrl-r848), or (d) medium supplemented with CpG-C (Ipg/ml, Invivogen, ref: tlrl-2395) in PBMC samples after dendritic cell enrichment with the lab-made method. Data representative of n>5 experiments.
[0044] FIGURE 7 depicts to scatter pots illustrating the percentages of pDCs, DC2s or DC3s expressing CD86 (7 A) and CD80 (7B) in the PBMC samples after enrichment with the lab-made method. Data representative of n>5 experiments. FIGURE 7A Ordinate: Percentage of pDCs, DC2s or DC3s expressing CD86 after 24 h of culture. Abscissa (from left to right): (a) Culture medium (control), (b) culture medium supplemented with Poly IC, (c) Culture medium supplemented with R848, (d) culture medium supplemented with CpG-C. FIGURE 7B Ordinate: Percentage of pDCs, DC2s or DC3s expressing CD80 after 24 h of culture. Abscissa (from left to right): (a) culture medium (control), (b) culture medium supplemented with Poly IC, (c) culture medium supplemented with R848, (d) culture medium supplemented with CpG- C. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Kruskal-Wallis test with Dunn’s post-test. Central horizontal lines represent means and error bars indicate SEM.
[0045] FIGURE 8 depicts to scatter plots illustrating the secretion of IL- 10 (8A) and IP-10 (8B) by the pDCs, DC2s or DC3s within the PBMC samples after enrichment with the lab-made method. Secretion measured by Cytometric Bead Array. Data representative of n>5 experiments. FIGURE 8A Ordinate: Quantity of IL- 10 secreted by pDCs, DC2s or DC3s after 24 h of culture expressed in pg/ml. Abscissa (from left to right): (a) culture medium (control), (b) culture medium supplemented with Poly IC, (c) culture medium supplemented with R848, (d) culture medium supplemented with CpG-C. FIGURE 8B Ordinate: Quantity of IP-10 secreted by pDCs, DC2s or DC3s after 24 h of culture expressed in pg/ml. Abscissa (from left to right): (a) Culture medium (control), (b) culture medium supplemented with Poly IC, (c) Culture medium supplemented with R848, (d) culture medium supplemented with CpG-C. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Kruskal-Wallis test with Dunn’s post-test. Central horizontal lines represent means and error bars indicate SEM.
[0046] FIGURE 9 depicts to scatter plots illustrating the percentages of DCs among live PBMC. Results representative of n=4. ***, P < 0.005; Repeated measures one-way ANOVA with Dunn’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of dendritic cells among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) 50.106 PBMC enriched with the lab-made method using Miltenyi LS Columns (130-042-401, 1 passing) (c) l,000.106 PBMCs enriched with the lab-made method using Miltenyi Automacs Pro (130-092-545, 2 passings).
[0047] FIGURE 10 depicts to scatter plots illustrating the percentages of DCs among live PBMC. Results representative of n=5. ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of dendritic cells among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0048] FIGURE 11 depicts to scatter plots illustrating the percentages of AS-DCs among live PBMC. Results representative of n=5. ns, not significant. Repeated measures oneway ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of AS-DCs among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0049] FIGURE 12 depicts to scatter plots illustrating the percentages of pDCs among live PBMC. Results representative of n=5. ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of pDCs among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0050] FIGURE 13 depicts to scatter plots illustrating the percentages of cDCls among live PBMC. Results representative of n=5. ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of cDCls among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0051] FIGURE 14 depicts to scatter plots illustrating the percentages of DC2s among live PBMC. Results representative of n=5. ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of DC2s among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0052] FIGURE 15 depicts to scatter plots illustrating the percentages of DC3s among live PBMC. Results representative of n=5. ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of DC3s among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies.
[0053] FIGURE 16 depicts to scatter plots illustrating the percentages of CD14+ DC3s among live PBMC. Results representative of n=5. *, P < 0.05; ns, not significant. Repeated measures one-way ANOVA with Bonferroni’s post-test. Central horizontal lines represent means and error bars indicate SEM. Ordinate: Percentage of CD14+ DC3s among live PBMCs. Abscissa: from left to right, (a) untouched PBMCs sample, (b) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD88 antibody alone, (c) PBMCs sample after enrichment with the lab-made method using the antibody combination with CD89 antibody alone, (d) PBMCs sample after enrichment with the lab-made method using the antibody combination with both CD88 and CD89 antibodies. [DETAILED DESCRIPTION]
Definitions
[0054] The terms used in this specification generally have their ordinary meanings in the art. Certain terms are discussed below, or elsewhere in the present disclosure, to provide additional guidance in describing the products and methods of the presently disclosed subject matter.
[0055] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form.
[0056] The following definitions apply in the context of the present disclosure.
[0057] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, “an antibody” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
[0058] Furthermore, the term “and/or”, where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a sentence such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone).
[0059] Furthermore, the term "optionally”, where used herein, is used to denote that an element or component place after the term optionally in the sentence is discretionary and may or may not be present within the described object or composition or combination. Thus, for instance, the term “optionally” as used in a sentence such as “A and, optionally B” herein is intended to denote that A is mandatory and B is discretionary and may or may not be present.
[0060] The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. [0061] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “including,” “consisting of,” and “consisting essentially of’ aspects and embodiments. The words “include” and “comprise,” or variations such as “includes,” “including,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of’ implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of’ implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the characteristic(s) of the stated elements. It is understood that the different embodiments of the disclosure using the term “comprising”, “including” or equivalents cover the embodiments where this term is replaced with “consisting of’ or “consisting essentially of’.
[0062] As used herein, the term “independently” refers to the capability or feature of an element or process to function, operate, or perform without being influenced, controlled, or reliant upon other interconnected elements or processes. For instance, in a given formulation, different antibodies may exist at diverse concentration levels, each concentration being unaffected by or not reliant upon the concentration of the others within the same formulation.
[0063] Within the disclosure, the term “significantly” used with respect to a change intends to mean that the observed change is noticeable and/or it has a statistic meaning.
[0064] As used herein the term "antibody" has the same meaning and will be used equally in the present description. The term "antibody" as used herein refers to isolated immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, such as VP1 capsid protein. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, L- CDR3-L and CDR1-H CDR2-H, CDR3-H, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
[0065] The term “antibody” includes, but is not limited to, monoclonal antibodies and human antibodies. The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). In some embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof is included in an antibody combination. In some embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof is directed against non-dendritic cells. In some embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof may be directed against T cells, B cells, Natural Killer (NK) cells, monocytes, granulocytes, red blood cells and/or platelets.
[0066] It is intended that all references to the term “antibody” or “antigen-binding fragment thereof’ as used herein, referred to the antibodies or fragments thereof as described in the present disclosure.
[0067] An "isolated" compound or entity, such as an antibody or antigen-binding fragment thereof, a vector or a cell, is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the use of the compound or entity, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the compound or entity is partially or substantially purified. In some embodiments, the compound or entity may be purified to greater than about 95% by weight of antibody as determined by the Lowry method, for example, more than 95%, 96%, 97%, 98% or 99% by weight of compound or entity.
[0068] The term “monoclonal antibody”, as used herein 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 on an antigen expressed by a cell.
[0069] As used herein, the term “antigen” refers to a molecule or a portion of a molecule capable of being bound by one or more antibodies. An antigen can have one or more than one epitope. For instance, in the context of the disclosure, an antigen may be an antigen such as a Complement receptor (CD) or a Toll-Like Receptor (TLR) expressed on the surface of a cell, specifically on the surface of a peripheral blood mononuclear cell. The AXL antigen, also known as the Tyrosine -protein kinase receptor UFO, can be identified by the UniProt number P30530. The Siglec6 antigen, also known as the Sialic acid-binding Ig-like lectin 6, can be identified by the UniProt number 043699. The XCR1 antigen, also known as the Chemokine XC receptor 1, can be identified by the UniProt number P46094.
[0070] As used herein, the term “complement receptor” or “CD” refers to a specific type of cell surface receptor protein that plays a key role in the regulation and activation of the complement system; a part of the immune system responsible for recognizing and eliminating pathogens and damaged cells. Complement receptors are proteins expressed on various cells, such as lymphocytes, neutrophils, and dendritic cells, facilitating the characterization of the cells in a context of in vitro studies. When the symbol “+” is used within a CD, for instance CD14+, it means that the antigen is expressed by the cell. When the symbol is used within a CD, for instance CD14-, it means that the antigen is not expressed by the cell. When the symbol “+/-” is used within a CD, for instance CD14+/-, it means that the antigen may (+) or may not (-) be expressed by the cell. For instance, a complement receptor (CD) can be CD3, CD88, CD89, CD56, CD16, CD19, CD20, CD15, CD66b, CD41, CD61 or CD235a.
[0071] As used herein, the symbol “+” means a positive expression. Thus, when the symbol “+” is applied to a complement receptor in the characterization of a cell, for instance CD14+ DC3 cell, it means that the antigen is expressed by the cell.
[0072] As used herein, the symbol means a negative expression. Thus, when the symbol is applied to a complement receptor in the characterization of a cell, for instance CD14- DC3 cell, it means that the antigen is not expressed by the cell.
[0073] The CDlc antigen, also known as the T-cell surface glycoprotein CDlc, can be identified by the UniProt number P29017. [0074] The CD2 antigen, also known as the T-cell surface antigen CD2, can be identified by the UniProt number P06729.
[0075] The CD3 antigen, also known as the T-cell surface glycoprotein CD3 delta chain, is a protein complex composed of two CD3s chains, one CD3y chain, one CD35 chain, one CD3^ chain, and a T-cell receptor (a/p or y/5) heterodimer. CD3s chain can be identified by the UniProt number P07766. CD3y chain can be identified by the UniProt number P09693. CD35 chain can be identified by the UniProt number P04234.
[0076] The CD5 antigen, also known as the T-cell surface glycoprotein CD5, can be identified by the UniProt number P06127.
[0077] The CD1 lb antigen, also known as the Integrin alpha-M, can be identified by the UniProt number Pl 1215.
[0078] The CD11c antigen, also known as the Integrin alpha-X, can be identified by the UniProt number P20702.
[0079] The CD 14 antigen, also known as the Monocyte differentiation antigen CD 14, can be identified by the UniProt number P08571.
[0080] The CD 15 antigen, also known as Sialyl Lewisx, can be identified by the CAS number 98603-84-0.
[0081] The CD 16 antigen encompasses the Fc receptors FcyRIIIa (CD 16a) and the FcyRIIIb (CD16b), identified by the UniProt numbers P08637 and the UniProt numbers 075015, respectively.
[0082] The CD19 antigen, also known as the B-lymphocyte antigen CD19, is a protein that can be identified by the UniProt number P15391.
[0083] The CD20 antigen, also known as the B-lymphocyte antigen CD20, is a protein that can be identified by the UniProt number Pl 1836.
[0084] The CD41 antigen, also known as the Integrin alpha- lib, can be identified by the UniProt number P08514.
[0085] The CD56 antigen, also known as the Neural cell adhesion molecule 1, is a protein that can be identified by the UniProt number P13591.
[0086] The CD61 antigen, also known as the Integrin beta-3, can be identified by the UniProt number P05106.
[0087] The CD66b antigen, also known as the Carcinoembryonic antigen-related cell adhesion molecule 8, can be identified by the UniProt number P31997.
