JP2019514870A - Methods and vaccine compositions for the treatment of B cell malignancies - Google Patents

Abstract

The present invention relates to methods and vaccine compositions for the treatment of B cell malignancies. Epstein-Barr virus (EBV) infection in humans is a major trigger for malignant and non-malignant B cell proliferation. We have shown that EBV-specific T lymphocytes can not proliferate properly when stimulated with CD70-deficient EBV-infected B cells, but expression of CD70 restores proliferation . In particular, the present invention comprises the steps of: i) preparing a sample of malignant B cells obtained from a subject, ii) isolating and culturing a group of malignant B cells from the sample of step i), iii) malignancy of step ii) A subject in need thereof, which comprises introducing into the B cell population a nucleic acid molecule encoding a CD70 polypeptide, and iv) administering a therapeutically effective amount of the malignant B cell population of step iii) to the subject. For the treatment of B cell malignancies in

Description

Field of the Invention:
The present invention relates to methods and vaccine compositions for the treatment of B cell malignancies.

Background of the invention:
Epstein-Barr virus (EBV) is a gamma-herpesvirus that infects most humans and has significant tropism for B lymphocytes. Importantly, EBV is known to be one of the most potent triggers of endogenously uncontrolled B cell proliferation and lymphoma formation. Rare genetic diseases in particular predispose to incomplete control of EBV infection, which results in virus-associated hemophagocytic syndrome, lymphoproliferative disorder (LPD) such as Hodgkin's lymphoma and non-Hodgkin's lymphoma ^1,2 . Currently, CTPS1, SH2D1A, MAGT1, ITK , and mutation of CD27 is associated with high expression rates of EBV infection, up to 90% of patients have developed a disease and lymphoma associated with persistent infection EBV ^{3 4, 5, 6, 7, 8} . These primary immunodeficiency studies have revealed important pathways involved in T cell responses to EBV-infected B lymphocytes and, more generally, to T cell function. In healthy individuals, the effectiveness of the immune response to EBV is in fact mainly dependent on the massive expansion of specific CD8 ⁺ cytotoxic T cells that eliminate EBV-infected B cells ^{9, 10} . In CTPS1, SH2D1A, and MAGT1 deficiency, CD8 ⁺ T cell responses to EBV-infected B lymphocytes are either cell-mediated cytotoxicity and / or proliferation of specific cytotoxic CD8 ⁺ T cells As a result of defects in the The syndrome X-linked lymphoproliferative syndrome (XLP) -1, characterized by EBV-induced hemophagocytic syndrome and development of B lymphoma, codes for the protein SAP associated with the signaling lymphocyte activation molecule (SLAM) Is caused by mutations in SH2D1A. In XLP-1, the cytotoxic response of CD8 ⁺ T cells to B cells infected with EBV is specifically impaired, as the activity of the SLAM receptor, which is dependent on SAP for the function of the SLAM receptor, is impaired And abnormal ^11,12,13 . MAGT1 Encodes a Transmembrane Mg ²⁺ Transporter Involved in TCR Signaling and Expression of NKG2D, a Key Cytolytic Activated Cell Receptor Expressed by CD8 ⁺ T Cells ^{14, 15} . Thus, the NKG2D and SLAM-SAP pathways represent key components of the immune response to EBV. ITK deficiency is caused by mutations in ITK, a well-characterized tyrosine kinase of the BTK family that is involved in T cell activation through its ability to activate PLC-γ1 ¹⁶ . In the absence of ITK, PLC-γ1 activation in response to TCR activation is impaired, resulting in a decrease in Ca ²⁺ mobilization ^{17, 18} . Itk deficient mice show incomplete CD8 ⁺ T cell proliferation during antiviral response ¹⁹ . However, the exact mechanism underlying the defective immune response to EBV in ITK deficient patients is unknown. Recently, CD17 deficiency has been identified in 17 patients who all developed lymphoproliferative disorders driven by EBV, which supports the important role of CD27 in immunity to EBV. The exact mechanism of action has not been described so far 5, ^{6, 20, 21, 22} . The ligand for CD27 CD70, some of the B lymphocytes and dendritic cells are expressed in subgroups of ²³ found transiently on most immune cells upon activation. CD70 is also present on a variety of cancer cells, including neoplasms that originate from B cells ²⁴ . CD27 and CD70 are respectively homodimer type I and homotrimer type II membrane protein ^21. Both belong to the tumor necrosis factor (TNF) superfamily. Studies in mouse models have established that the CD27-CD70 pathway plays an important role in the development and maintenance of T cell immunity, particularly during antiviral responses22, ^{25, 26, 27} .

Summary of the invention:
The present invention relates to methods and vaccine compositions for the treatment of B cell malignancies. In particular, the invention is defined by the claims.

Detailed Description of the Invention:
Epstein-Barr virus (EBV) infection in humans is a major trigger for malignant and non-malignant B cell proliferation. Although congenital defects in ITK and CD27 are characterized by an impaired immune response to EBV-infected B cells, the underlying pathological mechanisms have not yet been identified. Here, we report a patient suffering from EBV-induced recurrent B cell proliferation caused by a deficiency in CD70, a ligand for CD27, a costimulatory molecule of T cells. . We show that EBV-specific T lymphocytes can not proliferate properly when stimulated with CD70-deficient EBV-infected B cells, but expression of CD70 restores proliferation . Analysis of ITK-deficient lymphocytes demonstrates that CD70-triggered and CD27-mediated T cell proliferation is completely dependent on ITK, which couples CD27 to downstream signaling cascades, including calcium mobilization. Be done. Thus, the CD70-CD27-ITK pathway appears to be an important component of EBV-specific T cell immunity, more generally, for immune surveillance of B cells. Thus, CD70-CD27 represents an important target for inducing anti-tumor vaccination. Restoration or induction of expression of CD70 in some lymphoma cells lacking CD70 may represent a therapeutic approach to induce global and potent anti-tumor immunity.

  Therefore, the first object of the present invention is: i) preparing a sample of malignant B cells obtained from a subject, ii) isolating and culturing a group of malignant B cells from the sample of step i), iii) Introducing into the group of malignant B cells of step ii) a nucleic acid molecule encoding a CD70 polypeptide, and iv) administering to the subject a group of malignant B cells of step iii) of a therapeutically effective amount. For the treatment of B cell malignancies in a subject in need thereof.

  The term "B cell malignancy" as used herein includes any type of leukemia or lymphoma of B cells. The term "B cell lymphoma" refers to a cancer that arises in lymphoid cells derived from B cells. B cells are leukocytes that develop from the bone marrow and produce antibodies. They are also known as B lymphocytes. As B-cell malignancies, non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary exudative lymphoma, diffuse large B-cell lymphoma, perisplenic zone lymphoma, MALT (mucosal associated lymphoid tissue) lymphoma, Hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma (eg, various types of Hodgkin's disease, B-cell non-Hodgkin's lymphoma (NHL) and related lymphomas (eg, warden strain) Mucomacroglobulinemia (also called lymph plasma cell lymphoma or immunocytoma) or lymphoma of the central nervous system), leukemia (eg acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; B cell) Chronic lymphocytic leukemia (also called BCLL), hairy cell leukemia and chronic myeloblastic leukemia), Myeloma, including but not limited to multiple myeloma, Additional B-cell malignancies include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic band Lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, follicular lymphoma , Mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymus) large cell B cell lymphoma, intravascular large cell B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, gray zone lymphoma And B-cell proliferation of unknown grade, lymphomatous granulomatosis, and post-transplant lymphoproliferative disorder.

  In some embodiments, B cell lymphoma is caused by Epstein-Barr virus (EBV).

In some embodiments, the B cell lymphoma is a diffuse large B cell lymphoma with mutations in the gene encoding CD70. CD70 is indeed one of the most frequently mutated genes in diffuse large B-cell lymphoma ^{42, 44, 45} .

  The term "treatment" or "treating" as used herein refers to prophylactic or preventative treatment, as well as curative or disease modifying treatment (which is at risk of having the disease or suspected of having the disease). Refers to both the treatment of a patient as well as the treatment of a patient who has been diagnosed with a disease or suffering from a disease or medical condition, including the suppression of clinical recurrence. Treatment reduces the severity of a subject who has a medical disorder or who may eventually suffer from the disorder to prevent, cure, delay the onset of the disorder or a relapsing disorder Or to ameliorate one or more symptoms thereof, or to prolong the survival of the subject beyond the expected survival if not receiving such treatment. By "therapeutic regimen" is meant a treatment pattern of a disease, eg, a dosing pattern, used during treatment. The treatment regimen may include an induction regimen and a maintenance regimen. The phrases "induction regimen" or "induction period" refer to a treatment regimen (or part of a treatment regimen) used for initial treatment of a disease. The general goal of induction regimens is to provide patients with high levels of drug during the initial period of treatment regimens. The induction regimen may use (partially or totally) a "loading regimen", which involves administering more doses of the drug than the physician would use during the maintenance regimen, the physician It may include administering the drug more frequently than would be administered during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" is used, for example, for the maintenance of a patient during treatment of a disease, in order to keep the patient in remission for a long period (months or years) Refers to a treatment regimen (or part of a treatment regimen). Maintenance regimens may be continuous therapy (eg, regular intervals of dosing, eg, weekly, monthly, yearly, etc.) or intermittent therapy (eg, intermittent treatment, intermittent treatment) Treatment at relapse, or treatment upon reaching certain predetermined criteria [eg, manifestation of disease etc.] may be used.

  Samples of malignant B cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, and tumor tissue. Typically, B cells are separated by any number of techniques known to those skilled in the art, such as separation by FicollTM, a copolymer of sucrose and epichlorohydrin that can be used to prepare high density solutions. Can be obtained from a unit of blood collected from the subject. In some embodiments, cells from circulating blood of an individual are obtained by apheresis or leukapheresis. Products of apheresis typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells recovered by apheresis can be washed to remove plasma fractions, and the cells can be placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or lack many divalent cations, even if not all divalent cations. It is also good. Those skilled in the art will appreciate that the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., a Cobe 2991 cell processor) according to the manufacturer's instructions. It will be easy to understand. After washing, cells can be resuspended in various biocompatible buffers, such as, for example, PBS. Alternatively, undesired components of the apheresis sample may be removed and cells may be directly resuspended in culture medium. B cells can be isolated from peripheral blood or leukapheresis using techniques known in the art. For example, PBMCs can be isolated using Ficoll (TM) (Sigma Aldrich, St. Louis, MO), CD19 + B cells are known in the art, such as the Rosetta Tetramer Complex System (Stemcell Technologies, Vancouver, Canada) It can be purified by negative or positive selection using any of a variety of antibodies known in the art. Other isolation kits, such as R & D Systems' Human B Cell Isolation Kit (Minneapolis, Minn.) Are also commercially available.

