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WO2025083196A1 - Procédé de fabrication d'un produit de thérapie cellulaire et kits et dispositifs respectifs - Google Patents

Procédé de fabrication d'un produit de thérapie cellulaire et kits et dispositifs respectifs Download PDF

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WO2025083196A1
WO2025083196A1 PCT/EP2024/079474 EP2024079474W WO2025083196A1 WO 2025083196 A1 WO2025083196 A1 WO 2025083196A1 EP 2024079474 W EP2024079474 W EP 2024079474W WO 2025083196 A1 WO2025083196 A1 WO 2025083196A1
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cell
cellular therapy
cellular
binding
reagent
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Sabine RADISCH
Christian Eckert
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TQ Therapeutics GmbH
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TQ Therapeutics GmbH
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    • 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
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C12N5/0635—B lymphocytes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a method of manufacturing a cellular therapy product, wherein the cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell and/or wherein the cellular therapy product produces and/or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell.
  • the invention also relates to a kit, a device and a tubing to be used in the method.
  • a strategy providing time and cost benefit is the so-called “allogeneic approach” that takes advantage of novel gene editing methods to generate universal donor T cells from third parties in a way that they would not be rejected by the patient’s immune system but also would not induce a graft-versus-host reaction in the patient (3).
  • This requires excessive genetic engineering of the therapeutically active cells to circumvent both rejection (host-vs-graft) or anti-host (host-vs-graft) responses.
  • this approach may enable the manufacturing of hundreds of doses per batch, but it possesses an inherent risk of high clinical toxicity associated with unmet immunological hurdles (4).
  • the FDA halted all clinical trials using universal donor cells due to safety concerns (5).
  • T cells as an example in this context
  • Such delivery systems or nanocarriers can be produced by standardized methods rather easily, enabling a broader worldwide clinical application as compared to conventional cellular therapies.
  • Recent studies show some anti-tumoral responses in humanized mouse models but also method associated side effects comprising high immunogenicity of gene engineering delivery systems and the cargo, fast hepatic and renal clearance of administered delivery systems from body, potentially pre-existing anti-viral vector immunity, limited distribution of agents within the body which may require multiple injections and limit the overall therapeutic efficiency.
  • evidence for a certain degree of unspecific targeting of the gene delivery system towards non-T cells impacting dramatically the potential safety profile of the treatment (7-9).
  • WO 2019/217964 A1 and WO 2022/072885 A1 describe a closed-loop system for the modification of cells from patients, wherein a cell containing sample, e.g. a blood sample, from the patient enters the system and is first separated in a cell separation module. Within this cell separation module cells separated from the initial sample could optionally be enriched. After passing the cell separation module the cells are modified using modifying agents, e.g. therapeutic drugs. While no experimental data are reported, WO 2019/217964 A1 and WO 2022/072885 A1 describe that thereafter the modified cells can be enriched and then are reinfused to the patient. WO 2019/217964 A1 and WO 2022/072885 A1 also describe that these CAR T-Cells are to be activated, for example, by CD3 and CD28 costimulation prior to administration to the patient.
  • modifying agents e.g. therapeutic drugs
  • the international patent application publication WO 2023/122277 A2 discloses a rapid method of generating CAR T cells by simultaneous activating the T cells of a mononuclear cell sample and exposing the mononuclear cell sample to a viral vector for transducing T cells with a CAR construct.
  • the invention provides a method of manufacturing a cellular therapy product, wherein the cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell, and/or wherein the cellular therapy product produces and/or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell, comprising a) enriching via reversible immobilization (in a holding step) a cellular therapy starting cell obtained from a sample comprising the cellular therapy starting cell, using at least one second cellular surface receptor of the cellular therapy starting cell, and b) genetically modifying the cellular therapy starting cell using one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor and/or for expression and/or secretion of the polypeptide, thereby generating a precursor of the cellular therapy product, wherein the method takes place in a closed system.
  • the invention provides a use of a set of reagents for genetically modifying a cellular therapy starting cell, wherein the cellular therapy starting cell is immobilized on a stationary phase or a receptor binding reagent during the genetic modification or the use of the set of reagents for genetic modification of the a cellular therapy starting cell, wherein the gene modification takes place before eluting the cellular therapy starting cell from the stationary phase.
  • the invention provides a kit for manufacturing a cellular therapy product, wherein the cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell, and/or wherein the cellular therapy product produces and/or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell, the kit comprising a) one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor or of the polypeptide of the cell therapy product, and b) a receptor binding reagent comprising a binding site B and a binding partner C,
  • the binding site B comprised in the receptor binding reagent is capable of specifically binding to the second cellular surface receptor on a cellular therapy starting cell and wherein the bond between the binding site B of the receptor binding reagent and the second cellular surface receptor has a dissociation constant (KD) is of low affinity or in the range from about 10’ 3 to about 10’ 7 M,
  • KD dissociation constant
  • the binding partner C comprised in the receptor binding reagent is capable of reversibly binding to a binding site Z of an affinity reagent and the reversible bond between the binding partner C of the receptor binding reagent and the binding site Z of the affinity reagent has a dissociation constant (KD) in the range from about 10’ 2 to about 10’ 13 M, and
  • the stationary phase having the affinity reagent immobilized thereon
  • the affinity reagent comprises one or more binding sites Z, wherein said binding site Z forms a reversible bond with the binding partner C comprised in the receptor binding reagent, and wherein the binding site B of the receptor binding reagent binds to the second cellular surface receptor on the cellular therapy starting cell, to allow reversibly immobilizing the cell therapy starting cell on the stationary phase.
  • the invention provides a device for manufacturing a cellular therapy product, comprising an operating device and a tubing-set, wherein the tubing set is a closed and single-use tubing-set, for use in above mentioned method of manufacturing a cellular therapy product.
  • the invention provides a tubing having an affinity reagent immobilized on an inner surface of the tubing, wherein the affinity reagent comprises one or more binding sites Z, wherein said binding site Z forms a reversible bond with a binding partner C comprised in the receptor binding reagent, and wherein the binding site B of the receptor binding reagent binds to a (second) cellular surface receptor on a cellular therapy starting cell, thereby allowing reversibly immobilizing the cellular therapy starting cell on the tubing acting as stationary phase.
  • FIG. 1 is an illustration of the inventive CAR T cell generation strategies. Respective facilities, as well as the whole duration of CAR T cell generation including potential manufacturing steps until product infusion are depicted per strategy.
  • Figure 2 depicts an illustrative example of the inventive method using the inventive device for an extra-corporal fully automated cell/gene therapy.
  • the process runs automated on a dedicated device using a singleuse set and consists of the following steps: 1 ) collection of blood starting material (e.g., whole blood) directly from the patient, 2) short target cell specific hold-step via dedicated cell surface markers, 3) release and wash of target cells; 4) targeted genetic modification (e.g., non-viral CRISPR/Cas editing via e.g. electro-Zmechanoporation, incubation with loaded lipid nanoparticles), 5) in-process analytics (e.g., non-invasive analytics like viable cell number); 6) removal of residuals and re-infusion.
  • blood starting material e.g., whole blood
  • the process runs automated on a dedicated device using a singleuse set and consists of the following steps: 1 ) collection of blood starting material (e.g., whole blood) directly from the patient, 2) short target cell specific hold
  • Figure 3 depicts a further illustrative example of the inventive method using the inventive device for an extra-corporal fully automated cell/gene therapy.
  • the process runs automated on a dedicated device using a single-use set and consists of the following steps: 1 ) collection of blood starting material (e.g., whole blood) directly from the patient, 2) short target cell specific holdstep via dedicated cell surface markers and targeted genetic modification during the hold-step (e.g., non-viral CRISPR/Cas editing via incubation with loaded lipid nanoparticles), 3) release and wash of target cells; 4) in-process analytics (e.g., non- invasive analytics like viable cell number); 5) removal of residuals and re-infusion.
  • blood starting material e.g., whole blood
  • short target cell specific holdstep via dedicated cell surface markers and targeted genetic modification during the hold-step
  • targeted genetic modification during the hold-step e.g., non-viral CRISPR/Cas editing via incubation with loaded lipid nanoparticles
  • Figure 4 depicts the results of an example of the enrichment of CD3+ cells via reversible immobilization from whole blood starting material.
  • Fig. 4A depicts cell counts (Mio) in the starting whole blood material and in the enriched positive material.
  • Fig. 4B is showing CD3 target cell depletion,
  • Fig. 4C is showing purity and
  • Fig. 4D is showing yield. Depletion is calculated on target cells in starting material vs. target cells in negative fraction. Yield is calculated on target cells in starting material vs. target cells in positive fraction.
  • Fig. 4E shows flow cytometry data for two donors for whole blood starting material and positive fraction. Purity is measured via Flow Cytometry and pre-gated on single living CD45 cells.
  • Figure 5 depicts a TRAC Knock Out in selected CD3 T cells.
  • the graphic is showing the TRAC Knock Out using an RNP complex, composed of Cas9 Nuclease and gRNA directed against the TRAC locus for up to 12 days after mechanoporation of the selected and non-activated T cells.
  • FIG. 6 depicts a T cell transfection using transgene LNPs (GFP as mRNA cargo). CD3+ T cells were retained from whole blood and transfected with transgene LNPs.
  • Fig. 6A depicts Flow cytometry gating strategy for measuring transgene expression on LNP incubated and non-activated T cells.
  • Fig. 6B is showing the control where selected T cells are incubated in the incubator for 4 hours without LNPs.
  • Fig. 6C is showing transgene expression after 4 hours incubation with LNPs on column and subsequent elution of transfected cells.
