WO2021225532A1 - Récepteur antigénique chimérique et cellules modifiées portant le récepteur - Google Patents

Récepteur antigénique chimérique et cellules modifiées portant le récepteur Download PDF

Info

Publication number
WO2021225532A1
WO2021225532A1 PCT/TH2021/000018 TH2021000018W WO2021225532A1 WO 2021225532 A1 WO2021225532 A1 WO 2021225532A1 TH 2021000018 W TH2021000018 W TH 2021000018W WO 2021225532 A1 WO2021225532 A1 WO 2021225532A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
domain
seq
car
cells
Prior art date
Application number
PCT/TH2021/000018
Other languages
English (en)
Inventor
Usanarat ANURATHAPAN
Suradej HONGENG
Somsak PRASONGTANAKIJ
Amornrat Tangprasittipap
Suparerk BORWORNPINYO
Original Assignee
Mahidol University
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
Priority claimed from TH2001002539A external-priority patent/TH2001002539A/th
Application filed by Mahidol University filed Critical Mahidol University
Priority to CN202180031586.6A priority Critical patent/CN115461361A/zh
Publication of WO2021225532A1 publication Critical patent/WO2021225532A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present disclosure relates to an engineered chimeric antigen receptor, which is capable of interacting with Cluster of Differentiation 19 molecule (CD 19) expressed on the surface of B- lymphocytes or cells of B-lymphocytes lineage. Further, the present disclosure also includes genetically engineered cells, particularly immunogenic T cells, being conferred with the ability to express and carry the chimeric antigen receptor.
  • CD 19 Cluster of Differentiation 19 molecule
  • B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common childhood cancer and it has an overall good prognosis, with up to 90% of the patients being alive at 5 years after diagnosis. 1 However, for those who relapse on therapy, or who suffer chemotherapy -refractory disease, the outlook is poor. Allogeneic hemopoietic stem cell transplantation (allo-HSCT) still offers a chance for cure, but unfortunately relapse is common after allo-HSCT for leukemia. Patients who relapse ALL after allo-HSCT are very difficult to manage, and in addition to chemotherapy donor lymphocyte infusions have been advocated as an effective adjunct by many investigators, although available data do not support their use in patients with overtly active disease.
  • CD 19 is a highly attractive target for immunotherapy as its expression is virtually universally present on the blasts in B-cell ALL, but it is otherwise restricted to normal cells of the B lineage.
  • ' Modification of the T-cell receptors for the CD 19 anti- gen subsequently led to the development of chimeric antigen receptor (CAR) T-cell preparations for treatment of refractory B-cell ALL and advanced B-cell lymphomas.
  • CAR chimeric antigen receptor
  • the basic structure of the CAR-modified T cell is composed of an extracellular antigen-binding domain derived from a monoclonal antibody, and the signaling domains from the T-cell receptor to ren- der a capability of targeting a specific surface antigen(s).
  • the second- and third-generation CARs incorporate one or two co-stimulatory molecules such as CD28, 4- 1BB, or OX40, 4 which promotes in vivo expansion. 5
  • co-stimulatory molecules such as CD28, 4- 1BB, or OX40, 4 which promotes in vivo expansion.
  • another element of CARs a spacer domain (CH2CH3), appears to play an important role in mediating antitumor activity.
  • CRS cytokine release syndrome
  • W02020037066A1 and W02020051493A1 teach about potential use of any hinge or spacer domain originated from CD28, CD16A, CD8a, or Immunoglobulin G (IgG) in assembling of a CAR capable of binding on specific antigen, but the suitable type of Immunoglobulin G (IgG) has never been identified in these disclosures.
  • the present disclosure aims to provide a CAR configured to bind onto specific antigen expressed on a surface of a given cell type or cell lineages in deriving a beneficial outcome, particularly a therapeutic outcome, for a subject.
  • Further object of the present disclosure is directed to a CAR particularly designed to recognize and bind onto CD 19 expressed on the surface of B -lymphocytes or cells of B lineage applicable for a treatment of B-cell ALL.
