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WO2025008642A1 - Procédé - Google Patents

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WO2025008642A1
WO2025008642A1 PCT/GB2024/051766 GB2024051766W WO2025008642A1 WO 2025008642 A1 WO2025008642 A1 WO 2025008642A1 GB 2024051766 W GB2024051766 W GB 2024051766W WO 2025008642 A1 WO2025008642 A1 WO 2025008642A1
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cells
vector
transduced
sample
effector
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Siddhartha K. BHAUMIK
Louise WEBB
Agne GASPARAVICIUTE
Wei Wang
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Autolus Ltd
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Autolus Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • 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
    • A61K40/30—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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention provides a method for determining the potency of a viral vector preparation. More specifically, the present invention provides a method of determining the potency of a viral vector encoding a chimeric antigen receptor using a cell impedance-based real-time assay.
  • Adoptive cell therapy is a personalised therapy that involves administration to the subject of immune cells with activity directed against a specific disease related antigen.
  • CARs chimeric antigen receptors
  • Viral vectors are typically used in the manufacture of ACT drug products to transduce cells. Although vector batches are manufactured using standardised processes, there remains the possibility that individual vector lots may possess different biological activities. Vector potency is therefore a critical attribute that must be tested prior to the release of a batch of vector.
  • FIG. 1 - CD8 Cells transduced with higher concentrations of vector are better able to kill Target cells, up until a maximum percentage cytolysis is reached.
  • CD8 cells were serially transduced, on method day 1, with high to low concentrations of vector (i.e., at different Multiplicity of infections (MOIs)).
  • MOIs Multiplicity of infections
  • 100,000 total CD8 cells (Effectors) were seeded into 3 separate xCELLigence plates that had been pre-seeded on method day 6 with 50,000 Targets. The percentage cytolysis was normalised to Targets and non-transduced Effectors and assessed at the 24 hour co-culture time-point.
  • the graph shows the average ⁇ standard deviation (SD) when data points from all 3 plates are combined.
  • Figure 2 - A 4PL model fits the vector dose response curve.
  • Two batches of vector, PDE-B20042 and 21079/B were used to transduce CD8 cells.
  • T 1 and T2 Two separate but identical transduction series (T 1 and T2) were performed.
  • A The percentage transduction efficiency was assessed on method day 7 for Effectors transduced on day 1 at different MOIs. Non-linear regression was used to fit curves to the data points.
  • B The CAR density per CD8 cell was assessed on method day 7. Linear regression was used to fit lines through the data points.
  • C 100,000 total Effector cells per well were seeded, on method day 7, into two separate xCELLigence plates pre-seeded on method day 6 with 50,000 Targets per well.
  • PDE-B20042 was used both as the reference standard and as a test sample, together with 21079/B.
  • 21079/B provided the reference standard and a test sample, together with PDE-B20042.
  • the percentage cytolysis at 24 hours co-culture was assessed, with data normalised to targets and non-transduced Effectors (mock). Non-linear regression was used to fit curves to the data points.
  • Figure 3 The vector potency method can be used for stability testing.
  • CD8 cells were transduced with vector batch PDE-B20042 or 21079/B.
  • PDE- B20042 acted as the reference standard.
  • Vector 21079/B acted as the test sample in both runs.
  • vector 21079/B on day 1 was either: not treated, freeze-thawed four times, treated at 60 °C for 1 hour, 60 °C for 6 hours, 40 °C for 30 minutes, 40 °C for 1 hour, 40 °C for 2 hours, or 40 °C for 3 hours.
  • the percentage transduction efficiency was assessed on method day 7 for all transduction series.
  • the graph inserted at top right of the right hand graph shows a magnification of the percentage transduction efficiency for the top two vector concentrations.
  • (B) Six days post transduction, 100,000 transduced Effectors were seeded into an XCELLigence plate pre-seeded with 50,000 Target cells the day before. The percentage cytolysis of the Targets was normalised to Targets and non-transduced Effectors (mock) and assessed over time. Each stability run consisted of two plates. Each data point shows the average ⁇ SD of three replicates within the plate.