  • Fig. 6D is showing transgene expression after 4 hours incubation with LNP after T cell selection and subsequent elution.
  • Fig. 6A depicts Flow cytometry gating strategy for measuring transgene expression on LNP incubated and non-activated T cells.
  • Fig. 6B is showing the control where selected T cells are incubated in the incubator for 4 hours without LNPs
  • FIG. 6E is showing transgene expression after 4h LNP incubation and on day 1 (after 24h), day 3, day 5, day 7 and day 9 after LNP incubation as well as viable T cells in %. All data are pre-gated on single living CD45 cells and gated separately on CD4 T cells and CD8 T cells.
  • Figure 7 shows potent in vivo tumor cell killing resulting from the inventive cellular therapy product.
  • Fig. 7A shows a flow-diagram depicting an example of the inventive ultra-short process for extra-corporal ly generated CD19 CAR T cells comprising a brief CD3 hold/enrichment step from a 200ml whole blood draw, washing, CD19 CAR CRISPR/Cas KO/KI and subsequent infusion.
  • FIG. 7B shows in vivo expansion and functionality of the inventive ultra-short extra-corporally generated CAR T cells that were analyzed in a clinically relevant mouse model of lymphoma.
  • 6-8 week old NSG- SGM3 (NSGS) mice were injected intra venously with 0.5x106 Raji-fluc-GFP CD19+ tumor cells and subsequently intra peritoneally injected with CAR+ cells prepared according to the invention two days later (a total of 1x106 cells was administered as flat dose).
  • Conventionally manufactured CD19 CAR T cells using a standard lentivirus (LV) transfection and expansion process served as positive controls while mice only injected with PBS served as negative control group. Tumor growth/rejection was evaluated using MS bioluminescence.
  • LV lentivirus
  • mice treated with CAR+ cells manufactured according to the inventive method (TQ process) and standard LV control cells showed rapid expansion and high tumor reactivity with complete tumor clearance post day 20 (Fig. 7B middle and right columns).
  • control groups treated with PBS only showed uncontrolled tumor progression (Fig. 7B left column) with mice succumbing to the tumor starting around day 14.
  • Fig. 7C shows that TRAC-CAR knock-in and expression was confirmed after extended parallel in vitro culture by flow cytometry.
  • Fig. 7D shows a survival benefit of mice receiving the inventive cell products compared to PBS control. All mice that received the inventive cells survived and controlled the tumor while all control mice succumbed to the tumor starting around day 14.
  • Figure 8 shows a quantification of tumor growth/control, survival and in vivo expansion of CAR T cells as an exemplary inventive cellular therapy product that can be manufactured using the invention.
  • Fig. 8A shows quantification of tumor growth measured by average radiance [p/s/cm2/sr] post luciferin treatment is shown over time using Living image software (Perkin Elmer).
  • Fig. 8B shows that besides quantification of expanding transferred CAR T cells, residual Raji tumor cells were measured in blood using flow-cytometry.
  • Fig. 8C-G shows engraftment and in vivo expansion of total transferred T cells and CAR+ transferred T cells. For these experiments mice were bled weekly for ex vivo analysis using flow cytometry and staining for T cell subsets and CAR expression.
  • Fig. 8C shows a measurement of the total CD3+ CAR+ T cells, wherein the percentage of CAR+ cells in the blood reaches a maximum at day 14 post CAR T cell injection.
  • Fig. 8D shows the percentage of CD4+ CAR+ T cells in the blood and
  • Fig. 8E shows the percentage of CD8+ CAR+ T cells in the blood. Both cell types had their maximum at day 14 post CAR T cell injection.
  • Fig. 8F shows the percentage of CD4+ cells in the blood. The percentage of CD4+ T cells showed a decrease at day 14 post CAR T cell injection and reached a maximum at day 21 post CAR T cell injection.
  • Fig. 8G shows the percentage of CD8+ T cells in the blood.
  • mice injected with the CAR T cells prepared according to the invention showed an increase of percentage of CD8+ T cells in the blood post CAR T cell injection.
  • Figure 9 shows a schematic drawing of an example of the inventive device including an operating device, a single-use tubing set, process buffers and a set of reagents for gene modification.
  • the operating device will incorporate all non-disposable components needed to automatically operate the inventive method.
  • the device will only get operational via insertion of a single-use tubing set to the operating device.
  • the tubing set will comprise tubing, the cell retention reservoir as well empty bags for whole blood-, waste- and blood cell preparation collection. At the day of treatment, the tubing set will be connected to separately delivered liquid-containing bags as the respective reagents and process buffers.
  • the process unit operation with respect to associated process steps are depicted in the schematic. DETAILED DESCRIPTION OF THE INVENTION
  • the invention is directed to a method of manufacturing a cellular therapy product, wherein the cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell and/or wherein the cellular therapy product produces and/or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell.
  • This method comprises a) enriching via reversible immobilization (in a holding step) a cellular therapy starting cell obtained from a sample comprising the cellular therapy starting cell, using at least one second cellular surface receptor of the cellular therapy starting cell, and b) genetically modifying the cellular therapy starting cell using one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor and/or for expression and/or secretion of the polypeptide, thereby generating a precursor of the cellular therapy product.
  • the method takes place in a closed system.
  • the manufactured cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell.
  • the manufactured cellular therapy product produces and/ or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell.
  • the polypeptide is an amino acid chain having at least three amino acids connected by peptide bonds.
  • the polypeptide may be, as nonlimiting examples, a peptide hormone, an insulin, or a coagulation factor.
  • the cellular therapy starting cell for genetic modification for production and/or secretion of such a polypeptide is preferably a B cell.
  • the manufactured cellular therapy product comprises the first and the second alternative.
  • this reversible immobilization can be carried out as a single enrichment step and so obtained enriched sample (which comprise the cellular therapy starting cell) can still be subjected to genetic modification that results in the functional precursor of the cell therapy product - this precursor, once administered to a patient, will mature and develop into the functional cellular therapy product (see Example 3).
  • this enrichment step holding step even allows to use a whole blood sample obtained from a patient to be used for genetic modification without any prior treatment of the blood sample.
  • a patient such as critically ill patient can obtain his autologous precursor cell therapy product within a very short time such as about 90 minutes (or a bit longer, for example about 2 hours) and thus on-site in a hospital or doctor’s office.
  • This reduces the costs and administrative efforts of manufacturing a cellular therapy product significantly and thus allows to make cellular therapy available for any patient for whom cellular therapy is indicated as medically beneficial.
  • the nonactivated genetically modified cell such as a T cell, B cell or NK cells can be administered to the patient without such conventional activation. Also, this shortens the manufacturing time and simplifies the manufacturing process and respective closed-loop device.
  • PBMCs peripheral blood mononuclear cells
  • the so enriched CD8+ cells had a purity of only 68%, cf.
  • WO 2021/152178 A1 reports a cell isolation experiment that uses a liquid-handling device (no closed system) and in which biotin coated beads were incubated with strep-Tactin®/dextran polymers loaded with CD3 binding Fab fragments equipped with a streptavidin binding peptide (Strep-Tag®) in a column. Thereafter, PBMCs were added, the column was closed on both sides and incubated with rotating.
  • closed system or “closed-loop system” as used herein means the “closed system” as defined in Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products of 22.11.2017, Glossary No. 12 stating “A process system designed and operated so as to avoid exposure of the product or material to the room environment. Materials may be introduced to a closed system, but the addition must be done in such a way so as to avoid exposure of the product to the room environment (e.g. by means of sterile connectors or fusion systems). A closed system may need to be opened (e.g., to install a filter or make a connection), but it is returned to a closed state through a sanitization or sterilization step prior to process use.”
  • the inventive method allows manufacturing a cellular therapy product in a very short time period.
  • the duration of the inventive method may, for example, be between about 1 .5 hours to about 16 hours. However, it is also envisaged here that with further process optimization and automation, it may be possible to manufacture a cellular therapy product within less than about 1 .5 hours.
  • the drug target molecule can be any molecule on the surface of the drug target cell, provided it is able to get in contact with the first cellular surface receptor.
  • the drug target molecule can however also be a soluble molecule or a drug target cell associated molecule, which could be targeted by the polypeptide secreted by the cellular therapy product.
  • the drug target molecule is a molecule relevant in a human or animal disease.
  • One non-limiting example of such drug target molecules are cluster of differentiation (CD) molecules, especially CD19.
  • suitable drug target molecules include, but are not limited to, Claudin 6, IL13RA2, EGFRv3, a disialoganglioside GD2, prostate-specific membrane antigen, or CLDN18, as described below, or checkpoint inhibitors.
  • the drug target molecule may also be a coagulation factor as a non-limiting example of a soluble (secretory) drug target molecule.
  • the drug target cell can be any cell related to a disease, in particular the cell expresses a suitable drug target molecule on their surface.
  • the drug target cell may be a human or an animal cell.
  • the drug target cell may be a cell that is diseased, for example, a cancer cell, a cell affected by an autoimmune disease, or a cell infected by a virus or a bacterium.
  • Exemplary cancers from which the cancer cell is derived are solid tumors or hematological tumors.
  • solid tumors include, but are not limited to, germ cell tumors, epithelial ovarian cancer, desmoplastic small round cell tumor, gastric adenocarcinoma, colorectal cancer, endometrial carcinoma, breast cancer, glioblastoma, and neuroblastoma.
  • hematological tumors are, but are not limited to, acute or chronic myeloid leukemia, lymphoblastic leukemia, and lymphoma.
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus, diabetes, psoriasis, or rheumatoid arthritis to name only a few.