  The term "CD70" as used herein has its general meaning in the art and refers to a ligand for CD27 (eg Bowman MR et al., J. Immunol. 1994 Feb. 15; 152 ( 4): 1756-61). CD70 is also "CD70 molecule", "CD27L", "CD27LG", "TNFSF7", "tumor necrosis factor (ligand) superfamily member 7", "CD27 ligand", "CD70 antigen", "surface antigen CD70", It is also referred to as "tumor necrosis factor ligand superfamily member 7", "Ki-24 antigen" and "CD27-L". CD70 is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) ligand family. It induces the proliferation of co-stimulated T cells, enhances the generation of cytotoxic T cells, and contributes to T cell activation, surface antigens on activated T and B lymphocytes It is. It has also been suggested that CD70 plays a role in the regulation of B cell activation, in the cytotoxic function of natural killer cells, and in the synthesis of immunoglobulins (Hintzen RQ et al., J. Immunol. 1994). Feb. 15; 152 (4): 1762-73). An exemplary human amino acid sequence of CD70 is referenced under GenBank Accession No. NP-001243 (SEQ ID NO: 1):

SEQ ID NO: 1

  In some embodiments, the malignant B cells have been transformed with a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

  According to the present invention, the first amino acid sequence having at least 90% identity to the second amino acid sequence is 90: 91; 92; 93 to the second amino acid sequence. It means having the same rate of; 94; 95; 96; 97; 98; 99 or 100%. Sequence identity is frequently measured in units of percent identity (or percent similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988; Higgins and Sharp, Gene, 73: 237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., Appl. Biosci., 8: 155-165, 1992; and Pearson et al., Meth. Mol. Biol., 24: 307- 31, 1994. ing. Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed discussion of sequence alignment methods and homology calculations. For example, alignment comparisons can be performed using the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) (Internet Program® 1996, WR Pearson and University of Virginia, fasta 20 u 63 version 2.0 u 63, release date December 1996). ALIGN compares all sequences to each other, while LFASTA compares regions of local similarity. These alignment tools and their respective textbooks are available, for example, at the NCSA website on the Internet. Alternatively, for amino acid sequence comparisons longer than about 30 amino acids, Blast2 sequence functions can be used using the default BLOSUM62 matrix set to the default parameters (gap existence cost 11 and gap cost per residue 1) . When aligning short peptides (less than about 30 amino acids), alignment is performed using PAM30 matrix set to default parameters (open gap 9, penalty for extension gap 1) using Blast2 sequence function It should be. BLAST sequence comparison systems are available, for example, from the NCBI website; Altschul et al., J. Mol. Biol., 215: 403-410, 1990; Gish. & States, Nature Genet., 3: 266-272, 1993; Madden et al. Meth. Enzymol., 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997; and Zhang & Madden, Genome Res., 7: 649-. See also 656, 1997.

  The term "nucleic acid molecule" as used herein has its general meaning in the art and refers to a DNA or RNA molecule. However, the term refers to sequences comprising any of the known base analogs of DNA and RNA, such as 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (Carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyl Adenine, 1-Methyl pseudouracil, 1-Methylguanine, 1-Methylinosine, 2,2-Dimethylguanine, 2-Methyladenine, 2-Methylguanine, 3-Methylcytosine, 5-Methylcytosine, N6-Methyladenine 7-Methylguani 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, β-D-mannosylcoeosin, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, Uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxocine, pseudouracil, queocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, Uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosin, 2-thiocytosine, and sequences including but not limited to 2,6-diaminopurine.

  The term "transformation" as used herein refers to the introduction of "foreign" (i.e. external or extracellular) genes, DNA sequences or RNA sequences into a host cell, such that the host cell is introduced. Such genes or sequences will be expressed to produce the desired substance, typically the protein or enzyme encoded by the introduced gene or sequence. Host cells that receive and express DNA or RNA have been "transformed."

  Typically, transformation of malignant B cells with nucleic acid molecules uses viral vectors to transduce B cells. Examples of viral vectors include adenovirus-based vectors, adeno-associated virus (AAV) -based vectors, retrovirus vectors, retrovirus-adenovirus vectors, and vectors derived from herpes simplex virus (HSV), such as amplicon vectors, Such as, but not limited to, replication defective HSV and attenuated HSV (e.g., Krisky, Gene Ther. 5: 1517-30, 1998; Pfeifer, Annu. Rev. Genomics Hum. Genet. 2: 177-211, 2001). Reference, each of which is incorporated by reference in its entirety). In some embodiments, B cells are transduced with a retroviral vector, or a vector derived from a retrovirus. "Retroviruses" are envelopes that can infect animal cells and use reverse transcriptase to make DNA copies from their RNA genome at an early stage of infection, which is then typically integrated into the host genome It is an RNA virus having An example of a retroviral vector is a vector derived from the Moloney murine leukemia virus (MLV), a murine stem cell virus based retrovirus that provides long-term stable expression in target cells such as hematopoietic progenitor cells and their differentiated progeny Vectors (see, eg, Hawley et al., PNAS USA 93: 10297-10302, 1996; Keller et al., Blood 92: 877-887, 1998), hybrid vectors (eg, Choi, et al., Stem Cells 19) And complex retrovirus-derived vectors such as lentiviral vectors. As noted above, in some embodiments, lentiviral vectors are used. The term "lentivirus" refers to a complex retrovirus that can infect both dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV types 1 and 2), Visna maedi virus, Goat arthritis enteritis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency Viruses (BIV) and simian immunodeficiency virus (SIV). Lentiviral vectors can be derived from any one or more of these lentiviruses (e.g., Evans et al., Hum Gene Ther. 10: 1479-1489, 1999; Case et al., PNAS USA 96: 2988-). 2993, 1999; Uchida et al., PNAS USA 95: 11939-11944, 1998; Miyoshi et al., Science 283: 682-686, 1999; Sutton et al., J Virol 72: 5781-5788, 1998; and Frecha et al., Blood. 112: 4843-52, 2008, each of which is incorporated by reference in its entirety). In some embodiments, the retroviral vector comprises specific minimal sequences derived from a lentiviral genome, such as the HIV genome or the SIV genome. The lentiviral genome is typically in the 5 'terminal repeat (LTR) region, the gag gene, the pol gene, the env gene, auxiliary genes (eg nef, vif, vpr, vpu, tat, rev) and the 3' LTR region It consists of The viral LTR is divided into three regions called U3, R (repetitive sequences) and U5. The U3 region contains enhancer and promoter sequences, the U5 region contains a polyadenylation signal, and the R region separates the U3 and U5 regions. The transcribed R region sequences appear at both the 5 'and 3' ends of the viral RNA (e.g., "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, 2000); O Narayan, J. Gen. Virology. 70: 1617-1639, 1989; Fields et al., Fundamental Virology Raven Press., 1990; Miyoshi et al., J Virol. 72: 8150-7, 1998; No. 6,013,516, each of which is incorporated by reference in its entirety). The lentiviral vector may contain any one or more of these sequences of the lentiviral genome to modulate the activity of the vector as desired, or they are to reduce the pathological effects of lentiviral replication Or may contain deletions, insertions, substitutions, or mutations in one or more of these sequences in order to limit the lentiviral vector to a single infection. Typically, minimal retroviral vectors will comprise specific 5 'LTR and 3' LTR sequences, one or more genes of interest (to be expressed in target cells), one or more promoters, and RNA. Containing cis-acting sequences for packaging. Other regulatory sequences may also be included as described herein and known in the art. Viral vectors are typically cloned into plasmids that can be transfected into packaging cell lines, such as eukaryotic cells (eg, 293-HEK), and also sequences that are typically useful for plasmid replication in bacteria. Also includes. Typically, the nucleic acid molecule of interest is located between the 5 'LTR sequence and the 3' LTR sequence. In addition, the nucleic acid molecule of interest is preferably, for example, a transcriptional regulatory sequence, such as a promoter and / or an enhancer, to regulate expression of the gene of interest in a particular manner, once the gene has been incorporated into target cells. It is functionally related to other gene sequences. In some embodiments, useful transcriptional regulatory sequences are those that are highly regulated for activity both temporally and spatially. In some embodiments, viral vectors such as retroviral vectors use one or more heterologous promoters, enhancers, or both. In some embodiments, U3 sequences derived from the 5 'LTR of a retrovirus or lentivirus may be substituted in the viral construct with a promoter sequence or an enhancer sequence. In some embodiments, an "internal" promoter / enhancer located between the 5 'LTR and 3' LTR sequences of the viral vector is used and operably linked to the gene of interest. "Functional relationship" and "operably linked" mean that the gene is in the correct position and orientation with respect to the promoter and / or enhancer, but is not limited to this, thus expression of the gene can be It will occur when the enhancer comes in contact with appropriate regulatory molecules. Any which modulates (eg, increases, decreases) expression of the viral RNA genome in the packaging cell line, or modulates expression of the selected gene of interest in infected target cells, or both An enhancer / promoter combination may be used. A promoter is an expression control sequence formed by a DNA sequence that allows RNA polymerase binding and transcription to occur. The promoter is located upstream (5 ') of the start codon of the selected gene of interest (typically within about 100 to 1000 bp) and the coding polynucleotide sequence to which they are operably linked Untranslated sequences that control transcription and translation of The promoter may be inducible or constitutive. Inducible promoters initiate increased levels of transcription from DNA under the control of their promoters in response to some changes in culture conditions such as temperature changes. Various promoters are known in the art, as well as methods for operably linking the promoter to a polynucleotide coding sequence. Both natural promoter sequences and many heterologous promoters can be used to direct expression of the selected gene of interest. In some embodiments, the promoter is a heterologous promoter. Because they generally allow more transcription and higher yields of the desired protein as compared to the native promoter. In some embodiments, the promoter is selected among heterologous viral promoters. Examples of such promoters include polyoma virus, fowlpox virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and viruses such as simian virus 40 (SV40) And a promoter obtained from the genome of In some embodiments, the promoter is a heterologous mammalian promoter, such as an actin promoter, an immunoglobulin promoter, a heat shock promoter, or a promoter associated with the native sequence of the gene of interest. Typically, the promoter is compatible with target cells such as resting B lymphocytes, activated B lymphocytes, plasma B cells, or memory B cells. Non-limiting examples of constitutive promoters that can be used include promoters for ubiquitin, CMV promoter (see, eg, Karasuyama et al., J. Exp. Med. 169: 13, 1989), β-actin (eg, See, Gunning et al., PNAS USA 84: 4831-4835, 1987), and pgk promoters (eg, Adra et al., Gene 60: 65-74, 1987; Singer-Sam et al., Gene 32: 409-). 417, 1984; and Dobson et al., Nucleic Acids Res. 10: 2635-2637, 1982, each of which is incorporated by reference). Non-limiting examples of tissue specific promoters include the Ick promoter (e.g., Garvin et al., Mol. Cell Biol. 8: 3058-3064, 1988; and Takadera et al., Mol. Cell Biol. 9: 2173). -2180, 1989), myogenin promoter (Yee et al., Genes and Development 7: 1277-1289. 1993), and thy1 (see, for example, Gundersen et al., Gene 113: 207-214, 1992) Can be mentioned. Further examples of promoters include the ubiquitin-C promoter, human mu heavy chain promoter or Ig heavy chain promoter (eg MH-b12), and human kappa light chain promoter or Ig light chain promoter (eg EEK-b12) (which is B Functional in lymphocytes). In some embodiments, the promoter can be selected to allow inducible expression of the gene. Many systems for inducible expression are known in the art, including tetracycline responsive systems and lac operator-repressor systems. It is also contemplated that combinations of promoters can be used to obtain expression of the desired gene of interest. One skilled in the art will be able to select a promoter based on the desired gene expression pattern in the organism of interest and / or in the target cell.

