[go: up one dir, main page]

WO2024052318A1 - Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives - Google Patents

Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives Download PDF

Info

Publication number
WO2024052318A1
WO2024052318A1 PCT/EP2023/074271 EP2023074271W WO2024052318A1 WO 2024052318 A1 WO2024052318 A1 WO 2024052318A1 EP 2023074271 W EP2023074271 W EP 2023074271W WO 2024052318 A1 WO2024052318 A1 WO 2024052318A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
host immune
immune cell
car
domain
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/EP2023/074271
Other languages
English (en)
Inventor
Jean-Christophe BORIES
Bertrand ARNULF
Jean-Paul Fermand
Nathalie RODERS
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.)
Ifm Sas
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
Original Assignee
Ifm Sas
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
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 Ifm Sas, Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Cite filed Critical Ifm Sas
Priority to EP23765243.3A priority Critical patent/EP4583905A1/fr
Publication of WO2024052318A1 publication Critical patent/WO2024052318A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4222—CD38 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • PI proteasome inhibitors
  • IMIDs immunomodulators
  • R/R refractory/resistant diseases
  • New strategies based on the combination of PI, IMIDs and monoclonal antibodies (mAb) targeting the CD38 antigen, have significantly improve the prognosis (Dimopoulos et al., 2016). Nevertheless, most patients still relapse and MM remains an incurable disease.
  • Chimeric Antigen Receptors are hybrid molecules associating an extracellular portion, involved in antigen-recognition, with a transmembrane region fused with signalling domains including (in most cases) the CD3z and CD28 and/or the CD137 receptor, also referred to as 4- 1BB (Sadelain et al., 2013).
  • the CD3z provides an activation signal and the 4- 1BB region provides a co-stimulation signal, both of which are mandatory to mimic the physiological T cell mechanisms required for cytotoxicity, differentiation and persistence of T cells in vivo.
  • CAR T cells targeting the B cell maturation antigen (BCMA) have brought up to 80% response rates in MM patients (depending on studies), however, the median overall survival remains below 25 months (Munshi et al., 2021).
  • BCMA is detected on most post germinal centre B lineage cells, expression on plasma cells can vary through several mechanisms, including antigen loss or expression shedding, potentially leading to variable responses (Da Via et al., 2021; Laurent et al., 2015).
  • CD38 is a glycoprotein with cyclic ADP ribose hydrolase activities, which is expressed on tumor plasma cells (and normal plasma cells) as well as on other lymphoid and myeloid cell populations. It was originally identified as a T and B lymphocyte activation marker and was later shown to be expressed on multiple haematopoietic cells (HSC), including subsets of haematopoietic stem cells, NK cells and monocytes.
  • HSC haematopoietic cells
  • anti-CD38 CAR T cells have shown good anti-MM activities in pre-clinical models (Drent et al. Mol Ther.2017).
  • anti-CD38 CAR-T cell therapy might trigger side effects such as fratricide killing of activated CAR-T cells, as well as toxicity against myeloid cells, HSC or non-hematopoietic CD38 expressing cells (ie, endothelial cells).
  • CD38 represents a validated target for immune therapy in MM
  • improvements are needed to deliver safer and more efficient anti-CD38 CAR-T.
  • CAR-T cells targeting CD38 represent a potential alternative, but expression of CD38 on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such therapy.
  • the inventors developed DCAR, a double CAR system targeting CD38 and SLAMF7 (CS1) through split activation and co-stimulation receptors, respectively.
  • the inventors show that CRISPR/Cas9 inactivation of the CD38 gene enhances the anti-MM activity of DCAR-T in vitro. Edited DCAR-T developed strong responses specifically against MM cells expressing both the CD38 and the CS1 antigens in vitro and in vivo.
  • DCAR-T provides a safe and efficient alternative to treat MM patients.
  • the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.).
  • the percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%.
  • % identity is typically determined over the whole length of the query sequence on which the analysis is performed.
  • Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • engineered refers to an aspect of having been manipulated and altered by the hand of man.
  • engineered cell refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • the manipulation is or comprises a genetic manipulation.
  • a genetic manipulation is or comprises one or more of (i) introduction of a nucleic acid not present in the cell prior to the manipulation (i.e., of a heterologous nucleic acid); (ii) removal of a nucleic acid, or portion thereof, present in the cell prior to the manipulation; and/or (iii) alteration (e.g., by sequence substitution) of a nucleic acid, or portion thereof, present in the cell prior to the manipulation.
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a nucleic acid, and/or a particular form thereof
  • reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell.
  • the term “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or a mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a "polynucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • CD38 has its general meaning in the art and refers to the ADP- ribosyl cyclase/cyclic ADP-ribose hydrolase 1.
  • An exemplary amino acid sequence for CD38 is represented by SEQ ID NO:1.
  • the extracellular domain of CD38 ranges from the amino acid residue at position 43 to the amino acid residue at position 300 in SEQ ID NO:1.
  • SEQ ID NO:1 >sp
  • OS Homo sapiens
  • GN CD38
  • SLAMF7 is also known as CD2 subset 1, CD2-like receptor-activating cytotoxic cells (CRACC), Membrane protein FOAP-12, Novel Ly9, Protein 19A, and CD319.
  • An exemplary amino acid sequence for SLAMF7 is represented by SEQ ID NO:2.
  • the extracellular domain of SLAMF7 ranges from the amino acid residue at position 23 to the amino acid residue at position 226 in SEQ ID NO:2.
  • the term "cell” refers to any eukaryotic cell.
  • the cells are selected from the group consisting of multipotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (i.e. embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) or multipotent stem cell-derived differentiated cells of different cell lineages.
  • pluripotent i.e. embryonic stem cells (ES) or induced pluripotent stem cells (iPS)
  • ES embryonic stem cells
  • iPS induced pluripotent stem cells
  • the term “immune cell” refers to a cell that functions in an immune response or a progenitor, or progeny thereof.
  • the term “population” refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of human CAR-host immune cells comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the highly suppressive functions and which express the particular markers of interest).
  • T cell has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity.
  • T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility.
  • TCR T cell receptor for the antigen
  • There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells.
  • Cytotoxic T cells are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • chimeric antigen receptor has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signalling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • TCR T cell receptor
  • the chimeric antigen receptor of the present invention typically comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signalling domain.
  • the term “chimeric co-stimulatory receptor” or “CCR” refers to a specific type of chimeric antigen receptor (CAR) that mediates costimulation independently of activation. When expressed on host immune cells in combination with a CAR, the CCR is targeted to a second antigen.
  • CAR-T cell refers to a T lymphocyte that has been genetically engineered to express a CAR.
  • the T lymphocytes that are genetically modified may be "derived” or “obtained” from the patient who will receive the treatment using the genetically modified T cells or they may be “derived” or “obtained” from a different patient.
  • DCAR-T cell refers to a T lymphocyte that has been genetically engineered to express both a CAR and a CCR.