  • the present inventors provide herein a method for determining the potency of a viral vector preparation. More specifically, there is provided herein a method of determining the potency of a virus encoding chimeric antigen receptor. The method involves the use of a cell impedance-based real-time cell killing assay. As such, it is more closely aligned with the function of the final drug product.
  • the present invention provides a method of determining the relative potency of a viral vector encoding a chimeric antigen receptor (CAR), the method comprising: a. providing a sample viral vector to be analysed (sample vector) and a reference viral vector of known potency (reference vector); b. transducing separate Effector cell populations with the sample vector and reference vector at various multiplicities of infection (MOIs) to form a group of sample Effector cell populations and a group of reference Effector cell populations; c. conducting cell impedance-based real-time cell killing assays of Target cells using the sample Effector cell populations and the reference cell populations; d.
  • cytotoxicity versus MOI curve for the sample Effector cell populations and for the reference cell populations; e. comparing parallelism of the curves generated in step (d), wherein parallel curves indicates comparable data sets; f. determining the half maximal effective concentration (EC50) of the sample Effector cell population and the reference cell population; and g. comparing the EC50 of the sample Effector cell population and the reference cell population to determine the relative potency of the sample viral vector.
  • EC50 half maximal effective concentration
  • the viral vector is a retroviral vector or a lentiviral vector.
  • the CAR comprises an antigen binding domain, spacer domain, transmembrane domain, and intracellular signalling domain.
  • the CAR is specific for an antigen selected from the group comprising CD19, CD20, CD22, TRBC1, TRBC2, GD2, BCMA, CD33, CD123, and CLL1. More preferably the CAR is specific for CD19.
  • the Effector cells are T cells or NK cells. More preferably the Effector cells are CD8 positive T cells.
  • the Target cells are an immobilised suspension cell line. More preferably, the immobilised suspension cell line is immobilised via anti-CD40 antibody coated gold plates.
  • the Target cells may be selected from the group comprising Raji cells, Jurkat cells, and HEK293T cells.
  • the Target cells may also be an adherent cell line.
  • adherent cell lines include but are not limited to SKOV3 cells and adherent HEK293 cells.
  • the Effector to Target ratio (E:T ratio) in the cell impedance-based real-time cell killing assays may be 1 :1, 2:1 (preferred), 3:1, 4:1, 5:1, 8:1, 10:1 , or 12:1.
  • the E:T ratio is 2:1.
  • the E:T ration may be 10:1 , 20:1 , 30:1 , 40:1, 50:1 , 80:1, 100:1 , or 120:1
  • the transduction of Effector cells is carried out at a range of MOIs. This range may include an MOI of from 0.016 to 64.0. More specifically, the transduction of Effector cells is carried out at MOIs of 0.016, 0.063, 0.25, 1.0, 4.0, 16.0, and 64.0, or any combination of these values.
  • the antigen binding domain may bind to any desired antigen.
  • the antigen binding domain may bind to an antigen selected from the group comprising CD19, CD20, CD22, TRBC1, TRBC2, GD2, PSMA, CD33, CD123, CLL1 , FLT1, and Claudin.
  • the antigen binding domain of the CAR may comprise any suitable antigen binding structure, such as those derived from antibodies.
  • the antigen binding domain may comprise a single chain variable fragment (scFv), Fab fragment, domain antibody (dAb), or variable new antigen receptor (VNAR) domain.
  • a preferred antigen binding domain is the CAT19 binder described in
  • the spacer domain of the CAR may be derived from a number of sources, including but not limited to the CD8a stalk, CD28 co-stimulatory receptor or the immunoglobulin (IgG) Fc region.
  • the CD8a stalk is a well-characterised spacer and has been used in CARs taken to clinical trials and beyond to commercialisation.
  • the transmembrane domain of the CAR may be derived from CD28, which gives good receptor stability.
