WO2001036978A1 - Vaccins cellulaires : techniques de production, d'optimisation et de controle de la qualite - Google Patents

Vaccins cellulaires : techniques de production, d'optimisation et de controle de la qualite Download PDF

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Publication number
WO2001036978A1
WO2001036978A1 PCT/US2000/042213 US0042213W WO0136978A1 WO 2001036978 A1 WO2001036978 A1 WO 2001036978A1 US 0042213 W US0042213 W US 0042213W WO 0136978 A1 WO0136978 A1 WO 0136978A1
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cell
peptide
cells
major histocompatibility
histocompatibility complex
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PCT/US2000/042213
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English (en)
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Joachim L. Schultze
Robert H. Vonderheide
Lee M. Nadler
Britta Maecker
Michael Von Bergwelt-Baildon
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Dana-Farber Cancer Institute, Inc.
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Publication of WO2001036978A1 publication Critical patent/WO2001036978A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5052—Cells of the immune system involving B-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • This invention relates to cellular vaccines.
  • Immunization using a cellular vaccine is carried out by using an antigen presenting cell (APC) that has been "pulsed" with an antigen to stimulate T cells to be specific for that antigen.
  • APC antigen presenting cell
  • APCs stimulate T cells specific for a particular antigen by presenting the antigen, or a peptide of the antigen, on the surface of the APC in a complex with a major histocompatibility complex protein.
  • the invention provides a method for determining the cell surface density of a major histocompatibility complex protein (class I or class II) or other relevant cell surface molecules including but not limited to adhesion and costimulatory molecules such as CD54, CD58, CD80, and CD86 on a primary (e.g., a dendritic cell or a CD40-activated B cell) or artificial (e.g., a bead to which is bound a major histocompatibility complex protein) antigen presenting cell.
  • a primary e.g., a dendritic cell or a CD40-activated B cell
  • artificial e.g., a bead to which is bound a major histocompatibility complex protein
  • This method involves (a) providing a primary or artificial antigen presenting cell; (b) determining the cell surface area of the primary or artificial antigen presenting 5 cell; (c) determining the absolute amount of the major histocompatibility complex protein or other molecules (see above) on the surface of the primary or artificial antigen presenting cell; and (d) determining the ratio of the absolute amount to the cell surface area as a measure of the cell surface density.
  • Also included in the invention is a method for determining the cell surface l o density of a complex of a peptide and a major histocompatibility complex protein (class I or class II) on a primary (e.g. , a dendritic cell or a CD40-activated B cell) or artificial (e.g. , a bead to which is bound a major histocompatibility complex protein) antigen presenting cell.
  • This method involves (a) providing a primary or artificial antigen presenting cell; (b) determining the cell surface area of the
  • the invention also includes a kit for determining the cell surface density of a complex of a peptide and a major histocompatibility complex protein on a primary or artificial antigen presenting cell.
  • This kit can include (a) a means for detecting a major histocompatibility complex protein on the surface of a primary or artificial antigen presenting cell; and (b) a means for detecting a peptide in a 5 complex with a major histocompatibility complex protein on the surface of a primary or artificial antigen presenting cell.
  • the kit can also include a reference peptide that can be used as a positive control for peptide binding to a major histocompatibility complex protein, as well as ⁇ -2 microglobulin.
  • Also included in the invention is a method for identifying a compound that 0 increases the persistence of a major histocompatibility complex antigen (class I or class II)/peptide complex on the surface of a cell (e.g., a primary cell, such as a dendritic cell or a CD40-activated B cell) or an artificial antigen presenting cell.
  • a cell e.g., a primary cell, such as a dendritic cell or a CD40-activated B cell
  • an artificial antigen presenting cell e.g., a cell that 0 increases the persistence of a major histocompatibility complex antigen (class I or class II)/peptide complex on the surface of a cell (e.g., a primary cell, such as a dendritic cell or a CD40-activated B cell) or an artificial antigen presenting cell.
  • This method involves culturing the cell in the presence of the compound, and determining the length of time that the complex persists on the cell surface
  • the invention also includes a method of determining whether an antigen presenting cell (e.g., a dendritic cell, a CD40-activated B cell, or an artificial APC) is presenting an amount of peptide antigen that is sufficient for use of the cell in therapy.
  • an antigen presenting cell e.g., a dendritic cell, a CD40-activated B cell, or an artificial APC
  • This method involves determining the cell surface density of a complex of the peptide and a major histocompatibility complex protein.
  • l o Detection of a density of 100 molecules/ ⁇ m 2 or more identifies a cell that is sufficient for use in therapy.
  • polypeptide is a chain of amino acids linked to one another by peptide bonds.
  • a “protein” can be made up of one or more polypeptides, while a “peptide” is generally understood to be (or include) a 15 fragment of a polypeptide, and to consist of a chain of peptide bond-linked amino acids that is shorter in length than a full length polypeptide from which it may be derived.
  • an "antigen” is used herein to describe a molecule, such as a polypeptide, protein, peptide, or other molecule, that, when contacted with cells of the immune 0 system, ex vivo, in vitro, or in vivo, is capable of inducing an immune response specific for the antigen.
  • primary cell is meant a cell that is isolated from a subject and that is not capable of being propagated indefinitely in culture. That is, a primary cell is one that is not immortalized, for example, by prolonged culture or transformation 5 with a foreign nucleic acid molecule that results in immortalization.