  • cytotoxicity refers to ability of the host immune cell of the present invention to lyse target cells. Such cytotoxicity can be measured using standard techniques, e.g., by radioactively labeling the target cells. Typically said activity is measured according to the methods described in the EXAMPLE.
  • nucleic cytotoxicity means that the cytotoxicity is reduced by 50, 60, 70, 80, 85, 90, 95, 99 or 100%.
  • antigen has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in induction of an immune response.
  • Antigens according to the invention are typically, although not exclusively, peptides and proteins. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.
  • antibody and “immunoglobulin” have the same meaning, and will be used equally in the present invention.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
  • two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes three ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) to five ( ⁇ ⁇ ⁇ ⁇ ) domains, a variable domain (VH) and three to four constant domains (CH1, CH2, CH3 and CH4 collectively referred to as CH).
  • the variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site.
  • CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • Framework Regions refer to amino acid sequences interposed between CDRs. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al.
  • the correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system.
  • the CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.
  • the terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences.
  • human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • chimeric antibody refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.
  • a “chimeric antibody” is an antibody molecule in which (a) the constant region (i.e., the heavy and/or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Chimeric antibodies also include primatized and in particular humanized antibodies.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). (see U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • humanized antibody refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.
  • a humanized antibody contains minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the term "antibody fragment” refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region.
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single - chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • the term “specificity” refers to the ability of an antibody to detectably bind target molecule (e.g. an epitope presented on an antigen) while having relatively little detectable reactivity with other target molecules. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein.
  • affinity means the strength of the binding of an antibody to a target molecule (e.g. an epitope).
  • the affinity of a binding protein is given by the dissociation constant Kd.
  • Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • the affinity constant Ka is defined by 1/Kd.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • binding'' in the context of the binding of an antibody to a predetermined target molecule (e.g.
  • an antigen or epitope typically is a binding with an affinity corresponding to a K D of about 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10- 10 M or less, or about 10-11 M or even less.
  • the term “cancer” has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors.
  • the term “CD38-positive hematological malignancy” refers to a hematological malignancy characterized by the presence of tumor cells expressing CD38 including leukemias, lymphomas and myeloma.
  • CD38-positive hematological malignancies include precursor B-cell lymphoblastic leukemia/lymphoma and B-cell non-Hodgkin's lymphoma; acute promyelocytic leukemia, acute lymphoblastic leukemia and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), plasma
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the term "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the active agent depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a patient.
  • An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled inhibitor of the present invention, fragment or mini-antibody derived from the inhibitor of the present invention.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the human monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of a inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2
  • the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients.
  • the pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W.
  • the first object of the present invention relates to a host immune cell engineering to express a) a chimeric antigen receptor (CAR) that binds to CD38, wherein binding of the CAR to CD38 is capable of delivering an activation signal to the host immune cell, and b) a chimeric co- stimulating receptor (CCR) that binds to a second antigen, wherein binding of the CCR to the second antigen is capable of delivering a costimulatory signal to the host immune cell but does not alone deliver an activation signal to the host immune cell, wherein the host immune cell is capable of (i) exhibiting negligible cytotoxicity against cells that are single positive for CD38, and (ii) inducing cytotoxicity against cells that are positive for both CD38 and the second antigen.
  • CAR chimeric antigen receptor
  • CD38 chimeric co- stimulating receptor
  • the host immune cell is a hematopoietic cell from the lymphoid lineage that comprises B, T and natural killer (NK) cells.
  • the host immune cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells may be differentiated.
  • the host cell is a pluripotent stem cell (PSC). PSCs can be indeed be modified by a CAR and then can be used for deriving T cells (e.g. WO 2017100403).
  • PSCs include embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs).
  • iPSCs can be generated directly from adult cells (e.g., somatic cells).
  • iPSCs can be typically derived or generated by introducing a specific set of pluripotency-associated genes, or "reprogramming factors", into a given cell type.
  • Reprogramming factors include, but are not limited to, OCT4 (also known as "POU5FL”), SOX2, cMYC, and KLF4, which are also known as Yamanaka factors. See Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126 (4): 663-76.
  • the host cell is a hematopoietic stem cell.
  • HSC hematopoietic stem cell
  • the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity to self-renew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells.
  • Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin-CD34+CD38 ⁇ CD90+CD45RA ⁇ , Lin-CD34+CD38 ⁇ CD90 ⁇ CD45RA ⁇ , Lin-CD34+CD38+IL-3aloCD45RA ⁇ , and Lin- CD34+CD38+CD10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994).
  • the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life.
  • the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.
  • Chimeric antigen receptor (CAR) The chimeric antigen receptor (CAR) typically comprises an extracellular domain and an intracellular domain joined by a transmembrane domain.
  • the extracellular domain, expressed on the surface of the host immune cell comprises an antigen binding domain having binding affinity for CD38.
  • such antigen binding domain is an antibody, preferably a single chain antibody.
  • the antibody is a humanized antibody.
  • such antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFv.
  • such antigen binding domain is selected from a Fab and a scFv.
  • the antigen targeting domain when the antigen targeting domain is a scFv, the scFv can be derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mAb linked by a flexible linker.
  • the scFv retains the same specificity and a similar affinity as the full antibody from which it is derived.
  • the peptide linker connecting scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH–VL paring and antigen- binding sites.
  • Peptide linkers can vary from 10 to 30 amino acids in length.
  • the scFv peptide linker is a Gly/Ser linker. In some embodiments, the scFv is specific for an epitope located in the extracellular domain of CD38. In some embodiments, the scFv comprises a VH domain comprising i) the H-CDR1 as set forth in SEQ ID NO:3, ii) the H-CDR2 as set forth in SEQ ID NO:4 and iii) the H-CDR3 as set forth in SEQ ID NO:5, and, a VL domain comprising i) the L-CDR1 as set forth in SEQ ID NO:6, ii) the L-CDR2 as set forth in SEQ ID NO:7 and iii) the L-CDR3 as set forth in SEQ ID NO:8.
  • the scFv comprises a VH domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:9. In some embodiments, the scFv comprises a VL domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:10.