  • the disclosed CAR capitalizes on modified hinge or spacer domain for obtaining an improved killing efficiency towards B -lymphocytes or cells of B lineage to treat B-cell ALL or the like illnesses.
  • Still another object of the present disclosure is to derive a T cell capable of expressing and presenting the disclosed CAR that binding onto the compatible antigen available on a cell surface via the CAR leads to initiation of one or more preferred immunological reactions to eradicate cancerous cells relating to B-cell lymphomas.
  • More object of the present disclosure is to offer a viral vector incorporated with one or more polynucleotide sequences encoding for the peptides of the disclosed CAR.
  • the disclosed vector is fashioned to express the encoded the peptides which are further assembled into the disclosed CAR in a compatible transduced cell.
  • one of the embodiments of the present disclosure is a chimeric antigen receptor (CAR) comprising an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain.
  • CAR chimeric antigen receptor
  • variable heavy chain region connects to the intracellular domain through a spacer, which comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.
  • the spacer is Immunoglobulin (Ig) G2 -based.
  • variable light chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 2.
  • variable heavy chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 3.
  • the linker comprises an amino acid sequence as setting forth in SEQ ID No. 4.
  • the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5.
  • the transmembrane domain comprises an amino acid sequence as setting forth in SEQ ID No. 6.
  • the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from O ⁇ 3z- chain that the CD28 signaling domain and the cytoplasmic signaling domain may respectively comprises an amino acid sequence as setting forth in SEQ ID No. 7 and SEQ ID No. 8.
  • the disclosed CAR further comprises a leader sequence precedingly connected to the variable light chain region and the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9.
  • Another aspect of the present disclosure refers to a genetically engineered T-cell capable of expressing a chimeric antigen receptor that the chimeric antigen receptor comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain.
  • the variable heavy chain region of the chimeric antigen receptor in the engineered T cell connects to the intracellular domain through a spacer, which comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.
  • the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5.
  • the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from CD3 ⁇ chain.
  • the chimeric antigen receptor further comprises a leader sequence precedingly connected to the variable light chain region and the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9.
  • Another aspect of the present disclosure refers to a viral vector capable of promoting expression of a peptide encoding for chimeric antigen receptor in a transduced T cell.
  • the viral vector essentially comprises a polynucleotide sequence as setting forth in SEQ ID No. 10.
  • the viral vector is a lentiviral vector.
  • the encoded peptide comprises at least 90% similarity of an amino acid sequence setting forth in SEQ ID No. 11.
  • Fig. 1 is a schematic illustration of few embodiments of the CD 19 CAR with each of the constructs comprising a CD 19 scFV, a predetermined range of IgG2 spacer, a CD28 transmembrane domain, a CD28 intracellular domain, and CD3 ⁇ cytosolic domain;
  • Fig. 2 is a graph showing viable T-cell number after transduction with CAR having short spacer displayed the log phase after day 1 of transduction (similar to that of non-transduced cells) while CAR of full-spacer and intermediate-spacer entered log phase proliferation 2 days after transduction;
  • Fig. 3 is a graph showing percentage of transduction efficiency measured using QPCR with expression of the CAR CD28- CD3 ⁇ domain construct in genomic DNA being detected in CD 19 CAR T cells derived from different healthy donors using QPCR with specific primers targeting the CD28- CD3 ⁇ region, where * and # represent statistically significant differences at P ⁇ .05 when comparing with NT and the CAR full spacer construct respectively;
  • Fig. is an Agarose gel picture after QPCR with specific primers targeting CD28-CD3 ⁇ (the product size was 281 bp);