  • antigen presenting cell or “APC” is meant any cell that presents an antigen in the context of MHC on its cell surface, and that can induce antigen- specific, MHC-restricted T cells, such as cytotoxic T cells (CTLs).
  • CTLs cytotoxic T cells
  • APCs that can be used in the invention are activated B cells, activated T cells, 0 monocytes, macrophages, and dendritic cells.
  • APCs “professional APCs,” cells (e.g., epithelial cells or endothelial cells) that do not usually express MHC can be stimulated to do so, and thus can act as APCs, by treatment with cytokines, such as IFN- ⁇ and TNF.
  • cytokines such as IFN- ⁇ and TNF.
  • an "artificial APC” (and more throughout the manuscript) is a structure or scaffold that has an MHC protein and/or adhesion and costimulatory molecules on its surface, such that it can productively stimulate a T cell specific for an antigen when the MHC protein is bound to a peptide of the antigen.
  • artificial APCs are beads or lipid mycels coated with purified MHC proteins that are capable of binding to a specific peptide.
  • the APCs used in the invention are primary APCs or artificial APCs.
  • histocompatibility antigen is meant a molecule, such as a major histocompatibility complex (MHC) class I, MHC class II, or minor histocompatibility antigen, that mediates interactions of cells of the immune system with each other and with other cell types.
  • MHC class I antigens in APCs present processed antigens to cytotoxic T lymphocytes (CTLs), thus mediating antigen-specific, MHC-restricted CTL activation.
  • CTLs cytotoxic T lymphocytes
  • MHC proteins are of two classes, class I and class II, which differ from one another in structure and function.
  • MHC class I proteins consist of a membrane- spanning subunit having a large extracellular region, and this subunit associates with a second subunit, ⁇ -2 microglobulin.
  • MHC class II proteins consist of two membrane spanning subunits.
  • histocompatibility antigens include MHC class I antigens, such as HLA-A (e.g., Al , A2, A3, Al 1 , A24, A31, A33, and A38), HLA-B, and HLA-C, MHC class II antigens, such as HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, and HLA-DP, and minor histocompatibility antigens, such as HA-1.
  • generating CTLs is meant an in vivo, in vitro, or ex vivo process by which antigen-specific CTLs are activated (e.g., stimulated to grow and divide) and/or selected.
  • a peptide of an antigen is said to "specifically bind" to an MHC protein if the peptide adheres to a MHC protein under physiological conditions.
  • binding can be similar to that of a peptide antigen that is naturally processed and presented in the context of MHC in an antigen presenting cell.
  • cytotoxic T lymphocyte or antibody is said to "specifically recognize” an antigen or a peptide of an antigen if it binds to the antigen or peptide, but does not substantially bind to other, unrelated polypeptides or peptides.
  • a CTL is said to "specifically kill” a cell if it specifically recognizes and lyses a cell that expresses an antigen to which it has been activated, but does not substantially recognize or lyse cells not expressing the antigen.
  • Such a CTL is designated as a "antigen-specific CTL" herein.
  • an antigen (or a peptide of an antigen) is said to be "presented” if a peptide of the antigen is displayed on the extracellular surface of a cell (e.g., an antigen presenting cell), such that it can result in the in vivo, ex vivo, or in vitro generation of antigen-specific CTLs or the lysis of a cell expressing the antigen (e.g., a tumor cell) by an antigen-specific CTL.
  • the displayed antigen peptide is bound to a histocompatibility antigen.
  • the invention provides several advantages. For example, it facilitates the identification of conditions for generating optimal antigen-pulsed APCs, such as primary or artificial APCs, for use in therapeutic methods. It also provides a method for quality control of APCs to be used in therapy.
  • APCs can be, for example, dendritic cells and CD40-act ⁇ vated B cells, as well as any other cell that acts as an antigen presenting cell.
  • cells such as fibroblasts, endothehal cells, or tumor cells can be engineered and/or cultured under conditions that result in their having antigen presenting cell properties.
  • the method of the invention facilitates identification of such cells, optimization of their antigen presenting properties, and quality control of such cells for use in therapy.
  • the invention also enables the identification of compounds that enhance the therapeutic quality of antigen-pulsed APCs Such compounds can be used, for example, to treat an antigen-pulsed APC prior to administration and/or be administered with an antigen-pulsed APC.
  • Fig. 1 is a graph showing induction of CTL responses against peptides derived from viral and tumor antigens using CD40-B cells as APC. Specific lysis of T2 cells pulsed with (A) influenza MP58 peptide, (B) hTERT 1540 peptide, (C) HIN RTpol476 peptide, or (D) CD40-B cells pulsed with the hepatitis B core F18 peptide is shown here. Target cells were pulsed with specific peptide (•), irrelevant HLA-A2 binding control peptide (O) or no peptide (D).
  • Fig. 2 is a graph showing expansion of CD40-B cells.
  • A Overall expansion of 53 cultures from healthy individuals (red) and cancer patients (green). CD40-B cells were generated using CD40L transfected ⁇ IH3T3 cells and rhIL-4.
  • B, C Cultures from 11 healthy individuals (red) and 8 cancer patients (green) were grown in (B) pooled AB serum or (C) autologous plasma and expansion was normalized for CD19 + CD20 + cells.
  • Fig. 3 is a graph showing substitution of xenogeneic CD40L transfectants with recombinant, trimeric, soluble CD40L.