[0088] The CD88 antigen, also known as the C5a anaphylatoxin chemotactic receptor 1, is a protein that can be identified by the UniProt number P21730. [0089] The CD89 antigen, also known as the Immunoglobulin alpha Fc receptor, is a protein that can be identified by the UniProt number P24071.
[0090] The CD 123 antigen, also known as the interleukin-3 receptor, can be identified by the UniProt number P26951.
[0091] The CD141 antigen, also known as the Thrombomodulin, can be identified by the UniProt number P07204.
[0092] The CD 163 antigen, also known as the Scavenger receptor cysteine -rich type 1 protein Ml 30, can be identified by the UniProt number Q86VB7.
[0093] The CD172a antigen, also known as the Tyrosine-protein phosphatase nonreceptor type substrate 1, can be identified by the UniProt number P78324.
[0094] The CD235a antigen, also known as the Glycophorin-A, can be identified by the UniProt number P02724.
[0095] The CD303 antigen, also known as the C-type lectin domain family 4 member C, can be identified by the UniProt number Q8WTT0.
[0096] The CD304 antigen, also known as the Neuropilin- 1, can be identified by the UniProt number 014786.
[0097] The CLEC9A antigen or CD370 antigen, also known as the C-type lectin domain family 9 member A, can be identified by the UniProt number Q6UXN8.
[0098] As used herein, the term “Toll-Like Receptor” or “TLR” refers to a class of proteins primarily expressed by dendritic cells. Upon binding to these pathogen-associated molecular patterns (PAMPs), TLRs initiate signaling cascades that trigger immune responses from dendritic cells, including the production of cytokines, such as Interleukin-1 beta (IL-10) or Interferon gamma-induced protein 10 (IP-10). Each dendritic cell subset express particular class of TLR; dendritic cells of the pDC subset express TLR7 (UniProt number Q9NYK1) and TLR9; dendritic cells of the DC2 and DC3 subsets express, but are not limited to, TLR3 and TLR8.
[0099] The term “target cell” herein means any cell that may be targeted by an antibody combination of the present disclosure. A target cell also includes a cell expressing a target antigen that can be recognized by an antibody combination of the present disclosure. For instance, a target cell may be a T cell, a B cell, a NK cell, a monocyte, a red blood cell and/or a platelet.
[00100] As used herein, the term “enrichment” or “enriching” or equivalents, in the context of enriching a human cell sample with dendritic cells, refers to the isolation of dendritic cells from the cells presents in the human cell sample. It thus means that the percentage of the dendritic cells relative to the total number of cells in a human cell sample has increased in said sample. At the same time this means that the number of non-dendritic cells present in the human cell sample has decreased in said human cell sample. These non-dendritic cells which are depleted from the human cell sample may for example include T cells, B cells, NK cells, platelet, red blood cells, basophils, neutrophils and/or monocytes. The dendritic cell enrichment method of the present disclosure may lead to an enrichment of all the five dendritic cell subsets, specifically the AS-DC subset, the pDC subset, the cDCl subset, the DC2 subset and the DC3 subset. In some embodiments, the dendritic cell enrichment method of the present disclosure may lead to an enrichment of dendritic cells from 30% to 90% of the total cells within a given human cell sample. In some embodiments, the dendritic cell enrichment method of the present disclosure may lead to an enrichment of the DC3 subset, expressing at least the CD 14 antigen, from 5% to 15% of the total cells within a given human cell sample. In some embodiments, the dendritic cell enrichment method of the present disclosure may lead to a human cell sample depleted in CD88-positive (CD88+) cells and CD89-positive (CD89+) cells. In some embodiments, the dendritic cell enrichment method of the present disclosure may lead to a human cell sample depleted in HLA-DR-positive (HLA-DR+) myeloid cells other than dendritic cells.
[00101] As used herein, the term “human” refers to the species Homo sapiens, encompassing individuals belonging to the biological classification of humans. In the context of the present disclosure, “human” specifically denotes characteristics, biological components, or attributes unique to, or derived from, the human species. This may include but is not limited to, cells, tissues, proteins, or any other biological materials originating from or characteristic of humans.
[00102] As used herein, the term “magnetic beads” refers to solid nanoparticles typically composed of a magnetic core, often made from materials like iron oxide or magnetite, and coated with a surface layer that enables the binding or immobilization of specific target molecules such as proteins other biomolecules. Magnetic beads, in combination with antibodies, can be used for separation, purification, and isolation of cells from biological samples using magnetic forces. For instance, a magnetic bead may consist of MojoSort™ Human anti-APC Nanobeads purchased from BioLegend. In some embodiments, a magnetic bead may have a diameter from about 20 nanometer (nm) to about 800 nm. In some embodiments, a magnetic bead may have a diameter of about 20 nm, about 50 nm, about 75 nm, about 100 nm, about 115 nm, about 130 nm, about 145 nm, about 160 nm or about 800 nm. For instance, a magnetic bead that may be used in the present disclosure may have a diameter of about 130 nm. Human cell sample
[00103] The dendritic cell enrichment method according to the present disclosure is carried out on a cell sample from an individual, including dendritic cells and non-dendritic cells.
[00104] In some embodiments, an individual is a human.
[00105] In some embodiments, a cell sample may be a human cell sample.
[00106] In some embodiments, a human cell sample may be a whole blood sample. A whole blood sample may be obtained from an individual who may be healthy or sick. A whole blood sample may comprise a plurality of blood cells comprising the major types of blood cells including platelets, red blood cells and white blood cells.
[00107] As used herein, "red blood cells", also known as erythrocyte, refers to a type of non-nucleated cells found in the bloodstream and characterized by its biconcave disc shape. Red blood cell’s primary function is to deliver dioxygen (O2) to the body tissues and to carry carbon dioxide (CO2) to the lungs for exhalation/elimination. Red blood cells express the surface membrane antigen CD235a, also called Glycophorins A protein.
[00108] As used herein “blood platelets” or “platelets”, also known as thrombocytes, are small, disc-shaped non-nucleated cell fragments derived from megakaryocytes in the bone marrow. These cell fragments play a critical role in hemostasis and blood clotting mechanisms by adhering to damaged blood vessel walls, aggregating to form a plug at the site of injury, and releasing various factors essential for coagulation cascade activation, ultimately aiding in the cessation of bleeding. Blood platelets express the CD41, also known as Integrin allb (GPIIb), and/or the CD61 surface membrane antigen, also known as integrin 03 (GPIIIa). The CD41/CD61 complex is required for platelet aggregation and clotting.
[00109] As used herein “white blood cells”, also known as leukocytes, are a group of nucleated cells found in the bloodstream and lymphatic system of an individual. These white blood cells form a critical component of the human’s immune defense mechanism, functioning primarily to combat infections, diseases, and foreign substances by various means such as phagocytosis, antibody production, and cellular immune responses. White blood cells encompass several subtypes, including, but not limited to, neutrophils, lymphocytes (T cells, B cells, and NK cells), monocytes, dendritic cells, eosinophils, and basophils, each playing distinct roles in immune surveillance, response, and regulation within the human body. All white blood cells are produced and derived from hematopoietic stem cells in the bone marrow. All white blood cells express on their surface CD45 protein markers. [00110] In some embodiments, a human cell sample may be obtained from peripheral circulation, buffy coat, bone marrow and/or cord blood from an individual.
[00111] In some particular embodiments, a human cell sample may be obtained from peripheral circulation from an individual.
[00112] In some embodiments, a human cell sample may be an isolated fraction of a whole blood sample comprising or consisting essentially of a plurality of peripheral blood mononuclear cells (PBMCs).
[00113] Isolated PBMC sample may be derived from a whole blood sample by separated peripheral blood mononuclear cells from the other blood cells. Accordingly, isolated PBMCs comprise or consist essentially of peripheral blood cells having one round nucleus such as T cells, B cells, monocytes, Natural Killer (NK) cells, and dendritic cells. Non-nucleated blood cells may include red blood cells and platelets whereas multi-lobed nuclei blood cells may include neutrophils, basophils, and eosinophils. For example, an isolated PBMC sample may be obtained by density gradient centrifugation, in particular with the Ficoll-Paque method. Methods for obtaining isolated PBMCs from whole blood are well known in the art and taught, for instance, in Puleo et al. Bio Protoc. 2017 Jan 20;7(2):e2103; Lan et al. Curr Protoc Microbiol. 2007 Aug; or Fuss et al. Curr Protoc Immunol. 2009 Apr;Chapter 7:7.1.1-7.1.8. In some embodiments, an isolated PBMC sample may contain residual non-nucleated blood cells or multi-lobed nuclei blood cells that have not been depleted after the initial cell separation process.
[00114] In some embodiments, a human cell sample to be used in methods as disclosed herein may be an isolated PBMCs sample.
[00115] In some embodiments, a human cell sample to be used in methods as disclosed herein may be an isolated PBMCs sample derived from a whole blood sample.
[00116] An isolated PBMC sample may comprises T cells, B cells, NK cells, monocytes, and/or dendritic cells. In some embodiments, an isolated PBMC sample may contain a low concentration of residual granulocytes (basophils, neutrophils, eosinophils), red blood cells and/or platelets that have not been depleted after the initial cell separation process from whole blood cell.
Granulocytes
[00117] Granulocytes refer to a cell subset of white blood cells characterized by the presence of cytoplasmic granules. This category includes neutrophils, eosinophils, and basophils, each playing distinct roles in the immune response. [00118] Generally, during the isolation of PBMC through separation techniques such as ficoll gradient, the majority of granulocytes, including neutrophils, basophils, and eosinophils, are eliminated. However, a minor fraction of neutrophils and basophils from the granulocyte family may persist despite these methods.
[00119] Neutrophils can be characterized by the expression of at least the surface membrane CD15 antigen and/or the CD66b antigen.
[00120] Eosinophils can be characterized by the expression of at least the surface membrane antigen CCR3 (also known as CD193) and the absence of the CD16 antigen.
[00121] Basophils can be characterized by the expression of at least the CD88 surface membrane antigen, also named C5AR1.
T cells and NKT cells
[00122] T cells or blood T cells refer to a subset of lymphocytes derived from the thymus gland and circulating in the bloodstream, playing a pivotal role in adaptive immunity. These cells express T cell receptors (TCRs) on their surface and are involved in recognizing specific antigens presented by major histocompatibility complex (MHC) molecules. T cells encompass various subsets, including cytotoxic T cells, helper T cells, and regulatory T cells. Furthermore, T cells also encompass a heterogeneous group of cells called Natural killer T (NKT) cells, that share properties of both T cells and natural killer cells. These different subsets can be discriminated based on selective phenotypic extracellular markers, in combination with intracellular transcription factor and/or cytokine staining. The identification of these various phenotypic markers is well known in the art and highlight, for instance, by Donne Estipona. Biocompare. Life Science Article. 2020 or Mousset et al. Cytometry A. 2019 Jun;95(6):647- 654.
[00123] All the various T cell subsets, including NKT cells, can be characterized by commonly expressing at least the CD3 surface membrane antigen and/or the TCRa/p surface membrane antigen and the TCRy/6 surface membrane antigen.
[00124] NKT cells can also be characterized by the expression of at least the CD56 surface membrane antigen, also named NCAM, shared with NK cells.