  In some embodiments, the viral vectors provided herein (eg, retrovirus, lentivirus) are one or more selected viral glycoproteins, mainly to target selected cell types. Or "pseudotyped" using envelope proteins. Pseudotyping generally involves the incorporation of one or more heterologous viral glycoproteins onto cell surface viral particles, often allowing the viral particles to infect selected cells different from their normal target cells. Point to. "Heterologous" sequences are derived from viruses other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein coding region of the viral vector is genetically altered, such as by deletion, to prevent expression of its own glycoprotein. For illustrative purposes only, the envelope glycoproteins gp41 and / or gp120 derived from HIV-derived lentiviral vectors are typically deleted prior to pseudotyping with heterologous viral glycoproteins . In some embodiments, the viral vector is pseudotyped with heterologous viral glycoproteins that target plasma cells such as B lymphocytes. In some embodiments, viral glycoproteins allow selective infection or transduction of resting or quiescent B lymphocytes. In some embodiments, viral glycoproteins allow selective infection of activated B lymphocytes. In some embodiments, viral glycoproteins allow infection or transduction of both quiescent and activated B lymphocytes. In some embodiments, viral glycoproteins allow infection of B cell chronic lymphocytic leukemia cells. In some embodiments, the heterologous viral glycoprotein is derived from a measles virus, for example, a glycoprotein of measles virus of strain Edmonton. In some embodiments, the measles virus glycoprotein hemagglutinin (H), fusion protein (F), or both are pseudotyped (eg, Frecha et al., Blood. 112: 4843-52, 2008; And Frecha et al., Blood. 114: 3173-80, 2009, each of which is incorporated by reference in its entirety). In some embodiments, viral vectors are embedded, such as one or more variable regions (eg, heavy chain variable region and light chain variable region), which serve to target the vector to a particular cell type It contains an antibody binding domain.

  For example, Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, NY (1997)). And digestion with restriction endonucleases, as described in “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)), ligation, transformation, plasmid purification, PCR amplification, and It can be accomplished using any suitable genetic engineering manipulation technique known in the art, including but not limited to standard techniques of DNA sequencing.

Typically, B cells can be transduced using the viral vectors described herein using any of a variety of known techniques in the art (eg, Science 12 Apr. 1996 272: Blood 2009, 99: 2342-2350; Blood 2009, 113: 1422-1431; Blood 2009 Oct. 8; 114 (15): 3173-80; Blood. 2003; 101 (6): 2167-2174; See Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009)). For example, PBMC, B lymphocytes or T lymphocytes from a donor, and other B cell cancer cells, such as B-CLL cells, are isolated and serum free or 10% FCS and penicillin / streptomycin and / or other The cells may be cultured in RPMI 1640 (Gibco BRL Invitrogen, Auckland, New Zealand) or any other suitable medium, either supplemented with the appropriate supplements of In some embodiments, cells are seeded at 10 ⁵ cells in a 48-well plate, and the concentrated vector is at various doses that can be routinely optimized by those skilled in the art using conventional methods. Added. In some embodiments, B cells are RPMI supplemented with 10% type AB serum, 5% FCS, 50 ng / ml rhSCF, 10 ng / ml rhIL-15, and 5 ng / ml rhIL-2; If necessary, it can be transferred to MS5 cell monolayers in periodically refreshed medium. Other suitable media and supplements may also be used, if desired, as would be recognized by one skilled in the art.

  In some embodiments, as described herein, the B cells comprise a nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least a portion of the B cells. Contact with retroviral vector. In some embodiments, as described herein, the B cell comprises a nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least 20% of the B cells. Contact with retroviral vector. In some embodiments, the B cells comprise at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80% of the B cells. , 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% are contacted with the vector as described herein under conditions sufficient to transduce.

  Any of a variety of culture media may be used in the methods of the present invention as would be known to one skilled in the art (see, eg, Current Protocols in Cell Culture, 2000-2009 by John Willie and Sons, Inc. ). In some embodiments, media for use in the methods described herein include, but are not limited to, Iscoff's modified Dulbecco's medium (with or without fetal calf serum or other suitable serum) I will not. Exemplary media also include, but are not limited to, RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15, and X-Vivo20. In some embodiments, the culture medium may include a surfactant, an antibody, plasmanate, or a reducing agent (eg, N-acetyl-cysteine, 2-mercaptoethanol), or one or more antibiotics. Good. In some embodiments, IL-6, soluble CD40L, and crosslinking enhancers may also be used.

  In some embodiments, cells are cultured for 1 to 7 days. In some embodiments, cells are cultured for 7, 14, 21 days or more. Thus, cells under appropriate conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, The culture may be performed for 21, 22, 23, 24, 25, 26, 27, 28, 29, or more days. The cells may be replated, media and supplements may be added or replaced as needed using techniques known in the art. In some embodiments, the transduced B cells comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% of the cells. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% express the transgene of interest It can be cultured under conditions and for a sufficient time.

  Thus, a group of malignant B cells transformed with nucleic acid molecules encoding a CD70 polypeptide are particularly suitable for inducing anti-tumor vaccination. In particular, the cell population will promote T cell mediated immunity to tumor cells.

The malignant cells transformed according to the present invention may be administered alone, or as a vaccine composition in combination with a diluent and / or other components such as cytokines or cells. Briefly, the vaccine compositions of the present invention are a group of transformed malignant B cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; It may include oxidizing agents; chelating agents such as EDTA or glutathione; adjuvants such as aluminum hydroxide; and preservatives. The compositions of the invention are preferably formulated for intravenous administration. The vaccine compositions of the invention may be administered in a manner appropriate to the B cell malignancy to be treated. The dose and frequency of administration will be determined by the condition of the patient and such factors as the type and severity of the patient's malignancy, but the appropriate dose may be determined by clinical trials. Where a "therapeutically effective amount", an "anti-tumor effective amount", a "tumor suppression effective amount" or a "therapeutic amount" is indicated, the exact amount of the composition of the invention to be administered is: age, weight, tumor It can be determined by a physician in consideration of individual differences in size, degree of infection or metastasis, and condition of a patient (subject). Generally, a vaccine composition comprising B cells as described herein has a dose of 10 ⁴ to 10 ⁷ cells / kg of body weight, preferably 10 ⁵ to 10 ⁶ cells / kg of body weight. It can be administered (including all integer values within such range). The vaccine composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques generally known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by medical experts by monitoring the patient for signs of disease and adjusting treatment accordingly. Typically, in a test of related adoptive immunotherapy, antigen-specific T cells are administered to patients in approximately 2 × 10 ⁹ to 2 × 10 ¹¹ cells (eg, US Pat. No. 5,057,423) No.)). In some embodiments, a smaller number of inventive transduced B cells in the range of 10 ⁶ / kg (10 ⁶ to 10 ¹¹ per patient) may be administered. In some embodiments, B cells are administered to a subject at 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² cells. The vaccine composition may be administered multiple times at doses within these ranges. Administration of the vaccine composition to a subject may be performed by any convenient method, such as by aerosol inhalation, injection, digestion, infusion, implantation, or by implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intrathecal, intramuscular, intravenous (iv) injection, or intraperitoneally. In some embodiments, a vaccine composition of the invention is administered to a patient by intradermal or subcutaneous injection. In some embodiments, vaccine compositions as described herein are preferably administered by intravenous injection. The vaccine composition can be injected directly into the tumor or lymph node.