  • the generation of CD40-B cells was studied using (A) CD40L transfected NIH3T3 cells or (B) GMP-grade soluble trimeric CD40L under varying concentrations of clinical-grade rhIL-4 (shown here: 0 (•), 2 ( ⁇ ) and 10 ( ⁇ ) ng/ml). A representative experiment of three experiments is shown.
  • Fig. 4 is a graph showing quantification of cell surface molecules.
  • CD40- B cells were generated using tCD40L or sCD40L in the presence of IL-4 and Cs A and were coincubated with or without INF- ⁇ (added for four days before analysis). DC were generated by culture of monocytes with GM-CSF and IL-4.
  • A Surface expression of CD58, CD80, and MHC class I and MHC class II was measured by flowcytometry and normalized using the Quifikit ® to obtain the antigenic binding sites (ABS) per cell.
  • B Antibody binding sites per cell surface area was calculated to normalize for different sizes of different cell types or culture conditions. Median (lines), range (bars) and 95 th percentile (boxes) of five to nine experiments for each culture condition and cell surface molecule are shown.
  • Fig. 5 is a graph showing antigen loading of CD40-B cells.
  • A Specific binding of hepatitis B core peptide was demonstrated using HLA-A * 0201 + (left) and HLA-A * 0201 " (right) CD40-B cells. As negative control, auto fluorescence of CD40-B cells and isotype control is shown. Staining for MHC class I was used as positive control. Identical imaging parameters were used throughout the experiment.
  • B Binding affinity of the HLA-A2 binding peptides 1540 (•), F18 ( ⁇ ), and the pan-DR binding peptide PADRE ( ⁇ ) to CD40-B cells was determined by competition with the hepatitis B core F18-FITC reference peptide.
  • Fig. 6 is a graph showing that immature dendritic cells (DC) have a higher absolute expression of CD58 than the types of CD40-activated B cells under study.
  • DC dendritic cells
  • Fig. 7 is a graph showing that immature dendritic cells (DC) have a higher absolute expression of CD80 than CD40-activated B cells.
  • Fig. 8 is a graph showing that immature dendritic cells (DC) have a higher absolute expression of MHCI than CD40-activated B cells.
  • Fig. 9 is a graph showing that immature dendritic cells (DC) and CD40- activated B cells have similar surface expression of MHCII.
  • Fig. 10 is a graph showing that CD40-activated B cells and dendritic cells (DC) have a comparable density of CD58 molecules.
  • Fig. 11 is a graph showing that CD40-activated B cells have a higher density of surface CD80 molecules than dendritic cells (DC).
  • Fig. 12 is a graph showing that CD40-activated B cells have a higher density of surface MHCI than dendritic cells (DC).
  • Fig. 13 is a graph showing that CD40-activated B cells have a higher density of surface MHCII than dendritic cells (DC).
  • Fig. 14 is a graph showing a competition assay using HepB-FITC as a reference peptide. Inhibition of PadreAKX (square), 1540 (diamond), and HepB core F18 (sphere) on CD40-activated B cells.
  • antigen-pulsed APCs can be used as, or in the manufacture of, cellular vaccines.
  • antigen-pulsed APCs can be used ex vivo to stimulate antigen-specific T cells, which are administered to patients to induce an immune response against a cell (e.g., a tumor cell) or an organism (e.g., a viral or parasitic pathogen) that expresses the antigen.
  • antigen-pulsed APCs can be administered to patients directly, in whom they stimulate antigen- specific T cells.
  • APCs that present antigen/MHC complexes in a manner and in amounts that induce optimal T cell responses.
  • the methods and kits of the invention can readily be used for such quality control.
  • the invention includes a method for determining whether an APC has a sufficient cell surface density of MHC/peptide complex for use in therapy.
  • an APC has a cell surface MHC/peptide complex density of about 100 molecules/ ⁇ m 2 or more.
  • cells having the highest density possible, as determined using the methods of the invention can be used.
  • the methods and kits of the invention can also be used in research and development applications. For example, as antigens are discovered to have utility in lmmunotherapeutic methods, such as those described above, optimal conditions for producing APCs that present them can be determined using the methods and kits of the invention. For example, the condition variables described below can be used as guidance in the determination of optimal conditions for generating APCs for such antigens.
  • the invention also includes methods for identifying agents that enhance the quality of APCs.
  • an agent that maximizes the length of time that a peptide-MHC complex persists on the surface of a cell can be identified using the methods of the invention.
  • an APC presenting an antigen in the context of MHC is cultured in the presence of a candidate compound (e g., a small organic or inorganic molecule), and the length of time that the antigen/MHC complexes persists on the cell surface is compared to a control culture, lacking the compound.
  • a compound that is identified as enabling the complexes to persist longer on the surface than controls can then be tested in animal model systems for its efficacy in potentiating the effects of the cellular vaccination methods described herein.
  • compounds that act similarly to Brefeldin A which blocks the activity of the Golgi apparatus, and thus results in the persistence of existing MHC molecules on the cell surface, can be identified and used in this manner.
  • Compounds identified using this method can be used, for example, to treat APCs before use in activating T cells (ex vivo, in vitro, or in vivo), or can be administered with APCs to patients.
  • the density of the MHC class I or MHC class II molecules on the surface of an APC can be calculated by dividing the absolute amount of antigenic sites on the surface of the APC by the cell surface area of the APC
  • the cell surface area of an APC can be determined using an apparatus capable of determining cell numbers and cell size or volume, e.g. , a Coulter Counter.