  • the transmembrane domain from CD8a may also be used Each spacer may be combined with each transmembrane domain listed above.
  • the intracellular signalling domain may comprise CD3 endodomain.
  • the intracellular signalling domain may further comprise a costimulatory domain selected from the list comprising CD28 endodomain, 4-1 BB endodomain, and 0X40 endodomain.
  • the CAR may be introduced into the cell by transduction, for example using a retoviral or lentiviral vector.
  • Measurement of cell impedance may be used to monitor a variety of aspects of cell viability, such as senescence, cell death, and proliferation in a label-free and real-time manner.
  • monitoring is carried out using microtiter plates containing gold microelectrodes (for example using the xCELLigence system from Agilent). Examples of such systems are described in W02004/010102, W02005/047482, and W02005/077104.
  • a monolayer of Target cells is formed on the gold electrode surface.
  • the impedance of the system will change depending on the status of this cell monolayer. Drug products to be tested can be added to the microtiter plate well and will influence the Target cell monolayer.
  • the change in impedance is used as a readout for the state of the cells and is faster and more convenient than previous solutions such as the widely used chromium release assay, which requires the use of radioactive reagents. Since the Target cells must form a monolayer, cells that are adapted for growth in suspension cannot be used on the gold electrodes directly. However, this can be overcome by immobilising the cells onto the electrode using methods known in the art.
  • One such method involves the use of immobilised anti-CD40 antibodies, which will bind to CD40 molecules on the cell surface, effectively immobilising the cells.
  • Target cells A variety of cells may be used as Target cells, depending on the target of the intended drug product.
  • Target cells are selected from the group comprising Raji cells, Jurkat cells, and HEK293T cells.
  • a cell line in which expression of the target antigen as been knocked out may be useful as a control (for example, CD19 negative Raji cells).
  • suspension cell line these may be immobilised onto the electrode surface via any suitable immobilisation method.
  • suitable immobilisation method include, but are not limited to, the use of antibodies specific for the Target cells chemically immobilised to the electrode surface.
  • an antibody is an anti-CD40 antibody.
  • Target cells must be seeded into the plate of the cell impedance-based real-time assay device and should be allowed to reach confluence before addition of Effector cells.
  • Cells may be seeded at any suitable level, for example at 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 cells per well. Preferably cells are seeded at 50,000 Target cells per well. In some embodiments, such as when adherent cells are used, the level of seeding may be as low as 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, or 20,000 cells.
  • Target cells are maintained in culture by passaging every 2 or 3 days.
  • cells may be seeded 1 , 2, 3, 4, 5, or more days post passage.
  • cells are seeded 3 days post passage.
  • the Effector cells used in the assay will be the same as or similar to those used in the intended drug product. In some cases, this may be peripheral blood mononuclear cells (PBMCs) from a healthy donor. Alternatively, suitable cells may be obtained via a cell bank or other supplier.
  • PBMCs peripheral blood mononuclear cells
  • the Effector cells are NK cells or T cells.
  • the T cells are CD8 positive T cells.
  • a T cell line may be used.
  • Suitable T cell lines include, but are not limited to Jurkat cells, SUP-T1 cells, and TALL-104 cells.
  • Effector cells may be seeded into the wells of the cell impedance-based real-time assay device at a variety of EffectorTarget (E:T) ratios, including but not limited to 20:1, 16:1 , 8:1 , 5:1, 2:1, 1:1, and 0.5:1.
  • E:T EffectorTarget
  • the skilled reader will understand the concept of generating a dose-response curve.
  • a response is measured at various doses and then these values are plotted against each other.
  • the dose in question is the multiplicity of infection (MOI) of the vector and the response is the cytotoxicity of the resulting CAR- expressing cells against suitable Target cells.
  • MOI multiplicity of infection
  • the dose-response curve can be used to calculate the half maximal effective concentration (EC50) of the sample Effector cell population and the reference cell population; and comparing the EC50 of the transduced sample Effector cell population and the transduced reference cell population to determine the relative potency of the sample viral vector.