B cells
[00125] B cells or blood B cells refer to a subset of lymphocytes originating from the bone marrow and circulating in peripheral blood, characterized by the presence of surface B-cell receptors (BCRs) crucial for antigen recognition. B cells play a pivotal role in adaptive immunity by differentiating into plasma cells, producing antibodies, and facilitating immunological memory, thereby contributing significantly to the body's defense against pathogens and foreign substances.
[00126] B cells can be characterized by the expression of at least the surface membrane CD 19 antigen and/or the CD20 antigen. While the CD20 antigen is acquired during late stages of B -cell lymphogenesis and is then lost upon differentiation into plasma cells, CD 19 expression covers the entire spectrum of early B-cell genesis and maturation.
NK cells
[00127] Natural Killer Cells, also called NK cells or large granular lymphocytes (LGL), refer to a subset of cytotoxic lymphocytes belonging to the innate immune system and represent about 5% to 20% of all circulating lymphocytes in humans. NK cells originate, differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the blood circulation. NK cells are characterized by their ability to recognize and eliminate virally infected cells, tumor cells, and cells displaying stress-induced surface markers, without prior sensitization or the need for specific antigens. NK cells exert their cytotoxic functions through the release of cytolytic granules containing perforin and granzymes, inducing apoptosis in target cells. Additionally, NK cells modulate immune responses by secreting cytokines and chemokines, thereby contributing to the regulation of adaptive immune reactions.
[00128] NK cell subsets can be characterized by commonly expressing at least the surface membrane CD56 antigen and/or the CD16a antigen (FcyRIIIa) and/or the CD57 antigen.
[00129] NK cell subsets can also be characterized by the absence of expression of at least the T cell receptor (CD3).
Monocytes
[00130] Monocytes refer a type of white blood cell as part of the innate immune system. Monocytes are characterized by their large, kidney-shaped nucleic and a relatively abundant cytoplasm. Monocytes circulate in the bloodstream before migrating into tissues where they differentiate into macrophages or monocyte-derived dendritic cells (moDC). Their role includes phagocytosis of pathogens, cellular debris, and antigen presentation, contributing significantly to immune surveillance, inflammation regulation, and tissue repair processes within the body.
[00131] Generally, monocytes were classified following three subsets based on expression of the CD14 antigen and/or the CD16 antigen: classical monocytes expressing the CD 14 surface membrane antigen and lack the expression of the CD 16 marker; intermediate monocytes expressing both the CD 14 and CD 16 surface membrane antigens; and nonclassical monocytes expressing the CD 16 surface membrane antigen and lack the expression of the CD 14 antigen. The expression of the CD 14 antigen is shared with the DC3 subset.
[00132] All the three monocyte subsets also express the CD88 surface membrane antigen and/or the CD89 surface membrane antigen.
Dendritic cell subsets
[00133] The present disclosure aims at enriching a cell sample with all the identified dendritic cell subsets.
[00134] Dendritic cells are antigen-presenting cells of the immune system characterized by their capacity to capture, process, and present antigens to T cells, thereby initiating and regulating immune responses.
[00135] All the dendritic cell subsets can commonly be characterized by the expression, on their surface, of at least the CD45 and HLA-DR antigens. These antigens are shared with other white blood cells such as, for instance, monocytes. To distinguish and isolate dendritic cells from other white blood cells, specific markers are used for further characterization of dendritic cells.
[00136] Generally, dendritic cells can be classified into five distinct subsets, namely Axl-i- Siglec6+ dendritic cells (AS-DCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells 1 (cDCls), dendritic cell 2 (DC2s) and dendritic cells 3 (DC3s).
[00137] Axl+ Siglec6+ dendritic cells or AS-DCs or pre dendritic cells can be characterized by the expression, on its surface, of at least the Axl antigen.
[00138] In some embodiments, AS-DCs can be characterized by the expression, on its surface, of at least both the Axl and Siglec6 antigens.
[00139] In some embodiments, AS-DCs can be characterized by the expression, on its surface, of at least the Axl antigen, the Siglec6 antigen, the CD2 antigen and the CD5 antigen, and optionally at least one antigen among the CD 123 antigen, and the CDllc antigen.
[00140] In some embodiments, AS-DCs can be characterized by the absence of expression of at least the CD88 antigen.
[00141] AS-DCs can have the capacity to secrete interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10). [00142] Plasmacytoid dendritic cells or pDCs can be characterized by the expression, on its surface, of at least one antigen among the CD 123 antigen and the CD303 antigen, and optionally the CD304 antigen.
[00143] In some embodiments, pDCs can be characterized by the expression, on its surface, of at least the CD303 antigen.
[00144] In some embodiments, pDCs can be characterized by the expression, on its surface, of at least the CD 123 antigen.
[00145] In some embodiments, pDCs can be characterized by the absence of expression of the CDllc antigen, the AXL antigen and/or the CD88 antigen.
[00146] In some embodiments, pDCs can be characterized by the expression, on its surface, of at least the CD123 antigen and by the absence of expression of at least the CDllc antigen, the AXL antigen and the CD88 antigen.
[00147] pDCs can have the capacity to secrete interferon alpha and can have the capacity to secrete interferon gamma-induced protein 10 (IP- 10).
[00148] Conventional dendritic cells 1 or eDC 1 s can be characterized by the expression, on its surface, of at least one antigen among the CD141 antigen and the CLEC9A antigen, and optionally at least the XCR1 antigen.
[00149] In some embodiments, cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, the XCR1 antigen, and the CLEC9A antigen.
[00150] In some embodiments, cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, and optionally at least the CLEC9A antigen, and/or the CDllc antigen.
[00151] In some embodiments, cDCls can be characterized by the absence of expression of the CDlc antigen, the AXL antigen, and/or the CD88 antigen.
[00152] In some embodiments, cDCls can be characterized by the expression, on its surface, of at least the CD141 antigen, and by the absence of expression of the CDlc antigen, the AXL antigen and the CD88 antigen.
[00153] cDCls can have the capacity to secrete interleukin- 1 beta (IL- 10).
[00154] The conventional dendritic cell subset comprises two sub-populations which are dendritic cells 2 and dendritic cells 3.
[00155] Dendritic cells 2 or DC2s can be characterized by the expression, on its surface, of at least one antigen among the CDlc and CD11c antigens, and optionally at least one antigen among the CD172a and CDllb antigens, and by the absence of the expression of the CD163 antigen. [00156] In some embodiments, DC2s can be characterized by the expression, on its surface, of at least one antigen among the CDlc and CD11c antigens, and optionally at least one antigen among the CD172a and CD1 lb antigens, and by the absence of expression of the CD163 antigen.
[00157] In some embodiments, DC2s can be characterized by the expression, on its surface, of at least the CDlc and CD11c antigens, and by the absence of expression of the CD163 antigen.
[00158] In some embodiments, DC2s can be characterized by the expression, on its surface, of at least the CDlc antigen, and by the absence of expression of the CD 163 antigen.
[00159] In some embodiments, DC2s can be characterized by the expression, on its surface, of at least the CDllc antigen, and by the absence of expression of the CD163 antigen.
[00160] In some embodiments, DC2s can be further characterized by the absence of expression of the CD163 antigen, the AXL antigen and/or the CD88 antigen.
[00161] In some embodiments, DC2s can be further characterized by the absence of expression of the AXL antigen and/or the CD88 antigen.
[00162] In some embodiments, DC2s can be characterized by the expression, on its surface, of at least the CDlc and CD11c antigens, and by the absence of expression of the CD163 antigen, the AXL antigen and the CD88 antigen.
[00163] DC2s can have the capacity to secrete interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
[00164] Dendritic cells 3 or DC3s can be characterized by the expression, on its surface, of at least both the CD163 and CD11c antigens, and optionally at least one of the antigens among the CDlc antigen, the CD172a antigen, and the CD14 antigen.
[00165] In some embodiments, DC3s can be characterized by the expression, on its surface, of at least the CD163 antigen, the CDllc antigen and the CDlc antigen, and optionally at least one antigen among the CD172a antigen and the CD14 antigen.
[00166] In some embodiments, DC3s can be further characterized by the absence of expression of the AXL antigen and/or the CD88 antigen.
[00167] In some embodiments, DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CD163 antigen and the CDlc antigen; and by the absence of expression of the AXL antigen and the CD88 antigen.
[00168] In some embodiments, DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CD 163 antigen, the CDlc antigen, and the CD 14 antigen. [00169] In some embodiments, DC3s can be characterized by the expression, on its surface, of at least the CDllc antigen, the CDlc antigen and the CD163 antigen, and by the absence of expression of the CD 14 antigen.
[00170] DC3s can have the capacity to secrete interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
Antibody combination
[00171] According to the present disclosure, a human cell sample, as described above, may be exposed to an antibody combination specifically designated for enriching the human cell sample with dendritic cells. As shown in the example section, it is demonstrated that the use of an antibody combination as described herein as a negative selection technique allows to prepare a human cell sample which is enriched in all the five dendritic cell subsets as mentioned herein.
[00172] Thus, an object of the present disclosure relates to an antibody combination comprising a plurality of antibodies or fragments thereof directed against non-dendritic cell antigens.
[00173] In some embodiments, the antibody combination as described herein is comprised in a composition.
[00174] As used herein, the expression “directed against” refers to the ability of an antibody or antigen-binding fragment thereof to detectably bind an epitope present on a specific antigen, while having relatively no or little detectable reactivity with different antigen. The antibody or antigen-binding fragment thereof forms a complex with the specific antigen that is relatively stable under suitable conditions.
[00175] In the present disclosure, the term “antibody” encompasses an “antigen-binding fragment”.
[00176] In some embodiments, the antibodies described herein may be isolated antibodies.
[00177] In some embodiments, the antibodies may be monoclonal antibodies.
[00178] In some embodiments, the antibodies may be human antibodies or human engineered antibodies.
[00179] In some embodiments, the antibodies may be labelled with a detectable molecule and/or conjugated to magnetic beads.
[00180] In some embodiments, the antibodies may be labelled with allophycocyanin and conjugated to magnetic beads. [00181] In some embodiments, the antibodies may be directly conjugated with magnetic beads.
[00182] In some embodiments, each antibody or its antigen-binding fragment thereof may be, independently, formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00183] The preparation of an antibody combination as described herein and the conditions to prepare such a combination are well known in the art. Such an antibody combination as described herein may be prepared upon use to avoid antibody aggregation.
[00184] An antibody combination disclosed herein, for enriching a human cell sample with dendritic cells, may comprise at least a plurality of antibodies of groups (i), (ii), (iii), (iv), (v) and (vi) defined as:
[00185] (i) antibodies or antigen-binding fragments thereof directed against the CD3 antigen, and/or antibodies or antigen-binding fragments thereof directed against the TCRa/p antigen, and/or antibodies or antigen-binding fragments thereof directed against the TCRy/6 antigen;
[00186] (ii) antibodies or antigen-binding fragments thereof directed against the CD88 antigen and, optionally antibodies or antigen-binding fragments thereof directed against the CD89 antigen;
[00187] (iii) antibodies or antigen-binding fragments thereof directed against the CD56 antigen;
[00188] (iv) antibodies or antigen-binding fragments thereof directed against the CD 16 antigen;
[00189] (v) antibodies or antigen-binding fragments thereof directed against the CD 19 antigen, and/or antibodies or antigen-binding fragments thereof directed against the CD20 antigen; and
[00190] (vi) antibodies or antigen-binding fragments thereof directed against the CD 15 antigen, and/or antibodies or antigen-binding fragments thereof directed against the CD66b antigen.