  • the scFv comprises an amino acid sequence having 90% of identity with the amino acid sequence as set forth in SEQ NO:11.
  • transmembrane domain is typically a hydrophobic alpha helix that spans across the lipid bilayer of the cell membrane.
  • the transmembrane domain of the CAR thus functions to anchor the extracellular domain on the cell surface.
  • the transmembrane domain is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some embodiments is derived from any membrane -bound or transmembrane protein.
  • Transmembrane regions include those derived from (i.e.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a transmembrane domain is thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signalling domain(s) of the CAR.
  • a glycine-serine doublet may provide a suitable linker.
  • the transmembrane domain comprises the amino acid sequence having at 90% of identity with the amino acid sequence as set forth in SEQ ID NO:12.
  • the intracellular domain of the CAR is composed of an intracellular signalling domain.
  • the role of the intracellular signalling domain of the CAR is to produce an activation signal to the host immune cell as soon as the extracellular domain has recognized the antigen (i.e. CD38).
  • the intracellular signalling domain of the CAR triggers or elicits activation of at least one of the normal effector functions of the host immune cell.
  • intracellular signalling domain sequences that are of particular use in the invention include those derived from an intracellular signalling domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRI, DAP10, and DAP12.
  • the intracellular signalling domain in the CAR comprises a cytoplasmic signalling sequence derived from CD3 ⁇ .
  • the intracellular domain in the CAR comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:13.
  • the CAR of the present invention comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:14.
  • the extracellular domain expressed on the surface of the host immune cell, comprises an antigen binding domain having a binding affinity for second antigen.
  • the antigen binding domain has a binding affinity for a second antigen selected from the group consisting of G-Protein Coupled Receptor 5D (GPRC5D), CD138, NY- ESO1, CD19 and SLAMF7/CS1. More particularly, the antigen binding domain has a binding affinity for SLAMF7/CS1.
  • such antigen binding domain is an antibody, preferably a single chain antibody.
  • the antibody is a humanized antibody.
  • such antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFv.
  • such antigen binding domain is selected from a Fab and a scFv.
  • the antigen targeting domain is a scFv
  • the scFv is specific for an epitope located in the extracellular domain of SLAMF7.
  • the scFv comprises a VH domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:15.
  • the scFv comprises a VL domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:16.
  • the scFv comprises an amino acid sequence having 90% of identity with the amino acid sequence as set forth in SEQ ID NO:17.
  • transmembrane domain of the CCR is the same nature as for the CAR and is typically a hydrophobic alpha helix that spans across the lipid bilayer of the cell membrane.
  • the transmembrane domain is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some embodiments is derived from any membrane -bound or transmembrane protein. Transmembrane regions include those derived from (i.e.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a transmembrane domain is thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signalling domain(s) of the CCR.
  • a glycine-serine doublet may provide a suitable linker.
  • the transmembrane domain comprises the amino acid sequence having at 90% of identity with the amino acid sequence as set forth in SEQ ID NO:12.
  • the intracellular domain of the CCR is composed of an intracellular signalling domain. Contrary to the CAR, the role of the intracellular signalling domain of the CCR is not able to produce an activation signal per se to the host immune cell as soon as the extracellular domain has recognized the antigen (e.g. SLAMF7). The role of the intracellular signalling domain of the CCR is thus to deliver a costimulatory signal to the host immune cell but does not alone deliver an activation signal to the host immune cell. Accordingly, the intracellular signalling domain of the CCR comprises one or more intracellular domain(s) of a costimulatory molecule.
  • a costimulatory molecule can be defined as a cell surface molecule that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • the intracellular signalling portion of the above recited co-stimulatory domains can be used alone or in combination with other co-stimulatory domains.
  • the CCR can comprise one or more co-stimulatory domains from the group consisting of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • the intracellular domain of the CCR comprises the co-stimulatory domain of 4-1BB (CD137). In some embodiments, the intracellular domain of the CCR comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:18. SEQ ID NO:18> CD137 costimulating domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL In some embodiments, the CCR of the present invention comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:19.
  • a further object of the present invention relates to a method of preparing a host immune cell of the present invention, comprising the steps consisting of introducing into a host immune cell i) a polynucleotide that encodes for the CAR and ii) a polynucleotide that encodes for the CCR.
  • Use of bicistronic polynucleotides or vectors encoding for both the CAR and the CCR are particularly suitable for preparing the host immune cells of the present invention.
  • bicistronic refers to a polynucleotide or vector that comprises two cistrons, i.e. comprising two genes.
  • bicistronic or multicistronic vectors including, but not limited to, (1) multiple promoters fused to the open reading frames for the CAR and CCR; (2) insertion of splicing signals between units of CAR and CCR; fusion of the CAR and CCR whose expressions are driven by a single promoter; (3) insertion of proteolytic cleavage sites between the CAR and CCR (self-cleavage peptide); and (iv) insertion of internal ribosomal entry sites (IRESs).
  • IVSs internal ribosomal entry sites
  • the CAR and CCR are expressed in a single open reading frame (ORF), thereby creating a single polypeptide.
  • ORF open reading frame
  • an amino acid sequence or linker containing a high efficiency cleavage site is disposed between the CAR unit and the CCR unit.
  • “high cleavage efficiency” is defined as more than 50%, more than 70%, more than 80%, or more than 90% of the translated protein is cleaved. Cleavage efficiency may be measured by Western Blot analysis.
  • high efficiency cleavage sites examples include porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A), or a combination thereof.
  • the high efficiency cleavage site is P2A.
  • High efficiency cleavage sites are described in Kim J H, Lee S-R, Li L-H, Park H-J, Park J-H, Lee K Y, et al.
  • the host immune cell is transduced with a polynucleotide that encodes for the amino acid sequence as set forth in SEQ ID NO:20.
  • Physical methods for introducing a polynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the polynucleotide construct is introduced into the host immune cell by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus.
  • the vector is a retroviral. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines.
  • a retroviral vector In order to construct a retroviral vector, the polynucleotide is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components.
  • the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
  • Retroviral vectors are able to infect a broad variety of cell types.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
  • Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference.
  • the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell.
  • the gag, pol and env genes of the vectors of interest also are known in the art.
  • the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No.5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell.
  • control sequences' refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • nucleic acid sequence is a "promoter” sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • the polynucleotide is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell.
  • Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • a variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.
  • a variety of assays may be performed.
  • the host immune cell of the present invention is engineered such that it does not express CD38.
  • the method includes introducing into the host immune cell a genome- editing nuclease designed to edit the CD38 coding region, and culturing the host immune cell under conditions for the genome-editing nuclease to modify the CD38 coding region to inhibit the expression of CD38.
  • introducing the genome-editing nuclease into the host immune cell includes introducing into the host immune cell a polynucleotide that encodes the genome-editing nuclease.
  • the genome-editing nuclease includes a TALEN nuclease, a CRISPR-associated endonuclease, or a megaTAL nuclease.
  • the genome-editing nuclease is a CRISPR-associated endonuclease.
  • CRISPR/Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA molecule (gRNA) comprising a targeting sequence (which is capable of hybridizing to the genomic DNA target sequence), and sequence which is capable of encoding for the CRISPR- associated endonuclease.
  • gRNA guide RNA molecule
  • the CRISPR-associated endonuclease is a Cas9 nuclease.
  • the Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence.
  • the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms.
  • Streptococcus species such as thermophilus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms.
  • the wild type Streptococcus pyogenes Cas9 sequence can be modified.
  • the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized.”
  • a humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765.
  • the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as pX330, pX260 or pMJ920 from Addgene (Cambridge, MA).
  • the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of pX330, pX260 or pMJ920 (Addgene, Cambridge, MA).
  • Artificial CRISPR/Cas systems can be generated, using technology known in the art, e.g., that are described in U.S. Publication No. 20140068797, WO2015/048577, and Cong (2013) Science 339: 819-823.
  • CRISPR/Cas systems that are known in the art may also be generated, e.g., that described in Tsai (2014) Nature Biotechnol., 32:6569-576, U.S. Pat. Nos.8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359, the contents of which are hereby incorporated by reference in their entirety.
  • Such systems can be generated by, for example, engineering a CRISPR/Cas system to include a gRNA molecule comprising a targeting sequence that hybridizes to a sequence of the CD38 gene.
  • the gRNA comprises a targeting sequence which is fully complementarity to 15-25 nucleotides, e.g., 20 nucleotides, of the CD38 gene.
  • the 15-25 nucleotides, e.g., 20 nucleotides, of the CD38 gene are disposed immediately 5′ to a protospacer adjacent motif (PAM) sequence recognized by the Cas protein of the CRISPR/Cas system (e.g., where the system comprises a S. pyogenes Cas9 protein, the PAM sequence comprises NGG, where N can be any of A, T, G or C).
  • PAM protospacer adjacent motif
  • foreign DNA e.g., DNA encoding the CAR and CCR
  • the contacting of the cells with the endonuclease system is done ex vivo. In some embodiments, the contacting is done prior to, simultaneously with, or after said cells are modified to express both CAR and CCR.
  • Methods of therapy A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the host immune cells of the present invention.
  • the host immune cells of the present invention are particularly suitable for the treatment of cancer and more particularly for the treatment of CD38-positive hematological malignancies.
  • the host immune cells of the present invention are particularly suitable for the treatment of multiple myeloma and even more particularly for the treatment of multiple myeloma wherein the expression of B-cell maturation antigen (BCMA) is low.
  • BCMA B-cell maturation antigen
  • the population of host immune cells prepared as described above can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No.2003/0170238 to Gruenberg et al; see also US Patent No. 4,690,915 to Rosenberg.
  • most adoptive immunotherapies are autolymphocyte therapies (ALT) directed to treatments using the patient's own immune cells.
  • these therapies involve processing the patient's own lymphocytes.
  • the treatments are accomplished by removing the patient's lymphocytes and transforming said cells in the population of DCAR-T cells as above described.
  • DCAR- T cells are prepared with the DCAR of the present invention, these ex vivo cells are reinfused into the patient to enhance the immune system to kill tumor calls.
  • the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount.
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the host immune cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5x103/kg; and as high as 107/kg, preferably 108/kg.
  • the number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • the host immune cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a pharmaceutically acceptable carrier) in a treatment- effective amount.
  • a further object of the present invention relates to a pharmaceutical composition comprising a population of host immune cells of the present invention and a pharmaceutically acceptable carrier.
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the T cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5x103/kg; and as high as 107/kg, preferably 108/kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells.
  • the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • FIGURES Figure 1: CAR T constructs and expression. (a) Schematic diagram of the CAR construct.
  • First generation anti-CD38 CAR (1G) is comprised of a scFv specific for CD38(Fayon et al., 2021), linked to the human CD8 hinge and transmembrane regions, followed by the human CD3 zeta intracellular signaling domain.
  • the second generation anti-CD38 CAR (2G) is comprised of the anti-CD38 scFv linked to the human transmembrane and co-stimulation domain of CD28 and the activation domain from CD3 zeta.
  • the DCAR was designed as a bicistronic construct.
  • the CS1-specific scFv(Chu et al., 2014) is cloned up stream of the CD8 hinge and transmembrane regions and fused to the 4- 1BB signaling domain to form the CS1 co-stimulation Receptor (CCR).
  • CCR co-stimulation Receptor
  • the anti-CS1 CCR and 1G anti-CD38 segments are linked by a P2A self-cleaving peptide sequence.
  • CAR T cytotoxicity was determined by co-culturing MM.1Sluc cells for 4 hours in the presence of 1G, 2G or DCAR CAR-T cells at different effector:target (E:T) ratios. Cell viability was assessed by luciferase activity and normalized to un-Transduced T cells. Each E:T ratio shows the mean ⁇ SEM from triplicate experiments. Significance was determined by a 2-way anova with Sidak’s multiple comparison test. ns ⁇ 0.05 *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 **** ⁇ 0.0001
  • Figure 3 DCAR efficiency In vitro compared to “classic” CD38 targeting CAR-T cells.
  • CAR-T cytotoxicity was determined by co-culturing different myeloma cell lines (MM.1Sluc, KMS-11luc & HCI-H929luc) for 24 hours in the presence of 1G, 2G or DCAR CAR-T cells at different effector:target (E:T) ratios. Cell viability was assessed by luciferase activity and normalized to un-transduced-T cells (NT). Each E:T ratio shows the mean ⁇ SEM from triplicate experiments. Significance was determined by a 2-way anova with Turkeys’s multiple comparison test, (b) CAR-T cell proliferation capacity was determined by a 14-day co-culture on NIH3T3 cells expressing CD38 and CS1(NIH38CS1).
  • DCAR-T cells need both CD38 and CS1 for an “optimal/complete” anti-tumor response
  • CAR T cytotoxicity was determined by co-culturing differentially edited MM.1Sluc cells (wt, CD38ko, CS1ko and doubleko) for 24 hours in the presence of DCAR-T cells at different effector:target (E:T) ratios. Cell viability was assessed by luciferase activity and normalized to un-transduced-T cells.
  • E:T ratio shows the mean ⁇ SEM from triplicate experiments Significance was determined by a 2-way anova with Sidak’s multiple comparison test. The table shows the statistical analysis for E:T ratios 0.5 and 0.1.
  • CAR-T cell proliferation capacity was determined by a 14-day co-culture on NIH3T3 cells expressing differentially edited to express either no target (NIHwt), one of the targets (NIH38 and NIHCS1) or both targets (NIH38CS1).25.000 CAR-T cells were seeded on 100.000 NIH cells at day 0, and passed every 2-3 days on a new feeder layer. Cell number was determined using an automated cell counter.