  • cells relating to an infectious disease are possible as drug target cell.
  • the drug target cell may be a cell physiologically participating in blood coagulation or be a cell physiologically producing insulin.
  • Claudin 6 is a tight junction protein physiological only expressed during fetal organogenesis, but also expressed from several solid tumor cells rendering Claudin 6 as a suitable drug target for cancer therapy. Claudin 6 expression was observed especially in germ cell tumors, epithelial ovarian cancer and endometrial carcinoma.
  • CLDN6 is a safe target for cell therapy in solid tumors and does not raise concerns for either combining CAR-T cells with CARVac or for exploring higher doses of CLDN 6 CAR-T cells.” (10).
  • the method, kit, device, and tubing of the present invention are also suitable for manufacturing the above described CLDN6 CAR-T cells from autologous blood samples easier, faster and in a closed system compared the method described in (10).
  • CAR-T cells using, for instance, drug target molecules such as IL13RA2 or EGFRv3 in glioblastoma patients, the disialoganglioside GD2 in neuroblastoma, H3K27M-mutated diffuse midline gliomas, prostate-specific membrane antigen in metastatic prostate cancer, or CLDN18.2 in patients with gastrointestinal cancers (10).
  • the cellular therapy starting cell as mentioned herein can be a nucleated animal or human cell.
  • the cellular therapy starting cell may be a human white blood cell, for example a T cell, a B cell, or a NK cell. If such cells are used as starting material for the cellular therapy starting, these cells may be a non-activated T cell, a non-activated B cell or a non-activated NK cell.
  • the method described here has the advantage that it does not actively/deliberately activates lymphocytes. This is in contrast to the conventional methods and protocols for gene modifying of lymphocytes such as T cells or B cells.
  • WO 2023/122277 A2 discloses a rapid method for producing CAR T cells, wherein the used T cells are mandatory activated before gene modification.
  • Xin et al. did not mention in the actual review regarding CAR T cells the use of explicit non-activated T cells (see references 5, cited above).
  • the use of non-activated T cells is also not mentioned (reference 10).
  • the cellular therapy starting cell is obtained in a first step by enrichment via reversible immobilization from a sample comprising the cellular therapy starting cell.
  • the sample can be any cell containing sample as long as the desired cellular therapy starting cell used in the method is comprised in the sample.
  • the sample comprises a human bodily fluid.
  • the sample may comprise nucleated blood cells, the sample can, for example, be a whole blood sample or the sample can comprise or consist of white blood cells.
  • enrichment via reversible immobilization means that the cellular therapy starting cell is immobilized during enrichment and that this immobilization is or can be reversed after the enrichment of the cellular therapy starting cell took place.
  • the enrichment or hold step is carried out without prior cell separation, in particular without prior cell separation in a cell separation module comprising a cell separator configured to produce a fraction enriched in nucleated blood cells.
  • the sample comprising the cellular therapy starting cell
  • the binding site B comprised in the receptor binding reagent is capable of specifically binding to the second cellular surface receptor on the cellular therapy starting cell and wherein the bond between the binding site B of the receptor binding reagent and the second cellular surface receptor has a dissociation constant (KD) is of low affinity or in the range from about 10’ 3 to about 10’ 7 M,
  • KD dissociation constant
  • the binding partner C comprised in the receptor binding reagent is capable of reversibly binding to a binding site Z of an affinity reagent and the reversible bond between the binding partner C of the receptor binding reagent and the binding site Z of the affinity reagent has a dissociation constant (KD) in the range from about 10’ 2 to about 10- 13 M, and
  • a suitable solid phase for example, floating particles or a stationary phase, the solid phase or the stationary phase having the affinity reagent immobilized thereon,
  • the affinity reagent comprises one or more binding sites Z, wherein said binding site Z forms a reversible bond with the binding partner C comprised in the receptor binding reagent, and wherein the binding site B of the receptor binding reagent binds to the second cellular surface receptor on the cellular therapy starting cell, thereby reversibly immobilizing the cellular therapy starting cell on the stationary phase,
  • the competition reagent comprising a binding site, specifically binding to the binding sites Z of the affinity reagent;
  • the competition reagent onto the stationary phase, thereby allowing disruption of non-covalent reversible complexes formed between (a plurality of) the receptor binding reagent, the second cellular surface receptor and the affinity reagent; - recovering an enrichment sample from the eluate of the stationary phase, wherein the enrichment sample comprises the cellular therapy starting cell.
  • a chromatographic method is a fluid chromatography, typically a liquid chromatography.
  • the chromatography can be carried out in a flow through mode in which a fluid sample containing the cellular therapy starting cell to be enriched is applied, for example, by gravity flow or by a pump on one end of a column containing the chromatography matrix and in which the fluid sample exists the column at the other end of the column.
  • the column as used herein may also comprise a system or arrangement of columns.
  • the column or at least one column of the system of columns may have one or more obstacle(s).
  • the chromatography can also take place in a tubing or a part of the tubing wherein most of the conditions for the chromatography using the column applies.
  • the chromatography can also be carried out in a batch mode in which the chromatography material (stationary phase or solid phase) is incubated with the sample that contains the cellular therapy starting cell, for example, under shaking, rotating or repeated contacting and removal of the fluid sample.
  • the chromatography material stationary phase or solid phase
  • Any material may be employed as chromatography matrix in the context of the invention, as long as the material is suitable for the chromatographic isolation of cells.
  • a suitable chromatography material is at least essentially innocuous, i.e. not detrimental to cell viability (or the viability or stability of the biological entity), when used in a packed chromatography column under desired conditions for cell enrichment.
  • a chromatography matrix as used in the present invention remains in a predefined location, typically in a predefined position, whereas the location of the sample to be enriched and of components included therein, is being altered.
  • the chromatography matrix is a “stationary phase” in line with the regular understanding of the person skilled in the art that the stationary phase is the part of a chromatographic system through which the mobile phase flows (either by flow through or in a batch mode) and where distribution of the components contained in the liquid phase (either dissolved or dispersed) between the phases occurs.
  • the term “solid phase” means particles which are free floating in e.g. a bag comprising the sample.
  • the terms “chromatography matrix”, “solid phase”, and “stationary phase” are thus used interchangeable herein.
  • An example of the “solid phase” are particles such as freely movable beads that are added to a liquid sample, mixed with the sample and are then removed from the sample.
  • the use of magnetic beads is not considered in the invention.
  • the respective chromatography matrix has the form of a solid or semisolid phase, whereas the sample that contains the cellular therapy starting cell to be enriched is a fluid phase.
  • the mobile phase used to achieve chromatographic separation is likewise a fluid phase.
  • the chromatography matrix can be a particulate material (of any suitable size and shape) or a monolithic chromatography material, including a paper substrate or membrane.
  • the chromatography can be both column chromatography as well as planar chromatography.
  • columns allowing a bidirectional flow such as PhyTip® columns available from PhyNexus, Inc.
  • chromatography columns can also be used for column based/flow through mode based chromatographic separation of cells as described here. Columns generating a non-laminar flow may also be advantageously used. Thus, columns allowing a bidirectional flow and/or a non-laminar flow are also encompassed by the term “chromatography columns” as used herein.
  • the particulate matrix material may, for example, have a mean particle size of about 5 pm to about 200 pm, or from about 5 pm to about 400 pm, or from about 5 pm to about 600 pm.
  • the chromatography matrix may, for example, be or include a polymeric resin or a metal oxide or a metalloid oxide.
  • the matrix material may be any material suitable for planar chromatography, such as conventional cellulose- based or organic polymer-based membranes (for example, a paper membrane, a nitrocellulose membrane or a polyvinylidene difluoride (PVDF) membrane) or silica coated glass plates.
  • the chromatography matrix/stationary phase is a non-magnetic material or non-magnetizable material.
  • the non-magnetic or non-magnetizable chromatography stationary phase includes a plastic tubing.
  • Further non-magnetic or non-magnetizable chromatography stationary phases that are used in the art, and that are also suitable in the present invention, include derivatized silica or a crosslinked gel.
  • a crosslinked gel (which is typically manufactured in a bead form) may be based on a natural polymer, i.e. on a polymer class that occurs in nature.
  • a natural polymer on which a chromatography stationary phase is based is a polysaccharide.
  • a respective polysaccharide is generally crosslinked.
  • An example of a polysaccharide matrix is an agarose gel (for example, SuperflowTM agarose or a Sepharose® material such as SuperflowTM Sepharose® that are commercially available in different bead and pore sizes) or a gel of crosslinked dextran(s).
  • a further illustrative example is a particulate cross-linked agarose matrix, to which dextran is covalently bonded, that is commercially available (in various bead sizes and with various pore sizes) as Sephadex® or Superdex®, both available from GE Healthcare.
  • Sephadex® or Superdex® commercially available (in various bead sizes and with various pore sizes) as Sephadex® or Superdex®, both available from GE Healthcare.
  • Sephacryl® is also available in different bead and pore sizes from GE Healthcare.
  • a crosslinked gel may also be based on a synthetic polymer, i.e. on a polymer class that does not occur in nature.
  • a synthetic polymer on which a chromatography stationary phase for cell separation is based is a polymer that has polar monomer units, and which is therefore in itself polar.
  • Such a polar polymer is hydrophilic.
  • Hydrophilic (“water-loving”) molecules also termed lipophobic (“fat hating”), contain moieties that can form dipole-dipole interactions with water molecules.
  • Hydrophobic (“water hating”) molecules also termed lipophilic, have a tendency to separate from water.