  • the absolute amount of MHC molecules on the surface of a primary or artificial APC can be determined by using a detectably-labeled (e.g., fluorescent) MHC class I or MHC class II antibody, added in combination with a fluorescent antibody to a lineage-specific marker.
  • the number of fluorescent signals emitting l o from the cells can be analyzed using beads with standardized numbers of fluorochrome (e.g, the DAKO Quifikit).
  • the "setup beads,” provided with the DAKO Quifikit, can be analyzed using a flow cytometer to determine populations of fluorescently-labeled and unlabeled beads.
  • the "calibration beads” also provided with the DAKO Quifikit), which consist of five distinct fractions with
  • MFI mean fluorescent intensity
  • the APCs can then be analyzed using the same settings.
  • the MFI of the cells can be converted to the number of antigenic sites using the calibration beads for normalization.
  • the absolute amount of surface molecules binding the fluorescent antibody on the cells can be calculated 0 by entering the five pairs of MFI-number of antigenic sites data into a software program.
  • Cells other than the APCs of interest can be excluded from this analysis by gating on the cells binding the second antibody, specific for a lineage-specific marker.
  • the absolute amount of MHC molecules associated with a peptide on the 5 surface of a primary or artificial APC can be determined by contacting an APC with a detectably-labeled peptide, added in combination with an antibody to a lineage-specific marker, and analyzing the fluorescence emissions as is described above.
  • the peptide can be biotinylated, in which case a fluorescent Streptavidin antibody can be used for detection of the peptide.
  • the peptide can 0 be directly labeled with a fluorescent conjugate, e.g. , FITC/PE.
  • the APCs can also be co-incubated with or without a second, unlabeled peptide in a competition assay.
  • the efficiency of peptide presentation can be calculated by subtracting the MFI of APCs incubated with a competing peptide from the MFI of APCs incubated without the competing peptide.
  • APCs that can be used in the invention include, for example, CD40-activated B cells, monocytes, macrophages, and dendritic cells.
  • cytokine e.g., IFN- ⁇ or TNF
  • the method of the invention can be used with any cell that naturally has, or is engineered or induced to have, antigen presenting capabilities.
  • tumor cells can be engineered or induced to have such capabilities, and thus can be subject of the methods of the invention.
  • cells used in the method of the invention are primary cells, and cells for use in the invention can be isolated by standard methods that are well known in the art.
  • an artificial antigen presenting cell can be used in the invention.
  • Artificial antigen presenting cells can be generated by, for example, attaching purified MHC molecules, capable of binding to a specific peptide, to a structure or scaffold, such as a bead. Techniques for attaching purified molecules to supports, such as beads, which may be inert or bioactive, are well known in the art.
  • Antigens and peptides can be obtained from natural sources or can be synthesized chemically. Antigen or peptide concentrations used to pulse APCs can vary from 0.001 ⁇ g/ml to 1,000 ⁇ g/ml. Preferably, the antigens or peptides are known to stimulate T cell expansion when presented in the context of an MHC protein. If a particular antigen or peptide does not induce a T cell response due to, for example, low binding affinity to an MHC molecule or poor MHC-peptide complex stability, a corresponding heteroclitic peptide can be used. Heteroclitic peptides are peptides that induce T cells with similar specificity as a peptide of interest, but have a higher binding affinity to MHC molecules.
  • these peptides induce stronger T cell responses. Since the biology of binding of heteroclitic peptides to an MHC molecule can differ from the native, corresponding peptide, the parameters indicated below can be tested in a similar fashion.
  • the interaction of a peptide with any given MHC molecule is unique, and therefore the optimal conditions for contacting an APC with a peptide, and the stability of a peptide/MHC molecule complex can vary for different peptides. Identification of optimal conditions can be achieved by varying the parameters described below. These parameters influence the process of peptide binding, as well as the stability of the peptide/MHC complex, which is important for the immunogenicity of an APC loaded with an antigenic peptide.
  • the duration of incubation of an exogenous peptide with an APC is critical to the final amount of peptide that binds to a MHC molecule. This time can range, in general, between 10 minutes and 72 hours (e.g., 6 hours). It can be desirable to dissociate existing MHC/peptide complexes prior to incubation of an APC with a peptide of interest. Such dissociation can be achieved by maintaining the APC at a temperature other than 37 °C. In particular, the APC can be incubated at temperatures lower than 37°C, for example, from 4°C to 32°C. During incubation with the peptide of interest, the temperature can be varied to be optimal for the specific peptide.
  • the temperature range to be analyzed for each individual peptide, to optimize peptide loading will vary between 4°C and 37 °C.
  • the stability of a peptide/MHC complex should also be monitored after the incubation of an APC with a peptide, for up to 24 hours, at a temperature range of 4°C to 37°C. Loading of a specific peptide onto an MHC molecule can also be influenced by the composition of the culture medium used.
  • the influence of serum components, proteins, including proteases, or hpids on the stability of a peptide/MHC-complex can also be monitored. This can be done by incubating an APC, loaded with peptide, in culture medium containing different amounts of supplements (whole serum, proteins, or hpids) for up to 24 hours to determine optimal complex stability.
  • the efficiency and stability of peptide binding to an MHC molecule can also depend on the pH and CO 2 concentration during incubation of an APC with a peptide of interest. By varying CO 2 saturation concentration (range 2-10%), or by adding weak acids or bases, it can be determined whether peptide loading can be increased by varying the pH.