  • the graph represents the mean ⁇ SEM from triplicate experiments, significance was determined using a 2-way anova with Turkey’s multiple comparison test. Significance is indicated for the proliferation at day 14.
  • Cytokine secretion was analyzed by taking supernatant from the CAR-T cells co-cultured on the different NIH3T3 feeder cell lines for 4 days. Cytokine levels of interleukin (IL)-2 and tumour necrosis factor-alpha (TNF-a) were determined by a bead- based immunoassay. The graphs represent the mean ⁇ SEM from four separate experiments. Significance was determined using Mann-Whitney tests.
  • mice within each treated group indicated by bar graphs ⁇ SEM and statistical significance was determined using a 1-way anova with Turkey’s multiple comparison test.
  • f Cell count of B cells or CD38 positive
  • Graphs represent the graphs indicate the mean ⁇ SEM and significance was determined using an unpaired T-test. *p ⁇ 0.05 **p ⁇ 0.01, ***p ⁇ 0.001.
  • EXAMPLE Methods: Construction of CAR Lentiviral vectors DNA sequences encoding for the anti-CD38 CAR (1G and 2G) and the anti-CS1 CCR were chemically synthetized (GeneArt ThermoFisher). Linkage of 1G anti-CD38 CAR to the P2A motif and the anti-CS1 CCR was performed using In-Fusion® snap assembly (Takara Bio) according to the manufacturers protocol.
  • the pSFFV-Kana lentiviral vector was generated by replacing the ampicillin resistance gene of the pRRL–PGK–WPRE (a gift from Dr Hana Raslova, Villejuif, France) with the chemically synthetized Kanamycin resistance gene and by inserting the SFFV promoter in an EcoRV site, upstream of the PGK promoter. Insertion of the CAR and CCR coding sequences into the pSFFV-Kana vector was performed using In-Fusion® snap assembly. Cell lines The MM1.S, KMS11 and NCI-H929 MM cell lines have been previously described (Fayon et al., 2021).
  • MM.1S, KMS11 or NCI-H929 were transduced with either lentiviral particles produced from the pLenti CMV Puro LUC (Addgene #17477) or retroviral particles.
  • MM1.S inactivated for the CD38 and/or SLAMF7 (CS1) genes have been previously described (Fayon et al., 2021). All MM cell lines were cultured in complete RPMI medium: RPMI-1640 supplemented with 10% heat-inactivated foetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 nM glutamine (all from ThermoFisher) at 37°C + 5% CO2.
  • NIH3T3 cells were transduced with retroviral particles to obtain NIH3T3 feeder cells expressing CD38, CS1 or both targets to generate feeder cell lines.
  • NIH3T3 cells were cultured in DMEM medium: DMEM (manufacturer) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 nM glutamine at 37°C + 5% CO2.
  • HEK293-T cells were cultured under the same conditions as NH3T3 cells.
  • Lentiviral production HEK293T cells were co-transfected with lentiviral CAR vector and packaging plasmids (pMD2G and psPAX2, all from Addgene) using calcium phosphate precipitation method (ThermoFisher) following the manufacturer’s protocol. Lentiviral supernatants were collected at 72-hours post-transfection and concentrated using high-speed ultracentrifugation. To generate the lentiviral stocks, the resulting concentrated lentivirus batches were suspended in PBS and stored at -80°C. CAR-T cell production and Genome editing Primary peripheral blood cells (PBMCs) were obtained from the “Etableau für du Sang”.
  • PBMCs Genome editing Primary peripheral blood cells
  • PBMCs were isolated from cytapheretic residues by centrifugation on a Pancoll (Human; PANTM Biotech) gradient (2:1 ratio of diluted blood to Pancoll), using Leucosep tubes (ThermoFisher) according to the manufacturers protocol, and frozen at -80°C in RPMI 20% SVF medium containing10% DMSO.
  • PBMCs were activated with CD3/CD28 activation beads (ThermoFisher) in CTSTM OpTmizerTM T Cell Expansion SFM (Gibco) containing 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 nM glutamine, IL-2 (10ng/ml, BioLegend) and hIL-7 (1ng/ ⁇ l, BioLegend), futher indicated at OpTmizerC, at 37°C + 5% CO2 for 24hours, followed by genome editing by CRISPR/Cas9.
  • CTSTM OpTmizerTM T Cell Expansion SFM Gibco
  • ribo-nucleoprotein (RNP) complexes were generated by incubating single guide RNA (sgRNA) targeting CD38 (100 ⁇ M, Sigma-Aldrich) was complexed with the Cas9nuclease (ThermoFisher) at a 1:2 molecular ratios, respectively, for 20minutes at room- temperature.
  • the RNP complex was then electroporated with 5x106 activated T cells in 100 ⁇ L of 2M electroporation buffer (Chicaybam et al., 2013) in a 2B-Nucleofactor Unit (Lonza).
  • Transfected T cells were maintained in culture, using CD3/CD28 dynabeads in OpTmizer (containing 5ng/ml of IL-2 instead of 10 ng/ml; indicated form here as normal conditions ) at 37°C + 5% CO2, for another 24hours prior to transduction with lentiviral particles.
  • Lentiviral transduction was performed in culture plates coated with retronectin (15mg/ml, Takara Bio), T cells were combined with lentiviral particles at MOI10 or higher at 37°C + 5% CO2. After 24hours the medium was changed and cells were kept in culture under standard conditions.
  • mice were fixed in a 5% formalin solution (Sigma-Aldrich) in PBS prior to analysis. To determine reconstitution in the humanized mice cells were treated with Fc block (BD, #564220) for 10min at RT, followed by staining with Zombie UVTM (Biolegend, # 423107, 1/1000) according to the manufacturers protocol.
  • Fc block BD, #564220
  • Zombie UVTM Biolegend, # 423107, 1/1000
  • Cytotoxicity assay The cytotoxicity of CAR-T cells was determined by assessing tumour cell viability following a co-culture with CAR-T cells. Sixteen hours prior to the cytotoxicity assay, anti-CD3/CD28 beads were magnetically removed from the cultures, and CAR-T cells were mixed with luciferase expressing target cells at different effector to target (E/T) ratios.
  • luciferase activity was determined using Bright-GloTM Luciferase Assay System (Promega) and bioluminescence was measured with a CLARIOstar plate reader (BMG Labtech).
  • CAR-T cells were co- cultured in feeder cells at an initial ration of 1:4 (25000:100000) CAR-T to feeder ratio. Every 2-3 days the CAR-T were transferred to fresh feeder cells.
  • Cell proliferation was determined by counting the viable cells, death cells were excluded by trypan Blue (Invitrogen) staining, using the Countess II (Invitrogen). Cell numbers were quantified using ImageJ (ImageJ) software.
  • Cytokine production Cytokine secretion was determined by collecting the supernatant of the long-term proliferation assay. The supernatant was collected at day 4 and stored at -20°C.
  • MM1.S.luc xenograft model Six to 12-week-old NOD/SCID/IL-2Rg null mice were inoculated with 3x106 MM1.SLuc cells by tail vein injection (i.v) at day 0, followed, 14 days later, by infusion of 106 CAR-T. Bioluminescence was measured with the IVIS Imaging System (PerkinElmer) every 7 days after tumour injection.
  • the study contained 6 mice in each group which were injected with CAR-T cells generated from 3 different healthy donors. Data was further analysed using Aura imaging software (Spectral Instruments Imaging) Subcutaneous MM1.S.luc xenograft model MM1.Sluc cells (0.5x106 cells per mouse) were injected sub cutaneous on the back of NSG mice. MM1.Swt cells were injected in the left flank and MM1.SCS1ko cells were injected in the right flank of the mice. CAR-T cells (106 cells per mouse) were administered IV in the tail vein 4 days post tumor injection. Tumor progression was followed by luminescent imaging every 7 days using the IVIS Imaging System. The study contained 4 mice in each group which were injected with CAR-T cells originating from 2 different healthy donors.
  • IMDM IMDM (Gibco) completed with 20%BIT (StemcellTM technologies), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 nM glutamine, 50ng/ml SCF (Miltenyi Biotec), 50ng/ml FLT3 (Miltenyi Biotec) 20ng/ml TPO (Miltenyi Biotec) , 10ng/ml IL-3 (Miltenyi Biotec) for 16H at 37°C + 5% CO2.
  • the CD34+ cells (4 different donors) were pooled and cells (0.35x106 per mouse) were administered IV in the tail vein in sub lethally irradiated NSG mice.5-weeks post injection of CD34+ cells mice injected with CAR-T cells (106 cells per mouse) IV in the tail vein. Each CAR-T was produced from 2 different healthy donors. Blood was collected from the mice to monitor the reconstitution prior to CAR-T cell injection. Blood, serum and bone marrow were collected post CAR-T cell treatment for further analysis by flow cytometry. Prior to analysis blood and organ samples were passed on a Pancoll gradient (2:1 ratio of diluted blood to Pancoll).