  • EC50 half maximal effective concentration
  • the dose response curve for the sample and the control should be parallel to one another.
  • Parallelism can be determined using mathematical methods known to the skilled person. These methods may be carried out using commercial software, such as the QuBAS software provided by Quantics Biostatistics. CHIMERIC ANTIGEN RECEPTOR (CAR)
  • a classical chimeric antigen receptor is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site, as described in more detail below.
  • scFv single-chain variable fragment
  • mAb monoclonal antibody
  • a spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • TNF receptor family endodomains such as the closely related 0X40 and 4-1 BB which transmit survival signals.
  • Third generation CARs have also been described which have endodomains capable of transmitting activation, proliferation and survival signals.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • Lentiviral vectors may be employed. In this way, a large number of antigen-specific cells can be generated for adoptive cell transfer. When a CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.
  • CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.
  • the antigen binding domain is the portion of the CAR which recognizes antigen.
  • the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • the antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody.
  • the antigen binding domain may comprise a domain based on a protein/peptide which is a soluble ligand for a tumour cell surface receptor (e.g., a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell.
  • the antigen binding domain may be based on a natural ligand of the antigen.
  • the antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein/peptide.
  • a preferred antigen binding domain is the CAT19 binder described in WO20 16/139487.
  • CARs typically comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer domains traditionally used in CARs are derived from the CD8a stalk, CD28 co-stimulatory receptor and the immunoglobulin (IgG) Fc region.
  • the CD8a stalk is a well-characterised spacer and has been used in CARs taken to clinical trials and beyond to commercialisation.
  • the transmembrane domain is the sequence of the CAR that spans the membrane.
  • a transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues.
  • the transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention.
  • transmembrane domain of a protein can be predicted by those skilled in the art using bioinformatics tools such as the TMHMM algorithm (http://www.cbs. dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e. a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (for example as described in US 7052906 B1 which is incorporated herein by reference).
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the transmembrane domain from CD8a may also be used.
  • the endodomain is the signal-transmission portion of the chimeric receptor. It may be part of or associate with the intracellular domain of the chimeric receptor. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.
  • the most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed. Costimulatory signals promote T-cell proliferation and survival.
  • co-stimulatory signals There are two main types of co-stimulatory signals: those that belong the Ig family (CD28, ICOS) and the TNF family (0X40, 41 BB, CD27, GITR etc).
  • chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative I survival signal, or all three can be used together.
  • the endodomain may comprise:
  • an ITAM-containing endodomain such as the endodomain from CD3 zeta;
  • a co-stimulatory domain such as the endodomain from CD28 or ICOS;
  • a domain which transmits a survival signal for example a TNF receptor family endodomain such as OX-40, 4-1 BB, CD27 or GITR.
  • the chimeric receptor may comprise a signal peptide so that when it is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the signal peptide may be at the amino terminus of the molecule.
  • a “target antigen” as used herein refers to the antigen which the CAR has specificity for, i.e. , the antigen which the antigen binding domain of the CAR has been engineered to have specificity for.
  • a target antigen may be a disease associated antigen.
  • the target antigen may be associated with chronic infection.
  • the target antigen may be associated with autoimmunity.
  • a target antigen may be a tumour associated antigen e.g., a cancer related antigen.
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding an antigen as indicated therein.
  • a vector potency assay based on a real-time cell analyser was developed.
  • the analyser used was a cell impedance-based device, specifically the xCELLigence platform supplied by Agilent.
  • a lentiviral vector encoding an anti-CD19 CAR was used for development purposes.
  • the CAR tested is described in WO2016/139487 and comprises the CAT19 binder, CD8 stalk, CD8 transmembrane domain, and an endodomain comprising 4-1 BB and CD3zeta endodomain.