  • Fig. 5 are graphs illustrating antitumor activity of CAR T cells using a CFSE-7-AAD-based cytotoxicity assay for investigating effector to target cell ratios (E/T) in connection to different spacer length CD19 CAR T, where Raji, RS4, and Sup-B15 (CD19 expressed cells) were used as target cells, the Jurkat cell line and PHA blasts (CD 19 non-expressing cells) were used as negative controls, and non-transduced T cells (NT) were used as controls that the data is representative of three independent experiments performed with CAR T cells generated from five separate donors (*, #, and $ represent P ⁇ .05 compared with NT, Full, and Intermediate spacer length, respectively, whereas ** and *** strand for P ⁇ .01 and .001 when compared with NT);
  • Fig. 6 is a gel picture showing detected CD 19 CAR T cells in peripheral blood (PB) and bone marrow (BM) samples with BM being examined on day 14 of the CAR T-cell infusion (D denotes Day, + ve indicates transduced cells, and -ve represents non-transduced cells); and
  • Fig. 7 shows amino acid sequences of (a) SEQ ID No. 1, (b) SEQ ID No. 2, (c) SEQ ID No. 3, (d) SEQ ID No. 4, (e) SEQ ID No. 5, (f) SEQ ID No. 6, (g) SEQ ID No. 7, (h) SEQ ID No. 8, and (i) SEQ ID No. 9;
  • Fig. 8 shows (a) a polynucleotide sequence, denoted as SEQ ID No. 10, encoding for (b) one embodiment of a CAR, denoted as SEQ ID No. 11 , that the polynucleotide sequence can be incorporated into a viral vector.
  • the terms “approximately” or “about”, in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to oligonucleotides greater than 30 nucleotide residues in length.
  • chimeric antigen receptor or “CAR” used herein throughout the specification refers to a man-made construct or complex, assembled from one or more peptides, comprising multiple domains that at least two of domains forming the construct or complex are of different origins.
  • the disclosed CAR comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end- to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain.
  • an engineered T cell carrying the CD 19-specific CAR shall facilitate recognition of the cells of B-lymphocytes lineage by the engineered T cell and coupling thereof.
  • the coupling of the engineered T cell and cell of B- lymphocyte lineage may lead to activation of the T cell and release of corresponding immunoregulatory cytokines that finally results in killing or eradication of the recognized cells of B -lymphocytes lineage.
  • the disclosed CAR may include CAR of third or fourth generation
  • variable heavy chain region of the disclosed CAR connects to the intracellular domain through a spacer.
  • the spacer commonly serves to provide enough space between the antigen binding domain and the membrane plasma of the transduced cells such that the antigen binding domain can be fold intrinsically free from interruption from plasma membrane or any cellular components mounted on the membrane.
  • the length of the spacer may play a role in determining efficiency of the T-cell carrying the CAR for leukemia treatment
  • the inventors of the present disclosure discovered that the rightful length of the spacer in fact affect transduction efficacy, which can be another important factor to decide treatment efficiency of the engineered T cells.
  • the spacer of the disclosed CAR is of IgG2 origin or, more particularly, the spacer is human IgG2 origin.
  • the spacer can be a continuous segment or region derived from human IgG2, with or without further modifications, in some other embodiments of the disclosed CAR.
  • the spacer comprises a hinge region, a CH3 region, and a CH2 region interposing between the hinge region and the CH3 region.
  • the spacer comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.
  • the antigen binding domain is a single-chain variable fragment.
  • the antigen binding domain is essentially composed of a variable light chain region and a variable heavy chain region joined in tandem through a linker.
  • the linker employed in some of the embodiments can be flexible GS linkers essentially composed of glycine (Gly) and serine (Ser) residues. More preferably, a G4S or (Gly-Gly-Gly-Gly-Ser) n flexible linker which is resistant against cellular protease activities is employed to fuse the variable heavy chain region and variable light chain region together, where n can range from 1 to 10.
