WO2017191504A1 - Validation de lymphocytes t thérapeutiques - Google Patents

Validation de lymphocytes t thérapeutiques Download PDF

Info

Publication number
WO2017191504A1
WO2017191504A1 PCT/IB2017/000563 IB2017000563W WO2017191504A1 WO 2017191504 A1 WO2017191504 A1 WO 2017191504A1 IB 2017000563 W IB2017000563 W IB 2017000563W WO 2017191504 A1 WO2017191504 A1 WO 2017191504A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
csk
dummy
tcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2017/000563
Other languages
English (en)
Inventor
Else Marit Suso INDERBERG
Lars-egil FALLANG
June Helen MYKLEBUST
Sebastien Walchli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oslo Universitetssykehus hf
Original Assignee
Oslo Universitetssykehus hf
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oslo Universitetssykehus hf filed Critical Oslo Universitetssykehus hf
Publication of WO2017191504A1 publication Critical patent/WO2017191504A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/38—Applying electric currents by contact electrodes alternating or intermittent currents for producing shock effects
    • A61N1/39—Heart defibrillators
    • A61N1/3925—Monitoring; Protecting
    • A61N1/3931—Protecting, e.g. back-up systems
    • 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
    • A61K40/32—T-cell receptors [TCR]
    • 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
    • A61K40/4244—Enzymes
    • A61K40/4251—Kinases, e.g. Raf or Src

