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WO2024261339A1 - Matrice de stimulation des lymphocytes (lsm) et son utilisation pour la multiplication des populations de lymphocytes - Google Patents

Matrice de stimulation des lymphocytes (lsm) et son utilisation pour la multiplication des populations de lymphocytes Download PDF

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WO2024261339A1
WO2024261339A1 PCT/EP2024/067655 EP2024067655W WO2024261339A1 WO 2024261339 A1 WO2024261339 A1 WO 2024261339A1 EP 2024067655 W EP2024067655 W EP 2024067655W WO 2024261339 A1 WO2024261339 A1 WO 2024261339A1
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lymphocyte
cells
methacrylate
stimulatory
day
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Simone STEINER
Therese CHOQUETTE
Friedemann ÜBELE
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Tigen Pharma Sa
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    • 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1107—B cells
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    • C12N2510/00—Genetically modified cells
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • degradability/dissolvability may either be inducible i.e., in response to an agent (such as a protease or reducing agent) which may be added to the culture medium, or due to the intrinsic nature of the material of which said lymphocyte-stimulatory ligand(s), polymer and/or monomer are made.
  • agent such as a protease or reducing agent
  • the hydrogel forming matrix may be provided in any conceivable three-dimensional structure/shape suitable for displaying the one or more lymphocytestimulatory ligands to a target lymphocyte population.
  • the hydrogel forming matrix may be provided in the shape of a flat or spherical structure (i.e., in the form of one or more particles).
  • Respective structures may be configured for being immobilized on the (inner) surface of a container (e.g., a culture vessel) utilized for the cell culturing thereby facilitating a subsequent separation/removal of the LSM from the expanded lymphocyte population simply by transferring (e.g., decanting or pumping) the liquid culture medium comprising the lymphocytes into a different container.
  • a container e.g., a culture vessel
  • said hydrogel forming matrix is in the form of one or more particles (i.e., spherical particles).
  • (i-a) at least, with increasing preference, 120%, 130%, 140%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or more of the diameter of a lymphocyte, preferably of a T cell; and/or
  • (i-c) at most, with increasing preference, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less of the diameter of a lymphocyte, preferably a T cell; and/or
  • a polydispersity index of at least, with increasing preference, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.98 or 0.99, and most preferably 1 .0.
  • the polydispersity index (PDI) as referred to in the above embodiment is intended in respect to the diameter (/.e., the spherical diameter) of the particles.
  • Means and methods for determining the diameter (and therefrom the PDI) of particles are known and available in the art. For example, these parameters can be determined from instruments that use dynamic light scattering (DLS) or determined from electron micrographs.
  • DLS dynamic light scattering
  • lymphocyte-stimulatory llgand(s) on the surface of said hydrogel forming matrix may be suitably established by a multitude of different approaches and designs.
  • the lymphocyte-stimulatory ligand(s) may be non-covalently attached to the surface of the hydrogel forming matrix or any molecule comprised therein.
  • Particularly envisaged embodiments include the attachment of the lymphocyte-stimulatory ligand(s) through the inclusion of one member of an affinity pair into the lymphocyte-stimulatory ligand(s) and the inclusion of corresponding second member of the affinity pair into the hydrogel forming matrix.
  • affinity pairs include, without limitation: (strept)avidin-biotin, antibody-antigen, DNA-DNA, DNA binding protein- DNA, DNA-RNA, polysaccharide-lectin.
  • the lymphocyte-stimulatory ligand(s) is biotinylated and bound by a biotin-binding moiety, preferably by a biotin-binding (poly)peptide, such as avidin, streptavidin, or even more preferably by an anti-biotin antibody, that is attached, preferably chemically conjugated, to the surface of the hydrogel forming matrix.
  • a biotin-binding moiety preferably by a biotin-binding (poly)peptide, such as avidin, streptavidin, or even more preferably by an anti-biotin antibody, that is attached, preferably chemically conjugated, to the surface of the hydrogel forming matrix.
  • said particle(s) comprise a plurality of layers, wherein each layer is degradable and/or dissolvable, wherein preferably each layer is degradable and/or dissolvable at a different rate.
  • the hydrogel forming matrix or the layers comprise or consist of one or more water-dissolvable polysaccharides.
  • the hydrogel forming matrix of the degradable and/or dissolvable LSMs comprises molecules that will be released into the culture medium when the LSMs are degraded or dissolved. Such molecules may be media supplements that result may support expansion of lymphocytes.
  • the hydrogel forming matrix of the degradable and/or dissolvable LSMs comprises cytokines, such as, without limitation interleukin-2 (IL-2); and/or interleukin-7 (IL-7); and/or interleukin-12 (IL-12); and/or interleukin-15 (IL-15), or variants thereof.
  • said particle(s) has/have a magnetic or buoyant core.
  • said hydrogel forming matrix in addition comprises a first member of a binding pair, which upon provision of the second member of the binding pair forms a binding pair, wherein the first binding pair is not a lymphocyte-stimulatory ligand and is preferably an affinity tag.
  • binding pair means a member of a pair of entities which will bind to each other when brought into contact.
  • binding pairs include, without intention for being limiting, biotin/(strept)avidin, glutathione (GSH)/glutathione-S-transferase (GST), nickel-NTA/polyhistidine (Ni- NTA/polyHis), FLAG/anti-FLAG, HA/anti-HA, Myc/anti-Myc, and antibody-antigen pairs.
  • the hydrogel forming matrix comprised in the LSM comprises biotin (e.g., a biotinylated (poly)peptide and/or other molecule(s) to which biotin has been conjugated) displayed on its surface.
  • biotin e.g., a biotinylated (poly)peptide and/or other molecule(s) to which biotin has been conjugated
  • a culture vessel may then be employed which, at its inner surface, is coated with streptavidin, avidin, or another biotin-binding moiety (e.g., a biotin-binding antibody).
  • streptavidin avidin
  • avidin-binding moiety e.g., a biotin-binding antibody
  • said monomers in said polymer are connected by at least one disulfide crosslink, wherein said disulfide crosslink is susceptible for reduction by a reducing agent.
  • the hydrogel forming matrix comprises disulfide bonds (also referred to as disulfide crosslink) which presence is required for maintaining its structural integrity. Accordingly, upon the provision of a reducing agent at sufficient concentrations, said disulfide bonds will be disrupted (reduced) and the hydrogel forming matrix will degrade/dissolve.
  • disulfide bonds also referred to as disulfide crosslink
  • reducing agent refers to a chemical species that provides electrons to another chemical species.
  • reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris (2-carboxyethyl) phosphine (TCEP) and their related salts.
  • DTT dithiothreitol
  • 2-ME 2-mercaptoethanol
  • TCEP tris (2-carboxyethyl) phosphine
  • disulfide- containing hydrogels which degradation can be induced through application of reducing agents are described, e.g., in Lakes AL et al., Acta Biomater. (2016);68: 178-189;
  • the invention provides a method for expanding a population of lymphocytes, the method comprising the step(s) of:
  • expanding when used in connection with cells (/.e., lymphocytes), refers to increasing the number of cells in the cell population due to cell replication.
  • the term “expanding a population of lymphocytes” is intended to refer to the expansion of at least one initially provided lymphocyte to result in at least one further lymphocyte, wherein the resulting expanded lymphocyte population comprises at least two lymphocytes.
  • culture medium is intended to encompass any culture medium known in the art or described herein suitable for expansion of lymphocytes.
  • culture medium may hence interchangeably referred to as “expansion medium”.