  • MHC class I complexes are composed of a peptide, an alpha chain, and ⁇ -2 microglobulin.
  • Exogenously added ⁇ -2 microglobuhn can increase peptide binding under certain conditions and for particular peptides
  • the above-described methodology can be used to monitor whether the binding of a peptide of interest is significantly increased by the addition of exogenous ⁇ -2 microglobulin Changing one parameter may affect peptide binding to an MHC molecule on an APC. Therefore, once optimal conditions for one parameter have been defined, these conditions can be further optimized by varying the other parameters. Data relating to variation of parameters of the methods of the invention are provided below.
  • kits of the invention can include any combinations of the reagents discussed below.
  • the kits can include MHC class I and/or MHC class II antibodies, which are used to determine the number of particular MHC molecules on the surface of an APC.
  • the kits can also include, as controls, beads that have known numbers of antigenic sites and that bind fluorescently labeled antibodies.
  • MHC class I and MHC class II peptides e.g., HIN gag77, pol, influenza, tetanus, or PADRE
  • These peptides can be detectably-labeled, for example, they can be biotinylated.
  • the kit can include ⁇ -2 microglobulin for determination of the effect of this agent on peptide binding; reagent buffers and media; serum- free media for incubation; PBS for wash steps; buffers to vary the culture conditions, e.g. weak acids or basis to change pH; paraformaldehyde for cell fixation; 96-well plates; and software to support the analysis.
  • CD40-B cells were used as the sole APC to induce CTL responses to a wide range of antigens, including the tumor antigen human telomerase reverse transcriptase (hTERT, 6/7 donors), the viral antigens influenza A matrix protein (3/4), RT-pol from HIN (4/7), hepatitis B virus core antigen (1/1), and human papilloma virus 16 E6 protein (1/1).
  • the generation of CTL against the influenza A-derived peptide MP58 is a typical example for a memory response.
  • CD40-B cell system can prime naive T cells
  • CTL against viral neo-antigens from HIV and HBN were generated from sero-negative healthy individuals
  • CTL were raised against the RTpol476 peptide from HIN (Fig. 1C) and the F18 peptide from the hepatitis B core protein (Fig ID).
  • These CTL showed significant peptide-specific cytotoxicity against target cells loaded with RTpol476 (Fig. 1C) or F18 (Fig. ID) peptide after four ex vivo stimulations.
  • Direct visualization of peptide-specific CTL by tetramer analysis revealed that 3.79% of all CD8 + T cells m the expanded T cell pool were specific for RTpol476 (Fig. 1G).
  • CD40-B cells boost viral antigen-specific memory T cells, break T cell ignorance to the tumor antigen telomerase, and can also prime neo-antigen specific T cells ex vivo
  • PBMC peripheral blood mononuclear cells
  • CD40-B cells For therapeutic use of CD40-B cells, it is desirable to avoid the use of culture medium supplemented with pooled human AB serum.
  • Fig. 2B In seven cancer patients we substituted the pooled serum (Fig. 2B) with autologous plasma (Fig. 2C) and demonstrated no statistically significant difference of B cell expansion, suggesting that plasma can be used instead of pooled AB serum in this system.
  • Replacing the xenogeneic component (tCD40L) of the CD40-B cell system with another source of CD40L would significantly improve its clinical applicability.
  • sCD40L GMP-grade human trimeric soluble CD40L
  • sCD40L In the presence of recombinant IL-4 and cyclosporin A (CsA), sCD40L induced a significant B cell expansion (3 orders of magnitude), which did not differ from that observed for B cells stimulated with tCD40L (Fig. 3).
  • IL-4 was an important co-factor, since B cell expansion in cultures with tCD40L or sCD40L alone did not expand over the whole culture period and could not be substituted by other cytokines including IL-6 or TNF- ⁇ . Further increasing the concentration of IL-4 (>2 ng/ml) did not lead to increased B cell expansion (Fig. 3). Determination of cell surface molecules as surrogate for APC function ofCD40-B cells
  • CD40-B cells cannot be routinely used as an assay to control for efficient APC function of these cells, particularly in a clinical setting. Since sufficient expression of MHC, adhesion, and costimulatory molecules closely correlates with APC function (Bluestone, Immunity 2:555-559, 1995; Croft et al, C ⁇ t. Rev. Immunol. 17:89-118, 1997), phenotypic analysis of these cell surface molecules was applied as a fast and reliable surrogate readout for APC function of CD40-B cells.
  • CD40-B cells After normalization for cell size, however, the density of all four cell surface molecules was not significantly different on CD40-B cells and DC (Fig. 4B).
  • CD80 showed the lowest density on CD40-B cells (16-49 ABS/ ⁇ m 2 ), while the density of MHC class II was the highest (778-2,359 ABS/ ⁇ m 2 ).
  • CD40-B cells either generated with soluble CD40L or CD40L transfectants, consistently express high levels of cell surface molecules involved in APC function.
  • the normalized analysis presented here revealed no major differences in density between CD40-B cells and DC.
  • HLA-DR binding peptide PADRE Alexander et al, Immunity 1 :751-761, 1994
  • FITC-conjugated F18 peptide did not compete with the FITC-conjugated F18 peptide for HLA-A2 binding.
  • CD40-B cells are efficiently loaded with peptide under serum-free conditions without the need of ⁇ 2 m in a relatively short time at 37°C.