Definitions

  • compositions and methods for monitoring the safety of immunotherapy are provided herein.
  • inactive T-cells and uses thereof are provided herein.
  • TCR T-cell receptor
  • pMHC peptide-MHC complex
  • a very powerful negative regulator of TCR signal is a kinase, c-terminal SCR kinase (CSK) (4).
  • the activity of this kinase is restricted to the phosphorylation of the inhibitory tyrosine of SCR family kinases (5), but it can also regulate LYP, a tyrosine phosphatase, by binding it (6).
  • Lck is recruited to the vicinity of the CD3-TCR complex and phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) motifs. These domains, once phosphorylated, become anchoring points for the recruitment of the TCR- signalling machinery.
  • CSK is believed to tune down this signalling activation by
  • compositions and methods for monitoring the safety of immunotherapy are provided herein.
  • inactive T-cells and uses thereof are provided herein.
  • the present invention provides a method of monitoring T-cell based therapy, comprising: a) administering a dummy T-cell to a subject, wherein the dummy T-cell does not function to kill cells or promote an immune response, and wherein the dummy T-cell comprises a detectable label; and b) detecting the location of the dummy T-cell in the subject.
  • the dummy T-cell is a modified therapeutic T-cell. It will be understood that the term "dummy T-cell" refers herein to a T-cell that has been engineered so that normal T-cell function has been inhibited or ablated.
  • the inhibition of normal T-cell function refers to the inhibition of the ability of the T-cell to function to kill cells (i.e., inhibition of killer cell function) or to promote an immune response.
  • the dummy T-cell overexpresses C-terminal SRC kinase (CSK).
  • CSK is expressed in an amount sufficient to inhibit or ablate normal T-cell function as described above.
  • the dummy cell is engineered to overexpress CSK by introducing an expression vector comprising a promoter operably linked to a nucleic acid sequence encoding CSK into the dummy T-cell.
  • the nucleic acid encoding CSK encodes an amino acid sequence that has at least 80%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:6.
  • the CSK is a variant CSK
  • the variant retains the biological activity wild type CSK, i.e., phosphorylation of tyrosine residues located in the C-terminal end of Src-family kinases (SFKs) including, for example, SRC, HCK, FYN, LCK, LYN and YES 1.
  • SFKs Src-family kinases
  • the dummy T-cell is specific for a target epitope.
  • the dummy T-cell is a tumor infiltrating lymphocyte expressing a chimeric antigen receptor, or a T-cell expressing a heterologous T-cell receptor.
  • the method further comprises the step of administering a therapeutic T-cell to the subject.
  • the method further comprises the step of visualizing the location of the detectable label in subject.
  • the presence of said detectable label in a location distinct from the target location of a target epitope is indicative of non-specific binding by the dummy T-cell.
  • the dummy T-cells can be used to detect unspecific localization of the cells in vivo after parenteral administration. Unspecific localization may indicate that use of corresponding therapeutic T-cells would trigger unnecessary or harmful immune responses. However, if the dummy T-cells are localized at the desired site(s), as can be verified by the label, it would indicate that the immune response would be triggered at these sites. Accordingly, compositions comprising such dummy T-cells can be used in confirmation of specific localization of the cells in vivo after parenteral administration. The difference between a dummy T-cell and a corresponding therapeutic T- cell may be overexpression of CSK only.
  • the subject is a human subject or a non-human mammal.
  • the target site is a tumor site.
  • kits or system comprising: a) a dummy T-cell, wherein the dummy T-cell does not function to kill cells or promote an immune response, and wherein the dummy T-cell comprises a detectable label; b) a detection component for detecting the location of the dummy T-cell in the subject.
  • a genetically modified human T-cell comprising a detectable label and a heterologous DNA sequence encoding a membrane bound receptor specific for a desired target epitope, wherein the genetically modified T-cell overexpresses CSK in an amount sufficient for preventing significant TCR signaling, and wherein the membrane bound receptor for the desired target epitope is expressed in an amount sufficient for specific binding of the T-cell to the desired target.
  • the membrane bound receptor for the desired target may be expressed in an amount sufficient for specific localization of the T- cell after parenteral administration and the CSK may be overexpressed in an amount sufficient for preventing an immune response.
  • the membrane bound receptor is specific for a cancer antigen.
  • the membrane bound receptor is a T-cell receptor.
  • the genetically modified human T-cell is engineered to overexpress CSK by introducing an expression vector comprising a promoter operably linked to a nucleic acid encoding CSK into the T-cell.
  • the nucleic acid encoding CSK encodes an amino acid sequence that has at least 80%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:6.
  • the CSK is a variant CSK
  • the variant retains the biological activity wild type CSK, i.e., phosphorylation of tyrosine residues located in the C-terminal end of Src-family kinases (SFKs) including, for example, SRC, HCK, FYN, LCK, LYN and YES 1.
  • SFKs Src-family kinases
  • composition comprising the dummy T-cells above for use in confirmation of specific localization of the cells in vivo after parenteral administration, e.g. injection.
  • FIG. 1A-C Overexpression of CSK potently suppresses TCR-induced
  • CSK 2A GFP construct consists of a fusion of the CSK coding sequence where the STOP codon was removed and replaced by a picornavirus 2A coding sequence, followed by the GFP gene sequence. Upon transcription, one mRNA is produced and transcribed into two distinct proteins. PBMC-derived T-cells were transduced with control vector (GFP), CSK- GFP or mCSK-GFP. Incubation with aCD3 and aCD28 Abs were used to activate TCR, and signaling was detected by phospho-specific flow cytometry.
  • B Histogram overlays from one representative experiment.
  • the color code shows induced phosphorylation level, relative to unstimulated cells, using arcsinh transformation of the median fluorescence intensity.
  • FIG. 2A-B mCSK is more potent than CSK in suppressing TCR-induced distal signaling events.
  • PBMC-derived T-cells were transduced with therapeutic TcR (TCR specific for TGFpRII frameshift peptide, termed Radium 1 TCR). The transduced T-cells were then expanded and transiently transfected with GFP, CSK-GFP or mCSK-GFP mRNA.
  • SupTl cells were loaded with relevant (TGFpRII peptide), or irrelevant peptide (MARTlp) were used as APC1 and APC2, respectively and added to the T-cells to induce TCR signaling.