  • a sample vector was produced using standardised methods via transient transfection of plasmids encoding the vector components. Once produced, the vector was purified and stored at -80 °C.
  • CD8+ T cells were transduced with the vector at a range of different MOIs in order to generate populations of Effector cells expressing the anti-CD19 CAR.
  • CD8 cells were serially transduced, on method day 1, with high to low concentrations of vector (i.e. , at different MOIs). Since transduction was carried out using 1 million CD8 cells per 1 mL of viral supernatant, the MOI is numerically equivalent to the vector’s infectious titre in million Tll/mL. The cytotoxicity of these cells was tested against CD19 expressing Raji Target cells which were immobilised to the electrodes of the xCELLigence E-plate using anti- CD40 antibodies, which were prepared according to the manufacturer’s instructions.
  • Targets were seeded into anti-CD40 coated xCELLigence E-plates and were allowed to reach confluence before addition of Effector cells. It was found that seeding with 50,000 Target cells provided optimal results. Cells were seeded 3 days post passage. An Effector to Target ratio (E:T ratio) of 2:1 was used. Cells were co-cultured for 24 hours before the level of remaining Target cells was measured. This was repeated for three plates.
  • E:T ratio Effector to Target ratio
  • CD8 Cells transduced with higher concentrations of vector are better able to kill Target cells, up until a maximum percentage cytolysis is reached.
  • the vector potency method should detect when a vector sample has become destabilised and hence lost potency.
  • vector 21079/B was subjected to one of a variety of heat treatments: 60 °C for 1 hour, 60 °C for 6 hours, 40 °C for 30 minutes, 40 °C for 1 hour, 40 °C for 2 hours, or 40 °C for 3 hours. Freeze-thaw cycles were also conducted on samples.
  • the untreated 21079/B vector was used as a control, with the PDE-B20042 vector as the reference standard.
  • the untreated 21079/B vector reported potencies of 1.567 (plate 1) and 1.381 (plate 2) relative to the reference standard.
  • Treatment of vector 21079/B at 60 °C for either 1 hour or 6 hours destroyed the vector, as indicated by the inability of this heat- treated vector to transduce CD8 cells ( Figure 3A, left).
  • Heat-treatment at 40 °C was also able to reduce the relative potency of vector 21079/B.
  • a clear trend was observed whereby the relative potency of 21079/B decreased as the time of heat-treatment increased ( Figure 3B, bottom).

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Abstract

La présente invention concerne un procédé de détermination de la puissance relative d'un vecteur viral codant pour un récepteur antigénique chimérique (CAR), le procédé comprenant : a. la fourniture d'un vecteur viral d'échantillon à analyser (vecteur d'échantillon) et d'un vecteur viral de référence de puissance connue (vecteur de référence); b. la transduction de populations de cellules effectrices séparées avec le vecteur d'échantillon et le vecteur de référence à diverses multiplicités d'infection (MOI) pour former un groupe de populations de cellules effectrices d'échantillon transduites et un groupe de populations de cellules effectrices de référence transduites; c. la réalisation de dosages de destruction de cellules en temps réel basée sur l'impédance de cellule conductrice de cellules cibles à l'aide des populations de cellules effectrices d'échantillon transduites et des populations de cellules de référence transduites; d. la génération d'une cytotoxicité par rapport à la courbe MOI pour les populations de cellules effectrices d'échantillon transduites et pour les populations de cellules de référence transduites; e. la comparaison du parallélisme des courbes générées à l'étape (d), des courbes parallèles indiquant des ensembles de données comparables; f. la détermination de la demi-concentration effective maximale (EC50) de la population de cellules effectrices d'échantillon et de la population de cellules de référence; et g. la comparaison de l'EC50 de la population de cellules effectrices d'échantillon transduites et de la population de cellules de référence transduites pour déterminer la puissance relative du vecteur viral d'échantillon.
PCT/GB2024/051766 2023-07-05 2024-07-05 Procédé Pending WO2025008642A1 (fr)

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