  • Non-limiting examples include commercially available media, such as PRIME-XV (IrvineScientific), X Vivo (Lonza), Excellerate (R&D Systems), AIM V (Gibco), CTS Optimizer (ThermoFisher), LymphoOne T Cell Medium (Takara), Stemline, ATCC Media (LGC Standards), and ImmunoCult TM -XF T cell expansion media.
  • the culture medium may optionally comprise one or more further constituents, particularly preferred examples of which are described herein below.
  • collecting refers to the act of isolating/separating the lymphocytes from the culture medium and debris.
  • said collecting is carried out by means of one or more round(s) of centrifugation.
  • the collecting may be conducted by immunomagnetic methods (e.g., the MACS-system from Miltenyi Biotech, Bergish-Gladbach, Germany) or flow cytometry (e.g., by fluorescence activated cell sorting (FACS)).
  • FACS fluorescence activated cell sorting
  • the invention provides a method for producing an expanded lymphocyte population, the method comprising the step(s) of:
  • lymphocytestimulatory matrix (a) culturing a population of lymphocytes in a culture medium in the presence of the lymphocytestimulatory matrix (LSM) according to the first aspect of the invention, thereby expanding the lymphocyte population; and
  • said population of lymphocytes comprises or consists of T cells.
  • T cell or “T lymphocyte” refers to a certain type of immune cell which is produced or processed by the thymus gland, and which is characterized by expressing CD3 (CD3+) and a T Cell receptor (TCR+).
  • CD3 refers to the Cluster of Differentiation 3, a protein complex composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3E chains. These chains associate with a molecule known as the T cell receptor (TCR) and the -chain to generate an activation signal in T lymphocytes.
  • TCR T cell receptor
  • -chain, and CD3 molecules together comprise the TCR complex.
  • said population of lymphocytes comprises or consists of tumor-infiltrating lymphocytes” (TILs).
  • TILs tumor-infiltrating lymphocytes
  • TILs refers to mononuclear white blood cells that have left the bloodstream and migrated towards or into a tumor.
  • TILs may comprise or consist of T cells, B cells, NK cells and/or monocytes.
  • the TILs comprise or consist of tumor-infiltrating T cells.
  • the population of lymphocytes comprises or consists of T cells, wherein, for the culturing in step (a), said T cells are Initially provided as comprised in a biological sample.
  • the biological sample may be a peripheral blood sample, a lymph node sample or a tumor sample.
  • Respective samples may be, or may have been, collected, e.g., by a needle biopsy, optionally a core needle biopsy (CNB) or a fine-needle aspiration (FNA).
  • peripheral blood sample refers to a sample of whole blood obtained from a subject.
  • the “peripheral blood sample” comprises or consists of “peripheral blood mononuclear cells (PBMCs)”.
  • PBMCs peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cell
  • T cells, B cells and NK cells lymphocytes
  • monocytes monocytes
  • PBMCs may be obtained from whole blood samples by any suitable method known in the art.
  • PBMCs can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells.
  • PBMCs can also be isolated from a donor’s whole blood by leukapheresis.
  • the T cells can be derived from one or more T cell lines available in the art.
  • T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system, which replicates the human thymic environment to support efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells. Additional methods of isolating T cells are known in the art.
  • ATO artificial thymic organoid
  • said T cells are tumor-infiltrating T cells and/or genetically engineered T cell.
  • tumor-infiltrating T cell or “tumor-infiltrating T lymphocyte”, as used herein, refers to TILs of the T cell type.
  • LSM LSM according to the first aspect of the invention and related methods according to the second and third aspects of the invention is/are particularly intended, yet without limitation, for being utilized for establishing an expansion of tumor-infiltrating lymphocytes, preferably tumor-infiltrating T cells, preferably such originating from a tumor sample (e.g., a biopsy sample from a tumor) which has been isolated from a subject’s body (J.e., a cancer patient’s body).
  • a tumor sample e.g., a biopsy sample from a tumor
  • tumor-infiltrating lymphocytes preferably, tumor-infiltrating T cells
  • TILs tumor-infiltrating lymphocytes
  • these cells can typically be isolated only in sparse amounts.
  • Their expansion ex vivo by the herein disclosed methods hence overcomes a major hurdle for exploiting their potent intrinsic capacity to combat the specific tumor cells in adoptive cell therapy (without the need for prior in vitro sensitization and selection).
  • the population of lymphocytes comprises or consists of tumorinfiltrating T cells, and wherein said tumor-infiltrating T cells are provided for said culturing in step (a) as comprised in a tumor sample, wherein more preferably said tumor sample comprises or consists of one or more tumor fragment(s). That is, in corresponding preferred embodiments, the population of lymphocytes comprises or consists of tumor-infiltrating T cells, which, for the culturing in step (a), are initially provided as comprised in a tumor sample, wherein said tumor sample comprises or consists of one or more tumor fragment(s); wherein more preferably each tumor fragment has a size of 1-3 mm 3 .
  • tumor fragment(s) having a size deviating from this latter size range may also be suitably employed.
  • the tumor fragments may be digested as known in the art before they are employed in the method of the invention.
  • the LSM employed in the culturing is in the form of particles (i.e., LSM particles), wherein said particles have a mean spherical diameter in the range of 10-40 pm, more preferably in the range of 10-30 pm; and the population of lymphocytes provided for the culturing in step (a) comprises or consists of tumorinfiltrating T cells comprised in one or more tumor fragment(s); and the culturing in step (a) is conducted with about 10 5 - 10 7 LSM particles per one tumor fragment; and wherein more preferably each tumor fragment has a size of 1-3 mm 3 ; wherein even more preferably the culturing in step (a) is conducted using four to eight, more preferably six tumor fragments each having a size of 1-3 mm 3 and about 2x 10 6 LSM particles.
  • LSM particles i.e., LSM particles
  • the culturing in step (a) is conducted using: between, with increasing preference, 10-1000, 25-500, 50-250, 50-150, and most preferably between 50-100 tumor fragments; preferably each having a size of 1-3 mm 3 ; and
  • the population of lymphocytes are expanded as disclosed in WO 2019/086711 or WO 2022/229464, with the exception that antigen-presenting cells, in particular B cells, are replaced with the LSM particles according to the invention.
  • T cells include genetically modified TILs, chimeric antigen receptor (CAR) T-cells and/or TCR-engineered T cells.
  • CAR chimeric antigen receptor
  • the TILs may be genetically engineered prior to the expansion process.
  • TILs may be genetically engineered to express at least one transgene having the ability to modulate the immune system.
  • transgenes may encode cytokines, such as, without limitation IL-2 or variants thereof, or immune-modulating proteins.
  • the immune-modulating protein may be an immunostimulatory ligand, such as, without limitation, CD40L, or a molecule that inhibits the interaction between immune checkpoint receptors and their ligand.
  • the immune-modulating protein may be an immune checkpoint decoy that mimics the binding site of an immune checkpoint receptor (such as, without limitation PD-1 , CTLA-4, or LAG3) and/or sequesters their ligands (such as, without limitation, PD-L1 , PD-L2 or B7-1/B7-2).
  • an immune checkpoint receptor such as, without limitation PD-1 , CTLA-4, or LAG3
  • sequesters their ligands such as, without limitation, PD-L1 , PD-L2 or B7-1/B7-2).
  • chimeric antigen receptor T cell or alternatively “CAR T cell” are used herein interchangeably to refer to a T-cell that has been recombinantly modified to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the term “chimeric antigen receptor” (CAR) refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule.
  • the stimulatory molecule may be the zeta chain associated with the T cell receptor (TCR) complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, for example, from 4-1 BB (CD137), CD27 and/or CD28.