  • A One representative experiment.
  • FIG. 3A-C CSK inhibited TCR stimulation cellular consequences.
  • J76 cells were transduced with the indicated constructs and sorted. The expression of the DMF5 TCR (anti- CD3) and/or CSK 2A GFP were analyzed by flow cytometry.
  • B The same cells as in (A) were incubated with SupTl cells expressing single chain trimer (SCT) molecules presenting MARTI peptide (SCT-MART-lp) or an irrelevant peptide (SCT-irr) for 12 hours.
  • SCT single chain trimer
  • FIG. 4A-C TCR membrane localization is increased when CSK is overexpressed.
  • MART-1 mul timers and binding was analyzed by flow cytometry.
  • the Geometric mean was plotted and each cell population was split in GFP positive and negative (dark green and light green, respectively) in order to compare cells from the same tube. This is single staining representative of two separate experiments.
  • B The same experiment as in (A) was repeated but this time in the presence of dasatinib.
  • FIG. 5A-C Trogocytosis is not affected by CSK overexpression.
  • APC T2 cells
  • TGF RIIp relevant (TGF RIIp) or an irrelevant peptide (MART-lp, Mlp) O/N prior to co-incubation with J76 cells constitutively expressing TCR-Radiuml in combination with GFP (control), CSK 2A GFP or mCSK_2A_GFP. Presence of CD3 was then monitored on T2 cells and the Geometric mean plotted.
  • B Same as in (A) but GFP + cells (J76) were gated and the presence of HLA-A2 detected.
  • FIG. 6 TCR-dependent target cell attachment is increased by CSK.
  • T-cells were electroporated with mRNA encoding for Radium 1 TCR and either GFP or the
  • FIG. 7 CSK 2A GFP construct validation in T-cells.
  • CSK 2A GFP was transduced into T-cells from PBMC, GFP signal correlated with CSK expression detected by intracellular staining.
  • FIG. 8 T-cells transduction. Radium-1 expression as detected using specific V-beta antibody and analyzed by Flow cytometry.
  • FIG. 9 Gating strategy used for the MARTI multimer binding study. Green are dmf5 expressing T-cells and purple are DMF5/GFP (here mCSK) double positive population.
  • FIG. 10 Redirected T-cells expressing CSK or mCSK are unable to kill their targets.
  • PBMC derived T-cells were electroporated with the indicated constructs. Eight hours later they were incubated with target cells pre-loaded with Europium at an Effector to target ratio
  • FIG. 11 Amino acid sequence for tyrosine-protein kinase CSK [Homo sapiens], NCBI
  • variants and mutants when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.
  • conservative amino acid substitutions refers to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is
  • phenylalanine, tyrosine, and tryptophan unnatural amino acids like p-aminophenylalanine, a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan).
  • Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).
  • amino acid substitution the following nomenclature is used: Original amino acid, position, substituted amino acid.
  • substitution of lysine with alanine at position 573 is designated as "K573A” and the substitution of lysine with proline at position 573 is designated as K573P.
  • Multiple mutations are separated by addition marks ("+") or "/”, e.g., "Gly205Arg + Ser41 lPhe” or “G205R/S41 IF", representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively.
  • the relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".
  • the degree of identity between two amino acid sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
  • Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later.
  • the optional parameters 11644.000-EP7 used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • amino acid position corresponding to a position in a reference sequence and similar expression is intended to identify the amino acid residue that in the primary or spatial structure corresponds to the particular position in the reference sequence. The skilled person will appreciate that this can be done by aligning a given sequence with the reference sequence and identifying the amino acid residue that aligns with the particular position in the reference sequence.
  • the expression Xnnn is intended to mean an amino acid residue X located in a position corresponding to position nnn in HSA and the expression XnnnY is intended to mean a substitution of any amino acid X located in a position corresponding to position nnn in HSA with the amino acid residue Y.
  • the term "under conditions such that said subject generates an immune response” refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).
  • immune response refers to a response by the immune system of a subject.
  • immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T-cell activation (e.g., CD4+ or CD8+ T-cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion).
  • lymphokine e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine
  • macrophage activation e.g., dendritic cell activation
  • T-cell activation e.g., CD4+ or CD8+ T-cells
  • NK cell activation e.g., antibody
  • immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T-cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells.
  • an immunogen e.g., antigen (e.g., immunogenic polypeptide)
  • CTL cytotoxic T lymphocyte
  • B cell response e.g., antibody production
  • T-helper lymphocyte response e.g., T-helper lymphocyte response
  • DTH
  • an immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self-antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign).
  • immunogens that the subject's immune system recognizes as foreign
  • immune response refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) cell- mediated immune responses (e.g., responses mediated by T-cells (e.g., antigen-specific T- cells) and non-specific cells of the immune system) and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • innate immune responses e.g., activation of Toll receptor signaling cascade
  • T-cells e.g., antigen-specific T- cells
  • B cells e.g.
  • immuno response is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).
  • an immunogen e.g., a pathogen
  • acquired e.g., memory
  • immunogen refers to an agent (e.g., a cancer epitope) and/or portion or component thereof (e.g., a protein antigen)) that is capable of eliciting an immune response in a subject.
  • immunogens elicit immunity against the immunogen (e.g. , microorganism (e.g., pathogen or a pathogen product)).
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample.
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • sample as used herein is used in its broadest sense.
  • sample is used in its broadest sense. In one sense it can refer to a tissue sample. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include, but are not limited to blood products, such as plasma, serum and the like. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • a sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g.
  • a sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.