  • DNA sequences encoding the anti-CD381G CAR and the anti-CS1 CCR were cloned into a P2A-based bicistronic construct, which allows the expression of two separate polypeptides from a single gene ( Figure 1a). All sequences coding for 1G, 2G and DCAR were cloned under the control of the spleen focus-forming virus (SFFV) promoter into a lentiviral vector and were stably expressed on T cells isolated from healthy human donors. FACS analysis of transduced T cells stained with a anti human scFv revealed strong expression of the CARs and/or CCR transgenes in around 90% of infected T cells (data not shown).
  • SFFV spleen focus-forming virus
  • the DCAR construct allows co-expression of the anti-CD38 CAR and the anti-CS1 CCR, while 1G and 2G vectors induced a higher expression level of the anti-CD38 CAR at the surface of transduced T cells.
  • Deletion of CD38 in anti-CD38 DCAR-T enhances their cytotoxic activity against MM cells Since CD38 is upregulated on activated T cells, we investigated whether this process could interfere with the expression or the function of anti-CD38 CAR.
  • CD38 edited T cells from healthy donors were transduced with the different vectors, expanded in vitro for 7-14 days with anti-CD3/CD28 beads plus interleukine- 2 (IL-2) and IL-7, then co-cultured overnight with MM1.Sluc at various E:T ratios and the luciferase levels were measured to determine the cytotoxic activity.
  • IL-2 interleukine- 2
  • 1G anti-CD38 CAR-T and not transduced T cells displayed little or no proliferation over time. Furthermore, we used this NIH cells model to measure the production of IL-2 and tumour necrosis factor-alpha (TNF-a) by T cells transduced with the different constructs. After 96 hours in culture on NIH38CS1 production of IL-2 was much lower in 1G than in 2G anti-CD38 CAR-T cultures (18.73 pg/mL compare with 580 pg/mL) ( Figure 3c). Remarkably, DCAR-T secreted an intermediate level of IL-2 (183 pg/mL), while no production was detected in the culture medium of not transduced control T cells.
  • TNF-a was readily produced by all transduced T cells, however the levels were higher in 1G and 2G anti-CD38 CAR-T compared with DCAR-T (1.2 ⁇ g/mL, 2.7 ⁇ g/mL and 0.45 ⁇ g/mL, respectively as average) (Figure 3c). These results indicate that DCAR-T responded to stimulation by CD38 and CS1 expressing targets by secreting strong levels of IL-2 and lower levels of TNF-a compare to 1G and 2G anti-CD38 CAR-T, in vitro.
  • DCAR-T require expression of both CD38 and CS1 on target cells to deliver efficient responses
  • CRISPR/Cas9 mediated approach to inactivate the CD38 and/or SLAMF7 (encoding for CS1) genes in the MM1.Sluc cell line (Fayon et al., 2021) and used the edited cells to assay the cytotoxic activity of in vitro expanded CD38 edited DCAR-T cells.
  • DCAR-T efficiently killed un-manipulated MM1.Sluc cells (MM1.Swt), even at low E:T ratios (20% viability at E:T of 1:2) ( Figure 4a).
  • MM1.S cells inactivated for both CD38 and CS1 appeared significantly resistant to DCAR-T cell lysis as 60% of the cells remained viable at the E:T of 1:2, as average, and 90% at the E:T of 1:10 ( Figure 4a).
  • MM cells inactivated for CD38 MM1.S38ko
  • CS1 MM1.SCS1ko
  • NIH3T3 cells expressing CD38 (NIH38), CS1 (NIHCS1), CD38 and CS1 (NIH38CS1) or none (NIHwt) were used each cell line to support CD38 edited DCAR-T cell expansion in vitro.
  • DCAR-T stimulated with either NIHwt or NIH38 cells expanded very poorly ( Figure 4b).
  • culture on NIH38CS1 feeder cells triggered a robust proliferation of DCAR-T, with cells numbers increasing from 25000 at day 0 to more than 1.5x106 at day 14.
  • DCAR-T co- culture on NIHCS1 cells readily expanded, albeit at a lower level compare to those cultured on NIH38CS1 cells expressing both CD38 and CS1 antigens.
  • NIH3T3 cellular models to investigate in vitro cytokine production by edited DCAR-T in response to engagement of anti-CD38 CAR, anti-CS1 CCR or both.
  • the medium of DCAR-T cultured for 96 hours on NIHwt contained low concentrations of IL-2 and TNF- ⁇ ( Figure 4c).
  • DCAR-T produced weak levels of IL-2 and TNF-a when cultured on NIH38.
  • mice that received DCAR-T cells showed prolonged control of tumor growth compared with mice treated with 1G anti-CD38 CAR-T ( Figure 5c).
  • mice injected with CD382G and DCAR had a significant elongation of survival compared to NT and 1G treated mice ( Figure 5d).
  • DCAR-T as 2G anti-CD38 CAR-T induced efficient and long-term inhibition of MM tumor progression in vivo.
  • DCAR-T display low cytotoxicity against CS1 deficient MM cells in vivo
  • One major pit fall of anti-CD38 CAR-T approach is their potential off tumor activity, which could lead to sever side effects.
  • DCAR-T selectively control the tumor progression of cells bearing both CD38 and CS1, while preserving CD38 targets lacking expression of CS1 in vivo.
  • DCAR-T cells preserve haematopoietic cells in humanized mice
  • HSC human hematopoietic stem cells
  • mice were then randomized into 3 cohorts and injected with non-transduced T cells, 2G anti-CD38 CAR-T or DCAR-T, all inactivated for CD38. Blood samples were collected 1 day before CAR-T inoculation, 3 days after treatment as well as at day 7, when mice were sacrificed and analysed by flow cytometry.
  • DCAR-T has no detectable toxicity towards human haematopoietic cells in vivo.
  • DCAR a split double CAR setting targeting MM tumour cells via the recognition of CD38 by an activation receptor bearing the CD3z signalling domain and CS1 through a co-stimulation receptor containing the 4-1BB intracellular domain.
  • Expression of the DCAR anti-CD38 and anti-CS1 activation and co-stimulation chimeric antigen receptors is driven by an original kanamycin resistant lentiviral vector which can be approved for clinical assays.
  • CRISPR/Cas9 edition increases DCAR-T cells killing of MM cells in vitro.
  • CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma. Leukemia 28, 917-927.
  • Dimopoulos M.A., Oriol, A., Nahi, H., San-Miguel, J., Bahlis, N.J., Usmani, S.Z., Rabin, N., Orlowski, R.Z., Komarnicki, M., Suzuki, K., et al. (2016). Daratumumab, Lenalidomide, and Dexamethasone for Multiple Myeloma. N Engl J Med 375, 1319-1331. Dimopoulos, M.A., Richardson, P., and Lonial, S. (2022). Treatment Options for Patients With Heavily Pretreated Relapsed and Refractory Multiple Myeloma.
  • Bi38-3 is a novel CD38/CD3 bispecific T-cell engager with low toxicity for the treatment of multiple myeloma. Haematologica 106, 1193-1197. Kloss, C.C., Condomines, M., Cartellieri, M., Bachmann, M., and Sadelain, M. (2013).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Le traitement par lymphocytes T du récepteur antigénique chimérique (CAR) pour le myélome multiple (MM) ciblant l'antigène de maturation des lymphocytes B (BCMA) induit des taux de réponse globaux élevés. Cependant, une rechute se produit fréquemment et de nouvelles stratégies pour des cellules CAR-T ciblant des cellules MM s'imposent. Les cellules CAR-T ciblant CD38 représentent une alternative potentielle, mais l'expression de CD38 sur des cellules T activées et d'autres cellules hématopoïétiques augmente les problèmes d'efficacité et de sécurité d'une telle thérapie. Ici, les inventeurs ont développé DCAR, un système CAR double ciblant CD38 et SLAMF7 (CS1) par l'intermédiaire respectivement de récepteurs d'activation et de co-stimulation divisés. En outre, les inventeurs montrent que l'inactivation de CRISPR/Cas9 du gène CD38 améliore l'activité anti-MM de DCAR-T in vitro. Les DCAR-T éditées ont développé de fortes réponses spécifiquement à l'encontre des cellules MM exprimant à la fois les antigènes CD38 et CS1 in vitro et in vivo. Plus important encore, les inventeurs attestent que, contrairement à CAR anti-CD38, qui suscite une réaction immunitaire grave contre des cellules hématopoïétiques dans un modèle de souris humanisé, DCAR-T ne présente aucun signe de toxicité. En conséquence, la présente invention concerne de nouvelles cellules CAR-T à double division et des utilisations de ces dernières pour le traitement de malignités hématologiques CD38-positives.
PCT/EP2023/074271 2022-09-06 2023-09-05 Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives Ceased WO2024052318A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP23765243.3A EP4583905A1 (fr) 2022-09-06 2023-09-05 Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22306319.9 2022-09-06
EP22306319 2022-09-06