  • 4-1 BB CD137
  • CD27 CD27
  • CD28 CD28
  • Currently applied CAR designs are reviewed, e.g., in Sterner R.C. & Sterner, R.M. Blood Cancer J. 11 :69 (2021); Alnefaie A et al. Front Bioeng BiotechnoL 2022; 10:797440; Weinkove R et al., Clin Transl Immunology. 2019;8(5):e1049).
  • T Cell Receptor-engineered T-cell refers to a T cell which has been genetically engineered to express cancer-antigen specific T cell receptors (TCRs).
  • TCR cancer-antigen specific T cell receptors
  • Conventional T cells recognize MHC-presented antigens through their T cell receptor (TCR), a disulfide- linked heterodimer comprised of an a and chain.
  • TCR a/0 heterodimers further complex with CD3E/y/5/ subunits.
  • TCRs recognize enzymatically cleaved peptides that are presented at the cell surface by MHC molecules (pMHC).
  • TCR binding to cognate pMHC leads to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in intracellular regions of the CD3 subunits, which results in T cell activation and initiation of effector functions including proliferation, cytokine secretion, and cytolysis via secretion of perforin and granzyme.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • T cells are edited to express TCR a and chains that confer a desired specificity.
  • introduced TCR a and 0 chains dimerize and complex with endogenous CD3 components to form a functional TCR that redirects T cell specificity towards an antigen of interest.
  • the culture medium may be supplemented with:
  • - serum preferably AB serum, or a serum substitute
  • IL-2 interleukin-2
  • IL-7 interleukin-7
  • IL-12 interleukin-12
  • the hydrogel-based LSMs according to the invention are degradable and/or dissolvable.
  • the supplements, In particular the cytokines may also be comprised inside the hydrogels, to allow successive release of the cytokines into the culture medium.
  • the term “supplemented with”, as used herein in connection with a culture medium, means that the referred item(s) has/have been added to the culture medium.
  • the respective embodiments referring to a culture medium that has been “supplemented with” the referred item(s) may, hence, alternatively, also be referred to as a culture medium initially comprising the referred items.
  • known non-limiting IL-2 variants which may be employed for the herein envisaged purposes, include any of the following mutations alone or in combination: M1 (Q22V, Q126A, I129D, S130G), M2 (L18N, Q126Y, S136R, M3 Q13Y, Q126Y, I129D, S1230R), and/or M4 (L18N, Q22V, T123A, S130R).
  • the IL-2 variant may be any of the IL-2 variants disclosed in LVO 2011/063770 or US 8,759,486, which are fully incorporated herein by reference.
  • a CD3 agonist and/or CD28 agonist (preferably an agonistic anti-CD3 antibody and/or agonistic anti-CD28 antibody) is added to the culture medium at a time point between, with increasing preference, day 1 and day 19, day 2 and day 18, day 3 and day 17, day 4 and day 16, day 5 and day 15, day 6 and day 14, day 7 and day 13, day 8 and day 12, day 9 and day 11 , and most preferably at day 10, after the start of the culturing.
  • an activator such as a CD3 agonist and/or CD28 agonist (preferably an agonistic anti-CD3 antibody and/or agonistic anti-CD28 antibody), may be added to the culture medium more than once. That is, in certain preferred embodiments, an agonistic anti-CD3 antibody, such as OKT-3, may be added to the culture medium twice, wherein the second dose of the antibody is given 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the first dose. In certain embodiments, an agonistic anti-CD3 antibody, such as OKT-3, may be added to the culture multiple times, for example, in intervals of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
  • CD3 agonist is an agonistic anti-CD3 antibody and/or the CD28 agonist is an agonistic anti-CD28 antibody; and/or
  • - IL-2 at a concentration in the range of between, with increasing preference, about 10-15,000 lU/ml, about 250-12,000 lU/ml, about 500-10,000 lU/ml, about 1 ,000-8,000 lU/ml, about 1 ,500-6,000 lU/ml, about 2,000-4,000 lU/ml, most preferably about 3,000 lU/ml; and/or
  • feeder cells wherein preferably the feeder cells are comprised in the second culture medium at a cell count ratio of feeder cells to lymphocytes in a range of between, with increasing preference, 1 :1 and 100:1 , 2:1 and 90:1 , 3:1 and 90:1 , 4:1 and 80:1 , 5:1 and 70:1 , 6:1 and 60:1 , 7:1 and 50:1 , 8.1 and 45:1 , 10:1 and 40:1 ; 15:1 and 35:1 , 20:1 and 30:1 and most preferably 25:1 ; and optionally,
  • (i-c) harvesting at least a portion of the lymphocyte population obtained from (i-b) and culturing said portion in a third culture medium, wherein optionally the third culture medium does not comprise feeder cells and/or IL-2.
  • the lymphocyte population comprises or consists of tumor infiltrating lymphocytes (TILs); and/or
  • the portion of the lymphocyte population obtained from (i-a) which in step (i-b) is cultured in the second culture medium comprises at least, with increasing preference, 1x 10 5 , 2x 10 5 , 3x 10 5 , 4x 10 5 , 5x 10 5 , 6x 10 5 , 7x 10 5 , 8x 10 5 , 9x 10 5 , 1x 10 6 , 2x 10 6 cells; and/or
  • the portion of the lymphocyte population obtained from (i-b) which in step (i-c) is cultured in the third culture medium comprises at least, with increasing preference, 1x 10 5 , 2x 10 5 , 3x 10 5 , 4x 10 5 , 5x 10 5 , 6x 10 5 , 7x 10 5 , 8x 10 5 , 9x 10 5 , 1x 10 6 , 2x 10 6 cells, 3x 10 6 , 4x 10 6 cells.
  • the culturing in step (a) is conducted:
  • the cell count which by the herein disclosed method can be obtained will depend upon the specific kind of the lymphocytes which are to be expanded. For instance, where the lymphocyte population comprises or consists of CAR-T cells (or TCR-engineered T cells), a cell count of at least 1x 10 5 would be envisaged. However, in such instances, where the lymphocyte population comprises tumor-infiltrating lymphocytes, e.g., tumor-infiltrating T cells, much higher cell counts, i.e., up to or even exceeding 10 9 , are envisaged.
  • step (i) the culturing in step (i-a) is conducted:
  • step (ii) the culturing in step (i-b) is conducted:
  • step (iii) the culturing in step (i-c) is conducted: (i-ca) for a time period of at least, with increasing preference, 0.5, 1 , 2, 3, 4 days, most preferably at least 5 days; and/or
  • (i-cc) up to a time point until said lymphocyte population reaches a cell count of at least, with increasing preferences, 3x 10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x 10 6 , 8x 10 6 , 9x 10 6 , 1x 10 7 , 5x 10 7 , 1x 10 8 , 5x 10 8 , or most preferably of at least 1x 10 9 .
  • the methods further comprise after step (a), prior to optional step (b), depleting the LSM from the culture medium.
  • the LSM or hydrogel forming matrix comprised therein is degradable/dissolvable, and wherein said degradability/ dissolvability is due to susceptibility of the LSM or a component comprised therein towards a certain agent (e.g., a reducing agent and/or a protease), said depletion of the LSM from the culture medium may be effected by addition of said agent to the culture medium.
  • said LSM or hydrogel forming matrix comprised therein is not degradable/dissolvable
  • said depletion of the LSM from the culture medium may be effected by other means.
  • the LSM or hydrogel forming matrix are in the form of particles (/.e., spherical particles), which particles differ in their size compared to the target lymphocytes
  • said depletion may be effected by filtration or size exclusion chromatography.