[00191] In some embodiments, a human cell sample as described herein may contain a low concentration of residual red blood cells and platelets that have not been depleted after the initial cell separation process from whole blood cell. Therefore, the antibody composition as describe herein may further comprise antibodies or fragments thereof directed against antigens specific of platelets and/or red blood cells. [00192] In some embodiments, an antibody combination disclosed herein, for enriching a human cell sample with dendritic cells, may further comprise antibodies of groups (vii) and (viii) defined as:
[00193] (vii) antibodies or antigen-binding fragments thereof directed against the CD41 antigen, and/or antibodies or antigen-binding fragments thereof directed against the CD61 antigen; and optionally
[00194] (viii) antibodies or antigen-binding fragments thereof directed against the CD235a antigen.
[00195] Group (i) antibody
[00196] Antibodies or an antigen-binding fragments thereof of group (i) described herein may specifically bind to an antigen present at the surface of T cells, including the subgroup of NKT cells.
[00197] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may consist of an antibody or an antigen-binding fragment thereof directed against the CD3 antigen and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/5 antigen.
[00198] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may consist of an antibody or an antigen-binding fragment thereof directed against the CD3 antigen.
[00199] Antibodies or an antigen-binding fragments thereof from group (i), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00200] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00201] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1.5 pg, or about 2 |jg, or about 2,5 |jg, or about 3 |jg, or about 3,5 |jg, or about 4 |jg, or about
4.5 |jg, or about 5 |jg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00202] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 pg to about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00203] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of about 0,05 pg, or about 0,1 pg, or about 0,19 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00204] In some embodiments, an antibody or an antigen-binding fragment thereof of group (i) may be formulated in the antibody combination in a concentration of 0,192 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00205] For instance, an antibody or an antigen-binding fragment thereof of group (i) may consist of an anti-human CD3 Antibody purchased from BioLegend.
[00206] Group (ii) antibody
[00207] Antibodies or an antigen-binding fragments thereof of group (i) described herein may specifically bind to an antigen present at the surface of monocytes and basophils. The use of an antibody specific to both the CD88 antigen and the CD89 antigen allows for targeting and binding monocytes and basophils without binding to, and thus depleting, the DC3 subset.
[00208] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen.
[00209] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and an antibody or an antigen-binding fragment thereof directed against the CD89 antigen.
[00210] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD89 antigen. [00211] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen.
[00212] Antibodies or an antigen-binding fragments thereof from group (ii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00213] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00214] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may be formulated in the antibody combination in a concentration of about 0,001 pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00215] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,01 pg to about 3 pg, or from about 0,02 pg to about 2 pg, or from about 0,05 pg to about 1,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00216] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may be formulated in the antibody combination in a concentration of about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,1 pg, or about 1,2 pg, or about 1,3 pg, or about 1,4 pg, or about 1,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00217] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may be formulated in the antibody combination in a concentration of about 1 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00218] In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and formulated in the antibody combination in a concentration ranging from about 0,001 |jg to about 5 |jg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00219] pg. In some embodiments, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD89 antigen and formulated in the antibody combination in a concentration 0,05 pg to about 1,2 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00220] For instance, an antibody or an antigen-binding fragment thereof of group (ii) may consist of an anti-human CD88 Antibody purchased from BioLegend.
[00221] Group (iii) antibody
[00222] Antibodies or an antigen-binding fragments thereof of group (iii) described herein may specifically bind to an antigen present at the surface of NK cells and NKT cells.
[00223] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD56 antigen.
[00224] Antibodies or an antigen-binding fragments thereof from group (iii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00225] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration ranging from about 0,0011 pg to about 5 pg, per about 50xl06 peripheral blood mononuclear cells.
[00226] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of about 0,001pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00227] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 |jg to about 1 |jg, or from about 0,005 |jg to about 0,5 |jg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00228] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of about 0,05 pg, or about 0,1 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,33 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00229] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iii) may be formulated in the antibody combination in a concentration of 0,336 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00230] For instance, an antibody or an antigen-binding fragment thereof of group (iii) may consist of an anti-human CD56 Antibody purchased from BioLegend.
[00231] Group (iv) antibody
[00232] Antibodies or an antigen-binding fragments thereof of group (iv) described herein may specifically bind to an antigen present at the surface of NK cells and monocytes, such as intermediate monocytes and non-conventional monocytes.
[00233] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen.
[00234] Antibodies or an antigen-binding fragments thereof directed against the CD 16 antigen can be directed against both the CD 16a antigen and the CD 16b antigen.
[00235] In some embodiments, an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen can be an antibody or an antigen-binding fragment thereof directed against the CD 16a antigen and/or the CD 16b antigen.
[00236] An antibody or an antigen-binding fragment thereof from group (iv), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00237] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 peripheral blood mononuclear cells. [00238] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration of about 0,001 pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00239] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,01 pg to about 3 pg, or from about 0,02 pg to about 2 pg, or from about 0,02 pg to about 1 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00240] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration of about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, or about 0,55 pg, or about 0,6 pg, or about 0,65 pg, or about 0,67 pg, or about 0,7 pg, or about 0,75 pg, or about 0,8 pg, or about 0,85 pg, or about 0,9 pg, or about 0,95 pg, or about 1 pg, per about 50xl06 peripheral blood mononuclear cells.
[00241] In some embodiments, an antibody or an antigen-binding fragment thereof of group (iv) may be formulated in the antibody combination in a concentration of 0,675 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00242] For instance, an antibody or an antigen-binding fragment thereof of group (iv) may consist of an anti-human CD 16 Antibody purchased from BioLegend.
[00243] Group (v) antibody
[00244] Antibodies or an antigen-binding fragments thereof of group (v) described herein may specifically bind to an antigen present at the surface of B cells.
[00245] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen.
[00246] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen. [00247] Antibodies or an antigen-binding fragments thereof from group (v), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00248] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00249] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may be formulated in the antibody combination in a concentration of about 0,001 pg, about 0,01pg, about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about 1,5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about 4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00250] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 pg to about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00251] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may be formulated in the antibody combination in a concentration of about 0,01 pg, or about 0,05 pg, or about 0,1 pg, or about 0,12 pg, or about 0,15 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00252] In some embodiments, an antibody or an antigen-binding fragment thereof of group (v) may be formulated in the antibody combination in a concentration of 0,12 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00253] For instance, an antibody or an antigen-binding fragment thereof of group (v) may consist of an anti-human CD 19 Antibody purchased from BioLegend.
[00254] Group (vi) antibody [00255] Antibodies or an antigen-binding fragments thereof of group (vi) described herein may specifically bind to an antigen present at the surface of neutrophils.
[00256] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
[00257] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may consist of an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen.
[00258] Antibodies or an antigen-binding fragments thereof from group (vi), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00259] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00260] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about
1.5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about
4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00261] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 3 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 pg to about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00262] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration of about 0,01 pg, or about 0,05 pg, or about 0,1 pg, or about 0,12 pg, or about 0,15 pg, or about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells. [00263] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vi) may be formulated in the antibody combination in a concentration of 0,1 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00264] For instance, an antibody or an antigen-binding fragment thereof of group (vi) may consist of an anti-human CD 15 Antibody purchased from BioLegend.
[00265] Group (vii) antibody
[00266] Antibodies or an antigen-binding fragments thereof of group (vii) described herein may specifically bind to an antigen present at the surface of platelets.
[00267] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen.
[00268] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD41 antigen.
[00269] Antibodies or an antigen-binding fragments thereof from group (vii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00270] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00271] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of about 0,001pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 pg, or about 0,7 pg, or about 0,8 pg, or about 0,9 pg, or about 1 pg, or about
1.5 pg, or about 2 pg, or about 2,5 pg, or about 3 pg, or about 3,5 pg, or about 4 pg, or about
4.5 pg, or about 5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00272] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,001 pg to about 2 pg, or from about 0,001 pg to about 1 pg, or from about 0,001 |jg to about 0,5 |jg, or from about 0,01 |jg to about 0,25 |jg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00273] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of about 0,01 pg, or about 0,012 pg, or about 0,013 pg, or about 0,014 pg, or about 0,015 pg, or about 0,016 pg, or about 0,017 pg, or about 0,018 pg, or about 0,019 pg, or about 0,02 pg, or about 0,03 pg, or about 0,04 pg, or about 0,05 pg, or about 0,06 pg, or about 0,07 pg, or about 0,08 pg, or about 0,09 pg, or about 0,1 pg, or about 0,2 pg, or about 0,25 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00274] In some embodiments, an antibody or an antigen-binding fragment thereof of group (vii) may be formulated in the antibody combination in a concentration of 0,0125pg per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00275] For instance, an antibody or an antigen-binding fragment thereof of group (vii) may consist of an anti-human CD41 Antibody purchased from BioLegend.
[00276] Group (viii) antibody
[00277] Antibodies or an antigen-binding fragments thereof of group (viii) described herein may specifically bind to an antigen present at the surface of red blood cells.
[00278] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may consist of an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
[00279] Antibodies or an antigen-binding fragments thereof from group (viii), as described herein, can be formulated within the antibody combination to efficiently bind an antigen, forming a complex under suitable conditions. When used in a negative selection technique to enrich a cell sample in dendritic cells, this complex formed between the antibody and the antigen will subsequently be selected and removed from the cell sample using appropriate separation techniques.
[00280] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50x10 cells, in particular peripheral blood mononuclear cells.
[00281] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of about 0,001 pg, or about 0,01pg, or about 0,1 pg, or about 0,2 pg, or about 0,3 pg, or about 0,4 pg, or about 0,5 pg, or about 0,6 |jg, or about 0,7 |jg, or about 0,8 |jg, or about 0,9 |jg, or about 1 |jg, or about
1.5 |jg, or about 2 |jg, or about 2,5 |jg, or about 3 |jg, or about 3,5 |jg, or about 4 |jg, or about
4.5 |jg, or about 5 |jg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00282] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration ranging from about 0,001 pg to about 5 pg, or from about 0,1 pg to about 3 pg, or from about 0,2 pg to about 2 pg, or from about 0,2 pg to about 1 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00283] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of about 0,2 pg, or about 0,25 pg, or about 0,3 pg, or about 0,35 pg, or about 0,4 pg, or about 0,45 pg, or about 0,5 pg, or about 0,55 pg, or about 0,6 pg, or about 0,65 pg, or about 0,67 pg, or about 0,7 pg, or about 0,75 pg, or about 0,8 pg, or about 0,85 pg, or about 0,9 pg, or about 0,95 pg, or about 1 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00284] In some embodiments, an antibody or an antigen-binding fragment thereof of group (viii) may be formulated in the antibody combination in a concentration of 0,5 pg, per about 50xl06 cells, in particular peripheral blood mononuclear cells.
[00285] For instance, an antibody or an antigen-binding fragment thereof of group (viii) may consist of an anti-human CD235a Antibody purchased from BioLegend.