  • peptide-loadmg efficiency of primary APC for clinical application can be easily monitored using FITC-conjugated reference peptides
  • MM myeloma
  • FL fol cular lymphopma
  • B cells from PBMC were stimulated via CD40 using NIH3T3 cells transfected with tCD40L (Schultze et al , Proc. Natl. Acad. Sci. U.S.A. 92.8200- 8204, 1995) or sCD40L (kmdly provided by Immunex Corp. Seattle, Washington) (Morris et al , J. Biol. Chem 274 418-423, 1999) CD40L expression on NIH3T3 cells has been stable for more than 5 years with > 95% of tCD40L cells positive for CD40L. No other human molecules are expressed on tCD40L cells.
  • tCD40L cells were lethally irradiated (96Gy) and plated on 6-well plates (Costar, Cambridge, MA) at a concentration of 0.4 x 10 5 cells/well in medium containing 45% DMEM (Life Technologies, Rockville, MD), 45% F12 (Life Technologies), 10% FCS, 2mM glutamine (Life Technologies), and 15 ⁇ g/ml gentamicm (Life Technologies). After 12-18 hours culture, tCD40L cells were adherent and could be used for co-culture. Before adding PBMC, tCD40L cells were rinsed with PBS.
  • CD40-B cells were generated from PBMC by co- culturing whole PBMC at 2x10 6 cells/ml with tCD40L or 2 ⁇ g/ml sCD40L.
  • GMP-grade IL-4 (2 ng/ml, R&D Systems, Minneapolis, MN) and clinical-grade CsA (5.5xl0 "7 M) (Novartis, Basle, Switzerland) were two additional and crucial factors necessary to induce B cell expansion.
  • Iscove's MDM (Life Technologies) was supplemented with 10% human AB serum or autologous plasma, 5 mg/ml insulin (Sigma Chemical Co., St. Louis, MO), and 15 mg/ml gentamicin.
  • Cultured cells were transferred to new plates and either stimulated with fresh irradiated tCD40L cells or sCD40L (2 ⁇ g/ml) in the presence of freshly added IL- 4 and CsA every 3-5 days. Once the cultured PBMC were > 75% CD19 + they were cultured at concentrations of 0.75-1.5xl 0 6 cells/ml. A Coulter Counter Z2 (Beckman Coulter Inc, Fullerton, CA) was used to measure the total number of viable cells and the number of CD19 + B cells was analyzed by flow cytometry on days 0, 6, and twice weekly thereafter.
  • DCs were generated as previously described (Schultze et al, J. Clin. Invest. 100: 2757-2765, 1997). Briefly monocyte-enriched fractions were obtained from PBMC by rosetting over sheep red blood cells and subsequently cultured for 6-8 days with GM-CSF (50 ng/ml, Genzyme, Cambridge, MA) and IL-4 (10 ng/ml) in Iscove's MDM supplemented with 2% human AB serum,
  • GM-CSF and IL-4 were replenished on day 4 of culture.
  • peptide-specific CTL the native hepatitis B core peptide F18 (FLPSDFFPSV), the Pan-DR binding peptide PADRE (AKFVAAWTLKAAA), the 1540 peptide of hTERT (ILAKFLHWL) (Vonderheide et al, Immunity 10:673-679, 1999), RT-pol476 (ILKEPVGHV) of HIV, MP58 (GILGFVFTL) of influenza A, and a peptide of E6 HPV16 (F52, FAFRDLCIV).
  • FLPSDFFPSV native hepatitis B core peptide F18
  • PADRE Pan-DR binding peptide PADRE
  • ILAKFLHWL the 1540 peptide of hTERT
  • RT-pol476 ILKEPVGHV
  • MP58 GILGFVFTL
  • E6 HPV16 F52, FAFRDLCIV
  • APC were harvested from culture, washed 3 times and resuspended in IMDM at 1.25 x 10 6 cells/ml, and seeded into 96-well plates (200 ⁇ l/well). Peptide concentration, incubation time and temperature, pH of culture medium, serum concentration, and concentration of ⁇ 2 m were varied. After incubation with peptide under different conditions, cells were harvested, washed and resuspended in PBS containing 0.1% formaldehyde. Fluorescence analysis was performed immediately on a Coulter EPICS XL flow cytometer.
  • HLA-A*0201 + CD40-B cells were incubated with peptide (up to 10 ⁇ g/ml), irradiated (32 Gy) and added to purified autologous CD8 + T cells (> 85%) at a ratio of T cells:CD40-B cells - 4: 1 in RPMI containing 10% human AB serum, glutamme, gentamicin, and IL-7 (10 ng/ml, Endogen, Woburn, MA).
  • T cell cultures were harvested, washed, and restimulated with fresh peptide-pulsed CD40-B cells and IL-7. This was repeated on days 14, 21, and 28.
  • IL-2 was first introduced into the cultures at day 8 (50 IU/ml) and every 2-3 days thereafter.
  • 51 Cr-release assay (Vonderheide et al, Immunity 10:673-679, 1999) Targets were labeled with 51 Cr and 5x10 3 labeled cells/well were plated with various concentrations of effector cells. Percent cytotoxicity was calculated as the
  • RT-pol476, or HTLV-I Tax 1 1 were synthesized essentially as described ( Altman et al , Science 274:94-96, 1996) and conjugated to streptavidm ALEXA-488 or streptavidm PE (Molecular Probes, Eugene, OR). For staining, CTL lines were incubated with the tetramer for 15 minutes and CD8-PE or CD8-PC5 (Coulter) for 20 minutes at room temperature.