  • compositions and methods for monitoring the safety of immunotherapy are provided herein.
  • inactive T-cells and uses thereof are provided herein.
  • Cancer immunotherapy is the use of the immune system to treat cancer.
  • Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti -tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.
  • TAAs tumor-associated antigens
  • Passive immunotherapies enhance existing anti -tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.
  • Active cellular therapies usually involve the removal of immune cells from the blood or from a tumor. Those specific for the tumor are cultured and returned to the patient where they attack the tumor. Cell types that can be used in this way are natural killer cells, lymphokine-activated killer cells, cytotoxic T-cells and dendritic cells. The only US- approved cell-based therapy is Dendreon's Provenge, for the treatment of prostate cancer.
  • Adoptive T-cell therapy is a form of passive immunization by the transfusion of T- cells. They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.
  • APCs antigen presenting cells
  • TILs Tumor Infiltrating Lymphocytes
  • TILs Tumor Infiltrating Lymphocytes
  • TcR T-cell Receptor
  • CARs composed of an antibody linked to the signaling domain of the TcR
  • TcR Receptors
  • TcRs are not restricted to one peptide combined with one MHC, but can potentially recognize thousands of MHC-presented peptides. This implies that these molecules potentially can cross-react with sequence related antigens.
  • target specificity is the antigen only expressed in tumor tissue. The methods developed to test this have been limited with the result of severe conditions of the patients, including deaths after T-cell injection, due to unpredicted cross-reactivity or unexpected antigen distribution. Indeed, pre-clinical experiments include SCID mice curing and screening of healthy tissue in vitro. A recent review describes all the unexpected side effects observed since the first therapeutic T-cells were used (Hinrichs and Restifo. 2013 Nat. Biotech.). It shows the difficulty to mimic and predict cross-reactivity and antigen distribution.
  • a preferred in vivo assay is performed in the same setting as the clinical trial, but with safe version of the T-cells.
  • the strategy described herein is to inject the same T-cells as the therapeutic ones, but with a modification preventing the cells to signal and hence to kill (CD8) or provide help (CD4), and study their tissue distribution after injection. Therefore, these cells are called "dummy T-cells".
  • TcR or CAR a therapeutic molecule
  • the T-cells overexpress the C-terminal SRC kinase (CSK).
  • CSK C-terminal SRC kinase
  • TcR or CAR therapeutic molecule
  • the membrane bound receptor for the desired target is expressed in an amount sufficient for specific localization of the T-cell after parenteral administration. Therefore, these dummy T-cells will serve to analyze potential side effects (mainly wrong homing) in patient in a safe manner, prior to the adoptive transfer of the fully functional redirected T-cell, i.e. the corresponding therapeutic T-cell.
  • T-cells are engineered in vitro or ex vivo to overexpress CSK.
  • Overexpression of CSK in a T-cell means that the level of CSK enzyme is significantly increased compared to an unmodified T-cell. This can be achieved in many ways including modification of the native CSK-gene present in the T-cell or by introduction of a
  • heterologous sequence encoding CSK is significantly increased compared to an unmodified T-cell. This can be achieved in many ways including modification of the native CSK-gene present in the T- cell or by introduction of a heterologous sequence encoding mCSK.
  • a transgene that overexpresses CSK and a detectable label are introduced into a therapeutic T- cell.
  • the CSK is a variant or wild-type human CSK and has at least 80%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:6, the reference amino acid sequence for human CSK.
  • the CSK is a variant CSK, it will be understood that the variant retains the biological activity wild type CSK, i.e., phosphorylation of tyrosine residues located in the C-terminal end of Src-family kinases (SFKs) including, for example, SRC, HCK, FYN, LCK, LYN and YES1.
  • SFKs Src-family kinases
  • variants are sequences that have a defined identity to SEQ ID NO:6, as can be measured electronically by making use of algorithms such as PILEUP and BLAST.
  • PILEUP See, e.g., Higgins & Sharp, CABIOS 5: 151 (1989); Altschul S. F., W. Gish, W. Miller, E. W. Myers, D. J. Lipman. Basic local alignment search tool. J. Mol. Biol. 1990; 215:403-10.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/).
  • a “deletion” is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid.
  • a deletion can involve deletion of about two, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids.
  • a protein or a fragment thereof may contain more than one deletion.
  • an “insertion” or “addition” is that change in an amino acid or nucleotide sequences which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein.
  • “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini.
  • an insertion or addition is usually of about one, about three, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids.
  • a protein or fragment thereof may contain more than one insertion.
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.
  • a substitution may, for example, be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in CSK.
  • any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of CSK or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of CSK are included within the scope of the invention.
  • a skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may, for example, involve introducing a limited number of possible substitutions and determining their influence on the properties of the CSK and cells transformed therewith thus obtained.
  • the present invention is not limited to particular expression vectors for
  • CRK is expressed using any suitable vector or expression system.
  • the vectors of the present invention comprise a promoter operably linked to a nucleic acid sequence encoding a variant or wild-type CRK protein encoding by SEQ ID NO:6, or having at least 80%, 90%, 95%, 97%, 98%, 99% identify to SEQ ID NO:6.
  • CRK is expressed via a suitable eukaryotic expression vectors (e.g., commercially available vectors).
  • CRK is expressed via a viral vector (e.g., adeno, adeno-associated, or lenti-viral vectors). Suitable vectors are introduced into T-cells and expression is induced.
  • vectors include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40, phage DNA; vectors derived from combinations of plasmids and phage DNA, retroviral vectors and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is contemplated that any vector may be used as long as it is viable and can be expressed in T-cells.