  • Suitable synthetic polymers are polyacrylamide(s), a styrene- divinylbenzene gel and a copolymer of an acrylate and a diol or of an acrylamide and a diol.
  • An illustrative example is a polymethacrylate gel, commercially available as a Fractogel®.
  • a further example is a copolymer of ethylene glycol and methacrylate, commercially available as a Toyopearl®.
  • a chromatography stationary phase or solid phase may also include natural and synthetic polymer components, such as a composite matrix or a composite or a co-polymer of a polysaccharide and agarose, e.g.
  • a derivatized silica may include silica particles that are coupled to a synthetic or to a natural polymer.
  • Examples of such embodiments include, but are not limited to, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) silica and poly(N- isopropylacrylamide) grafted silica.
  • polystyrene can be used as a solid phase.
  • a chromatography matrix is employed as an affinity chromatography matrix.
  • An affinity chromatography matrix itself includes permanently bonded (usually covalently bonded) moieties that are capable to specifically bind a selected target.
  • a conventional affinity chromatography matrix may include an antibody that binds a particular given target.
  • an affinity chromatography matrix is generally designed such that itself is able to specifically bind the analyte or target that is to be enriched.
  • the affinity chromatography matrix itself is not designed to be capable of specifically binding the cellular therapy starting cell that is to be enriched.
  • the affinity chromatography matrix (stationary or solid phase) used in the present invention comprises an affinity reagent that has at least one or more binding sites Z that are able to specifically bind to a receptor binding reagent that is also employed in the present invention.
  • the receptor binding reagent When the receptor binding reagent is brought into contact with the affinity reagent, a reversible complex via the binding partner C of the receptor binding reagent and the one or more binding sites Z of the affinity reagent is formed.
  • this complex formation relies on non-covalent interactions between a ligand and its respective binding partner.
  • the affinity reagent contains one binding site Z that is able to form a reversible bond with the binding partner C, as long as the affinity reagent is present/provided on the affinity chromatography matrix in a sufficiently high surface density to cause an avidity effect when the complex between the receptor binding reagent and the affinity reagent is formed via the binding site and the binding partner C.
  • the affinity reagent comprises two or more binding sites Z for the binding partner C.
  • two or more receptor binding reagents are immobilized on the affinity chromatography matrix closely arranged to each other such that an avidity effect can take place if the cellular therapy starting cell having (at least two copies of) a second cellular surface receptor present in the sample, is brought into contact with the receptor binding reagent that have one or more binding sites B being able to bind the particular second cellular surface receptor.
  • an avidity (multimerization) effect similar to the one described in US patent 7,776,562, US patent 8,298,782 or International Patent application W002/054065 can take place for allowing a reversible immobilization of the cellular therapy starting cell on the affinity chromatography matrix.
  • the cellular therapy starting cell can be subsequently eluted under mild conditions under which the receptor binding reagent completely dissociates from the cellular therapy starting cell, thereby avoiding that the receptor binding reagent affects the functional status of the cellular therapy starting cell.
  • This enrichment of the cellular therapy starting cell via this affinity chromatography method thus does not only have the advantage that it allows for the enrichment of cellular therapy starting cell population (or any other biological entity described herein) without altering the functional status of the cellular therapy starting cell population that is defined by a common specific receptor molecule. Rather, this method also has the added advantage that it entirely abolishes the need to use magnetic beads for cell purification and thereby simplifies any further handling of the cell and opens the way to automatization of the isolation of cellular therapy starting cells.
  • a chromatography matrix is used that has an affinity reagent immobilized thereon.
  • the affinity reagent is able to bind a binding partner C that is included in a receptor binding reagent (see below).
  • a chromatography matrix may be an affinity chromatography matrix. It may also be a gel filtration matrix, to which the affinity reagent has been coupled.
  • the chromatography matrix is in some embodiments included in a chromatography column, for example packed therein.
  • a sample that is contacted with the chromatography matrix for example, loaded onto a column packed therewith, can likewise be depleted of the receptor binding reagent.
  • the receptor binding reagent is included in a sample that is contacted with a respective stationary phase, i.e. chromatography matrix.
  • the chromatography matrix (regardless of being used for affinity chromatography or for gel permeation) may subsequently be washed with a mobile phase, such as an aqueous medium, e.g. a buffer, in order to remove any matter that has not been immobilized on the chromatography matrix.
  • a mobile phase such as an aqueous medium, e.g. a buffer
  • Dissociation of the above described non-covalent complex, the formation of which immobilizes the cellular therapy starting cell on the affinity chromatography matrix may then be induced, for example, by a change in conditions.
  • a change in conditions may for instance be a change in the ionic strength of an aqueous mobile phase or a change in temperature.
  • a competition reagent is employed in order to induce dissociation of the reversible non-covalent complex between receptor, receptor binding reagent and affinity reagent.
  • the competition reagent is able to associate to the affinity reagent by occupying or blocking the binding site of the affinity reagent for the binding partner included in the receptor binding reagent.
  • the competition reagent By using a competition reagent with a particularly high affinity for the affinity reagent or by using an excess of the competition reagent relative to at least one of the cellular therapy starting cell and the receptor binding reagent (in this case, the competition reagent might also have a lower affinity to the binding site Z of the affinity reagent than the binding partner C of the receptor binding reagent) the non-covalent bonding between the receptor binding reagent and the multimerization reagent may be disrupted.
  • the cellular therapy starting cell is allowed to elute from the chromatography matrix, e.g. from the column into which the chromatography matrix is packed. The eluate is collected and the cellular therapy starting cell thereby collected.
  • the fluid phase used as the mobile phase in chromatography may be any fluid suitable for preserving the biological activity of the cellular therapy starting cell.
  • the fluid is a liquid.
  • the respective liquid is or includes water, for example in the form of an aqueous solution.
  • Further components may be included in a respective aqueous solution, for example dissolved or suspended therein.
  • an aqueous solution may include one or more buffer compounds. Numerous buffer compounds are used in the art and may be used to carry out the various processes described herein.
  • buffers include, but are not limited to, solutions of salts of phosphate such as phosphate buffered saline (PBS), carbonate, succinate, carbonate, citrate, acetate, formate, barbiturate, oxalate, lactate, phthalate, maleate, cacodylate, borate, cell culture medium, N-(2-acetamido)-2-amino- ethanesulfonate (also called (ACES), N-(2-hydroxyethyl)-piperazine-N'-2- ethanesulfonic acid (also called HEPES), 4-(2-5 hydroxyethyl)-1 -piperazine- propanesulfonic acid (also called HEPPS), lactated ringers solution, piperazine-1 ,4- bis(2-ethanesulfonic acid) (also called PIPES), (2-[Tris(hydroxymethyl)-methylamino]- 1 -ethansulfonic acid (also called PIPES), (2-
  • buffers include, but are not limited to, triethanolamine, diethanolamine, zwitterionic buffers such as betaine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris-(hydroxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-iminotris(hydroxymethyl)-methane (also called BISTRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (also called TRICINE), to name only a few.
  • TRIS tris-(hydroxymethyl)aminomethane
  • BISTRIS bis-(2-hydroxyethyl)-iminotris(hydroxymethyl)-methane
  • TRICINE Tris(hydroxymethyl)-methyl]-glycine
  • the buffer may further include components that stabilize the cellular therapy starting cell to be enriched, for example proteins such as (serum) albumin, growth factors, trace elements and the like.
  • proteins such as (serum) albumin, growth factors, trace elements and the like.
  • suitable mobile phase is within the knowledge of the person of average skill in the art and can be carried out empirically.
  • the strength of the binding between the receptor binding reagent and a second cellular surface receptor on the cellular therapy starting cell may not be essential for the reversibility of the binding of the cellular therapy starting cell to the affinity reagent via the receptor binding reagent. Rather, irrespective of the strength of the binding, meaning whether the dissociation constant (Kd) for the binding between the receptor binding reagent via the binding site B and the second cellular surface receptor is of low affinity, for example, in the range of a Kd of about 10’ 3 to about 10’ 7 M, or of high affinity, for example, in the range of a Kd of about 10’ 7 to about 1 x 1O’ 10 M, the cellular therapy starting cell can be reversibly stained as long as the dissociation of the binding of the receptor binding reagent via the binding site B and the second cellular surface receptor occurs sufficiently fast.
  • Kd dissociation constant
  • the dissociation rate constant (k O ff) for the binding between the receptor binding reagent via the binding site B and the second cellular surface receptor may have a value of about 3 x w 5 sec 1 or greater (this dissociation rate constant is the constant characterizing the dissociation reaction of the complex formed between the binding site B of the receptor binding reagent and the second cellular surface receptor on the surface of the cellular therapy starting cell).
  • the association rate constant (k on ) for the association reaction between the binding site B of the receptor binding reagent and the second cellular surface receptor on the cellular therapy starting cell may have any value.
  • the k O ff value of the binding equilibrium is advantageous to select the k O ff value of the binding equilibrium to have a value of about 3 x w 5 sec 1 or greater, of about 5 x w 5 se 1 or greater, such as about 1 x 10’ 4 sec 1 or greater, about 1.5 x 10’ 4 sec 1 or greater.
  • the second cellular surface receptor that is located on the cellular therapy product starting cell surface may be any molecule as long as it remains covalently or noncovalently bonded to the cell surface during a chromatographic separation process in a method according to the invention.
  • the second cellular surface receptor is a molecule against to which a receptor binding reagent may be directed.
  • the second cellular surface receptor is a peptide or a protein, such as a membrane receptor protein.
  • the second cellular surface receptor is a lipid, a polysaccharide or a nucleic acid.