  In some embodiments, the vaccine composition comprises an agent such as an antiviral agent, chemotherapy, radiation, an immunosuppressant such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, an antibody, or other immunoremoving agent Including, but not limited to, treatment with CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytotoxins, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, radiation, etc. The subject is administered with (eg, prior to, simultaneously with, or after) any number of related treatments that are not administered. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70 S6 kinase, which is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993; In some embodiments, a vaccine composition of the invention is administered to a patient with (eg, prior to, simultaneously with, or after) bone marrow transplantation, external beam radiotherapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH. It is administered. In some embodiments, a vaccine composition of the invention is administered after B cell ablation therapy, such as an agent that reacts with CD20. As an example of an antibody with specificity for CD20, "C2B8" (US Patent No. 5,736,137), now called "Rituximab" ("Rituxan®"), is hereby expressly incorporated by reference. Incorporated), a chimeric pan-B antibody targeting CD20; an yttrium- [90] -labeled 2B8 mouse antibody designated “Y2B8”, or MX-DTPA for chelation to yttrium- [90] "Ibritsumomab tiuxetan" Zevaline®, which is a mouse IgG1 kappa mAb covalently linked to (US Patent No. 5,736,137, expressly incorporated herein by reference); a mouse also referred to as "Tositumomab" Radioactive 131 I to generate IgG2a “BI”, “1311-B1” antibodies Optionally labeled (iodine 131 tositumomab, BexalTM) (US Pat. No. 5,595,721, expressly incorporated herein by reference); mouse monoclonal antibody “1F5” (Press et al. Blood 69 (2): 584-591 (1987)) and variants thereof, such as "framework spliced" or humanized 1F5 (WO 03/002607, Leung, S .; ATCC deposit No. HB-96450); mouse 2H7 and chimeric 2H7 antibodies (US Pat. No. 5,677,180, expressly incorporated herein by reference); also known as humanized 2H7, ocrelizumab (PRO-70769); Ofatumumab (AR), a fully human IgG1 (huMax-CD20) against a novel epitope on CD20 La) (Jemab, Denmark; WO 2004/035607 (US application Ser. No. 10 / 687,799, expressly incorporated herein by reference)); a fully humanized and optimized IgG1 mAb to CD20 A20 antibody or variant thereof, such as a chimerized or humanized A20 antibody (cA20, hA20 respectively) (US Application No. 10 / 366,709), which is hereby incorporated by reference. Immunomedicine, Inc. clearly incorporated in the specification; monoclonal antibody L27, G28-2, 93-1 B3, B-CI, or NU-B2 (Valentine et al, Leukocyte classification available from Leukocyte Classification International Workshop) III (McMichael, Ed., P. 440, Oxford Univ. rsity Press (1987)). Furthermore, suitable antibodies include, for example, antibody GA101 (Obinetuzumab), a third generation humanized anti-CD20 antibody from Biogen Idec / Genentech / Roche. Furthermore, it is a second-generation humanized antibody that is specific for glycosylation-optimized humanized CD20 antibody, BLX-301 from Biorex Therapeutics, or CD20 from Immunomedics. Composed of veltuzumab (hA20) or a dimer of veltuzumab bivalent anti-CD20 mAb and a pair of stabilized Fabs derived from miratuzumab, an anti-CD20 mAb that has been enhanced using InNexus Dynamic Cross-Linking Technology from Inexus Biotechnology Suitably, DXL 625, a derivative of veltuzumab, such as the bispecific hexavalent antibody of IBC Pharmaceuticals (Immunomedics), both of which are humanised anti-CD20 antibodies. Further suitable antibodies are BM-ca (humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38 (2): 335-44)), C2H7 (CD20 specific for CD20) Antibody (Mol Immunol. 2008 May; 45 (10): 2861-8)), PRO 131 921 (a third generation antibody specific for CD 20 developed by Genentech), Lady Tax (Dr. Reddy's Laboratories, Inc.) (Biosimilar form of rituximab), PBO-326 (biosimilar form of rituximab developed by Probiomed), biosimilar form of rituximab developed by Zenotech, TL-011 (by Teba) The biosimilar version of rituximab developed, CMAB 304 (Litz developed by Shanghai CP Guojian) Biosimilar form of shimab), Biosimilar form of rituximab developed by Sand Company (Novartis), SAIT-101 (Biosimilar form of rituximab developed by Samsung Biologics, Inc.), Biosimilar form of rituximab, developed by Intus Biopharmaceutics, CT-P10 (biosimilar form of rituximab developed by Celtrion), Biosimilar form of rituximab, developed by Biocad, ub Rituximab (LFB-R603, a mAb targeted by CD20 targeting gene transfer developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (Pfizer Inc.) Therefore, it is assumed that the biosimilar form of rituximab is developed), Lymphomun (Bi-20, trifunctional anti-CD20 antibody and anti-CD3 antibody developed by Trion Pharma), developed by Natco Pharma Biosimilar form of rituximab, Biosimilar form of rituximab developed by I-Bio, Biosimilar form of rituximab developed by Gedion Richter / Stada, Rituximab developed by Curaxys, Inc. Biosimilar form, biosimilar form of rituximab developed by Coherus Biosciences / Daiichi Sankyo, biosimilar form of rituximab developed by BioXpress, BT-D 004 (developed by Protheon Biosimilar form of rituximab), P-052 (biosimilar form of rituximab developed by Aprogen), biosimilar form of ofatumumab developed by BioXpress, MG-1106 (biosimilar form of rituximab developed by Green Cross) , IBI-301 (humanized monoclonal antibody to CD20 developed by Innovent Biologics), BVX-20 (humanized mAb to CD20 developed by Vaxnex), 20-C2-2b (by Immunomedics) Developed CD20 and a bispecific mAb-IFNα targeting human leukocyte antigen-DR (HLA-DR), MEDI-552 (developed by Medeumune / AstraZeneca), NeoRx (now Ponyard)・ Fermouth Anti-CD20 antibody / streptavidin conjugate developed by Tikalics Inc., a second generation anti-CD20 human antibody developed by Fabril (now MMRGlobal), a CD20 fragment developed by Tolbion / Emergent BioSolutions Antibodies specific for TRU-015, as well as other preclinical approaches by various companies and institutions.

  The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

The reduced cytolytic and proliferative responses to the patient's autologous CD70-deficient lymphoblastoid cell line (LCL) are restored by the expression of CD70 in LCL. (A) T of two control individuals (Control 1 and Control 2) and CD70 deficient patients (patients) against autologous LCL, as measured by Cr ⁵¹ release at the indicated effector to target (E: T) ratio Cellular cytotoxic response. T cells were cocultured with autologous LCL for 4 weeks prior to testing. Each one represents a representative of two independent experiments. Standard deviation is measured three times. (B) FACS histogram of CD70 expression on LCL of patients infected with empty lentiviral vector (empty vector) or vector containing cDNA encoding wild type CD70 (pLenti vector) . Each one is representative of 5 independent experiments. (C) Patient-derived T cells are co-infected with autologous LCL infected with empty lentiviral vector (empty vector) or vector containing cDNA encoding wild type CD70 (pLenti-CD70) Same as (a) except cultured. Standard deviation is measured twice. (D) Patients who were cocultured for 8 days in the presence of irradiated CD70 deficient LCL (empty vector) or LCL restored expression of CD70 (pLenti-CD70), HLA-A matched (control 1 HLA) -A01 ^* ) or not matched (Control 2 HLA-A02 ^* and Control 3 HLA-A03 ^* ) Proliferation of T cells derived from PBMC of a healthy control. Representative dot plots of the expression of CD25 was gated on dilution and CD3 ⁺ cells violet dye. Each one is representative of 5 independent experiments. One healthy control (control) and CD70 deficient patients (patients) using irradiated autologous CD70 expressing LCL cells (empty CRISPR or pLenti-CD70) or CD70 deficient LCL cells (CD70- CRISPR or empty vector) 2.) Analysis of the cytolytic activity of T cells grown for 8 to 15 days from PBMCs. The cytolytic activity of T cells is then compared to 0, 4, to a mixture of autologous CD70 expressing LCL cells (blue) or CD70 deficient LCL cells (red) as target cells with effector to target ratio of 1: 1. And tested for 12 hours. Residual target cells were assessed by FACS analysis. Data are shown as the ratio of cells normalized to the ratio at time point 0.

EXAMPLES Materials and Methods Ethics. Informed consent was obtained from the donor, the patient, and the patient's family. The test and protocol are in accordance with the 1975 Declaration of Helsinki and local laws and ethics guidelines of Comite de Protection des Personnes de l'Ile de France II (Hopital Necker-Enfants Malade, Paris).

  patient. Individuals with infectious mononucleosis were diagnosed with tonsil biopsies and confirmed using anti-VCA and anti-EBNA IgM positive serum tests. The main clinical phenotype of patients with ITK and CD27 deficiency was EBV-driven lymphoproliferation. Homozygous c. 113T> C, p. A patient carrier of L38P was born from a Kurdish-born consanguineous parent, suffered recurrent respiratory infection with low immunoglobulin levels, and developed EBV-associated lymphoma. Her younger brother and maternal uncle also carry the same harmful allele and show the same clinical phenotype. Homozygous c. 85C> T, p. The two patient carriers of R29C are born from blood relatives of Algerian origin. Abnormalities in the ITK gene were identified by WES, and the identified mutations were further verified by direct DNA sequencing. Homozygous c. 329 G> A, p. The W110X patient carrier was born from a consanguineous parent of Tunisian origin and suffered from lymphoproliferative disease driven by recurrent EBV. CD27 abnormalities were diagnosed based on the loss of expression of CD27 on B cells. The four exons of CD27 were further sequenced.

Exome sequencing and analysis. Exome acquisition was performed using Illumina's TruSeq exome enrichment kit and sequencing of 100 bp paired end reads with Illumina's HiSeq according to the manufacturer's protocol. A sequence of about 10 Gb was obtained for each subject, whereby 90% of the coding bases of the exome defined by the Common Coding Sequence (CCDS) project were covered by at least 10 readings. Adapter sequences and quality trimming leads are removed using the Fastx toolkit (http: // hannonlab. Cshl. Edu / fastx_toolkit /) and then using a custom script, then both mates are present Make sure to use only lead pairs that have The leads were aligned to hg19 using BWA 31 and duplicate reads were marked using Picard (http://picard.sourceforge.net/) and excluded from downstream analysis. Single nucleotide polymorphisms (SNVs) and short insertions and deletions (indels) override base alignment quality (BAQ) adjustments using sam tool (http://samtools.sourceforge.net/) pileup and varFilter 32 Were determined and then at least 20% of the leads were filtered for quality in order to require variant call support. Variants are annotated using both ANNOVAR 33 and custom scripts, whether they affect the protein coding sequence, and if they have been previously sequenced at the public database of exomes and at our facility It was identified whether it was found in 7566 exomes. The CD70 mutation (19: 6586078 G / A) identified in the patient, ie the homozygous nonsense mutation c. 535 C> T p. Arg179X is a 1.48 x 10 ^-5 (2 alleles out of 134 542) database of the exome aggregation consortium (ExAC) (http://exac.broadinstitute.org) and the database of our laboratory. ) Were present as heterozygous mutations. Other available public databases of exome have not been reported (dbSNP, 1000 genomes, NHLBI exome sequencing project (http://evs.gs.washington.edu/EVS/)).

  DNA sequencing. Genomic DNA from peripheral blood cells of patients, their parents, and other family members was isolated according to standard methods. Genomic DNA was amplified using oligonucleotide primers within introns flanking exon 3 of CD70, exon 1 of ITK, and exon 3 of CD27. PCR products are amplified using high fidelity Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer's recommendations, purified using the QIAquick gel extraction kit (QIAGEN), and ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Sequencing was performed according to the manufacturer's recommendations using a kit (Perkin Elmer) and analyzed using a 3500 × L Genetic Analyzer (Applied Biosystems). All recovered sequences were analyzed using 4peaks software (version 1.7.2; A. Griekspoor and T. Groothuis, http://mekentosj.com/4peaks/) or DNADynamo (Blue Tractor software) .

  Immunochemical test. Staining and in situ hybridization were performed on a Leica Bond Max autostainer (Leica Biosystems). The presence of EBV was demonstrated by in situ hybridization for small RNAs encoding regions 1 and 2 (EBER). Antibody used and dilution ratio: anti-CD70 antibody (rabbit polyclonal, 1/200, Abgent), anti-PAX5 antibody (clone DAK-PAX5, 1/30, Dako), anti-CD27 antibody (clone 137B4, 1) / 50, Abcam's), anti-CD8 antibody (clone C8 / 144B, 1/200, Dako), and anti-LMP1 antibody (clone CS. 1-4, 1/200, Dako).