  • the linker comprises an amino acid sequence as setting forth in SEQ ID No. 4.
  • the employed linker connects the amino-terminal of the variable light chain region to the carboxy- terminal of the variable heavy chain region.
  • these domains are generally derived respectively from light and heavy chains of immunoglobulin of Mus musculus, with or without further modifications depending on the embodiments of the disclosed CAR.
  • variable light chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 2.
  • variable heavy chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 3.
  • extracellular domain spanning across the variable light chain region, the linker and the variable heavy chain region comprises an amino acid sequence as setting forth in SEQ ID No. 5.
  • the disclosed CAR may further comprise a leader sequence or leader peptide sequence that assist expression of the CAR on the engineered T cell membrane after secretion from Golgi complex.
  • the leader peptide sequence precedingly connects to the variable light chain region for guiding the secreted chimeric peptide towards the surface membrane for expression.
  • the leader sequence may comprise an amino acids sequence, but not limited to, as setting forth in SEQ ID No. 9.
  • the intracellular domain is preferably TCR zeta chain or structure configured to facilitate transfer of stimulating signal to activate T cells upon reacting of the antigen binding domain towards the CD 19 located on the cells of B -lymphocytes lineage.
  • the intracellular domain of the comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from human CD3 ⁇ -chain .
  • the CD28 signaling domain comprises an amino acid sequence as setting forth in SEQ ID No. 7 while the cytoplasmic signaling domain comprises an amino acid sequence as setting forth in SEQ ID No. 8.
  • the transmembrane domain comprises an amino acid sequence as setting forth in SEQ ID No. 6, which is preferably originated from human CD28 specifically found on surface of the T cells.
  • a genetically engineered T-cell capable of expressing a CAR configured to couple with CD 19 found on cellular surface of cells of B- lymphocyte lineage that binding of the CAR onto the CD 19 invokes a cascade of immunoregulatory reactions leading to killing or eradication of the bound cells of B -lymphocyte lineage. It is an effort of the inventors of the present disclosure to disclose an effective means for the treatment of B-cell-ALL via cell therapy through assembling a CD 19-specific chimeric antigen receptor for cell.
  • the disclosed engineered T cell possesses better transduction efficacy and killing efficiency over the recognized or identified cells of B-lymphocyte lineage in view of the application of a spacer incorporated with modified CH3 domain of IgG2.
  • the disclosed engineered T cell comprises a plurality of chimeric antigen receptor, which essentially comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain.
  • variable heavy chain region connects to the intracellular domain through a spacer comprising an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.
  • the CAR of the disclosed engineered T cell may include a segment of leader sequence precedingly connected to the variable light chain region.
  • the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9 such that the secreted peptide or polypeptide of the CAR can be effectively expressed on the surface of the engineered T cells.
  • the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5, which spanning across a variable light chain region, a linker and a variable heavy chain region respectively comprise amino acid sequence as setting forth in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
  • the intracellular domain of the CAR expressed in the disclosed engineered T-cell comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from CD3 ⁇ -chain.
  • the disclosed viral vector capable of promoting expression of a peptide, which corresponds to one or more embodiments of the disclosed CAR mentioned above.
  • the disclosed viral vector is encoding for a chimeric antigen receptor which can be expressed in a compatible transduced T cell under a predetermined culturing condition.