  • a second cellular surface receptor that is a protein may be a peripheral membrane protein or an integral membrane protein. It may in some embodiments have one or more domains that span the membrane.
  • the second cellular surface receptor may be an antigen defining a desired cell population or subpopulation, for instance a population or subpopulation of blood cells, e.g. lymphocytes (e.g. T cells, T-helper cells, for example, CD4+ T- helper cells, B cells or natural killer cells), monocytes, or stem cells, e. g. CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells.
  • lymphocytes e.g. T cells, T-helper cells, for example, CD4+ T- helper cells, B cells or natural killer cells
  • monocytes e.g. CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells.
  • T-cells include cells such as CMV-specific CD8+ T-lymphocytes, cytotoxic T-cells, memory T-cells and regulatory T-cells (Treg).
  • Treg are CD4+CD25+CD45RA Treg cells and an illustrative example of memory T-cells are CD62L+CD8+ specific central memory T-cells.
  • the receptor may also be a marker for a tumor cell.
  • Typical second cellular surface receptors used herein are CD3, CD4, CD8 (T-cell), CD45 (Leukocytes), CD56 (NK cell), CD27 (B and T-Cell), CD28 (B and T-Cell), CD19 (B-cell), CD62L, and CD25 (T cell).
  • the binding partner C that is included in the receptor binding reagent includes biotin and the affinity reagent includes a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments the binding partner C that is included in the receptor binding reagent includes a biotin analog that reversibly binds to streptavidin or avidin, and the affinity reagent includes streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective biotin analog.
  • the binding partner C that is included in the receptor binding reagent includes a streptavidin or avidin binding peptide and the affinity reagent includes streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective streptavidin or avidin binding peptide.
  • the binding partner that is included in the receptor binding reagent may include a streptavidin-binding peptide Trp-Ser-His-Pro-GIn-Phe-Glu-Lys (SEQ ID NO. 10) and the affinity reagent may include a streptavidin mutein having the amino acid sequence Val 44 -Thr 45 -Ala 46 -Arg 47 (SEQ ID NO.
  • streptavidin binding peptides might, for example, be peptides such as the “Strep-tag®” with one binding module as described in US patent 5,506,121 , for example, or streptavidin binding peptides having a sequential arrangement of two or more individual binding modules as described in International Patent Publication WO 02/077018 or US patent 7,981 ,632.
  • streptavidin binding peptides having a sequential arrangement of two individual binding modules are also offered under the trademark Twin-Strep-tag® by IBA Lifesciences GmbH, Gottingen, Germany.
  • Further suitable streptavidin muteins that can be used in the present invention are described in International patent applications WO 2014/076277 A1 or WO 2017/186669 A1.
  • streptavidin muteins are commercially available from IBA Lifesciences GmbH Gottingen under the name Strep-Tactin® XT.
  • the streptavidin muteins of WO 2017/186669 may (a) have a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin (as set forth in SEQ ID NO: 9) and (b) comprises at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth in SEQ ID NO: 9.
  • These muteins could additional have one of the above mentioned mutations SEQ ID NO. 7 or 8.
  • the binding partner C comprised in the receptor binding reagent comprises an affinity tag
  • the affinity reagent comprises a suitable binding partner for the affinity tag, which may be an antibody or an antibody fragment, which is capable of binding to the affinity tag.
  • Non-limiting examples of such affinity tags are an oligohistidine, a polyhistidine, an immunoglobulin domain, a maltose-binding protein, a glutathione-S-transferase (GST), a chitin binding protein (CBP), a thioredoxin, a calmodulin binding peptide (CBP), a FLAG’-peptide, a HA-tag, a VSV-G-tag, and a HSV-tag.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • CBP a thioredoxin
  • CBP calmodulin binding peptide
  • FLAG’-peptide FLAG’-peptide
  • HA-tag a HA-tag
  • VSV-G-tag a VSV-G-tag
  • HSV-tag HSV-tag
  • the affinity reagent is an oligomer or a polymer of streptavidin or avidin or of any analog of streptavidin or avidin.
  • the binding site Z is the natural biotin binding of avidin or streptavidin.
  • the respective oligomer or polymer may be crosslinked by a polysaccharide.
  • oligomers or polymers of streptavidin or of avidin or of analogs of streptavidin or of avidin may be prepared by the introduction of carboxyl residues into a polysaccharide, e. g. dextran, essentially as described in Noguchi, A, et al., (11 ) in a first step.
  • streptavidin or avidin or analogs thereof may be linked via primary amino groups of internal lysine residue and/or the free N-terminus to the carboxyl groups in the dextran backbone using conventional carbodiimide chemistry in a second step.
  • cross-linked oligomers or polymers of streptavidin or avidin or of any analog of streptavidin or avidin may also be obtained by crosslinking via bifunctional molecules, serving as a linker, such as glutardialdehyde or by other methods described in the art.
  • the one or more binding sites of the receptor molecule binding reagent which specifically binds to the second cellular surface receptor, may for instance be an antibody, a fragment thereof and a proteinaceous binding molecule with antibody-like functions.
  • (recombinant) antibody fragments are Fab fragments, Fv fragments, 5 single-chain Fv fragments (scFv), a divalent antibody fragment such as an (Fab)2'-fragment, nanobodies, diabodies, triabodies (12), decabodies (13) and other domain antibodies (14).
  • one or more binding sites of the receptor molecule binding reagent may be a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein that is also known as "duocalin".
  • the receptor binding reagent may have a single second binding site, i.e., it may be monovalent.
  • monovalent receptor binding reagents include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody like binding properties or an MHC molecule.
  • monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.
  • an immunoglobulin Especially preferred are an immunoglobulin, a functional fragment of an immunoglobulin, a proteinaceous binding molecule with immunoglobulin-like functions, an aptamer and an MHC molecule as receptor binding reagent.
  • the reversible immobilization is carried out via two or more types of the second cellular surface receptor, for example, by sequential immobilization via the cell surface receptor CD3 as first type of second cellular surface receptor and CD4 as second type of second cell surface receptor.
  • enriching via reversible immobilization of the cellular therapy starting cell is carried out as single enrichment step, without prior cell separation, in particular without prior cell separation in a cell separation module comprising a cell separator configured to produce a fraction enriched in nucleated blood cells.
  • the affinity matrix is bound/covalently coupled to beads and the beads are incubated with the sample comprising the cellular therapy starting cell in a reservoir in which the beads are moved.
  • a reservoir may be a column capped on both sides, which is incubated on a shaker. The advantage of such a reservoir is that the beads are movable within the reservoir and more cells could be bound by the receptor binding reagent via the second cellular surface receptor.
  • the affinity reagent is immobilized on the inner surface of the tubing, or a portion thereof, by (means of) a covalent bond, whereby this part of the tubing may be columnar extended.
  • a portion is advantage as it could be used like a known column but within the closed system.
  • higher purity of enriched cellular therapy starting cells may be achieved compared to other methods.
  • the cellular therapy starting cell is genetically modified using one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor and/or for expression and/or secretion of the polypeptide, thereby generating a precursor of the cellular therapy product.
  • the enriched cellular therapy starting cell is genetically modified, such that the cell after the gene modification is capable of expressing the first cellular surface receptor and/or for expression and/or secretion of the polypeptide.
  • the cell is termed “precursor of the cellular therapy product” or “precursor cell of the cellular therapy product”, because directly after gene modification the cell does not express the modified gene. Only after some time the cell expresses the modified gene and so the precursor of the cellular therapy product changes to the cellular therapy product, the cell expressing the first cellular surface receptor and/or the polypeptide.
  • the used cell has three different terms depending on the process step of the inventive method.
  • the one or more reagent(s) used for the gene modification is a set of reagents for gene modification.
  • the set of reagents comprise a viral gene delivering system or a non-viral gene delivering system.
  • the viral gene delivering system can be any suitable viral gene delivering system known to the skilled person. Typical but not limiting examples of such viral gene delivering system are systems which use a retroviral transduction, a lentiviral transduction, or an adenoviral transduction. Such systems are commercially available.
  • the non- viral gene delivering system can be any suitable non-viral gene delivering system known to the skilled person.
  • Typical but non-limiting examples of such non- viral gene delivering system are electroporation, lipid nanoparticles, mechanoporation, ultrasound treatment, optical gene delivery, exosomes, amphiphilic peptides or a combination thereof.
  • lipid nanoparticles which is shown in Example 2.
  • the set of reagents comprises nucleic acids coding for proteins which facilitate the gene transfer, e.g. nucleases. Additional or instead of the nucleic acid the proteins themselves can be part of the set of reagents.
  • Typical non-limiting examples are a nucleic acid encoding a Transcription activator-like effector nuclease (TALEN), a Zinc-finger nuclease (ZFN), CRISPR and Cas, or transposases such as sleeping beauty, piggyback, Tn5 transposases.
  • Exemplary proteins are a Transcription activator-like effector nuclease (TALEN), a Zinc-finger nuclease (ZFN), Cas.
  • the gene construct as part of the set of reagents comprising a nucleic acid encoding the first cellular surface receptor and/or the polypeptide, wherein the nucleic acid can be DNA or RNA.
  • DNA includes any possible type of DNA, e.g. single stranded DNA, double stranded DNA.
  • RNA includes any possible type of RNA.
  • the gene construct comprises a nucleic acid encoding a chimeric antigen receptor (CAR), or a recombinant T cell receptor (TCR), and/or switch receptors or a bi-specific T cell engager (Bite), which is able to bind to the drug target molecule. It is known to the skilled person how to construct the chimeric antigen receptor (CAR) and the recombinant T cell receptor.