  Cell culture fluid. Whole blood samples were collected from patients and control donors. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Ficoll-pack density gradient (Linhoprep, Proteogenics) using standard procedures. Proliferation of T cell blasts was determined by adding Panserin 401 (Panbiotech) supplemented with 5% human male AB serum (BioWest), penicillin (100 U / ml), and streptomycin (100 μg / ml). It was obtained by incubating PBMC for 72 hours with phytohemagglutinin (PHA) (2.5 μg / ml, Sigma-Aldrich). After 3 days dead cells were removed by Ficoll-pack density gradient and blasts were maintained in culture medium containing IL-2 (100 or 1000 UI / ml). Burkitt's lymphoma large cell line obtained from ATCC and mouse thymoma L1210 were supplemented with heat-inactivated 10% fetal bovine serum (Gibco), penicillin (100 U / ml) and streptomycin (100 μg / ml) The cells were cultured in RPMI 1640 Glutamax medium (Invitrogen). Cells did not contain mycoplasma and were routinely tested for mycoplasma contamination.

EBV transformed LCL and EBV specific T cell lines. A lymphoblastoid cell line (LCL) transformed by EBV was made from fresh or frozen PBMCs of patient and control donors. PBMC were incubated with the supernatant from B95-8 cells in the presence of 1 μg / ml cyclosporin A (Sigma Aldrich) ⁴⁹ as described previously. EBV-specific T cell lines were generated from patient and control healthy donors. PBMCs were co-cultured with autologous LCL irradiated with 40 Gy at an effector to stimulator (E / S) ratio of 40: 1. After 9-14 days, viable cells were stimulated for 5-7 days with autologous LCL irradiated with 40 Gy at a 4: 1 E / S ratio. It was then analyzed for cytotoxicity.

  Gene expression analysis. Total mRNA was extracted from EBV-B cell lines and T cell lines of patient and control donors were activated with Pure Link RNA Mini kit (Life Technologies) and treated with DNaseI. Reverse transcription was performed using the SuperScript II first strand synthesis system (Life Technologies). Real-time quantitative PCR for CD70 and actin was performed in triplicate using the ViiA 7 real-time PCR system (Life Technologies), SYBR Select Master Mix (Life Technologies).

Cell cytotoxicity assay. The cytotoxic activity of CTL was assessed using a standard 4 hour Cr ⁵¹ release assay. Briefly, target cells of 1 × 10 ⁵ cells labeled with Cr ⁵¹ were incubated with effector cells for 4 hours at 37 ° C. at the indicated effector to target (E: T) ratio. Then, 30 μl of the supernatant was collected and counted on a MicroBeta Trilux and JET Counter (Perkin Elmer) using 180 μl OptiPhase Supermix cocktail (Perkin Elmer). The dissolution ratio (%) was calculated according to the formula: 100 × (experimental release-spontaneous release) / (maximum release-spontaneous release). Cytotoxic activity of T cells grown for 8 days from PBMCs was also labeled with LCL and CFSE expressing CD70 labeled with autologous cell trace violet at a 1: 1 effector to target ratio The CD70-deficient LCL was evaluated over 0, 4 and 12 hours in the co-culture solution. Residual target cells were assessed by FACS analysis. Data are shown as the ratio of cells normalized to the ratio at time point 0. Degranulation of CD8 ⁺ T cells was determined by analysis of the expression of CD107 / LAMP, a marker of exocytosis of lytic granules as previously described ⁸ .

  Stimulation and proliferation assay. The PHA-stimulated T cells were washed and cultured for 72 hours with IL-2 to synchronize the cells. The PHA-stimulated T cells or PBMC were then cultured for 4-8 days in complete medium alone or in the presence of anti-CD3 antibody / anti-CD28 antibody coated beads (Invitrogen). In co-culture proliferation experiments, anti-CD27 antibody (Clone LG. 3A10 manufactured by EBiosciences or O323 manufactured by BioLegend) (10 μg / ml) or ibrutinib (5 to 500 nM) was added to T cells or PBMC stimulated by PHA. (Serekchem) with or without 45 μg irradiated with 45 Gy, pre-incubated for 1 hour with or without 1 μg / ml anti-CD3 antibody (OKT3) Co-culture was performed in the presence of homologous LCL. Cells were co-cultured with a ratio of 1 T cell or PBMC to 10 LCL (1/10 ratio). Cell proliferation by labeling T cells or PBMCs with CellTrace violet dye (Violet Proliferation Dye 450, BD Biosciences) prior to stimulation or co-culture with LCL according to the manufacturer's instructions. It was monitored. After 8 days of culture, cells were harvested and CellTrace violet dye dilution was assessed by flow cytometry.

  Flow cytometry. Cell staining and flow cytometry based phenotypic analysis of PBMC, blast T cells, and cell lines was performed according to standard flow cytometry methods. The following mAbs (whose identification numbers are shown in parentheses), fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-cyanine 5 (PE-Cy5), phycoerythrin-cyanine 5.5 (PE-Cy5.5) Phycoerythrin-cyanine 7 (PE-Cy7), peridinin-chlorophyll (PerCP), peridinin-chlorophyll-cyanine 5.5 (PerCP-Cy 5.5), allophycocyanin (APC), allophycocyanin-cyanine 7 (APC-Cy7) , Allophycocyanin-Vio7 (APC-Vio7), alexa-700, brilliant violet 421 (BV 421), brilliant violet 510 (BV 510), brilliant violet 650 (BV 650), brili Anti-CD3 antibody (UCHT1), anti-CD4 antibody (OKT4), anti-CD8 antibody (RPA-T8), anti-CD14 antibody (M5E2), anti-CD19 antibody (HIB19), anti-CD25 antibody (BC96) Anti-CD27 antibody (LG.3A10, M-T271, O323), anti-CD28 antibody (CD28.2), anti-CD31 antibody (WM59), anti-CD56 antibody (HCD56), anti-CD70 antibody (113-16), anti-CD107a / B antibody (H4A3 / HAB4), anti-CD161 antibody (HP-3G10), anti-CD197 antibody (G043H7), anti-TCR V 7.2 antibody (3C10) (all purchased from Sony Biotechnology), anti-CD16 antibody (3G8) ), Anti-CD45RA antibody (HI100), anti-CD45RO antibody (UC) HL1), anti-CD95 antibody (DX2), anti-TCRαβ antibody, IgD (IA6-2), IgM (G20-127) (BD Biosciences), and anti-CD21 antibody (BL13), anti-TCRγδ antibody, anti-TCRVα24 It was conjugated to antibody (C15), anti-TCRVβ (manufactured by Beckman Coulter, Inc.). Binding of CD27 to cells expressing CD70 was evaluated by incubation with human CD27-muIg / biotin fusion protein (Ancel) according to the manufacturer's protocol and standard flow cytometry. Binding of CD27-muIg to cells was detected using streptavidin conjugated to PE (BD Biosciences). All data were collected on a FACS-CantoII or LSR-Fortessa cytometer (both from BD Biosciences) and analyzed using Flowjo version 10.0.8r1 software (Tree Star).

Calcium influx analysis. The Ca ²⁺ response was assessed by flow cytometry as previously described ⁸ . Briefly, cells were loaded with 5 μM Indo-1AM (Molecular Probes) in the presence or absence of 1 × PowerLoadTM (Thermo Fisher Scientific) and 50 nM ibrutinib and washed. , Cross-linked with 10 μg / ml F (ab ') ₂ rabbit-anti-mouse IgG, which is incubated with anti-CD4-APC mAb and anti-CD8-PE mAb and adsorbs only to humans in the presence or absence of 50 nM ibrutinib Stimulated with 5 μg / ml anti-CD3 antibody (OKT3), anti-CD27 antibody (clone LG.3A10, LG.7F9 (E Biosciences) or O323) or anti-CD28 antibody (CD28.2) Incubated with ionomycin (5 μM). Cells were analyzed using a FACS ARIA flow cytometer (BD Biosciences). The change in intracellular calcium concentration was determined from the indo-1 emission peak (indo-blue) at 485 nm of the unbound dye, and the indo-1 emission peak (indo-violet) at 405 nm when the indo-1 molecule was bound to calcium. Quantified by the shift to Ca ²⁺ influx data was obtained using kinetic analysis of the FlowJo software package (Tree Star). Intracellular Ca ²⁺ levels correspond to a normalized ratio of indo-1 emission peaks at 405 nm / 485 nm.

  Production and detection of cytokines. For intracellular staining of cytokines overnight with irradiated LCL cells previously incubated with 1 μg / ml OKT3 and in the presence of Brefeldin A (GolgiPlug, BD) It was overirritated. The cells were then fixed and permeabilized using the BD cytofix / cytoperm plus kit (BD Pharmingen) according to the manufacturer's instructions. The cells are then treated with PE / Cy7-anti-TNF.alpha. Antibody (mouse IgG1; MAb11), APC-anti-IFN.gamma. Antibody (mouse IgG1, 4S.B3), and an isotype-matched monoclonal antibody purchased from Bio Legend. Label and analyze by flow cytometry. Cells were then harvested, washed, stained with BV 785-anti-CD3 mAb, BV 650-PE-anti-CD8 mAb, and BV 510-anti-CD4 mAb and analyzed by flow cytometry.

Detection of T cells specific for EBV. EBV-specific CD8 ⁺ T cells derived from PBMC are co-cultured with LCL irradiated with 45 Gy for 8-10 days, and BV 785-anti-CD3 monoclonal antibody, BV650-PE-anti-CD8 monoclonal antibody, and BV510- Detection was done using a mixture of unlabeled EBV HLA-A2: 01 Pro5® pentamer (Proimmune) mixed with R-PE Pro5® fluorotag in addition to the anti-CD4 monoclonal antibody. EBV HLA-A2: 01 Pro5® pentamer mixture is derived from EBV LMP2 protein and BMLF1 protein, FLYALLL (residues 356-364 from LMP2), CLGGLLTMV (residues 426-434 from LMP2), Or contains three different pentamers presenting GLCTLVAML (residues 259-267 from BMLF-1). After their isolation, the activated phenotype of EBV-specific T cells is characterized using PE / Cy7-anti-CD25 antibody and FITC-CD45RA antibody. All dyeings are carried out according to the manufacturer's instructions.

Cell transfection and CRISPR Cas9 genome editing. Full length cDNAs encoding wild type CD70 and CD70 R179X were obtained by RT-PCR from blasts of control donors and patients, respectively. The cDNA was confirmed by sequencing and a bicistronic lentiviral expression vector (pLenti7.3 / V5-TOPO, Invitrogen) encoding green fluorescent protein (GFP) as a reporter or an N-terminal Flag tagged expression vector (p3XFLAG-myc) -CMV-26 (Sigma Aldrich). Patients' LCL were transduced with pLenti7.3 / V5-TOPO or empty vector containing wild type CD70 as previously described ⁴ . The lenti CRISPR plasmid was a gift from Feng Zhang (Adgene, plasmid no. 49535). All sgRNAs were designed using the MIT CRISPR Design Tool (http://crispr.mit.edu). Three pairs of 24 bp forward (F) and reverse (R) oligonucleotides targeting different sequences within exon 3 of CD70 clone the sgRNA sequence to the BsmbI site within the reverse oligonucleotide It was synthesized (Eurogentech) with a 4 bp overhang to allow, and a 4 bp overhang containing PAM sequences in forward oligonucleotides. The synthesized oligonucleotide pairs were annealed, phosphorylated, ligated into linear vectors, and transformed into Stbl3 bacteria (Life Technologies). sgRNA insertion was confirmed by Sanger sequencing using sequencing primers. The lenti CRISPR plasmid was transduced by infection with LCL and transfected by electroporation with large cells using the Nepa 21 electroporator (Neppagene). LCL CD70 positive group and CD70 negative group were enriched by selection.