  • the disclosed viral vector comprises a polynucleotide sequence or a base sequence as setting forth in SEQ ID No. 10. It is possible to further modify the encoding sequence of SEQ ID No. 10 to derive a CAR with improved or altered properties for the treatment of B-cell ALL, the like illness and/or any complications associated thereto.
  • more embodiments of the disclosed viral vector comprise a polynucleotide sequence or a base sequence of at least 90% similarity as of setting forth in SEQ ID No. 10.
  • the disclosed viral vector can be employed for the creation of the CD 19-specific chimeric-antigen-receptor T-cells, which in turn being applicable as a treatment for B-cell leukemia patients through any established protocol for leukemia cell therapy.
  • the peptide being encoded by the disclosed vector may comprise at least 90% similarity of an amino acid sequence as setting forth in SEQ ID No. 11.
  • the disclosed viral vector is a lentiviral vector.
  • one aspect of the present disclosure is associated to a genetically engineered T-cell as disclosed or described above for use as a medicament in a treatment of B-cell ALL.
  • the two CAR constructs were kindly provided by the Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, and Houston Cincinnati Hospital, Houston, TX, USA. They were ligated to produce second-generation anti-CD19 scFv-CD28- CD3 ⁇ .
  • the intermediate and short constructs had CH2 and CH2CH3 deletion, respectively.
  • the three CAR constructs were subcloned into a lentiviral vector backbone plasmid (pSin-EF2-Puro; Addgene, Watertown, MA, USA).
  • HEK 293T cells (5 x 10 6 cells per 10-cm dish) were plated into poly-D lysine (Sigma-Aldrich, St. Louis, MO, USA)-coated plates prior to co-transfection of envelope (pMD2.G) and packaging (psPAX2) plasmids with pSIN-CD19 CAR vector.
  • pMD2.G poly-D lysine
  • psPAX2 packaging plasmids with pSIN-CD19 CAR vector.
  • HEK 293T cells were transfected using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). Supernatants were collected 48 and 72 h post-transfection and filtered through 0.45-mih polyethersulfone filter (Merck Millipore, Burlington, MA, USA) to remove cell debris.
  • Lenti viruses were concentrated using Lenti-X Concentrator (Takara, Shiga, Japan) according to the manufacturer’s instructions. Briefly, the supernatants were mixed with Lenti-X Concentrator at a ratio 1:3 (Lenti-X Concentrator: supernatant) and the mixtures were incubated for 30 min at 4°C. Thereafter, the mixtures were centrifuged at 1500 x g for 45 min at 4°C. The pellet was suspended in Dulbecco’s phosphate-buffered saline (DPBS) (GIBCO; Thermo Fisher Scientific, Inc, Waltham, MA, USA) and stored at -80°C until use.
  • DPBS Dulbecco’s phosphate-buffered saline
  • PBMCs Human peripheral blood mononuclear cells
  • Ficoll-Paque GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA. The experiment was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (pro- tocol ID 03-59-11).
  • the PBMCs were then cultured in TexMACS GMP medium (MACS; Miltenyi Biotec) with 5% heat-inactivated human AB serum (Sigma- Aldrich Pte Ltd) and recombinant human IL2200 U (GMP grade, CellGenix; CellGenix Inc).
  • the Raji cell line (Burkitt’s lymphoma) was kindly provided by Assistant Professor Dachrit Nilubol.
  • the Jurkat cell line (acute T- lymphoblastic leukemia) was kindly provided by Professor Kovit Pattanapanyasat.
  • RS4 and Sup-B15 cell lines (acute lymphoblastic leukemia) were purchased from American Type Culture Collection (Manassas, VA, USA).
  • the Raji, RS4, and Jurkat cell lines were cultured in RPMI- 1640 medium with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin/streptomycin (GIBCO), whereas Sup-B15 was cultured in Iscove’s modified Dulbecco’s medium (Hyclone, GE Healthcare Life Sciences) with 20% FBS, 1% penicillin/streptomycin, and 0.05 mM 2- mercaptoethanol (Sigma- Aldrich).
  • FBS fetal bovine serum
  • GEBCO penicillin/streptomycin
  • PHA blast cells For the generation of allogeneic phytohaemagglutinin-stimulated (PHA) blast cells, 5 x 10 5 PBMCs were stimulated with 5 ⁇ g/mL PHA (Invitrogen; Thermo Fisher Scientific, Inc) in RPMI- 1640 medium with 10% FBS and 1% penicillin/streptomycin. A total of 100 U/mL IL-2 (PeproTech, Rocky Hill, NJ, USA) was added on day 2. PHA blast cells were cultured for 7 days. The medium and IL-2 were replaced every 2- 3 days. All cell cultures were maintained at 37°C in a fully humidified atmosphere of 5% CO2 in air.
  • PHA phytohaemagglutinin-stimulated