[00286] In some embodiments, an antibody combination disclosed herein, optionally comprised in a composition, may comprise or consist of:
[00287] (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, or an antibody or an antigen-binding fragment thereof directed against the TCRy/8 antigen;
[00288] (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
[00289] (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
[00290] (iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen; [00291] (v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen, or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
[00292] (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
[00293] In some embodiments, an antibody combination disclosed herein, optionally comprised in a composition, may comprise or consist of:
[00294] (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
[00295] (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
[00296] (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
[00297] (iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
[00298] (v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
[00299] (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
[00300]
[00301] In some embodiments, an antibody combination disclosed herein, may comprise or consist of:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen; and (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen.
[00302] In some embodiments, an antibody combination disclosed herein, may comprise or consist of:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen;
(vi) an antibody or an antigen-binding fragment thereof directed against the CD15 antigen; and
(vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen.
[00303] In some embodiments, an antibody combination disclosed herein, may comprise or consist of:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen;
(vi) an antibody or an antigen-binding fragment thereof directed against the CD15 antigen;
(vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen; and
(viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
[00304] In some embodiments, an antibody combination disclosed herein, may comprise or consist of:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen;
(vi) an antibody or an antigen-binding fragment thereof directed against the CD15 antigen; (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen; and
(viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
Dendritic cell enrichment method
[00305] The present disclosure also provides methods implementing an antibody combination as disclosed herein for preparing a human cell sample enriched in dendritic cells as described herein.
[00306] The methods for preparing a human cell sample enriched in dendritic cells as described herein may be called “lab-made method” in the examples.
[00307] In some embodiments, the methods are performed in vitro.
[00308] Methods of preparing a human cell sample enriched in dendritic cells as described herein are based on a specific negative selection to deplete non-dendritic cells from the cell sample and thereby obtaining a human cell sample enriched in dendritic cells. Therefore, a method disclosed herein successfully provides a human cell sample enriched in all the five dendritic cell subsets as described herein, in particular the newly described DC3 subset including the DC3 expressing at least the CD 14 antigen.
[00309] For instance, a method as described herein when implemented on an isolated PBMCs sample allow the depletion of non-dendritic white blood cells, such as granulocytes, T cells, B cells, NK cells, monocytes and when appropriate the remaining platelets and red blood cells.
[00310] It is demonstrated in the examples that the method as disclosed herein for preparing a human cell sample enriched in dendritic cells provides a cell sample which is enriched with greater than about 20% of recovery dendritic cells. In particular, the method as disclosed herein may provide a cell sample which is enriched from about 40% to about 90% of dendritic cells.
[00311] A method of preparing a human cell sample enriched in dendritic cells as described herein may use at least a human cell sample as described herein, such as an isolated PBMCs sample, and an antibody combination as described herein.
[00312] In some embodiments, an in vitro method for preparing a human cell sample enriched in dendritic cells may comprise at least the steps of: [00313] (a) reacting a human cell sample as described herein with an antibody combination as described herein, under conditions suitable for the binding of each antibody or its antigen-binding fragment thereof to the cells expressing at their surface one or more antigens against which the respective antibodies are directed;
[00314] (b) removing the cells bound by the antibodies from the human cell sample; and [00315] (c) recovering a human cell sample which is enriched in dendritic cells.
[00316] In some embodiments, an in vitro method for preparing a human cell sample enriched in dendritic cells may comprise at least the steps of:
[00317] (a) reacting a human cell sample as described herein with an antibody combination comprising (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen; (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen; (iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen; (v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen, under conditions suitable for the binding of each antibody or its antigen-binding fragment thereof to the cells expressing at their surface one or more of the (i) CD3 and/or TCRa/p and/or TCRy/6; (ii) CD88 and, optionally CD89; (iii) CD56; (iv) CD16; (v) CD19 and/or CD20; and (vi) CD15, and/or CD66b antigens;
[00318] (b) removing the cells bound by the antibodies from the human cell sample; and [00319] (c) recovering a human cell sample which is enriched in dendritic cells.
[00320] In some embodiments, the human cell sample may be in a cell-culture medium for human cells suitable for isolating the dendritic cells in the human cell sample.
[00321] In some embodiments, the method as described herein may include the use of a cell-culture medium for human cells suitable for isolating the dendritic cells in the human cell sample.
[00322] In some embodiments, the cell-culture medium may comprise dulbecco's phosphate-buffered saline (DPBS) and ethylenediaminetetraacetic acid (EDTA). [00323] In some embodiments, the cell-culture medium may further comprise bovine serum albumin (BSA) and/or heat inactivated human serum.
[00324] Step (a)
[00325] Conditions suitable for the binding of an antibody with a targeted antigen within a cell sample may be selected by a skilled person in the art depending to factors, such as the nature and concentration of the antibodies comprised in the antibody combination.
[00326] To selectively deplete the non-dendritic cells of the human cell sample, the antibodies or antigen-binding fragments thereof comprised in the antibody combination as described herein may be labelled with a detectable molecule and/or conjugated to magnetic beads.
[00327] In some embodiments, a detectable molecule may be, but is not limited to, biotin or a fluorochrome.
[00328] In some embodiments, a fluorochrome may be fluorescein or allophycocyanin (APC).
[00329] In some embodiments, antibodies or antigen-binding fragments thereof of the antibody combination may be labelled with allophycocyanin.
[00330] In some embodiments, magnetic beads may be directed against allophycocyanin.
[00331] In some embodiments, step (a) of reacting a human cell sample as described herein with an antibody combination may be implemented in two steps:
[00332] a first step (a) of reacting a human cell sample as described herein with an antibody combination wherein the antibodies are labeled with a detectable molecule, and
[00333] a second step (a) of reacting the human cell sample obtained at the end of the first step (a) with magnetic beads directed against the detectable molecule.
[00334] In some embodiments of the first step (a), the antibodies may be labeled with allophycocyanin, and the second step (a) the magnetic beads may be directed against allophycocyanin.
[00335] In some embodiments, the human cell sample may be incubated in suitable conditions for the binding of each antibody or its fragment thereof to antigens at the surface of the cells present in the human cell sample. For instance, the human cell sample may be incubated in the dark at about 4°C for about 10 minutes.
[00336] In some embodiments, the human cell sample may be incubated in suitable conditions for the binding of magnetic beads to labelled antibodies. For instance, the human cell sample may be incubated in the dark at about 4°C for about 15 minutes. [00337] In some embodiments, within step (a), it may be reacted a human cell sample as described herein with an antibody combination wherein the antibodies are directly conjugated with magnetic beads.
[00338] When needed, the samples are suspended with a cell-culture medium as described herein.
[00339] In some embodiments, the antibody combination as described herein is reacted with a human cell sample as described herein in a single step. Specifically, the antibody combination is introduced at once in the human cell sample in order to react the cells with the antibodies that are comprised in the antibody combination.
[00340] In some embodiments, the in vitro method for preparing a human cell sample enriched in dendritic cells as described herein may comprise a step (a) as described herein wherein the antibody combination, optionally comprised in a composition, is introduced at once in a human cell sample as described herein in order to trigger a reaction between the cells of the human cell sample with the antibodies of the antibody combination, under conditions suitable for the binding of each antibody or its antigen-binding fragment thereof to the cells expressing at their surface one or more antigens against which the respective antibodies are directed.
[00341] Step ( h>
[00342] The method disclosed herein may comprise a step of removing the cells bound by the antibodies from the human cell sample obtained at step (a).
[00343] Conditions and devices, of methods for preparing a human cell sample enriched in dendritic cells as described herein, for removing the cells bound by the antibodies from the human cell sample at step (a) are well known in the art. In particular, devices for removing the cells bound by the antibodies from the human cell sample may depend on the concentration of cells includes in the cell sample.
[00344] For instance, step (b) of the method as described herein may be implemented with a magnetic cell separation system.
[00345] A magnetic cell separation system is configured to retain cells conjugated with magnetic beads.
[00346] In some embodiments, a magnetic cell separation system may comprise a magnetic column.
[00347] In some embodiments, a magnetic cell separation system may consist of the autoMACS® Pro Separator, the AutoMACS® NEO Separator, the XS columns or the LS columns developed by Miltenyi Biotec. [00348] In some embodiments, a magnetic cell separation system may consist of the LS column developed by Miltenyi Biotec.
[00349] In some embodiments, the human cell sample obtained at step (a) are placed in a magnetic cell separation system for retaining the cells bound by the antibodies.
[00350] In some embodiments, the human cell sample obtained at step (a) are directed through a magnetic column within a magnetic cell separation system for retaining the cells bound by the antibodies. Such a column may be configured to retain magnetically conjugated cells, allowing for the separation of the target cell population from the unconjugated cells, such as the dendritic cells. The magnetic force immobilizes the conjugated cells, while unconjugated cells pass through, facilitating efficient isolation of dendritic cells.
[00351] Step (c)
[00352] The methods disclosed here may comprise a step of recovering a human cell sample which is enriched in dendritic cells at the end of step (b).
[00353] The human cell sample enriched in dendritic cells, obtained by the method as described herein, include the five dendritic cells subsets, in particular the Axl+ Siglec6+ dendritic cells (AS-DCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells 1 (cDCls), dendritic cell 2 (DC2s) and dendritic cells 3 (DC3s).
[00354] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein, include at least:
[00355] - AS-DCs expressing the Axl antigen,
[00356] - pDCs expressing at least one antigen among the CD 123 antigen and the CD303 antigen, and optionally the CD304 antigen;
[00357] - cDCls expressing at least one antigen among the CD141 antigen and the CLEC9A antigen , and optionally the XCR1 antigen;
[00358] - DC2s expressing at least one antigen among the CDlc antigen and the CD 11c antigen, and optionally at least one antigen among the CD172a antigen and the CDllb antigen, and lack the expression of the CD163 antigen; and
[00359] - DC3s expressing at least both the CD163 antigen and the CDllc antigen, and optionally at least one antigen among the CDlc antigen, the CD172a antigen, and the CD14 antigen.
[00360] In some embodiments, the method of the present disclosure may lead to an enrichment of AS-DCs from about 0.5% to about 2% of the total cells within a given human cell sample. [00361] In some embodiments, the method of the present disclosure may lead to an enrichment of AS-DCs from about 0.75% to about 2%, or from about 0.75% to about 1.75%, or from about 0.75% to about 1.5% of the total cells within a given human cell sample.
[00362] In some embodiments, the method of the present disclosure may lead to an enrichment of pDCs from about 5% to about 25% of the total cells within a given human cell sample.
[00363] In some embodiments, the method of the present disclosure may lead to an enrichment of pDCs from about 5% to about 20%, or from about 5% to about 15%, or from about 7% to about 13% of the total cells within a given human cell sample.
[00364] In some embodiments, the method of the present disclosure may lead to an enrichment of cDCls from about 0.4% to about 3% of the total cells within a given human cell sample.
[00365] In some embodiments, the method of the present disclosure may lead to an enrichment of cDCls from about 0.4% to about 2.75%, or from about 0.4% to about 2.5%, or from about 0.4% to about 2 %, from about 0.4% to about 1.5% of the total cells within a given human cell sample.
[00366] In some embodiments, the method of the present disclosure may lead to an enrichment of DC2s from about 2% to about 6% of the total cells within a given human cell sample.
[00367] In some embodiments, the method of the present disclosure may lead to an enrichment of DC2s from about 2.5% to about 6%, or from about 2.5% to about 5.5% of the total cells within a given human cell sample.