  • the gene construct comprises a nucleic acid encoding the polypeptide, which is able to bind to a drug target molecule either soluble or associated with a drug target cell.
  • the gene modification takes place after eluting the cellular therapy starting cell from the stationary phase. This is exemplified in Example 2.
  • the gene modification takes place before eluting the cellular therapy starting cell from the stationary or solid phase. Also, this is exemplified in Example 2.
  • the sample is a whole blood sample
  • the cellular therapy starting cell is a non-activated T cell
  • the set of reagents comprises a non-viral gene delivering system, a nucleic acid and/or protein encoding the CRISPR and Cas, and a nucleic acid encoding a chimeric antigen receptor (CAR) or a TCR, which is able to bind to the drug target molecule.
  • CAR chimeric antigen receptor
  • the sample is a whole blood sample
  • the cellular therapy starting cell is an activated or a non-activated B cell
  • the set of reagents comprises a non-viral gene delivering system, a nucleic acid encoding the CRISPR and Cas, and a nucleic acid encoding the polypeptide.
  • the sample is a whole blood sample
  • the cellular therapy starting cell is an activated or a non-activated T cell
  • B cell or NK cell and the set of reagents comprises a non-viral gene delivering system comprising a lipid nanoparticle.
  • the invention is directed to a use of a set of reagents for genetically modifying a cellular therapy starting cell as mentioned before and included herein by reference.
  • the cellular therapy starting cell is immobilized on a stationary phase or a receptor binding reagent as described in the first aspect.
  • the precursor of the cellular therapy product is eluted from the stationary phase or the receptor binding reagent after the genetic modification.
  • the invention is directed to a kit for manufacturing a cellular therapy product, wherein the cellular therapy product comprises a first cellular surface receptor which is able to bind to a drug target molecule (on the surface) of a drug target cell, and/or wherein the cellular therapy product produces and/or secretes a polypeptide which is able to bind to a drug target molecule either soluble or associated with a drug target cell, the kit comprising a) one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor or of the polypeptide of the cell therapy product, and b) a receptor binding reagent comprising a binding site B and a binding partner C,
  • the binding site B comprised in the receptor binding reagent is capable of specifically binding to the second cellular surface receptor on a cellular therapy starting cell and wherein the bond between the binding site B of the receptor binding reagent and the second cellular surface receptor has a dissociation constant (KD) is of low affinity or in the range from about 10’ 3 to about 10’ 7 M,
  • KD dissociation constant
  • the binding partner C comprised in the receptor binding reagent is capable of reversibly binding to a binding site Z of an affinity reagent and the reversible bond between the binding partner C of the receptor binding reagent and the binding site Z of the affinity reagent has a dissociation constant (KD) in the range from about 10’ 2 to about 10’ 13 M, and
  • the stationary phase having the affinity reagent immobilized thereon
  • the affinity reagent comprises one or more binding sites Z, wherein said binding site Z forms a reversible bond with the binding partner C comprised in the receptor binding reagent, and wherein the binding site B of the receptor binding reagent binds to the second cellular surface receptor on the cellular therapy starting cell, to allow reversibly immobilizing the cell therapy starting cell on the stationary phase.
  • the one or more reagent(s) comprising a gene construct for expression of the first cellular surface receptor or of the polypeptide of the cell therapy product are described above in the method section; this description is incorporated herein by reference.
  • the receptor binding reagent, the stationary phase, the solid phase and the affinity reagent are as described above in the method section; this description is incorporated herein by reference.
  • the one or more reagent(s) comprise a viral gene delivering system or a non-viral gene delivering system, a nucleic acid part encoding a Transcription activator-like effector nuclease (TALEN), a Zinc-finger nuclease (ZFN), or CRISPR and Cas, and a gene construct comprising a nucleic acid encoding the first cellular surface receptor or the polypeptide.
  • TALEN Transcription activator-like effector nuclease
  • ZFN Zinc-finger nuclease
  • CRISPR and Cas CRISPR and Cas
  • the cellular therapy starting cell can typically be a T cell, a B cell, or a NK cell and may be a non-activated T cell or a non-activated B cell.
  • the gene construct of the kit comprises a nucleic acid encoding a chimeric antigen receptor (CAR) or TCR, which is able to bind to the drug target molecule.
  • CAR chimeric antigen receptor
  • TCR chimeric antigen receptor
  • the gene construct of the kit comprises a nucleic acid encoding the polypeptide, which is able to bind to a drug target molecule either soluble or associated with a drug target cell. This is also described above in more detail; this description fully applies to the embodiments described in this paragraph.
  • the drug target cell can be a diseased cell, e.g. a cancer cell or a cell affected by an autoimmune disease. Also, cells relating to an infectious disease are possible as the drug target cell. This is described in more detail above; this description fully applies to the embodiments described in this paragraph.
  • the viral gene delivering system of the kit comprises a retroviral transduction system or a lentiviral transduction system. These systems are known to the skilled person and described above; this description fully applies to the embodiments described in this paragraph.
  • the non-viral gene delivering system of the system comprises an electroporation, a lipid nanoparticle, mechanoporation, ultrasound treatment, optical gene delivery, exosomes, amphiphilic peptides or a combination thereof.
  • kit is used in the method described above.
  • the invention is directed to a device for manufacturing a cellular therapy product, comprising an operating device and a tubing-set, wherein the tubing set is a closed and single-use tubing-set, for use in the methods described above (the description is incorporated herein by reference).
  • tubing and “tubing set” includes the term “cassette”.
  • tubing set is a closed system within the above definition, which fully applies to the embodiments described in this paragraph.
  • the operating device comprises a liquid handling control unit comprising one or more valve(s), a liquid pump, a reservoir holder, and optionally a reservoir rocker.
  • the operating device can further comprise a process unit, for operating process steps, comprising blood collection, second target cell hold-step (cell enrichment), second target cell release/elution, second target cell wash, genetic modification, precursor cell of a cellular therapy product wash and formulation, reinfusion of the precursor cell of a cellular therapy product by optionally controlling the valve and optionally comprising means for input of commands relating to the process steps and means for output of an information relating to the process steps.
  • the operating device may include every possible part for automatic execution of the inventive method and the above-mentioned process steps, excluding the tubing set.
  • the tubing-set comprises a system for blood withdrawal, a system for reinfusion of cells into the human body, a second target cell retention reservoir, a genetic modification unit, a second target cell washing and formulation unit, a blood reservoir, one or more bag containing process buffers, a bag containing a set of reagents for gene modification as describe above in the method and kit sections (which are incorporated herein by reference), a waste reservoir and a re-infusion reservoir, wherein the reservoirs and bags are fluidly connected by a closed tube system.
  • the closed tube system is a closed system within the above definition.
  • the second target cell retention reservoir and the genetic modification unit share the same space.
  • the second target cell retention reservoir may be a column or a part of a tubing as described elsewhere herein and the genetic modification takes place within the second target cell retention reservoir turning it to the genetic modification unit while being the same column or tubing part.
  • the blood reservoir is fluidly connected to the second target cell retention reservoir
  • the second target cell retention reservoir is fluidly connected to the bag containing process buffers and optionally to the waste reservoir
  • the second target cell retention reservoir is in a first alternative further fluidly connected with the genetic modification unit, in a second alternative, in the case that the gene modification of the cellular therapy starting cell takes place before eluting the cellular therapy starting cell from the stationary phase, the genetic modification unit is part of the second target cell retention unit in that both units share the same space as described above,
  • the second target cell retention unit is fluidly connected to the bag containing the set of reagents for gene modification or the gene therapy product, to the bag containing process buffers and optionally to the waste reservoir,
  • the second target cell retention unit is further fluidly connected to the second target cell washing and formulation unit,
  • the second target cell washing and formulation unit is fluidly connected to the bag containing process buffers and to the waste reservoir
  • the second target cell washing and formulation unit is further fluidly connected to reinfusion reservoir
  • the re-infusion reservoir is fluidly connected to the system for reinfusion of cells into the human body
  • fluidly connections comprise tubes and valves, which could be controlled by the process unit.
  • the second target cell retention reservoir can be a tubular expansion of the fluidly connection.
  • an inner side of the second target cell retention reservoir can be a stationary phase as specified and further described above in the method section, this description is incorporated herein by reference.
  • the second target cell retention reservoir may comprise one or more sieve(s), for example to hold back particles comprising the affinity matrix.
  • the second target cell retention reservoir may also comprise obstacles such as one or more pins or meshes.
  • the invention is directed to a tubing, which equals to tubing set, having an affinity reagent immobilized on an inner surface of the tubing, wherein the affinity reagent comprises one or more binding sites Z, wherein said binding site Z forms a reversible bond with a binding partner C comprised in the receptor binding reagent, and wherein the binding site B of the receptor binding reagent binds to a (second) cellular surface receptor on a cellular therapy starting cell, thereby allowing reversibly immobilizing the cellular therapy starting cell on the tubing acting as stationary phase.
  • the meaning and examples relating to the affinity reagent, the binding partner C, the receptor binding reagent, and the binding site B are described in detail in the method section above and fully apply to the embodiments described in this paragraph.
  • the affinity reagent can be immobilized on the inner surface of the tubing by a covalent bond.
  • the affinity reagent is an avidin, a streptavidin or a streptavidin mutein.
  • the streptavidin mutein may include the amino acid sequences Val 44 -Thr 45 -Ala 46 -Arg 47 (SEQ ID NO. 7) or lle 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO. 8), and/or a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin (as set forth in SEQ ID NO: 9) and/or (b) may comprise at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth in SEQ ID NO: 9.