  • chromosomal, nonchromosomal and synthetic DNA sequences e.g., derivatives of SV40, phage DNA; vectors derived from combinations of plasmids and phage DNA, retroviral vectors and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. It is contemplated that any vector may be used as long as it is viable and can be expressed in T-cells.
  • some embodiments of the present invention provide recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
  • the appropriate DNA sequence is inserted into the vector using any of a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
  • vectors include, but are not limited to, the following vectors: pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • Promoters useful in the present invention include, but are not limited to, the LTR or SV40 promoter, the phage lambda PL and PR, T3 and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of gene in prokaryotic or eukaryotic cells or their viruses.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • thymidine kinase thymidine kinase
  • recombinant expression vectors include origins of replication and selectable markers permitting transformation of the host cell (e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli).
  • transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
  • Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the present invention provides T-cells containing the above- described constructs. In some embodiments of the present invention, following
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • kits comprising the dummy T-cells described herein.
  • dummy T-cells are provided in a system with reagents and/or instruments to detect the presence of the dummy T-cells (e.g., imaging systems).
  • kits and systems further comprise one or more of instructions, controls, delivery systems, etc.
  • Dummy T-cells may target any antigen (e.g., cancer antigen) including but not limited to proteins, subunits, domains, motifs, and/or epitopes belonging to the following list of target antigens, which includes both soluble factors such as cytokines and membrane-bound factors, including transmembrane receptors: 17-IA, 4-lBB, 4Dc, 6-keto-PGF la, 8-iso-PGF2a, 8-oxo-dG, Al Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RUB, ADAM, ADAM 10, ADAM 12, ADAM 15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, a
  • HCMV UL Hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGF A, High molecular weight melanoma-associated antigen (HMW- MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL
  • Neurotrophin-3, -4, or -6 Neurturin, Neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGGl, OPG, OPN, OSM, OX40L, OX40R, pi 50, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PEC AM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL,
  • T-cell receptors e.g., T- cell receptor alpha/beta
  • TdT T-cell receptor alpha/beta
  • TECK T-cell receptor alpha/beta
  • TEM1 TEM5
  • TEM7 TEM7
  • TEM8 testicular PLAP-like alkaline phosphatase
  • TfR TGF
  • TGF-alpha TGF-beta
  • TGF-beta Pan TGF-beta Rl (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-betal, TGF- beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-be
  • TNFRSF7 CD27
  • TNFRSF8 CD30
  • TNFRSF9 4-1 BB CD137, ILA
  • TNFRSF21 DR6
  • TNFRSF22 DcTRAIL R2TNFRH2
  • TNFRST23 DcTRAIL Rl TNFRH1
  • TNFRSF25 DR3Apo-3, LARD, TR-3, TRAMP, WSL-1
  • TNFSF10 TRAIL Apo-2 Ligand, TL2
  • TNFSF11 TRANCE/RANK Ligand ODF, OPG Ligand
  • TNFSF12 TWEAK Apo-3 Ligand, DR3Ligand
  • TNFSF13 APRIL TALL2
  • TNFSF13B BAFF BLYS, TALL1, THANK, TNFSF20
  • TNFSF14 LIGHT HVEM Ligand, LTg
  • TNFSF15 T1A/VEGI
  • TNFSF18 GITR Ligand AITR Ligand, TL6
  • TNFSF1A TNF
  • targets refers not only to specific proteins and biomolecules, but the biochemical pathway or pathways that comprise them.
  • CTLA-4 as a target antigen implies that the ligands and receptors that make up the T-cell co-stimulatory pathway, including CTLA-4, B7-1, B7- 2, CD28, and any other undiscovered ligands or receptors that bind these proteins, are also targets.
  • target as used herein refers not only to a specific biomolecule, but the set of proteins that interact with said target and the members of the biochemical pathway to which said target belongs.
  • T2 cells were maintained in the same medium.
  • the packaging cells were the modified Human Embryonic Kidney cells-293, Hek-Phoenix (Hek-P) and they were grown in DMEM (PAA) with 10% FCS. HeLa cells were grown in the same medium.
  • T-cells were grown in CellGro DC medium (CellGenix, Freiburg, Germany) supplemented with 5% heat-inactivated human serum (Trina Bioreactives AG, Nanikon, Switzerland), 10 mM N-acetylcysteine (Mucomyst 200 mg/mL, AstraZeneca AS, London, UK), 0.01 M HEPES (Life Technologies, Norway) gentamycin 0.05 mg/mL (Garamycin, Schering-Plough Europe, Belgium). Dasatinib was from LC Laboratories (Woburn, MA, USA). The TGFbRII frameshift peptidem-139, RLSSCVPVA was provided by Norsk Hydro ASA, (Porsgrunn, Norway). The MART-1 peptide26-35 EAAGIGILTV was manufactured by Prolmmune Ltd (Oxford, UK) and MART- 1 dextramer was from Immudex (Kobenhavn, Denmark).
  • CSK cloning was performed by amplifying cDNA from PBMC isolated from an healthy donor using the following primers: 5'-CAC CAT GTC AGC AAT AC A GGC-3' (full length construct; SEQ ID NO: 1) or 3'-CAC CAT GGG CTG TGG CTG CAG CTC AC A CCC CGA AGA TGC AAT AC A GGC CG-5' (membrane targeted CSK with LCK myristoylation domain; SEQ ID NO:2) and
  • the amplicon was subsequently cloned into pENTR vector (Invitrogen, Carlsbad, CA, USA) and sequence verified (Eurofins MWG Operon, Ebersberg, Germany). It was then transferred into pMP71 (retroviral vector) or pCIpA102 (mRNA synthesis construct) as described in (2 .
  • TCR expression constructs were prepared by amplifying TCR-a and - ⁇ chains separately with specific primers and followed by a second PCR to fuse the TCR chains as a TCR-2A construct as described in (21) and in (Inderberg et al, manuscript submitted) for DMF5 and Radium- 1, respectively.
  • HLA-A2 -peptide single chain trimer (SCT) expression plasmids were constructed as described in (22). Peptide coding sequence exchange to create a TGFFbR2 frameshift peptide SCT (SCT-TGFbRIIp) was performed by site direct mutagenesis using these peptides: 5'- GGC CTC GAG GCT CGT CTG TCA TCA TGC GTT CCT GTG GCT GGC TGT GGC AGC-3' SEQ ID NO:4 and 5'-GCT GCC ACA GCC AGC CAC AGG AAC GCA TGA TGA CAG ACG AGC CTC GAG GCC-3' SEQ ID NO:5.
  • the in vitro mRNA synthesis was performed essentially as previously described (23). Anti-Reverse Cap Analog (Trilink Biotechnologies Inc., San Diego, CA, USA) were used to cap the RNA. The mRNA was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA).
  • T-cells from healthy donors were expanded using a protocol adapted for GMP production of T-cells employing Dynabeads CD3/CD28 as described in (23).
  • PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads® ClinExVivoTM CD3/CD28, ThermoFischer, Oslo, Norway) at a 3: 1 ratio in complete CellGro DC Medium with 100 U/mL recombinant human interleukin-2 (IL- 2) (Proleukin, Prometheus Laboratories Inc., San Diego, CA, USA) for 10 days. The cells were frozen and aliquots were thawed and rested in complete medium before transfection.