  • the LSM comprises a first member of a binding pair (/.e., which according to the above embodiments is not a lymphocytestimulatory ligand)
  • a depletion may be effected through provision of the second member of the binding pair on a capturing matrix, such as the surface of a culture vessel or to a chromatography column. Upon passing the culture medium over such a surface, the LSM will be captured and thereby be depleted from the culture medium.
  • Bioreactors for expanding lymphocytes include, but are not limited to, ADVA (from ADVA Biotech), WAVE Bioreactor (Cytiva), GRex (Wilson Wolff), Ori Bioreactor (Ori), and Cocoon (Lonza).
  • the population of lymphocytes may be expanded in an ADVA bioreactor as described in WO 2022/229464, which is incorporated herein in its entirety.
  • the bioreactor used for the expansion of the population of lymphocytes may be any of the bioreactors disclosed in Garcia-Aponte et al. (J Biol Eng. 2021 ; 15: 13), which is incorporated herein in its entirety.
  • the invention provides a population of lymphocytes obtained or obtainable by the method according to the second or third aspect of the invention.
  • at least 90% of the cells comprised in said population are CD3+ T cells; and preferably at least, with increasing preference, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the CD3+ T cells are CD8+ T cells.
  • CD3+ T cells has its general meaning in the art and refers to a subset of T cells which express CD3 on their surface.
  • CD3 refers to the Cluster of Differentiation 3, a protein complex composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3E chains. These chains associate with a molecule known as the T cell receptor (TCR) and the -chain to generate an activation signal in T lymphocytes.
  • TCR T cell receptor
  • the TCR, -chain, and CD3 molecules together comprise the TCR complex.
  • CD8+ T cell has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. They are MHC class l-restricted, and function as cytotoxic T cells. "CD8+ T cells” are also called “cytotoxic T lymphocytes (CTL)”, “T-killer cells”, “cytolytic T cells”, or “killer T cells”. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class l-restricted interactions.
  • T cells which express a certain selected surface marker e.g., CD3 and/or CD8 in a population of cells
  • a certain selected surface marker e.g., CD3 and/or CD8
  • the percentage of T cells expressing a certain surface marker may be determined by flow cytometry, using antibodies directed against that surface marker (CD3 and/or CD8) and/or other suitable T cellspecific surface markers.
  • the invention provides a population of lymphocytes, wherein at least 90% of the cells comprised in said population are CD3+ T cells; and wherein preferably at least, with increasing preference, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the CD3+ T cells are CD8+ T cells.
  • the invention provides a pharmaceutical composition comprising the population of lymphocytes according to the fourth or fifth aspect of the invention.
  • the population of lymphocytes of the invention is intended for use in adoptive cell transfer (ACT) therapy in humans. That is, the cells comprised in the population of lymphocytes are preferably suspended in a liquid that is suitable for injection into the human bodies. Suitable liquids for suspending the cells comprised in the population of lymphocytes include, without limitation, pharmaceutically acceptable buffers.
  • the pharmaceutically acceptable buffer may be a sodium chloride buffer. In certain embodiments, the pharmaceutically acceptable buffer may be a 0.9% NaCI buffer. In certain embodiments, the pharmaceutically acceptable buffer may be supplemented with at least 5%, 10%, 15% or 20% DMSO to allow freezing of the population of lymphocytes. In certain embodiments, the pharmaceutically acceptable buffer may comprise between 0 and 15% DMSO. That is, the pharmaceutically acceptable buffer may comprise 0.9% NaCI and 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% DMSO.
  • the pharmaceutical composition is substantially free of contaminants, in particular bacterial contaminants, such as mycoplasma.
  • bacterial contaminants such as mycoplasma.
  • the absence of bacterla/mycoplasma can be tested with devices or kits known in the art such as, without limitation, with a BacTec device and/or a MycoSeq kit. Further, it is preferred that the pharmaceutical composition is substantially free of endotoxins.
  • the term “medicament” is used herein interchangeably with the term “pharmaceutical composition” and relates to a composition suitable for administration to a patient, preferably a human patient.
  • the invention provides a population of lymphocytes (preferably human lymphocytes, more preferably human T cells (including tumor-infiltrating T cells) - which may or may not be further genetically engineered to express one or more desired peptides or receptors) for use as a medicament and methods of producing such populations of lymphocytes for such use.
  • the medicament/pharmaceutical composition may be administered to an allogenic recipient or to an autologous recipient.
  • the medicament/pharmaceutical composition may comprise non- allogenic lymphocytes (“off the shelf’ lymphocytes, as known in the art).
  • the donor and recipient are of the same species. It is preferred that the patient/recipient is a human.
  • the expanded population of lymphocytes are typically admixed with a pharmaceutically acceptable carrier excipient and/or diluent and the resulting composition is administered to a subject.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject or engineered cells.
  • suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • the carrier may be a solution that isotonic with the blood of the recipient. Compositions comprising such carriers can be formulated by well-known conventional methods.
  • the pharmaceutical compositions of the invention can further comprise one or more additional agents useful in the treatment of a disease in the subject.
  • the pharmaceutical compositions of the invention can further include biological molecules known to be advantageous to lymphocyte function or activity, including but not limited to cytokines (e.g., IL-2, IL-7, IL- 15, and/or IL-21), which promote in vivo cell proliferation and engraftment.
  • cytokines e.g., IL-2, IL-7, IL- 15, and/or IL-21
  • the population of lymphocytes of the invention can be administered in the same composition as the one or more additional agent or biological molecule or, alternatively, can be co-administered in separate compositions.
  • chemotherapeutic agents include an anthracycllne (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g.
  • General chemotherapeutic agents considered for use in combination therapies include anastrozole, bicalutamide, bleomycin sulfate, busulfan, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5-fluorouracil, flutamide, tezacitibine, Gemcitabine, hydroxyurea (Hydrea.RTM.), Idarubicin, ifosfamide
  • Anti-cancer agents for use in combination with the populations of lymphocytes of the invention include but are not limited to, anthracyclines; alkylating agents; antimetabolites; drugs that inhibit either the calcium dependent phosphatase calcineurin or the p70S6 kinase FK506) or inhibit the p70S6 kinase; mTOR inhibitors; immunomodulators; anthracyclines; vinca alkaloids; proteosome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKN kinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.
  • Exemplary antimetabolites include, without limitation, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6- thioguanine, fludarabine phosphate, pentostatin, pemetrexed, raltitrexed, cladribine, clofarabine, azacitidine, decitabine and gemcitabine.
  • alkylating agents include, without limitation, nitrogen mustards, uracil mustard, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, temozolomide, thiotepa, busulfan, carmustine, lomustlne, streptozocin, dacarbazine, oxaliplatin, temozolomide, dactinomycin, melphalan, altretamine, carmustine, bendamustine, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, altretamine, ifosfamide, prednumustine, procarbazine, mechlor
  • the invention provides a population of lymphocytes according to the fourth or fifth aspect of the invention or the pharmaceutical composition according to the sixth aspect of the invention for use as a medicament.
  • the population of lymphocytes according to the fourth or fifth aspect of the invention or the pharmaceutical composition according to the sixth aspect of the invention are envisioned as for use as a medicament in the treatment or prevention of any disease that benefits from such a treatment, including, but not limited to, cancers or precancerous conditions, infections, or autoimmune diseases.
  • the invention provides a population of lymphocytes according to the fourth or fifth aspect of the invention or the pharmaceutical composition according to the sixth aspect of the invention for use in treating cancer, an infection or an autoimmune disease.