Immunoblot and pull down assays. Anti-CD27 antibody (monoclonal antibody LG.3A10) or anti-CD3 antibody (monoclonal antibody OKT3) (1 μg) cross-linking cells (5 × 10 ⁶ cells / ml) with rabbit-anti-mouse IgG (2 μg / ml) Stimulated for the time indicated by / ml). The cells were then subjected to ⁸ 1% NP40, 50 mM Tris (pH 8), 150 mM NaCl, 20 mM EDTA, 1 mM Na ₃ VO ₄ , 1 mM NaF, and a complete protease inhibitor cocktail (Roche) as previously described. Dissolved in. Protein concentration was quantified by BCA assay (Bio-Rad). 80 μg of proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). For the test, the expressi membrane was blocked with milk or BSA for 1 hour before 90 minutes incubation with primary antibody. The following mAb and rabbit polyclonal antibodies were used for the immunoblot: anti-ITK antibody (clone 2F12), anti-phosphorylated PLC-γ1 antibody (clone D6M9S), anti-phosphorylated ERK1 / 2 (clone D13.14.4E) And anti-phosphotyrosine antibody (clone PY-100) were purchased from Cell Signaling, and anti-FLAG antibody (clone M2) and rabbit polyclonal anti-ACTIN antibody were from Sigma. Membranes were then washed and incubated with anti-mouse or anti-rabbit secondary antibodies conjugated to HRP from Cell Signaling and GE Healthcare, respectively. Pierce ECL Western blot substrate was used for detection. The binding assay using GST protein, ^50, unstimulated cells was performed using lysates from cells stimulated with anti-CD3 antibody or pervanadate salt as has been previously outlined shown . A fusion protein of human FYN, ITK and the SH3 domain of LCK and GST was obtained by RT-PCR and subcloned into pGEX-2T vector (GE Healthcare).

result:
Clinical and Immunological Features We investigated an 8-year-old male of Egyptian origin with chronic EBV infection and lymphoma. His parents were cousins. He was diagnosed with EBV-positive nodular sclerosis Hodgkin's lymphoma at 3 years and 8 months. He received chemotherapy and radiation and achieved a complete remission and has not relapsed since then. At 4.5 years of age, she had recurrent fever, lymph node swelling, and hepatosplenomegaly, which was associated with high loading of EBV in the blood, and polyclonal proliferation of B cells in the lymph nodes and spleen. . Symptoms resolved after initiation of anti-CD20 antibody therapy, which reduced the viral load of EBV, but relapsed required repeated injection of anti-CD20 antibody and eventually allogeneic hematopoietic stem cell transplantation. No other viral and bacterial infections were noted.

  Immunological tests were conducted prior to anti-CD20 antibody therapy at the age of 5 years, including the reduction in the number of NK cells, iNKT cells, MAIT cells, and memory B cells and the reduction of serum IgM and IgA levels. However, a few anomalies became apparent. The proliferation of T cells upon stimulation with CD3, PHA and antigen was normal. Based on these observations, the high susceptibility to EBV infection associated with early-onset Hodgkin's lymphoma was a consequence of congenital abnormalities, which was suspected to cause immunodeficiency in patients.

Identification of Homozygous Nonsense Mutations in CD70 In order to identify the genetic basis of immunodeficiency in patients, we first tested known autosomal genetic abnormalities that cause susceptibility to EBV infection. Mutations in ITK, CD27, MAGT1 and CTPS1 were not detected at all, and their causal role was excluded. We next performed whole exome sequencing (WES). Because our parents are related, we focused our analysis on homozygous genetic mutations. Only one homozygous mutation was discovered that is deleterious and predicted to be localized to the CD70 gene. This mutation, which consists of a nonsense mutation (p.R179X, c.535C> T) in the third exon, has a frequency of 1.48 × 10 ⁻⁵ and is based on the public database of exome and the database of our laboratory Were present only as heterozygous mutations in ⁵ (see methods). The patient is c. Although homozygous for the 535 C> T mutation, his parents and two healthy sisters were identified as heterozygous carriers. At the protein level, p. The R179X mutation removes the last 15 amino acids at the C-terminus of CD70. Three-dimensional structural modeling of human CD70 protein using the three-dimensional structure of the TNF-like receptor Apo2L / TRAIL (pdb 1DG6 and pdb 1D0G) as a ^{template results} in the absence of the final internal beta chain H in the truncated CD70 ^R179X protein It was shown to be lost. In particular, in Apo2L this chain is involved in the contacts formed between the various subunits of the homotrimer ²⁸ . Importantly, CD70 is known to be a ligand for the CD27 molecule. Deficiency of CD27 causes high susceptibility to EBV infection and related lymphoproliferative disorders 5, ^{6, 20} . Thus, we determined that CD70 is a strong candidate gene underlying patient immunodeficiency.

Loss of expression and loss of binding of CD70 mutein to CD27 c. The 535 C> T mutation was detected by qPCR in patient-derived T cells stimulated by PHA and EBV-transformed B cells (also called lymphoblastoid cell line (LCL)). It had no effect on the amount of CD70 mRNA (data not shown). At the protein level, anti-CD70 antibodies failed to detect CD70 by flow cytometry on the cell surface of patient PHA-stimulated T cells. In contrast, CD70 expression could be detected on day 8 in the fraction of T cells stimulated by PHA from healthy donors. This proportion of CD70 ⁺ T cells increased in culture and by day 15 a high proportion of T cells expressed CD70. Similarly, CD70 was not detected in LCL derived from patients, whereas LCL derived from healthy donors expressed high levels of CD70 on their surface. Incomplete expression of CD70 was confirmed by analyzing the binding ability of CD70 ^{R179X to} CD27. The fusion protein (Fc-CD27), which contains the extracellular domain of CD27, failed to bind on the surface of patient-derived PHA-T cell blasts and LCL, while on control cells. Its binding of was detected. Similar results were obtained when wild type CD70 or CD70 ^R179X protein was transiently expressed in HEK-293T cells. The expression of CD70 and CD70 ^R179X in HEK-293T cells was further examined by Western blot using N-terminal FLAG tagged CD70 form. The mutant CD70 ^R179X was weakly expressed compared to wild type CD70. In summary, these results show that p. It is shown that the R179X mutation impairs its expression and has a deleterious effect on its ability to recognize its cognate ligand, CD27. Therefore, we conclude that CD70 deficiency mimics CD27 deficiency and thus may be responsible for the high susceptibility of the patient to EBV infection.

High levels of CD70 on activated B cells and B cells infected with EBV
In order to better understand the role of CD70 in anti-EBV immunity, we first analyzed the expression of CD70 in peripheral blood mononuclear cells (PBMCs) of several healthy donors. In humans, expression of CD70 has been shown to be restricted to some dendritic cell and B cell subsets, but CD27 is expressed on most T cells and memory B cells ^23,29 . In fact, expression of CD70 is barely detectable in CD4 and CD8 T cells, monocytes, dendritic cells and neutrophils, with the exception being a small fraction of B cells. This is in contrast to the high levels of CD27 on the surface of CD4 ⁺ and CD8 ⁺ T lymphocytes as well as a small fraction of B cells. Expression of CD70 on B cells was rapidly upregulated upon activation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono). Three days after stimulation, more than 80% of B cells expressed large amounts of CD70 in contrast to T cells. Similar to activated B cells, all EBV-transformed B cell lines tested by the inventor expressed high levels of CD70. These data suggest that CD70 expression is inducible in B cells during the course of EBV infection. To test this possibility, we analyzed the expression of CD70 in the tonsils of two individuals with infectious mononucleosis. Staining of CD70 was observed on large cells that were positive for the Epstein-Bar-encoded RNA (EBER) and for the EBV-specific marker PAX5 respectively and on B cells, corresponding to EBV-infected B cells. Was. In contrast, CD27 expression was not associated with PAX5 and EBER staining and accumulated in cells localized to the T cell area. These data indicate that CD70 expression is upregulated in activated B cells and B cells infected with EBV, but most T cells express high amounts of CD27 in resting state . Therefore, the CD27-CD70 interaction can occur during the course of EBV infection between CD27-expressing T cells and CD70-expressing B cells, and EBV-activated B cells Can play an important role in T cell control.

Against autologous B cells transformed with EBV, in the reduced cytolytic responses mice patient T cells, CD70 and CD27, in antigen-specific T cell response plays an important role especially in the antiviral response ^{26 , 30} . Thus, T cell responses to EBV can be impaired in the absence of CD70. To test this hypothesis, we analyzed T cell responses to EBV from PBMC of patients or healthy controls co-cultured with irradiated LCL. The ratio of CD4 ⁺ , CD8 ⁺ as well as the expression of activation markers were comparable when analyzed after co-cultivation for 4 to 5 weeks. After 4 weeks of co-culture, the cytotoxic response of the cells by T cells and IFN-γ production were evaluated against EBV transformed autologous B cell lines (FIG. 1a). Compared to control T cell cultures, the patient's T cells show markedly reduced cytotoxic activity and IFN-γ production, which indicates an impaired T cell response to EBV. In order to formally demonstrate the link between the deletion of CD70 on EBV transformed B cells and the defective T cell response to EBV transformed B cells we Reconstitution experiments were carried out by transducing LCLs of E. coli with a lentiviral vector (or an empty vector) containing a cDNA encoding wild type CD70, thereby providing LCL derived from control donors. Surface expression of CD70 was induced to a level equivalent to that expressed (FIG. 1b). In these conditions, the cytolytic response of the patient's T cells to autologous LCL expressing CD70 recovered to a similar size as control T cells (FIG. 1c). In summary, these results demonstrate that deletion of CD70 on patient B cells impairs the T cell lytic response to EBV-infected B cells. However, while the original cytotoxic function of the patient's T cells was preserved, ectopic CD70 expression on target cells did not increase T cell killing.