  • Examples of such mutein include the muteins m 1 -9 or m302 as described in WO 2014/076277.
  • the invention is directed to a use of the tubing of the fifth aspect in a method of manufacturing a cellular therapy product as described above and the disclosure of which fully applies to the embodiments described in this paragraph.
  • Example 1 Cellular therapy starting cell enrichment
  • the cellular therapy starting cells from a whole blood sample these cells were selective and reversible immobilized.
  • an anti-CD3 affinity matrix was used.
  • the matrix was composed as following (calculated on 20mL matrix): 10mg of multimerized streptavidin mutein (Strep-Tactin® m2 (as described in US 6,103,493), SEQ Id. No. 8) was coupled to 8g initial dry weight epoxy-activated polystyrene resin (CY17030; Cytosorbents) using solvent-exposed primary amine groups, in phosphate buffered saline (PBS) buffer.
  • PBS phosphate buffered saline
  • 20mL final anti-CD3 functionalized matrix were filled into a reservoir bigger than 20mL to enable movement of the matrix during blood loading, non-target cell washing and release of the hold-back cellular therapy starting cells.
  • the reservoir including the matrix Prior to holding back the cellular therapy starting cells, the reservoir including the matrix was equilibrated with wash buffer (PBS with 0.5% HSA).
  • the above mentioned anti-CD3 Fab fragment is peptide-tagged and was produced recombinantly (see International Patent App. Pub. Nos. WO 2013/011011 and WO 2013/124474).
  • the Fab fragment was derived from the CD3 binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001 TM ; see also U.S. Patent No. 4,361 ,549). It contained the heavy chain variable domain and light chain variable domain of the anti-CD3 antibody OKT3 e. g. described in Figure 1 in (16).
  • an anti-CD19 affinity matrix is used.
  • the matrix is composed as following (calculated on 20mL matrix): 10mg of multimerized streptavidin mutein (Strep-Tactin® m2 (as described in US 6,103,493), SEQ Id. No. 8) is coupled to 8g initial dry weight epoxy-activated polystyrene resin (CY17030; Cytosorbents) using solvent-exposed primary amine groups, in phosphate buffered saline (PBS) buffer. Afterwards cellular therapy starting cell selection agent (e.g.
  • 20mL final anti-CD19 functionalized matrix is filled into a reservoir bigger than 20mL to enable movement of the matrix during blood loading, non-target cell washing and release of the hold-back cellular therapy starting cells.
  • the reservoir including the matrix Prior to holding back the cellular therapy starting cells, the reservoir including the matrix is equilibrated with wash buffer (PBS with 0.5% HSA).
  • wash buffer PBS with 0.5% HSA.
  • Enrichment of cellular therapy starting cells using a liquid-handling device CD3+ cells
  • CD3+ cells CD3+ cells from two independent fresh whole blood samples (2 - 3 h post blood withdrawal) were enriched using a commercially available liquid handling device with a closed system.
  • the liquid handling device includes the operating device and a tubing set wherein the blood reservoir, containing the whole blood sample, is fluidly connected to the second target cell retention reservoir and the second target cell retention reservoir is fluidly connected to the bag containing process buffers and optionally to the waste reservoir.
  • wash buffer phosphate buffered saline (PBS) buffer with 0,5% human serum albumin (HSA)
  • HSA human serum albumin
  • the short enrichment step of cellular therapy starting cells was finished by flushing a 200 pM D-Biotin buffer solution (phosphate buffered saline (PBS) buffer with 0,5% human serum albumin (HSA) and 200 pM D-Biotin) into the matrix containing reservoir.
  • Cellular therapy starting cell release was performed with a speed of 75 mL per minute (calculated onto a standard blood tubing) in 30mL pulses using a backward pulse after each step in total 3 times under gentle movement of the matrix. Further cellular therapy starting cell release steps were performed by flushing the matrix with 30mL D-Biotin buffer followed by an incubation step with stronger movement of the matrix for 3 min, repeating this step 7 times for Run 1 and double the amount for Run 2. During cellular therapy cells release step the complete volume was flushed through a device specific membrane including device specific parameters for automated target cell wash into a final volume of 20 ml. Thus, the cellular therapy starting cells were specifically enriched by a reversible immobilization on an anti-CD3 affinity matrix.
  • Fig. 4A depicts the cell count (total nucleated cell count, TNC) in million cells of the starting whole blood sample and of the enriched CD3+ T cells.
  • Fig. 4B shows the depletion
  • Fig. 4C the purity
  • Fig. 4D the yield of CD3+ cells. Depletion was calculated on CD3+ cells in the starting material vs. CD3+ cells in the negative fraction (flow-through, etc.). Yield is calculated on CD3+ cells in the starting material vs. CD3+ cells in the positive fraction (eluate). Purity is measured via Flow Cytometry and pregated on single living CD45 cells. The results show a yield of CD3+ cells of about 40 %, with a very high purity of 99,2 %.
  • the enrichment step of the inventive method is unique in its ability to enrich CD3+ cells with a high yield and high purity.
  • Fig. 4E shows flow cytometry analysis of the whole blood sample and the CD3+ cell fraction (eluate) of two independent donors. For each of the two blood samples the CD19+ cells and CD3+ cells (CD19 vs. CD3) were measured. The results show a significant enrichment of CD3+ T cells with a clear reduction of CD19+ cells in the CD3+ cell fraction compared to whole blood sample.
  • the yield of CD3+ cells were 48,5 % in the whole blood sample and 99,5 % in the CD3+ cell fraction whereas the yield of CD19+ cells was reduced from 4,3 % in the whole blood sample to 0,03 % in the CD3+ cell fraction (cf. Fig. 4E, donor 2).
  • the enrichment of CD3+ T cells using a commercially available liquid handling device results in a yield of about 99% CD3+ T cells in the eluate, showing a specific and highly pure T cell fraction for using in the gene modification step of the inventive method.
  • CD19+ cells from a fresh whole blood sample are enriched using a commercially available liquid handling peristaltic pump and commercially available tubing to operate the peristaltic pump (or any other liquid handling systems).
  • the needed tubing set is self-build and includes the blood reservoir, containing the whole blood sample, that is fluidly connected to the target cell retention reservoir and the target cell retention reservoir is fluidly connected to the bag containing process buffers and optionally to the waste reservoir.
  • 100mL of whole blood from a healthy human donor with anticoagulant is transferred into one bag. Subsequently the bag is connected to the tubing set and the blood is flushed stepwise into a reservoir containing anti-CD19 affinity matrix (prepared as describe above) where cellular therapy starting cells, the target cells, are immobilized for a short time period while non-target cells flow through the affinity matrix into another separate bag.
  • Blood loading is performed with a special speed (e.g. 20 mL per minute, calculated onto a standard blood tubing) in 15mL pulses, following a backward-pulse and incubation of blood under gentle moving conditions of the matrix particles after each 15mL pulse.
  • wash buffer phosphate buffered saline (PBS) buffer with 0,5% human serum albumin (HSA)
  • HSA human serum albumin
  • the short enrichment step of cellular therapy starting cells are finished by flushing a D-Biotin buffer solution (phosphate buffered saline (PBS) buffer with 0,5% human serum albumin (HSA) and e.g.
  • Cellular therapy starting cell release is performed with a speed of 75 mL per minute (calculated onto a standard blood tubing) in 30mL pulses using a backward pulse after each step in total multiple times (e.g. 3) under gentle movement of the matrix. Further cellular therapy starting cell release steps are performed by flushing the matrix with 30mL D-Biotin buffer followed by an incubation step with stronger movement of the matrix for a time period (3 min, repeating this step multiple times (e.g. 7). After cellular therapy cells release step the complete volume is washed using centrifugation for a final cell wash into a final volume of 20 ml. Thus, the cellular therapy starting cells will be specifically enriched by a reversible immobilization on an anti-CD19 affinity matrix.
  • the cellular therapy starting cells were washed and transferred into electroporation buffer by using a Counterflow Centrifuge System.
  • a protocol was used to achieve maximal cellular therapy starting cell yield and minimal cellular therapy starting cell output volume with the following parameters: Input cells 300m L, Wash Buffer WOOmL, Load Waste OmL, Pulsing Buffer 55m L, Air Filter OmL, Wash waste OmL, Output OmL.
  • Electroporation buffer KCI 5mM, MgCl2 15mM, Sodium Succinate 25mM, D-Mannitol 25mM, Na2HPO4 120mM.
  • Electroporation buffer KCI 5mM, MgCl2 15mM, Sodium Succinate 25mM, D-Mannitol 25mM, Na2HPO4 120mM.
  • Gene modification of cellular therapy starting cells via electroporation using CD3+ cells After cellular therapy starting cell wash and re-buffering into electroporation buffer the cellular therapy starting cells were immediately electroporated in 5x10 6 cellular therapy starting cells number steps using 14,5 pM of ribonucleoprotein (RNP) complexes with Streptococcus pyogenes (S.
  • RNP ribonucleoprotein
  • pyogenes Cas9 and 1 pg of a HDR template per 1x10 6 cellular therapy starting cells comprising 5’ scrambled bases, 5' Cas9 target sequence site (CTS), 5’ homology arm, gRNA target sequence, 3’ homology arm, 3' Cas9 target sequence site (CTS), 5’ homology arm, 3’ scrambled bases. Additionally, 1 pM XL-413 CDK inhibitor and 1 pM M-3814 DNA-PK inhibitor was added during or after electroporation. The gene modification was done essentially as described in (15).
  • the TRAC-targeting gRNA is 5’-AGAGTCTCTCAGCTGGTACA-‘3 (SEQ ID NO. 3).