Publications (1)

Publication Number Publication Date
WO2024052318A1 true WO2024052318A1 (fr) 2024-03-14

Family

ID=83508827

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/074271 Ceased WO2024052318A1 (fr) 2022-09-06 2023-09-05 Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives

Country Status (2)

Country Link
EP (1) EP4583905A1 (fr)
WO (1) WO2024052318A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220251234A1 (en) * 2019-07-16 2022-08-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity for cd38 and uses thereof

Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5786464A (en) 1994-09-19 1998-07-28 The General Hospital Corporation Overexpression of mammalian and viral proteins
US5994136A (en) 1997-12-12 1999-11-30 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
US6114148A (en) 1996-09-20 2000-09-05 The General Hospital Corporation High level expression of proteins
US20030170238A1 (en) 2002-03-07 2003-09-11 Gruenberg Micheal L. Re-activated T-cells for adoptive immunotherapy
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2015048577A2 (fr) 2013-09-27 2015-04-02 Editas Medicine, Inc. Compositions et méthodes relatives aux répétitions palindromiques groupées, courtes et régulièrement espacées
WO2017025323A1 (fr) * 2015-08-11 2017-02-16 Cellectis Cellules pour l'immunothérapie modifiées afin de cibler l'antigène cd38 et pour inactiver le gène cd38
WO2017100403A1 (fr) 2015-12-08 2017-06-15 Regents Of The University Of Minnesota Lymphocyte t humain dérivé de cellule souche pluripotente induite dérivée de lymphocyte t et procédés de fabrication et d'utilisation
WO2019111249A1 (fr) * 2017-12-05 2019-06-13 The Medical Research Infrastructure And Health Services Fund Of The Tel Aviv Medical Center Lymphocytes t comprenant des récepteurs antigéniques chimériques anti-cd38 et anti-cd138 et leurs utilisations
WO2021009263A1 (fr) * 2019-07-16 2021-01-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps présentant une spécificité pour cd38 et leurs utilisations
WO2022003158A1 (fr) * 2020-07-03 2022-01-06 Cellectis S.A. Procédé de détermination de la puissance d'un récepteur d'antigène chimérique exprimant des cellules immunitaires

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US5786464A (en) 1994-09-19 1998-07-28 The General Hospital Corporation Overexpression of mammalian and viral proteins
US5786464C1 (en) 1994-09-19 2012-04-24 Gen Hospital Corp Overexpression of mammalian and viral proteins
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
US6114148A (en) 1996-09-20 2000-09-05 The General Hospital Corporation High level expression of proteins
US6114148C1 (en) 1996-09-20 2012-05-01 Gen Hospital Corp High level expression of proteins
US5994136A (en) 1997-12-12 1999-11-30 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
US20030170238A1 (en) 2002-03-07 2003-09-11 Gruenberg Micheal L. Re-activated T-cells for adoptive immunotherapy
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8771945B1 (en) 2012-12-12 2014-07-08 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
WO2015048577A2 (fr) 2013-09-27 2015-04-02 Editas Medicine, Inc. Compositions et méthodes relatives aux répétitions palindromiques groupées, courtes et régulièrement espacées
WO2017025323A1 (fr) * 2015-08-11 2017-02-16 Cellectis Cellules pour l'immunothérapie modifiées afin de cibler l'antigène cd38 et pour inactiver le gène cd38
WO2017100403A1 (fr) 2015-12-08 2017-06-15 Regents Of The University Of Minnesota Lymphocyte t humain dérivé de cellule souche pluripotente induite dérivée de lymphocyte t et procédés de fabrication et d'utilisation
WO2019111249A1 (fr) * 2017-12-05 2019-06-13 The Medical Research Infrastructure And Health Services Fund Of The Tel Aviv Medical Center Lymphocytes t comprenant des récepteurs antigéniques chimériques anti-cd38 et anti-cd138 et leurs utilisations
WO2021009263A1 (fr) * 2019-07-16 2021-01-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps présentant une spécificité pour cd38 et leurs utilisations
WO2022003158A1 (fr) * 2020-07-03 2022-01-06 Cellectis S.A. Procédé de détermination de la puissance d'un récepteur d'antigène chimérique exprimant des cellules immunitaires