Impaired Proliferation of T Cells Specific to the Patient's EBV Next, we have shown that impaired T cytotoxicity and cytokine production against CD70 deficient autologous LCL in patients is specific to EBV specific T cell proliferation. The possibility of having been the result of a defect of. The patient's HLA genotyping showed that he was a carrier of HLA-A ^* 01 and no tetramer reagent was available to directly assess EBV-specific T cells. However, EBV-induced proliferating T cells in the patient's PBMCs, when cocultured with autologous LCL that restored expression of CD70, resulted in cell trace violet dye dilution and expression of the activation marker CD25. But were not detectable when cocultured with CD70 deficient parent LCL (FIG. 1 d, top panel). Proliferating T cells from healthy donors expressing HLA-A ^* 01 were also observed when co-cultured with LCL expressing CD70 of patient HLA-A ^* 01 , Was not observed when cocultured with LCL lacking HLA-A ^* 01 CD70 (upper middle panel). In contrast, in non-self conditions, only marginally proliferating T cells are detected in PBMC derived from healthy donor carriers of HLA-A ^* 02 and HLA-A ^* 03 alleles (Middle lower and lower panels).

Incomplete T Cell Proliferation of EBV-Specific T Cells in the Absence of CD70 on B Cells Next, to demonstrate the role of CD70 in anti-EBV immunity, CD70 deficient LCL can be used to generate healthy HLA -Derived from PBMCs of A ^* 02 individuals. ³¹ Most ^HLA-A * 02 individuals, EBV derived lytic cycle proteins BMLF1, to develop EBV-specific T cells to GLCTLVAMV peptide (also referred to as GLC epitope). CD70 deficient LCL was obtained by blunting expression of CD70 using nuclease Cas9 technology associated with CRISPR (clustered regularly spaced short palindromic repeats). LCL was transiently transfected with three different CRISPR-Cas9 constructs containing an RNA guide targeting exon 3 of CD70. After enrichment for CD70 negative cells by selection and selection, three stable CD70 deficient (CD70 KO) LCL cell lines were obtained (hereinafter referred to as CD70 KO- CRISPR 1, CD 70 KO- CRISPR 2, and CD 70 KO- CRISPR 3 Is called). These cell lines expressed comparable amounts of HLA-A ^* 02 molecules on their surface as compared to wild type parent LCL or LCL transfected with empty CRISPR-Cas9 constructs . We then used the health of HLA-A ^* 02 in co-culture experiments with irradiated autologous wild-type LCL (transfected with the empty CRISPR-Cas9 construct) or CD70KO- CRISPR1 LCL. The proliferation of donor EBV-specific T cells was examined. EBV-specific T cells were used on day 0 using a mixture of pentamers for GLC, CLG, and FLY epitopes, and after co-culture with wild-type LCL (empty CRISPR LCL) expressing CD70 It was detectable and the ratio increased 5 fold on day 8 of culture. Most of these cells expressed CD25 and were strongly proliferative (data not shown). In marked contrast, culture of autologous CD70 KO- CRISPR 1 LCL and T cells resulted in no proliferation of EBV-specific T cells. Similar results were obtained with PBMCs derived from two other healthy HLA-A ^* 02 donors (Control 2 and Control 3). No such proliferation was observed in T cells derived from EBV naive donors (Control 4). It was also observed that proliferation did not occur when HLA-A ^* 02 PBMC were co-cultured with both other CD70 deficient LCL, CD70 KO- CRISPR2 and CD70 KO- CRISPR3. Thus, our observations demonstrate that CD70 is an important factor required for the proliferation of EBV-specific T cells. In addition, control and patient T cells grown in the presence of autologous LCL expressing CD70 efficiently kill both LCL expressing CD70 and LCL lacking CD70. This indicates that the lack of CD70 itself does not eliminate T cell cytotoxic activity (FIG. 2). In contrast, T cells co-cultured with CD70 deficient autologous LCL show no killing activity, demonstrating that specific effector T cells require CD70 for proliferation.

CD70 on B Cells Provides a Costimulatory Signal Required for TCR-Mediated Proliferation Further advances our observations, proliferation in response to stimulation of TCR by B-LCL due to the lack of CD70. And to formally prove that activation is blunted, we have shown that after stimulation with irradiated LCL expressing or not expressing CD70 in the presence of anti-CD3 antibodies. T cell proliferative responses were analyzed. Importantly, activation of T cells in these conditions was not limited to HLA-A molecules expressed by irradiated LCL. At day 8, only a small percentage of T cells from patients or healthy donors are cocultured with CD70 deficient LCLs obtained by CRISPR or CD70 deficient LCLs derived from patients. Was detectable. In contrast, when PBMCs are co-cultured with LCL from patients with restored expression of CD70 or with LCL (empty CRISPR LCL) expressing CD70 from healthy donors Most of the T cells from both donors were able to divide. Most of these proliferating T cells expressed high levels of CD27 and CD25. Dividing T cells contained both CD4 ⁺ and CD8 ⁺ T cells, but the proportion of CD8 ⁺ cells was higher. These proliferating T cells exhibited an effector phenotype. Because most of them had the ability to secrete IFN-γα upon restimulation. The results were restored and expanded using T cells derived from different healthy donors with different HLA haplotypes, including HLA-A1, HLA-A2 and HLA-A3. Under these conditions, T cell proliferation is dependent on CD3 stimulation and in most individuals, most T cells achieve at least one division when cocultured with CD70 expressing LCL However, this was not achieved when cocultured with CD70 deficient LCL. Furthermore, this growth was substantially reduced by the addition of anti-CD27 antibody as expected. These results demonstrate that CD70, when expressed on B cells, provides an essential costimulatory signal for TCR-mediated T cell proliferation.

T cell proliferation mediated by CD27 is dependent on ITK As CD27, ITK and CD70 defects appear as phenocopy, we have found that CD27-CD70 and ITK are functional and molecular. It was temporarily established that it was possible to form various clusters. In order to test it, we first tested CD27 deficient and ITK deficient patients (one patient carrier of homozygous mutation p.W110X in CD27, homozygous mutation p.R29C in ITK) two patients with (1 patient ITK ^- and patient 2 ITK ^-), and one patient with homozygous mutations p.L38P in ITK (patient 3 ITK ^-) containing, newly described four CD70-dependent T cell proliferation of PBMCs derived from patients) was examined. By evaluating the possible effects of these mutations on the three-dimensional structural model of ITK, generated based on the three-dimensional structure of BTK (pdb: 1B55), as they have been previously reported ¹⁷ Similar to the R29H mutation, it has been shown that the PH domain can impair the ability to interact with phosphoinositol.

The W110X mutation abolished the surface expression of CD27 on patient PBMCs and T cell blasts. As expected, the proliferation of CD27 deficient T cell blasts in response to irradiated CD70 expressing B-LCL in the presence of anti-CD3 antibody was significantly reduced. The expression of ITK was strongly reduced in lysates from T cells of patients with R29C and L38P mutations. When examined, T cell proliferation of ITK deficient PBMC or T cell blasts in response to LCL expressing irradiated CD70 in the presence of anti-CD3 antibody was significantly reduced. Importantly, ITK deficient T cells expressed CD27 levels equivalent to control T cells or CD70 deficient T cells and showed normal proliferation in response to stimulation with CD3 or CD3 + CD28. Taken together, these data indicate that the CD27 triggered costimulatory signal on T cells when bound to CD70 on B cells is dependent on the tyrosine kinase ITK. Thus, the addition of ibrutinib, an inhibitor of TEC kinase ³² , including ITK, inhibited T cell proliferation mediated by LCL expressing CD70.

To further decipher the role of ITK in CD27 signaling, we next analyzed the activation events that occur after binding to CD27. Stimulation of human T cells with anti-CD27 antibodies or soluble CD70 has been reported to mobilize calcium to naive T cells and γδ T cells ^{33, 34} . In mice, ITK is also known to activate PLC-γ1 following TCR activation and subsequently to activate calcium influx ^16,18 . Therefore, calcium mobilization in T cells in response to stimulation with anti-CD3 or anti-CD27 antibodies was examined. T cells and T cell blasts derived from ITK deficient PBMC showed reduced calcium mobilization upon stimulation with CD3 as compared to cells derived from healthy donors, CD70 deficient patients or CD27 deficient patients. Stimulation of CD27 also induced calcium mobilization in T cell and T cell blasts derived from CD70 deficient patients and healthy donors. The magnitude of the calcium signal triggered by CD27 is often less important and consistently slower than the signal triggered by stimulation with CD3. In these conditions, stimulation with anti-CD28 antibody did not induce any calcium influx. In contrast, stimulation of ITK deficient T cells or CD27 deficient T cells by CD27 resulted in very weak calcium mobilization. Similarly, ibrutinib completely inhibited CD27-mediated calcium mobilization in T-cells and T-cell blasts from healthy donors, but CD3-mediated calcium mobilization was observed in ITK-deficient cells Partially blocked to a size equivalent to the one. These results demonstrate that ITK is required for CD27-mediated calcium mobilization.

The exact mechanism involved in CD27 signaling is still not clearly defined, but the role of TRAF molecules has been reported ³⁵ . Interestingly, CD27 contains several proline and tyrosine residues in its intracellular domain, which may form potential docking sites for the SH3 and SH2 domains of ITK, respectively. There is. In particular, the NH2-terminal region has a proline-containing sequence that shares similarities with the consensus sequence involved in the interaction with ITK's SH3 domain ³⁶ . These sequences in CD27 are highly conserved in mammals. Therefore, we examined whether ITK can functionally or directly interact with CD27. We first tested CD27's ability to trigger a tyrosine phosphorylation event. Tyrosine phosphorylated protein was detected in lysates from healthy donor control T cell blasts 15 minutes after stimulation with anti-CD27 antibody. These phosphorylation signals were partially distinct and delayed compared to the signals observed in response to stimulation with CD3. Interestingly, proteins with the size of ITK were phosphorylated upon stimulation with CD27 but not upon stimulation with CD3. Further analysis, such as phosphorylation of the signal ¹⁶ known to be ITK-dependent, eg PLC-γ1 and ERK1 / 2, results in both pathways being more intense when stimulated with CD27 than in response to stimulation with CD3 Was low, but was shown to be activated. Then, evidence of direct association between ITK and CD27, GST fusion protein containing the SH3 domain of ITK, FYN, or LCK, the most important SH3-containing tyrosine kinase involved in TCR activation It was searched by pull down assay using. CD27 was precipitated from PBMC and T cell blast lysates using GST containing the SH3 domain of ITK. In contrast, no association with GST alone or with GST containing the SH3 domain of FYN or LCK was observed or only moderate association was observed. These data strongly suggest that ITK interacts directly with CD27 through its SH3 domain and has the ability to couple CD27 to downstream signaling pathways.

Consideration:
In this study, we report for the first time the genetic defect of CD70 in patients with incomplete immunity to EBV infection, with uncontrolled lymphoproliferation of infected B cells. The present inventors decipher the pathway in which CD70 and its ligand CD27 are involved, and the functional cluster in which CD70, CD27 and ITK molecules play an important role in immune surveillance of B cells by T cells. Provide evidence that they are forming. When expressed on B cells, CD70 delivers signals to T cells through CD27 and ITK, which are necessary for maintenance of T cell proliferation and thus execution of effector programs.