  • the following methods were used to determine absolute numbers of surface molecules for CD 58, CD 80, MHC I, and MHC II, as is illustrated in Figs. 6-9.
  • the antigen presenting cells are harvested, washed at 1500 rpm for 5 minutes, resuspended in PBS 2% FCS, counted, and diluted to 2 x 10 6 cells/ml. 2 x 10 5 cells are used per test. To prevent unspecific binding, the cells are incubated with a blocking antibody at 4°C for 10 minutes. 100 ⁇ l of this solution is then transferred to each test tube and the fluorescent CD58, CD80, MHC-I, or MHC-II antibody is added in combination with a fluorescent antibody to a lineage specific marker at 4°C for 30 minutes. The cells are washed at 1800 rpm for 3 minutes and resuspended in 200 ⁇ l 0.1 % Paraformaldehyde.
  • the setup beads are analyzed first to calibrate for fluorescence labeled and unlabeled bead populations. Second, the calibration beads, which consist of five distinct fractions with known numbers of antigenic sites, are measured. Using the same settings for the mean fluorescence intensity (MFI) the cells are analyzed.
  • MFI mean fluorescence intensity
  • the MFI of the cells can be converted to the number of antigenic sites using the calibration beads for normalization. Entering the five pairs of MFI - number of antigenic sites into a calculation software its function can be determined. Using this function the absolute number of surface molecules on the cells binding the antibody can be calculated. By gating on the cells binding the second antibody, cells other than the ape of interest are excluded from the analysis. Determination of densities of cell surface molecules
  • the following methods were used to determine density of surface molecules for CD 58, CD 80, MHC I, and MHC II, as is illustrated in Figs. 10-13
  • the density of the surface molecules is calculated by dividing the absolute number of antigenic sites by the cell surface area.
  • the antigen presenting cells are harvested, washed at 1500 rpm for 5 minutes, and resuspended in PBS 2% FCS.
  • the cellular volume is determined using a Coulter Z2.
  • the surface area is calculated using the formula.
  • A V 2/3 x 4.84.
  • a competition assay was performed using the following method.
  • the CD40-B cells were premcubated for 30 minutes at 4°C in a 5% CO 2 atmosphere with increasing concentrations of the competitor peptide (0.3 to 20 ⁇ g/ml) and 1.5 ⁇ g/ml of ⁇ 2 m.
  • the reference peptide was added at a concentration of 0.2 ⁇ g/ml for 150 minutes.
  • the cells were washed and fixed in 0.1% Paraformaldehyde and analyzed by flowcytometry
  • the flowcytomet ⁇ c analysis and the calculation of the absolute number of MHC/Peptide complexes on the cell surface is performed analogous to the MHC-I/II analysis. Shown in Fig. 14 are the results from 1 of 4 experiments.
  • CD40 B cells were harvested, counted, and then plated m IMDM at 2 x 10 6 cells per ml in 24-well plates. They were incubated with 10 ⁇ g/ml of HepB Fitc peptide at 37°C for 60 minutes. The cells were washed 3 times in IMDM, diluted to 2.5 x 10 6 per ml, and coincubated with immature DC (2.5 x 10 6 per ml) in 200 ⁇ l of IMDM in a 96- well plate for 60 minutes.
  • the cells were then washed, fixed in 0.1 % Paraformaldehyde, and measured by flowcytomet ⁇ c analysis.
  • the amount of peptide transferred can be calculated by determining the number of FITC molecules present on the Dendritic cells using bead normalization analysis (see above).
  • dendritic cells generated from peripheral blood monocytes by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, and matured with CD40L and interferon-gamma, and CD40-activated B cells, were used.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CD40L and interferon-gamma CD40L and interferon-gamma
  • CD40-activated B cells Two sources of highly efficient APCs, dendritic cells, generated from peripheral blood monocytes by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, and matured with CD40L and interferon-gamma, and CD40-activated B cells.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CD40L and interferon-gamma CD40L and interferon
  • Immature dendritic cells were obtained by depleting peripheral blood monocytes of T cells, B cells, and natural killer cells by magnetic bead depletion. Mature dendritic cells were generated from the remaining cell fraction by stimulation with GM-CSF and interleukin-4 in IMDM, supplemented with 5% human AB serum, 50 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, 2 mM glutamine, and 15 ⁇ g/ml gentamicin at 37° C in 5% CO 2 .
  • the primary APCs (immature dendritic cells and CD40-activated B cells) were harvested, washed at 1,500 rpm (300 x g) for 5 minutes, and resuspended in phosphate- buffered saline containing 2% fetal calf serum (PBS 2% FCS), counted, and diluted to 2 x 10 6 cells/ml. The amount of cells that was used in each test was 2 x 10 5 cells/ml. To prevent non-specific binding, the cells were incubated with a blocking antibody at 4°C for 10 minutes.
  • a blocking antibody at 4°C for 10 minutes.
  • One hundred ⁇ l of the cell solution was transferred to a test tube, and fluorescent MHC class I or MHC class II antibody was added, in combination with a fluorescent antibody to a lineage-specific marker, at 4°C for 30 minutes.
  • the cells were then washed at 1,800 rpm for 3 minutes and resuspended in 200 ⁇ l 0.1 % paraformaldehyde.