  • T cells were stimulated, using antihuman CD3 (MACS; Miltenyi Biotec) and anti-CD28 (MACS; Miltenyi Biotec) monoclonal antibodies, and then cultured in TexMACS GMP medium with 5% heat-inactivated human AB serum and recombinant human IL2200 U (GMP grade). Transduction was carried out on day 3.
  • Activated T cells were transduced using RetroNectin (Takara, Shiga, Japan). RetroNectin was used at a concentration of 20 ⁇ g/mL in DPBS to coat the 24-well plate.
  • the plate was incubated for 2 h at room temperature and blocked with 2% heat-inactivated human AB serum (Sigma- Aldrich) in DPBS. The plate was then washed once with DPBS. Lentiviruses were added into 500 ⁇ L complete TexMACS GMP medium and added into the coated well. The coated plate containing viruses was centrifuged at 2000 x g at 32°C for 1.5 h. Thereafter, 105 activated T cells in 500 ⁇ L complete TexMACS GMP medium with IL-2 at a final concentration of 100 U/mL were added to each well. The complete medium and IL-2 were replaced every 2-3 days. Transduced T cells were cultured for 6 days for cytotoxicity assay.
  • DNA was extracted from transduced CD 19 CAR T cells and non-transduced T cells using GenUP gDNA Kit (Biotechrabbit, Hennigsdorf, Ger- many). Quantitative polymerase chain reaction (QPCR) was performed to determine the copy number of transgene, using QPCR Green Master Mix LRox (Biotechrabbit), and it was performed with the CLX96 Real-Time PCR Detection System (Bio-Rad, Singapore), according to the manufacturer’s instructions. The present disclosure used primers that were specific for the CD28CD3 ⁇ domain, such that the PCR would display the same product regardless if the DNA came from cells transduced with a full-, intermediate-, or short-length spacer domain.
  • QPCR Quantitative polymerase chain reaction
  • Each reaction contained 100 ng of genomic DNA, and each experiment was performed in triplicate.
  • the CAR copy number quantification was based on a comparison with a standard curve.
  • Genomic DNA of non-transduced T cells was spiked with plasmid containing transgene in a serial dilution to obtain a standard curve.
  • the CAR copy number of all constructs was calculated before co-culture with target cells. All constructs expressed different transduction efficiency with approximately 13%, 41%, or 21% for full, intermediate, or short spacer, respectively (Fig. 3).
  • the QPCR products of genomic DNA from CAR T cells and non-transduced T cells (NT) cells were subjected to agarose gel electrophoresis. The size of the respective CAR products was about 281 bp, whereas, as expected, the NT control cells did not express the CAR construct as shown in Fig. 4.
  • the cytotoxicity assay was performed using flow cytometry -based analysis. Effector cells (T cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen; Thermo Fisher Scien- tific, Inc) at a concentration of 1 mM. Effector cells were co-cultured with all target cells at ratios of 1:1, 10:1, and 20:1 (Effector: Target) overnight. Control samples, which only had target cells, were used to assess spontaneous cell death. On the following day, cells were collected and stained with 5 ⁇ L of 7-AAD (7-amino-actinomycin D) (eBioscience; Thermo Fisher Scientific, Inc).
  • 7-AAD 7-amino-actinomycin D
  • the present disclosure monitored CD 19 CAR T cells in peripheral blood and bone marrow specimens by RT-PCR as described in Section 2.5 by using the same primers specific for the CD28CD3 ⁇ domain.
  • the minimal residual disease (MRD) panel is a 14-color panel with 27 leukemia- associated markers including CD37, CD164, CD58, CD304, CD20, CD81, CD200, CD24, CD10, CD38, CD44, CD79b, CD49f, CD10, CD123, CD73, CD13, CD33, CD15, CD86, CD66c, NG2(7.1), CD72, CD19, CD10, CD34, and CD45.
  • immature B cells are gated by using CD 19, CD79a, CD 10, CD34, CD45, FSC, and SSC.
  • An immunophenotypic pattern of gated immature B cells is compared to the template of hematogones.
  • the residual mature B cells are used as an internal control.
  • MRD is considered as a positive result when there are two or more aberrancies compared with the normal template and MRD pattern at diagnosis.
  • the lower limit of detection is 0.01% of all nucleated cells.
  • CD 19 CAR T cells were cytotoxicity experiments performed with three B cell AFF cell lines: Raji, RS4, and Sup-B15. The specific cytotoxicity of unsorted CD 19 CAR T cells against lymphoma cells was evaluated with the flow cytometry- based assay.