  • IL-2 human interleukin-2
  • T-cells were washed twice and resuspended in CellGro DC medium (CellGenix GmbH) to 70 ⁇ 10 6 cells/mL.
  • the mRNA was mixed with the cell suspension at 100 ⁇ g/mL, and electroporated in a 4-mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc., Hawthorne, NY, USA).
  • T-cells were transferred to complete culture medium at 37°C in 5% CO2 overnight.
  • Viral particles produced as described in (21 were used to transduce cells (cell line or activated T-cells) as follows: Spinoculation was performed with 1 Volume of retroviral supernatant in a 12-well or a 24-Well culture non-treated plate (Nunc A/S, Roskilde, Denmark) pre-coated with retronectin (20 ⁇ g/mL, Takara Bio. Inc., Shiga, Japan). T-cells were spinoculated twice whereas all the other cell lines were spinoculated only once. Cells were then harvested with PBS-EDTA (0.5 mM) and grown in their medium or in X-vivo 20, 5% HS, 100 U/mL IL-2 and 2 ng/mL IL-15 for T-cells. Cells were used for experiments after 3 to 7 days.
  • T-cells were stimulated for 5 hours with APCs, loaded or not with the indicated peptide, at a T-cell to target ratio of 2: 1 and in the presence of BD
  • TCR/CSK_2_GFP or GFP expressing J76 cells were incubated with T2 cells that had been O/N loaded with the indicated peptide at saturating concentration (5 ⁇ ) at 1 : 1 ratio for 4 hours. Cells were then extensively washed and stained for flow analysis (see below) using specific antibodies.
  • Trogocytosis was measured by determining the increase of signal of the transferred markers.
  • the following antibodies were used: ⁇ 3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4- eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8- PE-Cy7 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA).
  • Cells were washed in flow buffer (FB, phosphate buffered saline (PBS) with 2% human bovine serum albumin (BSA) and 0.5 ⁇ EDTA).
  • FB flow buffer
  • PBS phosphate buffered saline
  • BSA human bovine serum albumin
  • dextramer and antibody staining cells were incubated for 30 minutes at RT with the recommended dilution in FB. If fixed, cells were incubated in FB containing 1% paraformaldehyde. All antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA).
  • T-cell binding to cognate APC was performed as follows: 0.15x 10 6 HeLa cells were seeded into Glass Bottom 6-Well Plates (MatTek Corp., MA, USA); after 12 hours, they were transfected with SCT constructs (1 ⁇ g DNA/well) and left another 24 hours. Then 0.6x 10 6 T-cells were added, left for 35 minutes and cells were washed once or not with RPMI before confocal microscopy. Images were taken on an Olympus Fluoview 1000 IX-81 inverted confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) ) using 40X objective lens. HeLa cells were identified by their morphological characteristics whereas T-cells were traced by GFP expression. Ten images per condition were acquired; Image J software (Bethesda, MA, USA) was used to analyze images. The number of cells per frame was counted; plotting and statistical analysis were performed using GraphPad prism software (La Jolla, CA USA). Results
  • TCR signalling in T-cells transduced with a TGFpRII-frameshift specific TcR was next tested in a more physiological setting, using antigen presenting cells (APC) loaded with relevant (TGFpRII) or irrelevant (MARTI) peptide.
  • APC antigen presenting cells
  • T-cells were cultured alone or with APC cells for 5 or 15 minutes and then subjected to phospho-specific flow cytometry measurements. The presence of relevant APCs induced phosphorylation of ERK in a fraction of CD8+ and CD8- T-cells, whereas the presence of irrelevant APCs did not (Fig. 2A, B).
  • ERK became phosphorylated in the highest fraction of T-cells with 40% and 22% positive CD8T- cells 5 and 15 min post APC stimulation, respectively (Fig 2B).
  • mCSK the APC-induced phosphorylation of ERK was almost completely blocked.
  • the lack of blockade in a few cells could be due to that not all T-cells overexpressed mCSK.
  • overexpression of CSK had limited effect (Fig 2B).
  • TCR-negative cell line J76 (20). transduced with MARTI -specific TcR DMF5 (25). These cells were super infected with CSK and GFP + /CD3 double positive cells were sorted in order to obtain a pure population. A non-TCR transduced control population expressing CSK only was prepared and sorted (Fig. 3A). We then analyzed IL-2 release upon incubation with APC. Equal numbers of the transduced J76 cells were incubated with SupTl cells expressing Single-Chain Trimer (SCT) fused to the MARTI peptide (MARTlp) or an irrelevant peptide.
  • SCT Single-Chain Trimer
  • T-cells isolated from PBMC previously transduced with a TGF RII- frameshift specific TcR (Radium 1 TCR, Inderberg et al. submitted). This TCR could be detected by staining with a specific anti-V 3 antibody (Fig. 8).
  • TGF RIIp TGF RIIp
  • T-cell stimulation was determined by detection of the degranulation marker CD107a (Fig. 3C). Similar to the J76 experiment, the presence of CSK decreased the TCR-dependent stimulation, but was unable to block it.
  • TCR TCR signaling dependent
  • HeLa cells expressing single chain trimer (SCT) molecules fused to the relevant antigenic peptide (TGF- ⁇ , SCT-TGF) or exposing an irrelevant peptide (MART-lp, SCT-M1) were used as APCs.
  • T-cells were incubated with SCT-HeLa cells for 35 minutes and plates were washed or not before the number of bound T- cells were counted per image.
  • SCT-HeLa cells were incubated with SCT-HeLa cells for 35 minutes and plates were washed or not before the number of bound T- cells were counted per image.
  • Safeguard systems such as inducible suicide genes, in which the redirected T-cells can be stopped before fatal events happen have been proposed earlier (9). However, the time of response might still represent a challenge. It is therefore becoming obvious that predictions or in vitro testing of off-target toxicity for a therapeutic TCR are not sufficient for safety evaluation.
  • alternative methods to test a TCR in vivo the use of soluble TCR combined with a tracer is an attractive one, but the lack of all cellular companion proteins (co-receptors) might hinder proper detection.
  • Another method could be the use of transient systems such as mRNA electroporation (40). Nevertheless the transient TCR expression might not allow for detection of accumulation of the redirected T-cells at an inappropriate location.
  • Emery Emery, L. Litzky, A. Bagg, B. M. Carreno, P. J. Cimino, G. K. Binder-Scholl, D. P.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des compositions et des procédés pour surveiller la sécurité d'une immunothérapie. En particulier, l'invention concerne des lymphocytes T inactifs et leurs utilisations.
PCT/IB2017/000563 2016-05-03 2017-05-03 Validation de lymphocytes t thérapeutiques Ceased WO2017191504A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662331049P 2016-05-03 2016-05-03
US62/331,049 2016-05-03