[00368] In some embodiments, the method of the present disclosure may lead to an enrichment of DC3s from about 14% to about 25% of the total cells within a given human cell sample.
[00369] In some embodiments, the method of the present disclosure may lead to an enrichment of DC3s from about 15% to about 25%, or from about 17% to about 25%, or from about 17% to about 22% of the total cells within a given human cell sample.
[00370] In some embodiments, the human cell sample enriched in dendritic cells may include at least one conventional dendritic cell expressing at least the antigens CDllc, CD 163 and CD 14.
[00371] In some embodiments, the method of the present disclosure may lead to an enrichment of DC3s expressing at least the antigens CDllc, CD163 and CD14 from about 5% to about 15% of the total cells within a given human cell sample. [00372] In some embodiments, the human cell sample enriched in dendritic cells may include at least one conventional dendritic cell expressing the CDllc and CD163 antigens and lacking the expression of the CD 14 antigen.
[00373] In some embodiments, the human cell sample enriched in dendritic cells, may include the least one conventional dendritic cell secreting interleukin- 1 beta (IL- 10) and/or interferon gamma-induced protein 10 (IP- 10).
[00374] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include non-dendritic cells.
[00375] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include CD88-positive (CD88+) cells and/or CD89-positive (CD89+) cells.
[00376] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include HLA-DR-positive (HLA-DR+) myeloid cells other than dendritic cells.
[00377] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include basophils, neutrophils and monocytes.
[00378] In some embodiments, the human cell sample enriched in dendritic cells, obtained by the method as described herein does not include T cells, B cells, NK cells, platelet, red blood cells, basophils, neutrophils and/or monocytes.
Use of the antibody combination
[00379] The present disclosure also provides uses of an antibody combination as disclosed herein in a method for preparing a human cell sample enriched in dendritic cells as described herein.
[00380] In some embodiments, the uses are performed in vitro.
[00381] It is disclosed the use of an antibody combination as disclosed herein in a method for preparing a human cell sample enriched in dendritic cells as described herein.
[00382] The present disclosure also relates to the specific use of an antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, in a method for preparing a human cell sample enriched in dendritic cells. [00383] In some embodiments, the antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally the antibody or an antigenbinding fragment thereof directed against the CD89 antigen as described herein, may be used in a method for preparing a human cell sample enriched in dendritic cells as described herein.
[00384] In some embodiments, the human cell sample enriched in dendritic cells, using an antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, may include at least one conventional dendritic cell expressing at least the antigens CDllc, CD163 and CD14.
[00385] In some embodiments, the human cell sample enriched in dendritic cells, using an antibody or an antigen-binding fragment thereof directed against the CD88 antigen, as described herein and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, may include the least one conventional dendritic cell secretes interleukin-1 beta (IL-10) and/or interferon gamma-induced protein 10 (IP-10).
[00386] In some embodiments, the human cell sample enriched in dendritic cells, using an antibody or an antigen-binding fragment thereof directed against the CD88 antigen as described herein, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen as described herein, may include at least one conventional dendritic cell expressing at least the antigens CDllc, CD 163 and CD 14, and secreting interleukin- 1 beta (IL-10) and/or interferon gamma-induced protein 10 (IP-10).
[00387] Embodiments of the methods for preparing a human cell sample enriched in dendritic cells as disclosed herein are applicable to the present uses.
Kit-of-parts
[00388] The present disclosure also provides a kit-of-part useful for enriching a human cell sample in dendritic cells. Such a kit may be suitable to implement the uses and methods disclosed herein.
[00389] In some embodiments, a kit-of-part may include a plurality of antibodies of groups (i), (ii), (iii), (iv), (v) and (vi) as described herein.
[00390] In some embodiments, a kit-of-part may further include a plurality of antibodies of groups (vii) and (viii) as described herein.
[00391] In some embodiments, a kit-of-part may include:
(A) (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/0 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen,
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen,
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen,
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen and/or an antibody or a fragment directed against the CD20 antigen, and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen; and
(B) instructions for preparing a human cell sample enriched in dendritic cells.
[00392] In some embodiments, a kit-of-part may include:
(A) (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/8 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen,
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen,
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen,
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen and/or an antibody or a fragment directed against the CD20 antigen,
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen;
(vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen; and
(viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen; and
(B) instructions for preparing a human cell sample enriched in dendritic cells. [00393] The antibodies of the kit are packaged in suitable containers allowing their conservation over time. Such containers are known in the art.
[00394] The antibodies may be packaged individually or in combination.
[00395] It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which at least one limitation, element, clause, descriptive term, etc., from at least one of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, on any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
[00396] Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., they also encompass embodiments consisting, or consisting essentially of, such elements, features, etc. For purposes of conciseness, those embodiments have not, in every case, been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
[00397] The publications and other reference materials referenced herein for describing the background of the disclosure and providing additional detail regarding its practice are hereby incorporated by reference.
[00398] Without limiting the present disclosure, examples are described below for the purpose of illustration.
[EXAMPLES]
EXAMPLE 1
[00399] In the present examples, it is compared the performances of two widely used panDC enrichment kits (Miltenyi and STEMCELL), as well as the dendritic cell enrichment method (lab-made method) of the present disclosure. It is demonstrated that the lab-made method significantly enriches the total dendritic cell fractions from human blood samples while maintaining the full diversity of the different dendritic cell subsets compared to the two widely used pan-DC enrichment kits.
PART 1: MATERIALS AND METHODS
Human Peripheral blood mononuclear cells (PBMCs)
[00400] Fresh leukapheresis blood samples (Cytapheresis rings) from 5 healthy donors were obtained from Etablissement Francais du Sang. Donors provided written informed consents under an ethically-approved convention and according to national and european regulations.
[00401] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples by centrifugation at 800 x g for 15min at 20°C with no brakes, on a density gradient (Lymphoprep, Stemcell, #07811). After centrifugation, the PBMC ring is gently recovered by pipetting and cells are wash three time in PBS lx by centrifugating at 200 x g for 10 min at 4°C to remove platelets.
Buffer PBS+
[00402] For about 500 mF of buffer PBS+, 500mL of DPBS IX (GIBCO® no calcium, no magnesium - ThermoFishcer Ref: #14190094) is supplemented with 2mM EDTA UltraPure 0.5M, pH 8 (Invitrogen. Ref: #15575020) and 1% Heat inactivated human serum (Sigma- Aldrich. Ref: #H3667).
Cells counting within the PBMCs sample
[00403] To prepare fresh PBMCs sample for counting, it is added 500 mF of cold DPBS IX (GIBCO® ThermoFishcer Ref: #14190094), to top up the PBMC sample tube. The obtained solution is mixed thoroughly and set aside an aliquot for counting. Then, the total PBMCs is counted using an optical microscope (Malassez chamber) during centrifugation of the PBMCs sample tube at 200 x g for 10 minutes at 4°C. After centrifugation, the supernatant is gently discarded and the PBMC pellet is resuspended at a concentration of 50xl06 cells/ml with a cold solution of PBS+.
Antibody combination preparation for the lab-made method
[00404] An antibody combination is prepared to target and then excluded non-dendritic cells from the PBMCs sample. Specifically, the antibody combination is composed to specifically exclude Lineage (non-dendritic cells), namely T cells expressing the CD3 marker, B cells expressing the CD19 and CD20 markers, NK cells and NKT cells expressing the CD56 marker and monocytes and basophils expressing the CD88 marker.
[00405] The antibodies comprised in the antibody combination are labeled with a fluorescent molecule, Allophycocyanin (APC).
[00406] Specifically, the APC-conjugated antibody combination is prepared for a sample comprising 50xl06 PBMC by mixing, in a separate tube, the antibodies as described in Table 1. Some variability in the determination of antibody concentrations is acceptable, as long as it does not compromise the efficiency of dendritic cell enrichment which is determined by the skilled person. Using a concentration of the anti-CD89 and anti-CD41 antibodies higher than the recommended levels in a dendritic cell enrichment method may potentially result in the depletion of dendritic cells.
[00407] When it is used a greater concentration than 50xl06 PBMC, the antibody concentration is adjusted accordingly. When it is used a lower concentration than 50xl06 PBMC, is can be used the antibody concentration recommended for processing 50xl06 PBMC.
[00408] The antibodies were purchased from BioLegend.
Table 1: APC-coniugated antibody combination for PBMC concentration of 50xl06
“pl”: microliter microgram
“ml”: milliliter
[00409] The antibody combination is kept in dark before processing with the lab-made method.
DC Enrichment pourcentages and absolute number
[00410] After DC enrichment using either the lab-made protocol, the Miltenyi kit or the Stemcell methods, 20pl of cell suspension was set aside to evaluate absolute number of cells using an optical microscope. Remaining cells were stained zombie NIR Fixable Viability Kit (Biolegend, 423106) washed, and then incubated for 30 minutes at 4°C with CD3 FITC (Biolegend, 300306), CD56 FITC (Biolegend, 318304), CD20 FITC (BD, 555622), CD88 FITC (Biolegend, 344306), CD19 FITC (Biolegend, 302206), HLA-DR AF700 (Biolegend, 327014), CD88 PE Dazzle 594 (Biolegend, 344318), CD14 BUV 737 (BD, 612763), CDl lc BUV 395 (BD, 563787), CDlc PE Cy7 (Biolegend, 331516), CD163 BV 711 (Biolegend, 333630), CD141 BV 421 (Biolegend, 344114), CD123 BV 650 (Biolegend, 306020) and AXL PE (R&D, FAB154P). Cells were analyzed on BD Fortessa (BD Biosciences).
Dendritic cell subsets purification using flow cytometry cell sorting
[00411] After DC enrichment using the lab-made protocol, the different DC subsets were purified by flow-cytometry cell sorting. Cells were stained for 30 minutes at 4°C with CD88 PE- Dazzle-594 (Biolegend, 344318), AXL PE (thermofisher, 12-1087-42), CD141 BV421 (Biolegend, 344114), CD123 AF700 (Biolegend, 306040), CDlc FITC (Biolegend, 331518), CDl lc APC Fire750 (Biolegend, 337240) and CD163 BV711 (Biolegend, 333630). 7-AAD (BD, 559925) was used to exclude dead cells, and the APC-conjugated antibodies used for DC enrichment served as a lineage antibody cocktail for sorting. DC were isolated based on the expression and co-expression of DC subset-specific combination of surface markers; (AS-DC (CD88-AXL+), pDC (CD88-AXL- CDl lc-CD123+), cDCl (CD88-AXL-CDl lc+CDlc-CD141+), DC2 (CD88-AXL- CD1 lc+CDlc+CD163-), and DC3 (CD88-AXL-CD1 lc+CDlc+ CD163+). DC were isolated using BD FACSAria™ III Cell Sorter (BD Biosciences).
Isolated dendritic cell subsets stimulation
[00412] For studying the properties and functionalities of each dendritic cell subsets, they are stimulated in vitro to trigger immune responses. Stimulation is allowed thought interaction with Toll-Like Receptors (TLR) on the surface of dendritic cells and TLR agonists, such as Poly IC which is a TLR3 agonist (Invivogen, tlrl-pic), R848 which is a TRL7/8 agonist (Invivogen, tlrl-r848) or CpG-C which is a TLR9 agonist (Invivogen, tlrl-2395).