  • the invention provides a method for treating cancer, an infection, or an autoimmune disease in a subject in need thereof, the method comprising administering to the subject the population of lymphocytes according to the fourth or fifth aspect of the invention or the pharmaceutical composition according to sixth aspect of the invention.
  • cancer or “proliferative disease”, as used herein, means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art, including, without limitation, all types of tumors, lymphomas, and carcinomas.
  • Non-limiting examples of such cancers include colorectal cancer, brain cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, renal cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, melanoma, skin cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, cervical cancer, bladder cancer, lymphoma, chronic or acute leukemia (such as B, T, and myeloid derived), sarcoma, lung cancer and multidrug resistant cancer.
  • a “precancerous condition” refers to a condition, tumor or lesion involving abnormal cells which are associated with an increased risk of developing into cancer.
  • infection refers to state wherein a pathogen, such as bacteria or a virus, has invaded a subject’s body.
  • autoimmune disease refers to a disorder that results from an autoimmune response.
  • An autoimmune disease is the result of an inappropriate and excessive response to a selfantigen.
  • autoimmune diseases include but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain- Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroid
  • treatment generally mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of partially or completely curing the disease or condition, and/or adverse effect attributed to the disease or condition.
  • treatment covers any treatment of a disease or condition in a subject and includes: (a) preventing and/or ameliorating a proliferative disease (preferably cancer) from occurring in a subject that may be predisposed to the disease; (b) inhibiting the disease, i.e., arresting or slowing its development, such as slowing or inhibiting cancer progression; (c) relieving the disease, i.e., causing regression of the disease, such as the repression of cancer; and/or (d) preventing, inhibiting or relieving any symptom or adverse effect associated with the disease or condition.
  • the term “treatment” as used herein relates to medical intervention of an already manifested disorder, e.g., the treatment of a diagnosed cancer.
  • the treatment or therapy may be performed alone or in combination with appropriate treatment protocols for the particular disease or condition as known in the art.
  • suitable treatment protocols include but are not limited to, administration of pain medications, administration of chemotherapeutics, therapeutic radiation, and surgical handling of the disease, condition or symptom thereof.
  • the treatment regimens disclosed herein encompass the administration of the population of lymphocytes of the invention (or the pharmaceutical composition comprising the population of lymphocytes of the invention) together with none, one, or more than one treatment protocol suitable for the treatment or prevention of a disease, condition or a symptom thereof, either as described herein or as known in the art.
  • Administration “in combination” or the use “together” with other known therapies encompasses the administration of the medicament/pharmaceutical composition of the invention before, during, after or concurrently with any of the co-therapies disclosed herein or known in the art.
  • the pharmaceutical composition/medicament disclosed herein can be administered alone or in combination with other therapies or treatments during periods of active disease, or during a period of remission or less active disease.
  • the population of lymphocytes of the invention can be administered in an amount or dose that is higher, lower or the same than the amount or dosage where each therapy or agent would be used individually, e.g., as a monotherapy.
  • the administered amount or dosage of the lymphocyte therapy, and/or at least one additional agent or therapy is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the corresponding therapy(ies) or agent(s) used individually.
  • the population of lymphocytes of the invention may further be rendered resistant to chemotherapy drugs that are used as standards of care as described herein or known in the art.
  • Engineering such resistance into the population of lymphocytes of the invention is expected to help the selection and expansion of such engineered lymphocytes in vivo in patients undergoing chemotherapy or immunosuppression.
  • the population of lymphocytes of the invention may undergo robust in vivo expansion upon administration to a patient and may remain persist in the body fluids for an extended amount of time, preferably for a week, more preferably for 2 weeks, even more preferably for at least one month.
  • the population of lymphocytes of the invention may also be additionally engineered with safety switches that allow for potential control of the cell therapeutics.
  • Such safety switches of potential use in cell therapies include (but are not limited to) the engineering of the cells to express targets allowing antibody depletion (e.g., truncated EGFR; Paszkiewicz et al., J Clin Invest 126(2016), 4262- 4272), introduction of artificial targets for small molecule inhibitors (e.g., HSV-TK; Liang et al., Nature 563(2018), 701-704) and introduction of inducible cell death genes (e.g., icaspase; Minagawa et al., Methods Mol Biol 1895(2019), 57-73).
  • antibodies depletion e.g., truncated EGFR; Paszkiewicz et al., J Clin Invest 126(2016), 4262- 4272
  • introduction of artificial targets for small molecule inhibitors e.g., HSV-TK; Liang et al., Nature 563(2018), 701-704
  • the administration of the population of lymphocytes of the invention may be carried out in any convenient manner, including by injection, transfusion, implantation, or transplantation.
  • the population of lymphocytes of the invention or corresponding pharmaceutical compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally.
  • the population of lymphocytes of the invention or corresponding pharmaceutical compositions are to be administered to a patient by intradermal or subcutaneous injection.
  • the population of lymphocytes of the invention or corresponding pharmaceutical compositions are to be administered by intravenous injection.
  • the population of lymphocytes of the invention or corresponding pharmaceutical compositions are to be administered by intravenous infusion.
  • the population of lymphocytes of the invention may be administered in effective doses.
  • the effective dose may be either one or multiple doses, and sufficient to produce the desired therapeutic effect.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient’s size, body surface area, age, the particular compound/medicament to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the population of lymphocytes of the invention may be administered to the subject at a dose of 10 4 to 10 10 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight.
  • the lymphocytes may be administered in such a way that an upscaling of the lymphocytes to be administered is performed by starting with a subject dose of about 10 5 to 10 6 cells/kg body weight and then increasing to dose of 10 10 cells/kg body weight.
  • the effective dose may be readily determined by the skilled person and/or may be calculated based on the stage of the malignancy, the health of the subject, and the type of malignancy. In the situation where multiple doses are administered, that dose and the interval between the doses may be determined based on the subject's response to therapy.
  • the lymphocytes or corresponding pharmaceutical compositions may be administered multiple times at these dosages.
  • the population of lymphocytes or the pharmaceutical composition according to the invention is used in autologous cell therapy, in particular for the treatment of cancer. That is, it is preferred herein that the lymphocytes comprised in the population of lymphocytes or the pharmaceutical composition according to the invention are obtained by expanding a sample of lymphocytes that has been obtained from a subject suffering from cancer. Subsequently, the expanded population of lymphocytes, preferably in the form of a pharmaceutical composition, may be infused back into the same subject.
  • the lymphocytes in the composition of lymphocytes specifically attack the subject’s tumor.
  • the lymphocytes may be expanded in the presence of an antigenic peptide that has previously been identified as being present in the subject’s tumor.
  • the invention relates to a method for treating cancer in a subject, the method comprising the steps of: a) surgically removing a tumor from a subject or taking a biopsy from a subject’s tumor; b) identifying at least one tumor antigen in the tumor sample obtained in step (a); c) expanding lymphocytes comprised in the tumor sample obtained in step (a) with the method according to the invention, wherein the lymphocytes are expanded in the presence of at least one antigen that has been identified in step (b) to be present in the tumor sample; and d) infusing the expanded lymphocytes into the subject from which the tumor sample has been obtained.
  • tumor antigen refers to an antigen that is uniquely or differentially expressed by a tumor cell, whether intracellular or on the tumor cell surface (preferably on the tumor cell surface), compared to a normal or non-neoplastic cell.