Publications (1)

Publication Number Publication Date
WO2017191504A1 true WO2017191504A1 (fr) 2017-11-09

Family

ID=65365756

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2017/000563 Ceased WO2017191504A1 (fr) 2016-05-03 2017-05-03 Validation de lymphocytes t thérapeutiques

Country Status (1)

Country Link
WO (1) WO2017191504A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020043903A1 (fr) * 2018-08-31 2020-03-05 Invectys Méthode pour évaluer la fonctionnalité des car
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014184744A1 (fr) * 2013-05-13 2014-11-20 Cellectis Procédés de production, par génie génétique, d'un lymphocyte t hautement actif à vocation immunothérapeutique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014184744A1 (fr) * 2013-05-13 2014-11-20 Cellectis Procédés de production, par génie génétique, d'un lymphocyte t hautement actif à vocation immunothérapeutique

Non-Patent Citations (48)

* Cited by examiner, † Cited by third party
Title
ALMASBAK, H. ET AL: "Transiently redirected T cells for adoptive transfer", CYTOTHERAPY, vol. 13, 2011, pages 629 - 640
ALMASBAK, H.; M. LUNDBY; A. M. RASMUSSEN: "Non-MHC-dependent redirected T cells against tumor cells.", METHODS IN MOLECULAR BIOLOGY, vol. 629, 2010, pages 453 - 493
ALTSCHUL S. F. ET AL: "Basic local alignment search tool.", J. MOL. BIOL., vol. 215, 1990, pages 403 - 410, XP002949123, DOI: doi:10.1006/jmbi.1990.9999
AUCHER, A. ET AL: "Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells.", BLOOD, vol. 111, 2008, pages 5621 - 5628
BURKHARDT, J. K.; W. A. COMRIE: "Action and traction: cytoskeletal control of receptor triggering at the immunological synapse", FRONTIERS IN IMMUNOLOGY, 2016, pages 7
CAMERON, B. J. ET AL: "Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells", SCIENCE TRANSLATIONAL MEDICINE, vol. 5, no. 197, 7 August 2013 (2013-08-07), pages 197RA103, XP055107711, DOI: doi:10.1126/scitranslmed.3006034
CHEN, L.; D. B. FLIES.: "Molecular mechanisms of T cell co-stimulation and co-inhibition", NATURE REVIEWS, vol. 13, 2013, pages 227 - 242, XP055150254, DOI: doi:10.1038/nri3405
CHOW L M ET AL: "Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.", NATURE 09 SEP 1993, vol. 365, no. 6442, 9 September 1993 (1993-09-09), pages 156 - 160, XP002772225, ISSN: 0028-0836 *
CHOW, L. M. ET AL: "Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk", NATURE, vol. 365, 1993, pages 156 - 160, XP002772225
DANIEL-MESHULAM, I. ET AL: "Enhanced antitumor activity mediated by human 4-1BB-engineered T cells", INT J CANCER, vol. 133, 2013, pages 2903 - 2913
DAVIS, D. M.: "Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response", NATURE REVIEWS, vol. 7, 2007, pages 238 - 243
DE FELIPE, P. ET AL: "E unum pluribus: multiple proteins from a self-processing polyprotein.", TRENDS IN BIOTECHNOLOGY, vol. 24, 2006, pages 68 - 75, XP025052212, DOI: doi:10.1016/j.tibtech.2005.12.006
DEPONTIEU, F. R. ET AL: "Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 106, 2009, pages 12073 - 12078, XP055079804, DOI: doi:10.1073/pnas.0903852106
EBERT, P. J. ET AL: "MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade", IMMUNITY, 2016
HEEMSKERK, M. H. ET AL: "Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region", BLOOD, vol. 102, 2003, pages 3530 - 3540, XP055070635, DOI: doi:10.1182/blood-2003-05-1524
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151
HINRICHS, C. S.; N. P. RESTIFO: "Reassessing target antigens for adoptive T-cell therapy", NATURE BIOTECHNOLOGY, vol. 31, 2013, pages 999 - 1008, XP055243629, DOI: doi:10.1038/nbt.2725
HINRICHS; RESTIFO, NAT. BIOTECH., 2013
HUDRISIER, D. ET AL: "Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner", J IMMUNOL, vol. 166, 2001, pages 3645 - 3649, XP002397053
IRISH, J. M.; N. KOTECHA; G. P. NOLAN.: "Mapping normal and cancer cell signalling networks: towards single-cell proteomics", NATURE REVIEWS. CANCER, vol. 6, 2006, pages 146 - 155, XP055160605, DOI: doi:10.1038/nrc1804
JOHNSON, L. A. ET AL: "Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes", J IMMUNOL, vol. 177, 2006, pages 6548 - 6559, XP055216481, DOI: doi:10.4049/jimmunol.177.9.6548
JONES, B. S.; L. S. LAMB; F. GOLDMAN; A. DI STASI: "Improving the safety of cell therapy products by suicide gene transfer", FRONTIERS IN PHARMACOLOGY, vol. 5, 2014, pages 254
KJETIL TASKEN: "Negative regulation of T-cell receptor activation by the cAMP-PKA-Csk signalling pathway in T-cell lipid rafts", FRONTIERS IN BIOSCIENCE, vol. 11, no. 1, 1 January 2006 (2006-01-01), US, pages 2929, XP055391629, ISSN: 1093-9946, DOI: 10.2741/2022 *
LINETTE, G. P. ET AL: "Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma", BLOOD, vol. 122, 2013, pages 863 - 871
LISSINA, A. ET AL: "Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 340, 2009, pages 11 - 24, XP025991295, DOI: doi:10.1016/j.jim.2008.09.014
LIU, Z.; Z. LI.: "Molecular imaging in tracking tumor-specific cytotoxic T lymphocytes (CTLs).", THERANOSTICS, vol. 4, 2014, pages 990 - 1001, XP055279416, DOI: doi:10.7150/thno.9268
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
OKADA, M.: "Regulation of the SRC family kinases by Csk", INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES, vol. 8, 2012, pages 1385 - 1397
OKOYE, I. ET AL: "T cell metabolism. The protein LEM promotes CD8(+) T cell immunity through effects on mitochondrial respiration", SCIENCE (NEW YORK, NY, vol. 348, 2015, pages 995 - 1001, XP002753926, DOI: doi:10.1126/science.aaa7516
OSBORNE, D. G.; S. A. WETZEL.: "Trogocytosis results in sustained intracellular signaling in CD4(+) T cells", J IMMUNOL, vol. 189, 2012, pages 4728 - 4739
PALMER, D. C. ET AL: "Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 212, 2015, pages 2095 - 2113, XP055391183, DOI: doi:10.1084/jem.20150304
PIEPENBRINK, K. H. ET AL: "The basis for limited specificity and MHC restriction in a T cell receptor interface", NATURE COMMUNICATIONS, vol. 4, 2013, pages 1948
RICE ET AL.: "EMBOSS: The European Molecular Biology Open Software Suite", TRENDS IN GENETICS, vol. 16, 2000, pages 276 - 277, XP004200114, DOI: doi:10.1016/S0168-9525(00)02024-2
ROYBAL, K. T. ET AL: "Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits", CELL, vol. 164, 2016, pages 770 - 779, XP029416808, DOI: doi:10.1016/j.cell.2016.01.011
SCHOENBORN, J. R. ET AL: "Feedback circuits monitor and adjust basal Lck-dependent events in T cell receptor signaling", SCIENCE SIGNALING, vol. 4, 2011, pages RA59
SEWELL, A. K.: "Why must T cells be cross-reactive?", NATURE REVIEWS, vol. 12, 2012, pages 669 - 677
TAN, Y. X. ET AL: "Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling", NATURE IMMUNOLOGY, vol. 15, 2014, pages 186 - 194
TORGERSEN, K. M. ET AL: "Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, 2001, pages 29313 - 29318
VANG, T. ET AL: "Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 34, 2004, pages 2191 - 2199
VANG, T. ET AL: "LYP inhibits T-cell activation when dissociated from CSK", NATURE CHEMICAL BIOLOGY, vol. 8, 2012, pages 437 - 446
WALCHLI, S. ET AL: "A practical approach to T-cell receptor cloning and expression.", PLOS ONE, vol. 6, 2011, pages E27930
WALSENG, E. ET AL: "Soluble T-cell receptors produced in human cells for targeted delivery.", PLOS ONE, vol. 10, 2015, pages E0119559
WEI, F. ET AL: "Strength of PD-1 signaling differentially affects T-cell effector functions", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 110, 2013, pages E2480 - E2489, XP055142199, DOI: doi:10.1073/pnas.1305394110
WEI, P. ET AL: "Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells", NATURE, vol. 488, 2012, pages 384 - 388
WEICHSEL, R. ET AL: "Profound inhibition of antigen-specific T-cell effector functions by dasatinib", CLINICAL CANCER RESEARCH : AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 14, 2008, pages 2484 - 2491, XP002568969, DOI: doi:10.1158/1078-0432.CCR-07-4393
WERLEN, G.; E. PALMER.: "The T-cell receptor signalosome: a dynamic structure with expanding complexity", CURRENT OPINION IN IMMUNOLOGY, vol. 14, 2002, pages 299 - 305, XP004350478, DOI: doi:10.1016/S0952-7915(02)00339-4
WOLCHINSKY, R. ET AL: "Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes", J IMMUNOL, vol. 192, 2014, pages 2109 - 2119
WU, C. Y. ET AL: "Synthetic biology approaches to engineer T cells", CURRENT OPINION IN IMMUNOLOGY, vol. 35, 2015, pages 123 - 130