  • the effector cells were CD 19 CAR T cells of three different constructs — full, intermediate, and short length, as shown in Figure 1.
  • NT served as negative control T cells. All three constructs of CAR T cells exhibited significantly increased cytotoxicity against all B-cell lines when compared with NT (35-50% vs ⁇ 10%). Different target cells showed different response to CD 19 CAR T cells.
  • CAR design is an ongoing process in order to optimize clinical efficacy and minimize clinical toxicity of such treatment.
  • Different costimulatory molecules such as CD28, 4- IBB, and 0X40 have been utilized to test CAR T cell efficacy against tumor cells.
  • Other structural units, such as the CH2CH3 spacer domain, have been reported to play a role in persistence and survival of CAR T cells in vivo.
  • the present disclosure investigated a second-generation anti-CD 19 CAR T cells containing three different lengths of the spacer region; full length (CH2CH3 spacer), “intermediate” (-/CH3 spacer), and “short” (-/- spacer).
  • the present disclosure expressed all three different lengths of the spacer region CAR constructs on the T cells, with intermediate length showing the highest transduction efficiency.
  • CD 19 CAR T cells with full, intermediate, or short spacers did not have a higher percentage of specific Jurkat cell lysis when compared with that of NT, whereas all three types of CD 19 CAR T cells had higher percentages of specific lysis of Raji, RS4, and Sup-B 15 cells at an E:T ratio of 10:1 as compared with the killing effect of NT.
  • the short CD 19 CAR T cells displayed the highest killing efficiency against all CD 19-expressing target cells despite its transduction efficiency being less than that of the intermediate CD 19 CAR T cells.
  • the full and intermediate CD 19 CAR T cells each displayed a fraction of specific lysis that was similar to that of the short CD 19 CAR T cells.
  • their killing efficiencies against the RS4 and Sup-B 15 cells, which express relatively less CD 19 on their cell surfaces were lower than their killing efficiencies against the Raji cells.
  • the difference in CAR T-cell killing potency could be an effect of the presence of target antigens and the intensity of antigen expression.
  • the modified T cells expressing any of the three constructs had higher efficiencies for eradicating CD 19-expressing cells compared with NTs.
  • the modified CAR T cells expressing any of the three constructs did not kill allogeneic PHA blasts.
  • the disclosed CAR-expressing T cells appear to be specific against CD 19-expressing cells and have minimal killing effects on CD 19-negative allogeneic cells.
  • the present disclosure planned to investigate allogeneic CD 19 CAR T cells in relapsed/refractory B- cell ALL patients in a phase I-II setting as bridging therapy to allo-HSCT, followed by a posttransplant CAR T-cell boost as consolidation in these high-risk patients, because the present disclosure was concerned about the possible deleterious effect of the pretransplant conditioning chemotherapy on the CAR T population that was administered as salvage therapy prior to HSCT.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un récepteur antigénique chimérique (CAR) comprenant un domaine extracellulaire ayant un domaine de liaison à l'antigène spécifique à CD19 devant être disposé sur une surface d'une membrane de plasma d'un lymphocyte T modifié, le domaine de liaison à l'antigène comprenant une région variable de chaîne légère et une région variable de chaîne lourde se liant d'une manière de bout en bout par l'intermédiaire d'un lieur, un domaine intracellulaire pour réguler l'activation du lymphocyte T modifié; et un domaine transmembranaire d'ancrage sur la membrane plasmique du lymphocyte T modifié et l'interposition entre le domaine extracellulaire et le domaine intracellulaire. De préférence, le domaine extracellulaire se lie au domaine intracellulaire par l'intermédiaire d'un espaceur lgG2.
PCT/TH2021/000018 2020-05-08 2021-05-05 Récepteur antigénique chimérique et cellules modifiées portant le récepteur WO2021225532A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180031586.6A CN115461361A (zh) 2020-05-08 2021-05-05 嵌合抗原受体和携带该受体的经修饰的细胞