  • a tumor antigen may be present in or on a tumor cell and not typically in or on normal cells or non-neoplastic cells (e.g., only expressed by a restricted number of normal tissues, such as testis and/or placenta), or a tumor antigen may be present in or on a tumor cell in greater amounts than in or on normal or non- neoplastic cells, or a tumor antigen may be present in or on tumor cells in a different form than that found in or on normal or non-neoplastic cells.
  • TSA tumor-specific antigens
  • TAA tumor-associated antigens
  • CT cancer/testis
  • tumor antigens include, without limitation, p-human chorionic gonadotropin ( HCG), glycoprotein 100 (gp100/Pmel17), carcinoembryonic antigen (CEA), tyrosinase, tyrosinase-related protein 1 (gp75/TRP-1), tyrosinase-related protein 2 (TRP-2), NY-BR-1 , NY-CO-58, NY-ESO-1, MN/gp250, idiotypes, telomerase, synovial sarcoma X breakpoint 2 (SSX2), mucin 1 (MUC1), antigens of the melanoma-associated antigen (MAGE) family, high molecular weight melanoma-associated antigen (HMW-MAA), melanoma antigen recognized by T cells 1 (MARTI), Wilms' tumor gene 1 (WT1), HER2/neu, mesothelin (MSLN), alpha-fetoprotein (AFP),
  • Tumor antigens may also be subject specific (e.g., subject specific neoantigens; see, e.g., U.S. patent 9, 115,402, and international patent application publication numbers WO 2016/100977, WO 2014/168874, WO 2015/085233, and WO 2015/095811)
  • subject specific neoantigens see, e.g., U.S. patent 9, 115,402, and international patent application publication numbers WO 2016/100977, WO 2014/168874, WO 2015/085233, and WO 2015/095811
  • the population of lymphocytes for use in the treatment of cancer comprises Neo-TILs.
  • Neo-TILs are tumor-infiltrating lymphocytes, preferably T cells, which specifically recognize a neoantigen.
  • Neo-TILs may be specifically expanded by contacting tumor samples or T cells obtained from tumor samples with a neo-antigenic peptide as described in more detail herein. It is preferred that the presence of the neoantigen has been confirmed in the patient which receives the population of lymphocytes comprising the Neo-TILs.
  • Neoantigens result from somatic mutations in tumor cells and are thus expressed only in tumor cells but not in normal cells. Because normal cells do not express neoantigens, they are considered non-self by the immune system.
  • neoantigens are ideal targets for therapeutic cancer vaccines and T cell-based cancer immunotherapy.
  • synthetic neoantigen drugs can be designed according to the situation of tumor cell mutation to achieve the effect of treatment.
  • the antigens presented are neoantigens retrieved by sequencing tumors or peripheral blood cells or other potential sources of antigens of the patient to be treated (e.g., a tumor sample or sample of infected tissue) and identified by a relevant algorithm.
  • a relevant algorithm include, e.g., Neon (NeonTherapeutics) and Achilles (Achilles Therapeutics).
  • Neon NeonTherapeutics
  • Achilles Achilles Therapeutics
  • the identification of neoantigens in tumor samples has been disclosed, without limitation, in 1 0 2017/106638, WO 2011/143656, WO 2017/011660, WO 2018/213803 or WO 2021/116714, which are fully incorporated herein by reference.
  • Neo-antigenic peptides that may be used in the method according to the invention are disclosed in WO 2016/187508, which is fully incorporated herein by reference.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends on.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C,
  • a or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a cell” can mean “one or more cells”) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • the term “about” as used herein refers to a value within 10% of the underlying parameter (/.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (/.e., “about 1 , 2 and 3” refers to about 1 , about 2 and about 3).
  • a weight of “about 100 grams” can include weights between 90 grams and 110 grams.
  • FIG. 1 G-Rex conditions - overview. Five different G-Rex conditions were set up for this experiment. Each with six tumor fragments and identical media composition. Together with the tumor fragments either, no Bcells, ACT_EP_Bcells, ACT_Bcells + ArtQC Bcells, ArtQC Bcells, or ACT_EP_Bcells without IL-12 were seeded.
  • FIG. 1 Experimental design. The distinct stages of TIL expansion using tumor fragments as starting material. The different media compositions used for the different stages of TIL expansion are laid out in Table 1. Abbreviations: D: day; IL-2: lnterleukin-2; OKT3: anti-CD3 antibody Muromonab-CD3; M: million; REP: rapid expansion protocol.
  • FIG. 1 G-Rex®10M Open System.
  • the G-Rex 10M has a 10 cm 2 gas permeable membrane surface area with 100 mL media capacity. It is built to expand 5 million cells into between 200 to 400 million cells in about 10 days with no medium exchange.
  • Figure 4 Total viable cell count after harvest. Comparison of the total number of cells of all conditions; Cell count on D10 - Reseeding of 2x10e6 cells, Cell count on D15 - Reseeding of 4x10e6 cells, cell count after final harvest.
  • Figure 5 Cell viability. Comparison of the cell viability after the harvests for all conditions.
  • Figure 6 Viable cell count interpolation ( Figure 6A) and Cell diameter ( Figure 6B).
  • (A) Calculated possible viable cell count without harvests on D10 and D15 and with no limitation in growth by the G- Rex bioreactor.
  • (B) Comparison of the cell diameter after the harvests for all conditions.
  • Figure 7 Lactate per timepoint. Lactate levels for each condition as an indicator for cell proliferation.
  • Figure 8 Accumulated lactate over time. The accumulated lactate level over time shows the total amount of lactate excretion of the cells.
  • Figure 9 Visual comparison of the 10M G-Rex bioreactors.
  • D20 Visual confirmation of higher lactate levels in the conditions C and D containing ArtQC Bcells (Yellow colour of the cell Media).
  • FIG. 10 Accellix results (TBNK cartridge) showing the major cell populations per group.
  • FIG 11 Accellix results (T cell cartridge) showing the T cell populations per group.
  • Figure 12 Summary of Accellix results (TBNK cartridge and T cell cartridge).
  • Figure 13 Cell viability (A) and cell size (B) at post-cryopreservation were assessed using NC-202 and compared with the ones at pre-cryopreservation. One or two independent experiments were performed for pre- and post-cryopreservation, respectively. Mean ⁇ SD values were shown.
  • Figure 14 Cells were thawed and assessed for apoptosis-necrosis using Annexin-V/7-AAD. The percentages of different cell populations (necrosis, late apoptosis, early apoptosis and non-apoptotic) were shown in A. The difference in viability pre-cryopreservation versus post-cryopreservation from NC- 202 data (Y-axis) was plotted against the percentage of non-apoptotic cells in B.
  • FIG. 15 Cells were thawed and tested for IFNg release capacity following CD3 stimulation overnight.
  • the TIL batches (A) were cultured overnight with IL-2 only or with additional stimulation with 1 , 10, 100 or 500 ng/mL of anti-CD3 antibody (B).
  • Activation cocktail PMA+ionomycin was used as positive control (C).
  • Figure 16 Cells were left unstimulated (unstim) or stimulated (stim) with anti-CD3 antibody, and cell proliferation was assessed at day 7, day 14 and day 21 (X-axis). Interpolated cell numbers (A), fold expansion (B), cell viability (C) and cell diameter (D) were shown (Y-axis).
  • Figure 17 Cells were left unstimulated (e.g. A0) or stimulated with anti-CD3 antibody (e.g. A3).
  • the Glucose (Y-axis) and Lactate (X-axis) levels were quantified from cell supernatant at day 4 (A), day 7 (B), day 14 (C) and day 21 (D).
  • the viable cell numbers was assessed at day 7 (E), day 14 (F) and day 21 (G), (Y-axis). Lactate levels were quantified from cell supernatants (X-axis).