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11421228B2 (en) 2018-03-15 2022-08-23 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11608500B2 (en) 2018-03-15 2023-03-21 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
WO2020043903A1 (fr) * 2018-08-31 2020-03-05 Invectys Méthode pour évaluer la fonctionnalité des car
CN112912727A (zh) * 2018-08-31 2021-06-04 因维克蒂斯股份有限公司 评估car功能性的方法

Similar Documents

Publication Publication Date Title
US12359001B2 (en) Method for producing polypeptide hetero-oligomer
KR102867311B1 (ko) 증강된 혈청 반감기를 위한 조작된 항체 fc 변이체
RU2758952C1 (ru) Способ получения полипептидного гетеромультимера
DK2552955T3 (en) Antibodies with modified affinity to FCRN that promote antigen clearance
US8569460B2 (en) Antibody constant domain regions and uses thereof
US20190358263A1 (en) Compositions and Methods for Cell Therapy
US20220380463A1 (en) Engineered ph-dependent anti-cd3 antibodies, and methods for their generation and use
KR20220035369A (ko) 고 친화도 항-cd3 항체, 및 이의 생성 및 사용 방법
WO2017191504A1 (fr) Validation de lymphocytes t thérapeutiques
CN119630793A (zh) 在观察到细胞死亡的组织中特异性地起作用的分子
KR20250023497A (ko) 조작된 fcriib 선택적 igg1 fc 변이체 및 이의 용도
US20250163166A1 (en) Antibody comprising igg fc region variant, and use thereof
RU2832079C2 (ru) Сконструированные ph-зависимые антитела к cd3, а также способы их получения и применения
WO2025203029A1 (fr) Dispositif de mise en prise et de rétention de cellules tri-spécifiques de tricer
AU2010229337B8 (en) Antibody constant domain regions and uses thereof
RU2021138150A (ru) Сконструированные ph-зависимые антитела к cd3, а также способы их получения и применения
RU2021138136A (ru) Высокоаффинные антитела к cd3 и способы их получения и применения
RU2024136954A (ru) Сконструированные ph-зависимые антитела к cd3, а также способы их получения и применения

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17727685

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17727685

Country of ref document: EP

Kind code of ref document: A1