Non-Patent Citations (39)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Immunology", 1992, GREENE PUBLISHING ASSOC.
"Genbank", Database accession no. KM099231.1
"Handbook of Growth Factors", 1994, CRC PRESS, article "Hematopoietic Growth Factors and Cytokines", pages: 1 - 2
"Remington's Pharmaceutical-Sciences", 1980, MACK PUBLISHING CO.
ALHAJ HUSSEN, K.VU MANH, T.P.GUIMIOT, F.NELSON, E.CHABAANE, E.DELORD, M.BARBIER, M.BERTHAULT, C.DULPHY, N.ALBERDI, A.J. ET AL.: "Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis", IMMUNITY, vol. 47, 2017, pages 680 - 696
BOYIADZIS, M.M.DHODAPKAR, M.V.BRENTJENS, R.J.KOCHENDERFER, J.N.NEELAPU, S.S.MAUS, M.V.PORTER, D.L.MALONEY, D.G.GRUPP, S.A.MACKALL,: "Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance", J IMMUNOTHER CANCER, vol. 6, 2018, pages 137
CHICAYBAM, L.SODRE, A.L.CURZIO, B.A.BONAMINO, M.H.: "An efficient low cost method for gene transfer to T lymphocytes", PLOS ONE, vol. 8, 2013, pages e60298, XP055873396, DOI: 10.1371/journal.pone.0060298
CHU, J.DENG, Y.BENSON, D.M.HE, S.HUGHES, T.ZHANG, J.PENG, Y.MAO, H.YI, L.GHOSHAL, K. ET AL.: "CS 1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma", LEUKEMIA, vol. 28, 2014, pages 917 - 927, XP055133640, DOI: 10.1038/leu.2013.279
COHEN ADAM D: "CAR T Cells and Other Cellular Therapies for Multiple Myeloma: 2018 Update", AMERICAN SOCIETY OF CLINICAL ONCOLOGY EDUCATIONAL BOOK. AMERICAN SOCIETY OF CLINICAL ONCOLOGY. MEETINGUNITED STATES2013,, vol. 38, 23 May 2018 (2018-05-23), pages e6 - e15, XP002790576, ISSN: 1548-8756, DOI: 10.1200/EDBK_200889 *
CONG, SCIENCE, vol. 339, 2013, pages 819 - 823
DA VIA, M.C.DIETRICH, O.TRUGER, M.ARAMPATZI, P.DUELL, J.HEIDEMEIER, A.ZHOU, X.DANHOF, S.KRAUS, S.CHATTERJEE, M. ET AL.: "Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma", NAT MED, vol. 27, 2021, pages 616 - 619, XP037424517, DOI: 10.1038/s41591-021-01245-5
DALEY ET AL., FOCUS, vol. 18, 1996, pages 62 - 67
DIMOPOULOS, M.A.ORIOL, A.NAHI, H.SAN-MIGUEL, J.BAHLIS, N.J.USMANI, S.Z.RABIN, NORLOWSKI, R.Z.KOMARNICKI, M.SUZUKI, K. ET AL.: "Daratumumab, Lenalidomide, and Dexamethasone for Multiple Myeloma", N ENGL J MED, vol. 375, 2016, pages 1319 - 1331, XP055964850, DOI: 10.1056/NEJMoa1607751
DIMOPOULOS, M.A.RICHARDSON, P.LONIAL, S: "Treatment Options for Patients With Heavily Pretreated Relapsed and Refractory Multiple Myeloma", CLIN LYMPHOMA MYELOMA LEUK, 2022
DRENT ET AL., MOL THER., 2017
FAYON, M.MARTINEZ-CINGOLANI, C.ABECASSIS, A.RODERS, N.NELSON, E.CHOISY, C.TALBOT, A.BENSUSSAN, A.FERMAND, J.PARNULF, B. ET AL.: "Bi38-3 is a novel CD38/CD3 bispecific T-cell engager with low toxicity for the treatment of multiple myeloma", HAEMATOLOGICA, vol. 106, 2021, pages 1193 - 1197
GLOBERSON LEVIN ANAT ET AL: "Treatment of Multiple Myeloma Using Chimeric Antigen Receptor T Cells with Dual Specificity", vol. 8, no. 12, 1 December 2020 (2020-12-01), US, pages 1485 - 1495, XP055836127, ISSN: 2326-6066, Retrieved from the Internet <URL:http://dx.doi.org/10.1158/2326-6066.CIR-20-0118> DOI: 10.1158/2326-6066.CIR-20-0118 *
GURNEY MARK ET AL: "CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide", vol. 107, no. 2, 30 December 2020 (2020-12-30), IT, pages 437 - 445, XP093005506, ISSN: 0390-6078, Retrieved from the Internet <URL:https://haematologica.org/article/download/haematol.2020.271908/74086> DOI: 10.3324/haematol.2020.271908 *
HARLOW ET AL.: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
K H CHEN ET AL: "A compound chimeric antigen receptor strategy for targeting multiple myeloma", LEUKEMIA, vol. 32, no. 2, 27 September 2017 (2017-09-27), London, pages 402 - 412, XP055530334, ISSN: 0887-6924, DOI: 10.1038/leu.2017.302 *
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1987, US DEPARTMENT OF HEALTH AND HUMAN SERVICES
KATSAROU AFRODITI ET AL: "Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence", SCI. TRANSL. MED.SCIENCE.ORG AT EUROPEAN PATENT OFFICE ON SEPTEMBER, 8 December 2021 (2021-12-08), pages 1962, XP055966551 *
KIM JHLEE S-RLI L-HPARK H-JPARK J-HLEE K Y ET AL.: "High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice", PLOS ONE, vol. 6, no. 4, 2011, pages e18556, XP055175691, DOI: 10.1371/journal.pone.0018556
KLOSS, C.C.CONDOMINES, M.CARTELLIERI, M.BACHMANN, M.SADELAIN, M: "Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells", NAT BIOTECHNOL, vol. 31, 2013, pages 71 - 75, XP055130697, DOI: 10.1038/nbt.2459
LANITIS, E.POUSSIN, M.KLATTENHOFF, A.W.SONG, D.SANDALTZOPOULOS, R.JUNE, C.H.POWELL, D.J., JR.: "Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo", CANCER IMMUNOL RES, vol. 1, 2013, pages 43 - 53, XP055170367, DOI: 10.1158/2326-6066.CIR-13-0008
LAURENT, S.A.HOFFMANN, F.S.KUHN, P.H.CHENG, Q.CHU, Y.SCHMIDT-SUPPRIAN, M.HAUCK, S.M.SCHUH, E.KRUMBHOLZ, M.RUBSAMEN, H. ET AL.: "gamma-Secretase directly sheds the survival receptor BCMA from plasma cells", NAT COMMUN, vol. 6, 2015, pages 7333
MAHER, J.BRENTJENS, R.J.GUNSET, G.RIVIERE, I.SADELAIN, M: "Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor", NAT BIOTECHNOL, vol. 20, 2002, pages 70 - 75, XP002588277
MORGAN, G.J.WALKER, B.A.DAVIES, F.E.: "The genetic architecture of multiple myeloma", NAT REV CANCER, vol. 12, 2012, pages 335 - 348, XP055103788, DOI: 10.1038/nrc3257
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MULLER, METH. ENZYMOL., vol. 92, 1983, pages 589 - 601
MUNSHI, N.C.ANDERSON, L.D., JR.SHAH, N.MADDURI, D.BERDEJA, J.LONIAL, S.RAJE, N.LIN, Y.SIEGEL, D.ORIOL, A. ET AL.: "Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma", N ENGL J MED, vol. 384, 2021, pages 705 - 716, XP055951668, DOI: 10.1056/NEJMoa2024850
NEEDLEMAN, SAUL BWUNSCH, CHRISTIAN D: "A general method applicable to the search for similarities in the amino acid sequence of two proteins", JOURNAL OF MOLECULAR BIOLOGY, vol. 48, no. 3, 1970, pages 443 - 53, XP024011703, DOI: 10.1016/0022-2836(70)90057-4
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RAFIQ, S.HACKETT, C.S.BRENTJENS, R.J.: "Engineering strategies to overcome the current roadblocks in CAR T cell therapy.", NAT REV CLIN ONCOL, vol. 17, 2020, pages 147 - 167, XP037114979, DOI: 10.1038/s41571-019-0297-y
REICHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
SADELAIN, M.BRENTJENS, R.RIVIERE, I: "The basic principles of chimeric antigen receptor design", CANCER DISCOV, vol. 3, 2013, pages 388 - 398, XP055287277, DOI: 10.1158/2159-8290.CD-12-0548
TAKAHASHI, KYAMANAKA, S: "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", CELL, vol. 126, no. 4, 2006, pages 663 - 76
TSAI, NATURE BIOTECHNOL., vol. 32, no. 6, 2014, pages 569 - 576

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220251234A1 (en) * 2019-07-16 2022-08-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity for cd38 and uses thereof
US12319745B2 (en) * 2019-07-16 2025-06-03 Inserm (Institut National De La Sante Et De La Recherche Medicale) Antibodies having specificity for CD38 and uses thereof

Also Published As

Publication number Publication date
EP4583905A1 (fr) 2025-07-16

Similar Documents

Publication Publication Date Title
JP7621344B2 (ja) 線維芽細胞活性化タンパク質（ｆａｐ）を標的とすることによる腫瘍組織の破壊の方法
AU2017371517B2 (en) Engineered natural killer cells and uses thereof
JP7433230B2 (ja) 細胞を培養するための無血清培地配合物およびその使用の方法
US20250136697A1 (en) Methods and compositions comprising b7h3 chimeric antigen receptors
JP2019516352A (ja) Ｆｌｔ３に対するキメラ受容体及びその使用方法
US20220356247A1 (en) ROR1 specific chimeric antigen receptors and their therapeutic applications
KR20230129979A (ko) 수지상 세포 활성화 키메라 항원 수용체 및 이의 용도
EP4022035A1 (fr) Méthodes et compositions pour la modification et l&#39;administration de lymphocytes
US20240123068A1 (en) Cd19 binders, car-t constructs comprising the same, and methods of using the same
CA3231615A1 (fr) Recepteurs antigeniques chimeriques comprenant un domaine de signalisation du recepteur de l&#39;interleukine-9
CA3225252A1 (fr) Recepteurs de lymphocytes t modifies fusionnes a des domaines de liaison d&#39;anticorps
US20240226154A9 (en) Car-t constructs comprising a novel cd19 binder combined with il18 and methods of using the same
WO2024052318A1 (fr) Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives
CN112661857B (zh) 一种嵌合抗原受体及其用途
US20250177442A1 (en) Boosting chimeric antigen receptor cells in the blood
US20240335539A1 (en) Modulation of bcl-2 to enhance chimeric antigen receptor cancer immunotherapy efficacy
US20240366672A1 (en) Chimeric antigen receptor and use thereof
US20240358834A1 (en) Novel cd19-targeting polypeptides and cd19-directed immunotherapies
US20240122981A1 (en) CCR4-Targeting Chimeric Antigen Receptor Cell Therapy
TW202521566A (zh) 一種靶向cd38和cll1的雙特異性嵌合抗原受體及其應用
WO2025006561A2 (fr) Méthodes de traitement utilisant des immunothérapies ciblant cd19
EP4580658A1 (fr) Procédé de génération de cellules car-t plus efficaces
WO2024153124A1 (fr) Lymphocyte t primaire modifié et son utilisation
HK40108209A (zh) Ccr4靶向嵌合抗原受体细胞疗法
HK40075954A (en) Disrupting tumor tissues by targeting fibroblast activation protein (fap)

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: 23765243

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023765243

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023765243

Country of ref document: EP

Effective date: 20250407

WWP Wipo information: published in national office

Ref document number: 2023765243

Country of ref document: EP