  • the coefficient of determination (R 2 ) is indicated in each graph.
  • FIG. 18 Cells were left unstimulated (Unstim, top) or stimulated with anti-CD3 antibody (aCD3, bottom). The proportion of T cells, NKT, NK and “ILC” cells were accessed at day 0 and day 21 using Accellix.
  • FIG. 19 Cells were left unstimulated (Unstim, top) or stimulated with anti-CD3 antibody (aCD3, bottom).
  • the composition of T cells (CD4+, CD8+, CD8- CD4-, CD8+ CD4+) were accessed at day 0, day 7, day 14 and day 21.
  • Figure 20 The table showed the percentage of positive cell populations for each combination of markers.
  • the heatmaps displayed the absolute (A) or relative (B*) expression levels of antibodies/CD markers (in columns) between the 4 conditions (in rows).
  • x_scaled (x - m) / s, where x is the absolute percentage, m is the mean percentage and s is the standard deviation for a given marker.
  • ACT_EP_B cells Autologous expanded, activated and electroporated B cells are commonly used to co-stimulate T cells in tumor infiltrating lymphocyte (TIL) manufacturing processes.
  • the B cells are isolated from autologous apheresis material and activated in vitro with IL-4 2 and CD40L 3 . Once activated, the B cells are electroporated with mRNAs coding for OX40L 4 , 4-1BBL (CD137L) 5 and eventually IL-12 6 for expression of these markers.
  • the ACT_EP_B cells are seeded together with tumor fragments at the start of the process and provide help to the TILs when they migrate out of the tumor fragments.
  • B cells may alternatively be isolated and expanded from peripheral blood mononuclear cells (PBMCs) instead of the autologous apheresis material.
  • PBMCs peripheral blood mononuclear cells
  • the autologous B cells are a critical raw material in the TIL manufacturing process.
  • the use of the autologous B cells increases the needed resources and complexity of the process and is dependent on patient apheresis.
  • a suitable surrogate for autologous ACT_EP_B cells with a more controllable starting material can be a substantial advantage for TIL manufacturing.
  • Synthetic hydrogel cells were manufactured, that were originally intended for use as controls and for instrument calibration in flow cytometry (FACS).
  • these synthetic hydrogel cells can be employed as a replacement for the ACT_EP_B cells, as the markers OX40L (CD134L) and 4-1 BBL (CD137L) can be attached to their surface and therefore be presented to T cells in a similar manner as by endogenous B cells.
  • the experiment described herein compared the effect of one batch of synthetic hydrogel cells modified with OX40L and 4-1 BBL (from hereon called “ArtQC Bcells”) on TIL expansion in G-Rex®10M bioreactors to electroporated B cells (ACT_EP_B cells) under different conditions.
  • IL-4 and IL-21 promote B cell proliferation, class switch recombination, and differentiation into plasma cells or germinal centres B cells (see, e.g., review article Cyster JG, Allen CDC. B Cell Responses: Cell Interaction Dynamics and Decisions. Cell. 2019;177(3):524-540).
  • CD40L is a member of the TNF superfamily and is expressed primarily by activated T cells, as well as activated B cells and platelets. CD40L engages CD40 receptor expressed by B cells and antigen presenting cells. CD40 signalling of B cells promotes germinal centre formation, immunoglobulin isotype switching, somatic hypermutation of the immunoglobulin to enhance affinity for antigen, and the formation of long-lived plasma cells and memory B cells (see, e.g., review article Elgueta R et al., Molecular mechanism and function ofCD40/CD40L engagement in the immune system. Immunol Rev. 2009;229(1): 152-72).
  • OX40L interacts with the 0X40 receptor on the surface of T cells.
  • 0X40 has co-stimulatory functions during T cell activation, mediating the survival and expansion of both CD4+ and CD8+ T cells.
  • 0X40 is also involved in controlling effector and memory T cell responses (see, e.g., review article Fu Y et al., Therapeutic strategies for the costimulatory molecule 0X40 in T-cell- mediated immunity. Acta Pharm Sin B. 2020;10(3):414-433).
  • the co-stimulatory receptor 4-1 BB (CD137) transduces the activation signal of NF-KB, which induces preferential expansion and activation of CD8+ T cells (see, e.g., Dai Q et al., 4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-lncorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells. Front Immunol. 2020; 11 :539654; Kim AM J et al., 4-1 BB: A promising target for cancer immunotherapy. Front Oncol. 2022;12:968360).
  • IL-12 promote proliferation and cytotoxicity of CD8+ T cells (see, e.g., review article Jia Z et al., IL12 immune therapy clinical trial review: Novel strategies for avoiding CRS-associated cytokines. Front Immunol. 2022; 13:952231).
  • the inventors aimed to explore the potential of the ArtQC Bcells in TIL manufacturing, by investigating their ability to mimic the function of ACT_EP_B cells in stimulating the growth of the TIL. Additionally, the inventors investigated the viability and phenotype of the expanded TILs.
  • the objective of this study was to explore the potential of ArtQC Bcells, containing the surface markers OX40L and 4-1 BBL, in activating TILs in manufacturing processes for cancer therapies.
  • the inventors aimed to explore whether synthetic cells could present certain proteins to T cells and trigger their activation, leading to their proliferation.
  • the inventors sought to develop a more efficient and controlled manufacturing process.
  • the inventors aimed to explore whether ArtQC Bcells can activate TILs in a controlled manner.
  • the inventors obtained custom-made synthetic ArtQC Bcells containing OX40L (CD134L) and 4-1BBL (CD137L) from Slingshot Biosciences, Inc., meant for the development of FACS IPCs.
  • the proteins on the ArtQC Bcells match costimulatory receptors (0X40 and 4-1 BB) on the surface of T cells and stimulate their proliferation.
  • the inventors conducted experiments in vitro using G- Rex®10M bioreactors.
  • the inventors co-cultured tumor fragments containing TILs either with ArtQC Bcells, activated B cells (ACT_BCs) or activated electroporated B cells (ACT_EP_BCs) and observed the response of the target cells upon activation.
  • ACT_BCs activated B cells
  • ACT_EP_BCs activated electroporated B cells
  • the G-Rex bioreactors had either no B cells, ACT_EP_BCs (6x10e6), ACT_BCs (3x10e6) + ArtQC Bcells (1x10e6), ArtQC Bcells (2x10e6) or ACT_EP_BCs w/o IL-12 (6x10e6).
  • the experiment was run over a period of 20 days.
  • Figure 1 presents a differentiated picture of the setup for the compared conditions.
  • the cells were seeded for the pre-rapid expansion protocol (Pre-REP) on DO (Day 0) together with the tumor fragments in 50 ml of complete cell media containing RPM1 1640 Medium, human male AB serum and Penicillin-Streptomycin (10,000 U/ml). Additionally, the cell culture was stimulated with 6000 lll/ml of human IL2.
  • Pre-REP pre-rapid expansion protocol
  • TILs were cryopreserved in CS10 for further analysis.
  • the following media compositions were used for the distinct stages of TIL expansion using tumor as starting material to ensure optimal growth conditions.
  • the three variants are laid out in Table 1 .
  • Feeder cells are irradiated allogeneic PBMCs from healthy donors that are needed to support TIL activation and expansion (see, e.g., Forget MA et al., Activation and propagation of tumorinfiltrating lymphocytes on clinical-grade designer artificial antigen-presenting cells for adoptive immunotherapy of melanoma. J Immunother. 2014;37(9):448-60).