  • Fifty ⁇ l of DAKO Quifikit 'setup' and 'calibration' beads were transferred to a test tube, and washed in phosphate buffered saline (PBS) at 1,800 rpm for 3 minutes.
  • PBS phosphate buffered saline
  • the beads were resuspended in 100 ⁇ l PBS 2% FCS and incubated with a fluorescent conjugate for 30 minutes at 4°C. The beads were then washed in PBS, for 3 minutes at 1,800 rpm, and resuspended in 200 ⁇ l of 0.1% paraformaldehyde. The total amount and density of MHC class I or MHC class II molecules on the surface of the APCs were determined as is described above.
  • the APCs were harvested, washed three times in serum- free IMDM at 1,500 rpm for 5 minutes, resuspended in IMDM (alternatively, various serum-free and serum-based media, such as RPMI and AIMV, can be used), counted, and diluted to 2.5 x 10 6 cells/ml. One hundred ⁇ l cells were transferred to a 96-well plate for each test.
  • Biotinylated peptides (MHC I hTERT peptide, HT540; MHC class II tetanus-derived peptide, TT830; and MHC class II PADRE-AKX peptide) and FITC-conjugated peptides (Hepatitis B core F18; TT-FITC 830) were diluted to concentrations of 100 ⁇ g/ml in serum-free IMDM and ⁇ -2 microglobulin ( ⁇ 2m) was added at a concentration of 1.5 ⁇ g/ml.
  • ⁇ 2m microglobulin
  • the cells were again harvested, washed at 1,500 rpm for 5 minutes, resuspended in PBS 2% FCS, counted, and diluted to 2.5 x 10 6 cells/ml. The amount of cells that was used in each test was 2.5 x 10 5 cells/ml.
  • One hundred ⁇ l of the cell solution was transferred per test tube and a fluorescent Streptavidin antibody was added in combination with a lineage-specific marker at 4°C for 30 minutes.
  • the cells were washed at 1,800 rpm for 3 minutes, and resuspended in 0.1 % paraformaldehyde.
  • the total amount and density of MHC class I or MHC class II molecules, in a complex with the biotinylated peptide, on the surface of the APCs were determined as is described above.
  • MHC class I molecules The density of MHC class I molecules was higher on CD40-act ⁇ vated B cells (400-600 molecules/ ⁇ m 2 ) than on dendritic cells (80-120 molecules/ ⁇ m 2 ).
  • the Pan DR epitope (PADRE) peptide and the tetanus toxoid-denved peptide (TT830) were detected at a density of up to 100 molecules/ ⁇ m 2 on mature dendritic cells and CD40-activated B cells, and 4- to 6-fold less on immature dendritic cells. Similar results were obtained with the MHC class I peptide HT540, derived from the universal tumor antigen, hTERT, which is the catalytic subunit of human telomerase.
  • DC Dend ⁇ tic cells
  • DC Dendritic cells
  • CD40-act ⁇ vated B-cells were incubated with biotinylated htert-peptide for 2, 4, 6, and 18 hours with different concentrations of b2m concentrations of b2m and then labeled with Streptavidm-FITC
  • the table shows the mean fluorescence intensity for the various conditions Vfe experiments
  • CD40-act ⁇ vated B cells were incubated with biotinylated hTERT for 2, 4, 6, and 12 hours in AIMV, IMDM, B cell medium (HS), DC medium (HS), and then labeled with Streptavidm-FITC.
  • the Table shows the mean fluorescence intensity and the viability of the cells under the vanous conditions. 1/1 exp
  • CD40-act ⁇ vated b-cells were incubated with different concentrations of biotinylated htert-peptide and the labeled with
  • CD40-act ⁇ vated b-cells were incubated with biotinylated htert-peptide and FITC-labeled flu-peptide for 4 with media with different pH values
  • the table shows the MFI.
  • CD40-act ⁇ vated b-cells were incubated with FITC-labeled flu-peptide for 2, 4, 6, 12, and 18 hours. 0, 3, and 25 ⁇ g/ml of b2m were added The table shows the MFI for these conditions
  • CD40-act ⁇ vated b-cells were incubated with FLU-Fitc peptide at different concentrations for 2, 4, 6, 12, and 18 hours.
  • the table shows the MFI
  • CD40-act ⁇ vated b-cells were incubated with the htert peptide at different temperatures for 2. 4, and 6 hours.
  • the table shows the MFI Table 12
  • CD40-act ⁇ vated b-cells were incubated with the FLU-Fitc peptide at different temperatures for 2, 4, and 6 hours
  • the table shows the MFI
  • CD40-act ⁇ vated b-cells were incubated with biotinylated htert-peptide for 6 hours with varying b2m concentrations and MHCI antibodies
  • the table shows the MFI

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Abstract

Cette invention concerne des techniques permettant de déterminer directement la densité de molécules à complexe majeur d'histocompatibilité (CMH) et de complexes CMH/peptides sur les surfaces de cellules présentatrices d'antigènes. Sont également décrites des kits permettant la mise en oeuvre de ces techniques.
PCT/US2000/042213 1999-11-15 2000-11-15 Vaccins cellulaires : techniques de production, d'optimisation et de controle de la qualite WO2001036978A1 (fr)

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US5612185A (en) * 1992-10-14 1997-03-18 Board Of Regents, The University Of Texas System Method for identifying tumor cells in cell cycle arrest

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