The patient showed recurrent B cell lymphoproliferation with high viral load of EBV and developed Hodgkin's lymphoma associated with EBV. The susceptibility to infection appears to be limited to EBV. This is because, to date, we have not suffered from other viral or bacterial infections. Because it is based on one, and in addition, young patients, it is clearly difficult to conclude firmly on the basis of the clinical results associated with the CD70 deficiency. However, defects in the gene for CD27, the only known ligand for CD70, have been reported in 17 patients 5, ^{6, 20} . In particular, they all developed EBV related disorders as an initial and major clinical manifestation, including lymphoproliferative disorders such as Hodgkin's lymphoma and diffuse large B cell lymphoma. Therefore, the clinical symptoms of CD70 deficient patients are similar, if not identical, to those associated with the loss of CD27. Thus, loss of CD70 appears to be a phenotypic copy of the loss of CD27. The phenotypes of CD27 deficient and CD70 deficient mice are also equivalent and characterized by an antiviral response in which both were ^{abolished 22, 25, 27} . Interestingly enough, the clinical phenotype associated with ITK deficiency is also very similar to that of CD27 and CD70 deficiency ⁷ .

Our results indicate that the CD70-CD27 system represents an important factor in the immune response to EBV in humans. We have shown that CD70 on EBV-infected B cells provides an important signal to EBV-specific CTL for CTL proliferation. We observed that CD70 was strongly upregulated in activated B cells and EBV infected B cells. CD70 is also constitutively expressed by many EBV-dependent and EBV-independent B cell malignancies ²⁴ . The unique predisposition for EBV infection with CD27 deficiency, CD70 deficiency, and ITK deficiency appears to result from the unique tropism of EBV on B cells and the ability of EBV to induce B cell proliferation and transformation. An efficient in vivo immune response to EBV requires rapid and extensive expansion of effector cells to provide a commensurate amount of CTL to eliminate highly proliferating EBV infected B cells. Thus, the CD70-CD27-ITK system is an important component of this response.

Many studies in mice have described the role of the CD27-CD70 system in the development and maintenance of CD8 ⁺ T cell responses, including the establishment of long-term memory. Recent descriptions of CD70 deficient mice have confirmed that CD70 is of particular importance in the initiation of early CD8 ⁺ T cell responses ²⁶ . Thus, acquisition of T cell immunity against EBV infected B cells appears to be defective in CD70 deficient patients, which does not result in proper amplification and expansion of EBV specific effector CD8 ⁺ T cells in vitro Correlate with. However, memory T cell immunity to EBV appears to be conserved in the absence of CD70. This is because specific anti-EBV T cells can be amplified from the patient's PBMC when cocultured with autologous LCL expressing CD70. Taken together, these results indicate cell division of activated T cells without affecting their cytotoxic ability to perform T cell cytotoxic and IFN-γ producing functions and differentiate into memory cells. Point out the specific role of the CD70-CD27 interaction in triggering

According to previous studies, our observations demonstrate that CD27 behaves as a costimulatory molecule for TCR-dependent lymphocyte activation ²⁹ . The co-signals delivered by CD27 are essential for sustained T cell proliferation as highlighted in recent years ³⁷ . Furthermore, we have shown that the proliferation-related co-signals downstream of CD27 are strictly dependent on ITK which can bind to the cytoplasmic tail of CD27 through the SH3 domain of ITK. It is well established that ITK is directly involved in TCR signaling by its ability to activate PLC-γ1, Ca ²⁺ influx, and ERK kinase ^{16, 18} . Thus, binding of CD27 also triggers activation of PLC-γ1, Ca ²⁺ influx, and ERK kinase, suggesting quantitative amplification of TCR intensity by CD27 as suggested29 ^{, 37} . More work is warranted to decipher the precise molecular requirements of ITK in CD27 signaling and how it cooperates with TCR signaling to drive cell division.

While our observations have established that CD70 on B cells is a key actor in T cell immunity to proliferating B cells, CD70 also has a subset of activated T cells and It is also expressed on other hematopoietic cells, including dendritic cells. In various models previously tested, priming and maintenance of CD27-dependent T cell responses has been shown to be involved in interactions with CD70 expressed on dendritic ^{cells38, 39} . Thus, it may also be considered that the absence of CD70 on dendritic cells is responsible for the uncontrolled EBV infection in the patient, particularly during the priming phase. Because CD27- and ITK-deficient patients did not develop great susceptibility to other infections, CD70 on dendritic cells is not absolutely required for immune responses to other pathogens It is. Partial overlap with other TNFR family members such as 4-1BB and OX40 expressed by T cells signals delivered by CD27 when bound to their ligands during interaction with dendritic cells And similar signals could be provided to T cells ^{29, 40} .

Interestingly enough, the mechanisms underlying defective immunity against EBV in SAP deficiency (ie XLP-1 syndrome) caused by SH2D1A mutations are distinctly different, with CD27-CD70-ITK-deficiency With a different clinical phenotype than that associated. In SAP deficiency, the impaired interaction of T cells and B cells through the SLAM receptor plays a central role in susceptibility to EBV infection, resulting in CD8 ⁺ T cells in most patients ³ blood cells associated with amplification phagocytosis syndrome (i.e. HLH) is effected. Our results show that the CD27-CD70-ITK system also plays an important role in T cell-B cell interactions during the course of EBV infection. However, impaired control of EBV infection in patients with CD27-CD70-ITK-deficiency did not immediately cause HLH. In the absence of SAP, effector-memory EBV-specific CD8 ⁺ T cells have been shown to show a reduced ability to kill EBV-infected B cells ¹¹ , ¹² , while cell proliferation is incompletely activated Normal or even increased due to cell death induced by ¹³ , which is in sharp contrast to CD27-CD70-ITK-deficiency. The pathogenesis of X-linked lymphoproliferative syndrome is thought to result from immune pathologies triggered by CD8 ⁺ T cells that can not kill B cell targets. This is consistent with the pathophysiology of HLH, which is known to be dependent on the accumulation of activated T cells producing large amounts of IFN-γ as a result of the inability to kill infected cells. There are ⁴¹ . Conversely, in CD27-CD70-ITK-deficiency, the accumulation of activated T cells during the course of EBV infection is insufficient, which results from its impaired ability to proliferate. These distinct molecular defects depend on two key steps in the induction of T cell responses to EBV: CD70 / CD27 / ITK and SLAM / SAP, which are different molecules / pathways for their implementation. Point out cell proliferation and cytotoxic effector function.

Taken together, our observations indicate that CD70 on B cells signals abnormal B cell proliferation to T cells, even in the absence of EBV, as a trigger for B cell proliferation. Suggest that it acts as a semaphore. This fits well with the observation that somatic mutations in CD70, including large deletions, have been identified in B cell lymphomas such as diffuse large B cell lymphoma and Burkitt's lymphoma ^{42, 43 44} . In particular, recent reports have identified CD70 as one of the most frequently mutated genes in a series of diffuse large B-cell lymphomas ⁴⁵ . Accumulation of mutations in CD70 may indicate a mechanism for malignant B cells that circumvents immune surveillance by T cells. Our data and previous studies in mice show that ectopic expression of CD70 on non-lymphoid tumor cells or expression of CD70 in B cells (as reported by the inventors) is due to their expression of CD70. ^{46, 47, 48} regardless of whether it can elicit T cell-mediated immunity to tumor cells. Thus, CD70-CD27 may represent an important target for inducing anti-tumor vaccination. Restoration or induction of expression of CD70 in some lymphoma cells lacking CD70 may represent a therapeutic approach to elicit global and potent anti-tumor immunity.

References:
Throughout the present application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated herein by reference to the present disclosure.

Claims (10)

  i) preparing a sample of malignant B cells obtained from a subject, ii) isolating and culturing a group of malignant B cells from the sample of step i), iii) forming a group of malignant B cells in step ii) Introducing a nucleic acid molecule encoding a polypeptide, and iv) administering to the subject a malignant B cell population of step iii) of a therapeutically effective amount, of a B cell malignancy in a subject in need thereof. Treatment.   B-cell malignancies include non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, perisplenic zone lymphoma, MALT (mucosal associated lymphoid tissue) lymphoma, Hair cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma (eg, various types of Hodgkin's disease, B-cell non-Hodgkin's lymphoma (NHL) and related lymphomas (eg, Waldenstrom) Macroglobulinemia (also called lymph plasma cell lymphoma or immunocytoma) or lymphoma of the central nervous system, leukemia (eg acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; B cell chronic) Lymphocytic leukemia (also called BCLL), hairy cell leukemia and chronic myeloblastic leukemia), myeloma For example, multiple myeloma), small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymph plasma cell lymphoma, perisplenic zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous trait Cell tumor, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinum (thymus) Large cell B cell lymphoma, endovascular large cell B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, gray zone lymphoma, B cell proliferation of unknown grade, lymphomatous granulomatosis, and post transplant lymph The method of claim 1, wherein the method is selected from the group consisting of proliferative disorders.   3. The method of claim 2, wherein B cell lymphoma is caused by Epstein-Barr virus (EBV).   3. The method of claim 2, wherein the B cell lymphoma is a diffuse large B cell lymphoma with a mutation in the gene encoding CD70.   2. The method of claim 1, wherein the malignant B cells are transformed with a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1.   6. The method of claim 5, wherein the introduction of the nucleic acid molecule is performed using a retroviral vector.   A vaccine composition comprising a group of malignant B cells transformed with a nucleic acid molecule encoding a CD70 polypeptide.   8. The vaccine composition of claim 7, wherein the malignant B cells are transformed with a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1.   The vaccine composition of claim 7 for use in the treatment of B cell malignancies.   B-cell malignancies include non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, perisplenic zone lymphoma, MALT (mucosal associated lymphoid tissue) lymphoma, Hair cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma (eg, various types of Hodgkin's disease, B-cell non-Hodgkin's lymphoma (NHL) and related lymphomas (eg, Waldenstrom) Macroglobulinemia (also called lymph plasma cell lymphoma or immunocytoma) or lymphoma of the central nervous system, leukemia (eg acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; B cell chronic) Lymphocytic leukemia (also called BCLL), hairy cell leukemia and chronic myeloblastic leukemia), myeloma For example, multiple myeloma), small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymph plasma cell lymphoma, perisplenic zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous trait Cell tumor, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinum (thymus) Large cell B cell lymphoma, endovascular large cell B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, gray zone lymphoma, B cell proliferation of unknown grade, lymphomatous granulomatosis, and post transplant lymph A vaccine composition for use according to claim 7, selected from the group consisting of proliferative disorders.

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