  • Anti-CD3 monoclonal antibodies supports T-cell activation in the presence of IL-2 (see, e.g., review article Wang X, Riviere I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics. 2016;3:16015).
  • G-Rex cell culture system (G-Rex®10M Open System) from Wilson Wolf (USA) to compare the cell growth in different conditions.
  • Lactate 5 excretion is an important marker for the cell metabolism and can give insights on cell proliferation (activated and proliferating cells release lactate).
  • ArtQC Bcells started off with low levels of lactate, very similar to the baseline condition G-Rex with only tumor fragments, both conditions ended the experiment with the highest lactate >20mmol/L.
  • both conditions containing electroporated B cells (B, E) build up more lactate, indicating a greater initial stimulation of the TILs after coming out of the tumor.
  • CD3+ T cells The proportion of CD3+ T cells, NKT cells, NK cells, and Innate Lymphoid Cells (ILC) are presented in Figure 10, while Figure 11 shows the frequency of T cells based on CD4 and CD8 expression (Accellix TBNK-16 NL and T cell cartridge).
  • the population of CD3 positive cells was lower in the conditions containing ArtQC Bcells compared to all other conditions.
  • the distribution of the major cell populations shifted on the final harvest (D20), where the percentage of CD3 positive cells was the highest with ArtQC Bcells (95.1% and 94.7%) in comparison to the other conditions (62.0% - 88.2%).
  • the percentage of NK cells decreased from 33.7% and 36.7% on the activation day (D10) to 2.1% and 2.7% at the time of harvest on D20.
  • Figure 10, Figure 11, and Table 3 summarize the cell populations obtained in the different conditions.
  • the cell expansion in the bioreactors containing ArtQC Bcells was slow at the beginning, following the pattern of the “tumor only”
  • the condition with ArtQC BC generated a pure CD3+ product containing a high CD8+ T cell population.
  • Frozen cells from final harvest of G-Rex (Day 20) from condition A (no B cells), C (tumor + Non EP B cells), D (tumor + ArtQC Bcells) and E (tumor + EP B cells without IL-12) were thawed and used for characterization.
  • Condition B had very few cryopreserved cells and was therefore not tested.
  • T cell activation in response to anti-CD3 stimulation
  • T cell phenotypes (2 panels)
  • Cell viability at thawing was generally lower compared to the viability at pre-cryopreservation for all conditions.
  • Cell viability post-cryopreservation for ArtQC Bcells was highest compared to other conditions.
  • Cell diameter post-cryopreservation was higher than pre-cryopreservation, but did not differ much between conditions.
  • TILs from all conditions produced IFNy in an anti-CD3 dose-dependent manner. Compared to the TILs alone (condition A), the IFNy levels released upon anti-CD3 stimulation were lower in the presence of EP-Bcells (condition E) and even lower with artQC Bcells (conditions C and D).
  • Anti-CD3 OKT antibody Biolegend Cat 317326 was added (stimulation-stim) or not (unstimulated condition-unstim) in the wells at 30 ng/mL final concentration.
  • Glucose and Lactate was measured from the supernatant and 1 mL of the culture medium was replaced with fresh medium (AIM- V 5% hABS, 100 U/mL Pen/strep and 3000 lU/mL of IL-2, for all contitions).
  • Cells were harvested and reseeded at day 7 and day 14.
  • Cells counts, Accellix and glucose/lactate measurement were performed at day 7, day 14 and day 21 using NC-202, Accellix, Glucose Unio and Lactate Scout 4 instruments, respectively. Interpolated cell numbers were calculated as if there were no intermediate harvests and re-seeding due to limitations in cell numbers by the used wells.
  • TILs previously cultured with ArtQC Bcells displayed superior proliferative capacity compared to TILs cocultured with EP Bcells without IL-12 (condition E) until at least day 14, both at baseline or in response to anti-CD3 stimulation.
  • the cell viability in condition C and D remained high and comparable to other conditions. From day 14 to day 21, cell expansion was decreased in all conditions.
  • TILs obtained with previous co-culture with art B cells have high proliferative capacity compared to TILs cocultured with B cells.
  • Cells were left unstimulated (e.g. AO) or stimulated with anti-CD3 antibody (e.g. A3).
  • the Glucose (Y- axis) and Lactate (X-axis) levels were quantified from cell supernatant at day 4 ( Figure 17A), day 7 (Figure 17B), day 14 (Figure 17C) and day 21 ( Figure 17D).
  • the coefficient of determination (R 2 ) is indicated in each graph.
  • TILs obtained with previous coculture with Art-BC have higher proliferative capacity and responded faster to stimulation, compared to TILs alone or TILs cocultured with B cells.
  • TILs obtained with previous co-culture with Art-BC have higher proliferative capacity and responded faster to stimulation, compared to TILs alone or TILs cocultured with B cells.
  • T cells were left unstimulated (Unstim, Figure 18 (top)) or stimulated with anti-CD3 antibody (aCD3, Figure 18 (bottom)).
  • the proportion of T cells, NKT, NK and “ILC” cells were accessed at day 0 and day 21 using Accellix.
  • the conditions where TILs were cocultured with ArtQC BC increase the number of T cell population, and reduce the percentage of NKT cells.
  • the cells populations at day 21 are similar between conditions when cells are stimulated with anti-CD3.
  • the condition with non-EP BC + ArtQC BC and ArtQC BC when stimulated with anti-CD3 have a slightly increase of NKT cells.
  • T cells were left unstimulated (Unstim, Figure 19 (top) or stimulated with anti-CD3 antibody (aCD3, Figure 19 (bottom)).
  • the composition of T cells (CD4+, CD8+, CD8- CD4-, CD8+ CD4+) were accessed at day 0, day 7, day 14 and day 21 .
  • CD8+ cells were similarly high between conditions and increased over time (>90%) throughout 21 days of culture, with or without anti-CD3 stimulation.
  • the percentages of CD4+, CD8- CD4-, CD8+ CD4+ remained low in all conditions.
  • TILs from previous co-culture with ArtQC B cells displayed similar T cell populations composition in restimulation/proliferation assay compared to TILs coculture with EP B cells.
  • TILs in conditions C and D displayed less differentiated phenotype (CD28+, CD27- CD28+), while TILs in conditions A and E (without Art BC) displayed more effector/differentiated/senescent phenotype (CD57+, KLRG1+, CD57+ KLRG1+, CXCR3+) (see Figure 20 and Table 6).
  • ArtQC Bcells support T cell proliferation without driving into differentiation and senescent phenotype like in normal process.

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Abstract

La présente invention concerne une matrice de stimulation des lymphocytes (LSM) contenant ou consistant en : (i) une matrice de formation d'hydrogel ; et (ii) un ou plusieurs ligands de stimulation lymphocytaire affichés à la surface de ladite matrice de formation d'hydrogel, ledit ou lesdits ligands de stimulation lymphocytaire comprenant ou consistant en OX40L (CD134L). L'invention concerne également l'utilisation de la LSM dans des procédés pour multiplier ou produire une population de lymphocytes, ainsi qu'une population de lymphocytes obtenue ou pouvant être obtenue selon ces procédés. La présente invention concerne également des compositions pharmaceutiques contenant de tels lymphocytes et, plus particulièrement, des utilisations médicales et des traitements appliquant ou utilisant les populations de lymphocytes divulguées et les produits pharmaceutiques connexes.
PCT/EP2024/067655 2023-06-23 2024-06-24 Matrice de stimulation des lymphocytes (lsm) et son utilisation pour la multiplication des populations de lymphocytes WO2024261339A1 (fr)

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