[00413] An immune response can be triggered through, for instance, dendritic cell activation (TLR agonists stimulate dendritic cells, prompting them to mature and become more functional to capture, process, and present antigens to immune cells) or via the production of cytokines and inflammatory factors, such as IP- 10 or IL- 10.
[00414] Thus, purified fractions of DCs subsets are seeded at 5xl05 cells/mL in a round bottom 96-well plate and stimulated in vitro using PolylC, R848 or CpG-C, at concentration of 25pg/ml, Ipg/ml, and Ipg/ml, respectively respectively, in a culture medium comprising RPMI 1640 Medium, GlutaMAX (Life Technologies) with 10% Fetal Calf Serum (Hyclone) lOOU/mL Penicillin/ Streptomycin (Gibco), MEM Non-Essential Amino Acids (Gibco) and 1 mM Sodium Pyruvate (Gibco).
[00415] After 24h stimulation at 37°C and 5% CO2 with saturating humidity, supernatants of samples were collected and DCs cytokines from dendritic cell supernatants were quantified using Cytometric Beads Array (CBA) on BD Fortessa (BD Biosciences). Cells were also recovered for flowcytometry analysis.
DC stimulation analysis by flow-cytometry:
[00416] DC were first stained with Zombie Aqua Fixable Viability Kit (Biolegend, 423102), washed, and then incubated for 30minutes at 4°C with CD80 BV786 (BD, 564159), CD86 BV650 (Biolegend, 305428), HLA-DR BUV395 (BD, 564040). Cells were analyzed on BD Fortessa (BD Biosciences).
Statistical analysis
[00417] Results were obtained from at least n=5 experiments and donors. **, P < 0.01; ***, P < 0.005; Repeated measures one-way ANOVA with Dunn’s post-test. PART 2: THE DENDRITIC CELLS ENRICHMENT METHODS
[00418] In the present example, it is compared the performances of two widely used panDC enrichment kits Miltenyi (ref: 130-100-777) and STEMCELL (ref: #19251), as well as the lab-made dendritic cells enrichment method of the present disclosure. These three methods being negative-selection magnetic-enrichment-based.
[00419] Briefly, several PBMCs samples (50.106 PBMCs) purified from healthy donor blood samples were either left untouched (control) or enriched with dendritic cells using the lab- made dendritic cells enrichment method of the present disclosure or using the commercial kits from Miltenyi (ref: 130-100-777) or from STEMCELL (ref: #19251), according the manufacturer’s instructions.
[00420] Each sample enriched with dendritic cells subsets is subsequently analyzed to determine the frequencies and absolute counts of total dendritic cells subsets (defined as Lineage-negative, HLA-DR-positive), and compared to untouched samples using flowcytometry. Furthermore, the properties and functionalities of each dendritic cell subset in each sample, enriched with the lab-made method, are examined by triggering immune responses in vitro.
StemCell kit protocol (ref: #19251)
[00421] The StemCell kit protocol used for the comparison of the kits follow the instructions of the manufactured protocol “The Big easy” (Catalog #18001) without the optional additional separation).
[00422] Briefly, for the preparation of one sample of 5xl07 PBMCs. The sample is prepared at a concentration of 5xl07/mL of PBMCs. Then, the PBMCs sample is supplemented with 30pL/ml of anti-human CD32 (FcR) Blocker (#14551C) to block undesired immune reactions. Subsequently, 50pl/ml of Pan-DC Pre-Enrichment Cocktail Component A (EasySep™ - #19251C.2) and 50pl/ml DC Enrichment Cocktail Component B (EasySep™ - #19060C) are added to the sample. The sample is then mixed and incubated at room temperature for 30 minutes.
[00423] Magnetic particles (EasySepTM D - #19250) are vortexed for 30 seconds before being added to the sample at a concentration of IOOi.i l/mL. The sample is then mixed and subsequently incubated at room temperature for 10 minutes. After incubating, the sample is top up to 5mL with PBS containing 2% FBS and 1 mM EDTA and mix by gently pipetting up and down 2-3times. Then the sample is placed into the magnet ("the Big Easy" EasySep™ Magnet, #18001) and incubated at room temperature for 5 minutes. After incubating, the magnet is picked up and the enriched dendritic cells are poured off into a new 14 mL tube for further in vitro analysis.
Miltenyi kit protocol (ref: #130-100-777)
[00424] The Miltenyi kit protocol used for the comparison of the kits follow the instructions of the manufactured protocol Order no. 130-100-777 (Miltenyi Biotec B.V. & Co. KG) with the use of LS column (130-042-401) on a QuadroMACS separator (130-090-976).
[00425] Briefly, 0 ,5xl08 PBMC, purchased from EFS, are prepared at a concentration of 108/ml of PBMCs. Then, the PBMCs samples are supplemented with 50pL/ml of human antibody (FcR) Blocker Reagent. Subsequently, lOOpl/ml of Pan DC Biotin- Antibody Cocktail is added to the sample. The sample is then mixed and incubated at room temperature for 5 minutes. After incubation, 400 pl/ml of buffer (PBS supplemented with 0.5% BSA and 2mM EDTA) is added.
[00426] Then, 100 pL/ml of Pan DC MicroBead- Antibody Cocktail are added to the sample. The sample is then mixed and subsequently incubated at room temperature for 5 minutes. After incubating, the PBMCs are washed by adding lOml/ml of buffer and centrifuge at 300xg for 10 minutes. After centrifugation, the supernatant is gently discarded and the PBMC pellet is resuspended at a concentration of 108 cells/ml with buffer.
[00427] Then, the separation of the dendritic cells and non-dendritic cells from the samples is carried out with LS Columns on a QuadroMACS Separator.
[00428] Dendritic cells are recovered and subsequently analyzed in vitro.
Lab-made dendritic cells enrichment protocol
[00429] Firstly, samples, purchased from EFS, are prepared at a concentration of 50xl06 /ml of PBMCs. Then, the PBMCs samples are supplemented with 20pL/ml of Human TruStain FcX (Biolegend, ref: 422302) to block undesired immune reactions. The samples are then shaken to mix the components. Subsequently, 50,24q l/ml of the APC-conjugated antibody combination is added to the PBMCs samples and mix again. The PBMCs samples are then incubated in the dark at 4°C for 10 minutes. After incubating, the PBMCs samples are top up with cold PBS+ and the samples are centrifugated at 450 x g for 5 minutes at 4°C. The supernatant is gently discarded after centrifugation, and the PBMC pellet is resuspended at a concentration of 50xl06 cells/ml with a cold solution of PBS+. [00430] After having stained non-dendritic cells with antibodies of the APC conjugated antibody combination, human anti-APC nanobeads (MojoSort™ - BioLegend - #480090) are vortexed to resuspend before being added to the PBMCs sample at a concentration of 50 pl/ml. The samples are then mixed and subsequently incubated in the dark at 4°C for 15 minutes. After incubating, the free nanobeads within the samples are washed by topping up the samples with cold PBS+ and centrifugated the samples during 5 minutes at 450 x g at 4°C. After centrifugation, the supernatant is gently discarded and the PBMC pellet is resuspended at a concentration of 50xl06 cells/ml with a cold solution of PBS+.
[00431] After having labelled the non-dendritic cells with the magnetic nanobeads, the separation of the dendritic cells and non-dendritic cells from the samples is carried out with either LS Columns on a QuadroMACS Separator (when enriching 50x106 cells), or 2 consecutive times on the AutoMACS® Pro Separator (when enriching lOOOxlO6 cells, Miltenyi Biotech - ref #130-092-545).
[00432] Dendritic cells are recovered and subsequently analyzed in vitro.
PART 3: RESULTS
DC enrichment
[00433] From Figures 1A and IB, it is demonstrated that all the three DC enrichment methods led to significant and similar increased in total DC frequencies in all biological samples following the dendritic cell enrichment of PBMCs samples (48% of DCs with the Lab-made method, 59,9% of DCs with the Miltenyi kit, and 46,7% of DCs with the StemCell kit).
[00434] However, the use of the StemCell kit protocol led to the retrieval of lower counts of total dendritic cells, suggesting a significant loss in total dendritic cell numbers with the method (Figure 1C).
[00435] At the level of each DC subset, we observed that both the Lab-Made and Miltenyi methods led to high recovery of AS-DC (Figure 2), pDC (Figure 3) and cDCl (Figures 4) absolute cell numbers with non-signification loss of cells, as compared to untouched samples, while the use of the StemCell kit method led to significant loss of the aforementioned DC subsets (Figures 2, 3 and 4).
[00436] Although the Miltenyi kit performed better than Lab-Made and StemCell in recovering DC2 cells (Figure 5B), the Lab-Made method shows better performances in recovering total DC3 cells (Figure 5C), particularly the recently identified CD14+DC3 subset (Figure 5D), which was systematically eliminated by the Miltenyi or STEMCELL commercial kits (Figure 5D).
[00437] Altogether, this systematic comparison of the different DC enrichment methods showed major differences in the recovery of DC subsets. Importantly, the Lab-Made DC enrichment method provides better performances in retrieving the recently identified DC3 CD 14+ subset, as compared to the commercial StemCell and Miltenyi DC enrichment methods.
Phenotypical and functional plasticity of the different DC subsets enriched with the Lab-Made enrichment method
[00438] To evaluate the phenotypical and functional plasticity of the different DC subsets enriched with the Lab-Made enrichment method, it was subsequently isolated each DC subsets by flow-cytometry cell sorting, based on the expression and absence of expression of DC subsetspecific surface markers (Figure 6).
[00439] It was stimulated in vitro the purified DC subsets with different Toll-Like Receptors (TLR) agonists, namely PolylC (TLR3 agonist), R848 (TRL7/8 agonist) and CpG-C (TLR9 agonist), or without any stimulated agonist (control medium - Figure 6).
[00440] pDC
[00441] The expression of co-stimulatory molecules CD80 and CD86 by pDCs is observed upon stimulation with R848 and CpG-C, but not following Poly-IC stimulation (Figures 7A and 7B). This observation aligns with the absence of TLR3 expression by pDCs (Liu, Annual Review of Immunology 2005 23:1, 275-306
[00442] Activation of pDCs was associated with the secretion of the chemokine IP- 10 in response of R848 and CpG-C stimulation (Figure 8B). No secretion of IL- 10 in response to Poly IC, R848 or CpG-C is observed (Figure 8A).
[00443] DC2
[00444] The expression of co-stimulatory molecules CD80 and CD86 by DC2 is observed upon stimulation with Poly IC and R848, but not following CpG-C stimulation (Figures 7A, and 7B). Activation of DC2s was associated with the secretion of the chemokine IP- 10 in response to Poly IC (Figure 8B), and with the secretion of IL-10 in response to Poly IC and R848 (Figure 8A).
[00445] DC3
[00446] The expression of co-stimulatory molecules CD80 and CD86 by DC3 is observed upon stimulation with Poly IC and R848, but not following CpG-C stimulation (Figures 7A, and 7B). Activation of DC3s was associated with the secretion of the chemokine IP-10 in response to Poly IC (Figure 8B), and with the secretion of IL- 10 in response to R848 (Figure 8A).