  • TRAC exon 1 target site 5'-ATTCACCGATTTTGATTCTC- 3' (SEQ ID NO. 6)
  • the gene modified cellular therapy starting cells which are now a precursor of the cellular therapy product, were washed and transferred into NaCI buffer.
  • the mechanoporation was used for mechanical disruption to permeabilize cell membranes of CD3+ cells for intracellular delivery of the RNP complex.
  • the Gateway system of Portal Biotechnologies was utilized to deliver cargo into purified CD3+ cells using a target cell affinity matrix.
  • the Gateway device squeezes the cells by mechanical pressure through a microchip with pores which leads to a disrupted cell membrane and enables cargo to enter the cell.
  • the process where cells are squeezed by pressure is called “boosting”.
  • the aim of the experiments was to knock-out the TRAC (T-cell receptor alpha chain constant) locus of CD3+ cells using the Gateway system.
  • the knock-out was performed by delivering an RNP complex, composed of Cas9 Nuclease and gRNA directed against the TRAC locus, into the cell.
  • Fig. 5 mechanoporation experiments were performed (Fig. 5) using a microchip with pore size of 5 pm.
  • RNA For Mechanoporation, selected CD3+ cells were resuspended in serum-free medium (Opti-MEM). 2 - 5 Mio CD3+ cells in 50 pl were used per boost.
  • Opti-MEM serum-free medium
  • 2 - 5 Mio CD3+ cells in 50 pl were used per boost.
  • tracrRNA and crRNA were mixed leading to a concentration of 40 pM, respectively.
  • the Cas9 nuclease is diluted to a concentration of 21 pM in sterile PBS and slowly added to the gRNA (tracrRNA + crRNA). The mixture is then incubated for 15 min at RT for assembly. 3 pl RNP complex per 1 Mio cells is added to the boosting sample.
  • the sample volume being boosted is adjusted to 100 pl.
  • the cells were boosted with 8 psi (pound-force per square inch) and collected in a tube. Between each sample, a wash of the microchip with 100 pl OptiMEM is performed. Pre-warmed complete medium was added to the cells post boost and cells were transferred to a cell culture plate. Cells were cultured for up to 12 days at 37°C and knock-out frequency was analyzed using flow cytometry. Cellular therapy starting cell wash and re-buffering using B cells
  • the cellular therapy starting cells are washed and transferred into buffer for genetic modification, e.g. electroporation buffer, by using a Centrifuge System.
  • buffer for genetic modification e.g. electroporation buffer
  • cellular therapy starting cells via e.g. electroporation using B cells
  • a ribonucleoprotein (RNP) complex (composed of Cas9 and guide RNA) is used to knock out the specific gene in the cell.
  • an HDR dsDNA template is added to the electroporation reagents to insert the gene of interest into the IGHG1 locus (e.g. enzyme like insulin ⁇ GFP reporter).
  • IGHG1 locus e.g. enzyme like insulin ⁇ GFP reporter.
  • a whole blood sample (e.g. 200ml) is collected into one bag and directly flushed into a reservoir containing anti-CD19 affinity matrix where cellular therapy starting cells, the target cells, are immobilized for a short time period while non-target cells flow through the affinity matrix into another separate bag.
  • non-target cells After complete loading of the whole blood non-target cells are washed out with wash buffer.
  • the cellular therapy cells are immediately genetically modified while staying on the anti-CD19 affinity matrix using e.g. LNPs.
  • the LNPs are flushed onto the anti-CD19 affinity matrix and incubated with the CD19 cells on the matrix.
  • Incubation of LNPs with the cellular therapy cells on the anti-CD19 affinity matrix can be either done with a coincubation with an activation reagent or without an activation reagent. After the incubation period the genetically modified cells are released from the anti-CD19 affinity matrix by flushing D-Biotin buffer into the matrix containing reservoir. After release of the cellular therapy cells, they are washed and reinfused into the human body after an optional in-process-analytics step. Transfection of CD3+ cells using liquid nanoparticles (LNP) after elution from the stationary phase
  • LNP liquid nanoparticles
  • CD3+ cells were selected from whole blood using a cell affinity matrix.
  • LNPs containing the desired information were generated by Pantherna Therapeutics GmbH.
  • 1 Mio CD3+ cells were resuspended in cell culture medium without antibiotics and 15 pg LNPs were added to the cells. The total sample volume was adjusted to 500 pl per well. Cells were then incubated for at least 4 h up to 9 d. Analysis (cell number, viability, GFP expression, phenotype) was performed on different time points, starting with the earliest time point at 4 h post transfection and the last time point on day 9. T cells for data in Fig. 6B and Fig.
  • T cells in Fig. 6E are analyzed after 4h incubation and on day 1 (after 24h), day 3, day 5, day 7 and day 9 after LNP incubation.
  • CD3+ cells were selected from 1 ml whole blood using a cell affinity matrix in a pipette tip.
  • LNPs containing the desired information were generated by Pantherna Therapeutics GmbH.
  • CD3+ cells were flushed with cell culture medium without antibiotics and 15 pg LNPs were added to the pipette tip in 60 pl volume.
  • Cells were then incubated for 4 h in the incubator while staying on the pipette tip. After 4 h cells where eluted from the resin in the pipette tip by using 0,1 mM D-Biotin and analyzed. Analysis (cell number, viability, GFP expression, phenotype) was performed and data are shown in Fig. 6C and Table 1 .
  • Example 3 Determination of functionality of the cellular therapy product in vivo
  • the expression and expansion of the target cells was studied in a clinically relevant humanized murine lymphoma model.
  • NSG-SGM3 6-8 week old NSG-SGM3 (NSGS) mice were injected intravenously with 0.5x10 6 Raji- fluc-GFP CD19+ tumor cells. Two days later these mice were intraperitoneally injected with the precursor of the cellular therapy product. A total of 10x10 6 precursor cells of the cellular therapy product were administered as flat dose. TRAC-CAR knock-in and expression was confirmed after extended parallel in vitro culture by flow cytometry (cf. Fig. 7C). Conventionally manufactured CD19 CAR T cells using a standard lentivirus (LV) transfection and expansion process served as positive controls while mice only injected with PBS served as negative control group. All groups consisted of 3-4 mice. The mice were monitored each 7 days by tumor growth imaging using MS bioluminescence and measuring in vivo expansion of the cellular therapy product in blood. Therefore, mice were bled weekly for ex vivo analysis using flow cytometry.
  • LV lentivirus
  • mice treated with precursor of the cellular therapy product and standard LV control cells showed rapid expansion and high tumor reactivity with complete tumor clearance post day 20 (Fig. 7B middle and right columns).
  • control groups treated with PBS only showed uncontrolled tumor progression (Fig. 7B left column) with mice succumbing to the tumor starting around day 14.
  • Fig. 7D depicting a Kaplan-Meier analysis, mice injected with PBS control died around 14 days post tumor cell injection whereas mice injected with the precursor of the cellular therapy product (TQ process 1 ) or standard LV control cells survived until the end of the experiment (measured until day 21 ).
  • the residual Raji tumor cells were measured in blood using flow-cytometry. The results are shown in Fig. 8B.
  • the amount of tumor cells is high compared to the tumor cell amount in mice treated with the precursor of the cellular therapy product (TQ process) or standard LV control cells.
  • mice were bled weekly for ex vivo analysis using flow cytometry and staining for T cell subsets and CAR expression.
  • Fig. 8D and Fig. 8F only CD4+ T cells were measured. The result shown in Fig. 8F, is also true for CAR+ CD4+ T cells (see Fig. 8D). The CAR+, CD 4 + T cells plateaus around day 14 after CAR cell injection, whereas the total CD4+ T cells rose until end of experiment (21 days post CAR cell injection).
  • Fig. 8E and Fig. 8G only CD8+ T cells were measured. The result shown in Fig. 8G, is also true for CAR+ CD8+ T cells (see Fig. 8E).
  • the CAR+, CD 8 + T cells plateau around day 14 after CAR cell injection.
  • the percentage of total CD8+ T cells in the blood in the experiment with CAR T cells prepared according to the invention rose until end of experiment (21 days post CAR cell injection), whereas the percentage of CD8+ T cells of the positive control experiment is unchanged.

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Abstract

La présente invention concerne un procédé de fabrication d'un produit de thérapie cellulaire, le produit de thérapie cellulaire comportant un premier récepteur de surface cellulaire pouvant se lier à une molécule cible de médicament (à la surface) d'une cellule cible de médicament et/ou le produit de thérapie cellulaire produisant et/ou sécrétant un polypeptide pouvant se lier à une molécule cible de médicament soluble ou associée à une cellule cible de médicament. Ce procédé comporte les étapes suivantes : a) enrichissement par immobilisation réversible (dans une étape de maintien) d'une cellule de départ de thérapie cellulaire obtenue à partir d'un échantillon comprenant la cellule de départ de thérapie cellulaire, à l'aide d'au moins un deuxième récepteur de surface cellulaire de la cellule de départ de thérapie cellulaire ; et b) modification génétique de la cellule de départ de thérapie cellulaire à l'aide d'un ou de plusieurs réactifs comprenant une construction génétique pour l'expression du premier récepteur de surface cellulaire et/ou pour l'expression et/ou la sécrétion du polypeptide, générant ainsi un précurseur du produit de thérapie cellulaire, le procédé se déroulant dans un système fermé.
PCT/EP2024/079474 2023-10-18 2024-10-18 Procédé de fabrication d'un produit de thérapie cellulaire et kits et dispositifs respectifs Pending WO2025083196A1 (fr)

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