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TH2001002539A TH2001002539A (th) 2020-05-08 ตัวรับแอนติเจนแบบไคเมอริกของทีเซลล์ที่ใช้จับจำเพาะกับแอนติเจนชนิดcd19สำหรับใช้ปฏิบัติต่อผู้ป่วยโรคมะเร็งเม็ดเลือดขาวชนิดบีเซลล์โดยการรักษาด้วยเซลล์บำบัด
TH2001002539 2020-05-08

Publications (1)

Publication Number Publication Date
WO2021225532A1 true WO2021225532A1 (fr) 2021-11-11

Family

ID=78468207

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/TH2021/000018 WO2021225532A1 (fr) 2020-05-08 2021-05-05 Récepteur antigénique chimérique et cellules modifiées portant le récepteur

Country Status (2)

Country Link
CN (1) CN115461361A (fr)
WO (1) WO2021225532A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015105522A1 (fr) * 2014-01-13 2015-07-16 Forman Stephen J Récepteurs d'antigènes chimères (car) comprenant des mutations dans la région de l'espaceur fc et procédés pour leur utilisation
WO2018064626A1 (fr) * 2016-09-30 2018-04-05 Baylor College Of Medicine Conception de lymphocytes t du récepteur d'antigène chimerique adaptatif
WO2019023396A1 (fr) * 2017-07-25 2019-01-31 Board Of Regents, The University Of Texas System Récepteurs antigéniques chimériques améliorés et leurs utilisations
WO2019209991A1 (fr) * 2018-04-26 2019-10-31 Baylor College Of Medicine Cellules effectrices immunitaires et adaptateurs moléculaires comprenant un complexe antigène-cytokine pour une immunothérapie efficace
WO2020020841A1 (fr) * 2018-07-23 2020-01-30 Ospedale San Raffaele S.R.L. Combinaison d'un inhibiteur de glycosylation avec une thérapie cellulaire à base de car pour le traitement du cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015105522A1 (fr) * 2014-01-13 2015-07-16 Forman Stephen J Récepteurs d'antigènes chimères (car) comprenant des mutations dans la région de l'espaceur fc et procédés pour leur utilisation
WO2018064626A1 (fr) * 2016-09-30 2018-04-05 Baylor College Of Medicine Conception de lymphocytes t du récepteur d'antigène chimerique adaptatif
WO2019023396A1 (fr) * 2017-07-25 2019-01-31 Board Of Regents, The University Of Texas System Récepteurs antigéniques chimériques améliorés et leurs utilisations
WO2019209991A1 (fr) * 2018-04-26 2019-10-31 Baylor College Of Medicine Cellules effectrices immunitaires et adaptateurs moléculaires comprenant un complexe antigène-cytokine pour une immunothérapie efficace
WO2020020841A1 (fr) * 2018-07-23 2020-01-30 Ospedale San Raffaele S.R.L. Combinaison d'un inhibiteur de glycosylation avec une thérapie cellulaire à base de car pour le traitement du cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUEDAN, S. ET AL.: "Engineering and Design of Chimeric Antigen Receptors", MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT, vol. 12, 15 March 2019 (2019-03-15), pages 145 - 156, XP055605656, [retrieved on 20210806], DOI: 10.1016/J.OMTM. 2018.12.00 9 *
WATANABE, N. ET AL.: "Fine-tuning the CAR spacer improves T- cell potency", ONCOIMMUNOLOGY, vol. 5, no. 12, 8 November 2016 (2016-11-08), pages 1 - 14, XP055402999, [retrieved on 20210806], DOI: 0.1080/2162402X.2016.1253656 *

Also Published As

Publication number Publication date
CN115461361A (zh) 2022-12-09

Similar Documents

Publication Publication Date Title
AU2018204208B2 (en) Method and compositions for cellular immunotherapy
US20210220404A1 (en) Chimeric antigen receptors and uses thereof
CN110248669B (zh) 工程化天然杀伤细胞及其用途
US11400115B2 (en) Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy
CN111448215B (zh) 抗-hla-a2抗体及其使用方法
WO2018132695A1 (fr) Récepteurs antigéniques chimériques ciblant tim-1
WO2020068702A1 (fr) Protéines de récepteur chimérique et leurs utilisations
CN115151635A (zh) 用编码新的嵌合抗原受体的基因转导的基因修饰nk细胞系及其用途
WO2018111340A1 (fr) Procédés de détermination de la puissance et de la fonction proliférative de lymphocytes t à récepteur antigénique chimérique (car)
JP2024517863A (ja) 細胞を刺激し、形質導入する方法
US20210060070A1 (en) Adoptive cell therapy and methods of dosing thereof
WO2021225532A1 (fr) Récepteur antigénique chimérique et cellules modifiées portant le récepteur
US20230019849A1 (en) Method for Preparing CD7-Negative, CD3-Positive T Cells
NZ714574B2 (en) Method and compositions for cellular immunotherapy
NZ726162B2 (en) Method and compositions for cellular immunotherapy
NZ714574A (en) Method and compositions for cellular immunotherapy

Legal Events

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

Ref document number: 21800888

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21800888

Country of ref document: EP

Kind code of ref document: A1