[00447] Consequently, it was previously stated in the art that DC2 differed from DC3 by the constitutive expression of CD86. However, it is shown that both DC2 and DC3 subsets overexpressed CD80 and CD86 after stimulation with Poly-IC and R848 (Figure 6, 7A and 7B). This observation aligns with the absence of TLR9 expression by both DC2 and DC3 subsets (Hornung et al. J Immunol. 2002.).
[00448] In contrast to pDCs, both DC2 and DC3 subsets were capable of secreting IL- 10 (in response to R848) or IP-10 (in response to Poly IC) (Figure 8A, 8B), although DC3s secreted higher levels of these two molecules than DC2s. Furthermore, the DC2 subset differed from DC3 subset by their capability to secrete IL-10 in response to PolylC (Figure 8A), suggesting functional differences between those DC2 and DC3 subsets depending on the type of stimulation.
PART 4: CONCLUSIONS
[00449] To conclude, the comparison between two widely utilized commercial DC- enrichment kits and the Lab-Made DC-enrichment method outlined in this disclosure highlighted significant disparities in recovering diverse contemporary human dendritic cells populations.
[00450] While the two commercial kits resulted in significant loss of the recently identified DC3 subset, the Lab-Made DC-enrichment method effectively retrieved this particular DC3 subset enabling downstream purification and functional analysis. Stimulation of the different DC populations with TLR agonists revealed both phenotypical and functional diversification among DC subsets, highlighting shared and subset-specific responses.
[00451] Hence, the lab-made method of the disclosure is a solution to enrich biological cell samples with all the DC subsets, in particular AS-DC, pDC, cDCl, DC2 and DC3 including CD 14+ DC3.
[00452] Furthermore, the lab-made method of the disclosure allows enriching biological cell samples with all dendritic cell subsets without causing a significant loss in total cell numbers.
EXAMPLE 2: Lab-made enrichment method upscaling test
[0001] In the present example, it is compared the use of the lab-made method on a sample concentration of 50.106 PBMC and L000.106 PBMC.
[0002] After anti-APC magnetic beads labelling with the lab-made method, 50.106 PBMC were enriched using Miltenyi LS Columns (130-042-401, 1 passing), and L000.106 PBMC using Miltenyi Automacs Pro (130-092-545, 2 passings). [0003] From figure 9, it is shown that the lab-made method demonstrates consistent performance, whether applied to samples with 50.106 PBMC or l,000.106 PBMC.
EXAMPLE 3: Anti-CD88 and Anti-CD89 antibodies usage in the antibody combination to enrich a sample in all the dendritic cell subsets
[0004] In the present example, it is compared three antibody combinations as described in Table 1 (comprising anti-CD3 antigen antibodies, anti-CD56 antigen antibodies, anti-CD16 antigen antibodies, anti-CD19 antigen antibodies, anti-CD15 antigen antibodies, anti-CD41 antigen antibodies and anti-CD235a antigen antibodies) wherein it comprised an anti-CD88 antibody, or both the anti-CD88 and anti-CD89 antibodies; or an anti-CD89 antibody.
[0005] PBMC of healthy donors were either left untouched or Pan-DC enriched using the lab-made method with the antibody combinations comprising either an anti-CD88 antibody alone (20pl for 50.106 PBMC), anti-CD89 antibody alone (20pl for 50.106 PBMC), or a combination of both the two anti-CD89 and anti-CD88 antibodies (lOpl of each for 50.106 PBMC) to deplete monocytes.
[0006] The lab-made method is described in example 1 above.
[0007] From figure 10, it is shown that dendritic cells are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti- CD88 and anti-CD89 antibodies.
[0008] From figure 11, it is shown that AS-DCs are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies.
[0009] From figure 12, it is shown that pDCs are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies.
[0010] From figure 13, it is shown that cDCls are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies.
[0011] From figure 14, it is shown that DC2s are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies. [0012] From figure 15, it is shown that DC3s are equally enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies.
[0013] From figure 16, it is shown that the newly identified CD 14+ DC3 subset is more significantly enriched with the use of only anti-CD88 antibodies in the antibody combination compared to the use of both anti-CD88 and anti-CD89 antibodies or the use of anti-CD89 antibodies alone.
[0014] In conclusion, the lab-made method is more efficient for enriching a human cell sample with all the dendritic cell subsets with the use of an antibody combination comprising anti-CD88 antibodies without anti-CD89 antibodies.
[REFERENCES]
Donne Estipona. Biocompare, A Guide to T Cell Markers. An overview of phenotypic markers for T lymphocytes. November 13, 2020
Fuss IJ, Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2009 Apr;Chapter 7:7.1.1-7.1.8. doi: 10.1002/0471142735.im0701s85. PMID: 19347849.
Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, Segura E, Tussiwand R, Yona S. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol. 2014 Aug;14(8):571-8. doi: 10.1038/nri3712. Epub 2014 Jul 18. PMID: 25033907; PMCID: PMC4638219.
Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdbrfer B, Giese T, Endres S, Hartmann G. Quantitative expression of toll -like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol. 2002 May l;168(9):4531-7. doi: 10.4049/jimmunol.l68.9.4531. PMID: 11970999.
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Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB. Comprehensive Phenotyping of T Cells Using Flow Cytometry. Cytometry A. 2019 Jun;95(6):647-654. doi: 10.1002/cyto.a.23724. Epub 2019 Feb 4. PMID: 30714682.
Puleo A, Carroll C, Maecker HT, Gupta R. Isolation of Peripheral Blood Mononuclear Cells Using Vacutainer® Cellular Preparation Tubes (CPT™). Bio Protoc. 2017 Jan 20;7(2):e2103. doi: 10.21769/BioProtoc.2103. PMID: 34458433; PMCID: PMC8376597.
Villani AC, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J, Griesbeck M, Butler A, Zheng S, Lazo S, Jardine L, Dixon D, Stephenson E, Nilsson E, Grundberg I, McDonald D, Filby A, Li W, De Jager PL, Rozenblatt-Rosen O, Lane AA, Haniffa M, Regev A, Hacohen N. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017 Apr 21;356(6335):eaah4573. doi: 10.1126/science.aah4573. PMID: 28428369; PMCID: PMC5775029.

Claims

[CLAIMS]
1. An antibody combination, for enriching a human cell sample with dendritic cells, comprising:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
2. The antibody combination according to claim 1 , wherein the antibody combination further comprises (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen; and optionally (viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
3. The antibody combination according to claim 1 or 2, wherein the antibody combination comprises (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen; (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen; (iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen; (v) an antibody or an antigen-binding fragment thereof directed against the CD19 antigen; and (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen.
4. The antibody combination according to any one of claims 1 to 3, wherein the antibody combination comprises (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen; (ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen; (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen; (iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen; (v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen; (vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen; (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen; and (viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
5. The antibody combination according to any one of claims 1 to 4, wherein each antibody or its antigen-binding fragment thereof is independently formulated in the combination in a concentration ranging from about 0,001 pg to about 5 pg, per about 50xl06 cells.
6. The in vitro use of the antibody combination according to any one of claims 1 to 5, in a method for preparing a human cell sample enriched in dendritic cells.
7. An in vitro method for preparing a human cell sample enriched in dendritic cells comprising at least the steps of:
(a) reacting a human cell sample with the antibody combination according to any one of claims 1 to 5, under conditions suitable for the binding of each antibody or its antigen-binding fragment thereof to the cells expressing at their surface one or more antigens against which the respective antibodies are directed;
(b) removing the cells bound by the antibodies from the human cell sample; and
(c) recovering a human cell sample which is enriched in dendritic cells.
8. The method according to claim 7, wherein the human cell sample is in a cell-culture medium for human cells suitable for isolating the dendritic cells in the human cell sample, and, optionally, wherein the cell-culture medium comprises dulbecco's phosphate-buffered saline (DPBS) and ethylenediaminetetraacetic acid (EDTA); and, optionally, bovine serum albumin (BAS) and/or heat inactivated human serum.
9. The method according to claim 7 or 8, wherein each antibody or its antigen-binding fragment thereof comprised in the combination is labelled with a detectable molecule and/or is conjugated to magnetic beads.
10. The method according to any one of claims 7 to 9, or the use according to claim 6, wherein the human cell sample enriched in dendritic cells includes at least Axl+ Siglec6+ dendritic cells (AS-DCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells 1 (cDCls), dendritic cells 2 (DC2s) and/or dendritic cells 3 (DC3s).
11. The method according to any one of claims 7 to 10, or the use according to claim 6 or 10, wherein:
- AS-DCs express at least the Axl antigen,
- pDCs express at least one antigen among the CD 123 antigen and the CD303 antigen, and optionally the CD304 antigen;
- cDCls express at least one antigen among the CD141 and CLEC9A antigens, and optionally at least the XCR1 antigen;
- DC2s express at least one antigen among the CDlc and CDllc antigens, and optionally at least one antigen among the CD172a and CDllb antigens, and lack the expression of the CD163 antigen; and
- DC3s express at least both the CD163 and CDllc antigens, and optionally at least one antigen among the CDlc antigen, the CD172a antigen, and the CD14 antigen.
12. The method according to any one of claims 7 to 11, or the use according to claim 6, 10 or 11, wherein the human cell sample enriched in dendritic cells includes at least dendritic cells expressing the CDllc, CD163 and CD14 antigens; and optionally the said dendritic cells further secrete interleukin-1 beta (IL-10) and/or interferon gamma-induced protein 10 (IP-10).
13. The method according to any one of claims 7 to 12, or the use according to claims 6 or 10 to 12, wherein the human cell sample consists of a whole blood sample or a peripheral blood mononuclear cell (PBMC) fraction thereof.
14. A kit-of-part useful for enriching a human cell sample in dendritic cells, including:
(A) (i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/0 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen, (iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen,
(iv) an antibody or an antigen-binding fragment thereof directed against the CD 16 antigen,
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen, and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen; and
(B) instructions for preparing a human cell sample enriched in dendritic cells.
15. Use of an antibody or an antigen-binding fragment thereof directed against the CD 88 antigen, and optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen, in a method for preparing a human cell sample enriched in dendritic cells.
16. The antibody combination according to any one of claims 1 to 5, wherein the antibody combination is comprised in a composition.
17. An antibody combination, for enriching a human cell sample with dendritic cells, consisting of:
(i) an antibody or an antigen-binding fragment thereof directed against the CD3 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRa/p antigen, and/or an antibody or an antigen-binding fragment thereof directed against the TCRy/6 antigen;
(ii) an antibody or an antigen-binding fragment thereof directed against the CD88 antigen and, optionally an antibody or an antigen-binding fragment thereof directed against the CD89 antigen;
(iii) an antibody or an antigen-binding fragment thereof directed against the CD56 antigen;
(iv) an antibody or an antigen-binding fragment thereof directed against the CD16 antigen;
(v) an antibody or an antigen-binding fragment thereof directed against the CD 19 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD20 antigen; and
(vi) an antibody or an antigen-binding fragment thereof directed against the CD 15 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD66b antigen.
18. The antibody combination according to claim 1, wherein the antibody combination further includes (vii) an antibody or an antigen-binding fragment thereof directed against the CD41 antigen, and/or an antibody or an antigen-binding fragment thereof directed against the CD61 antigen; and optionally (viii) an antibody or an antigen-binding fragment thereof directed against the CD235a antigen.
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