TRAC Gene Research Articles

TCRab Deficient CAR T-Cells Targeting CD123: An Allogeneic Approach of Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia (AML)

The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products.In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML).Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency.Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR.We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested.Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients. DisclosuresGaletto:Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

419. Allogenic CAR T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR's to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition to CAR expression, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). To identify an effective CAR targeting CD123, we have screened multiple antigen recognition domains in the context of several different CAR architectures to identify candidates displaying activity against cell lines expressing variable levels of the CD123 antigen. Furthermore, experiments in an AML mouse model using anti-CD123 CAR T cells demonstrate important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T cells from a single healthy donor will thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients.

Allogenic T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating cancer. However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis. To overcome this limitation, we have developed an allogeneic based platform for large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donors. This platform utilizes Transcription Activator-Like Effector Nuclease (TALENTM) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRαβ negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the capacity to perform efficient multiplex genome editing using TALENTM offers the possibility of simultaneously rendering cells resistant to standard chemotherapy or to tumor evasion mechanisms.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). In a first step, we have screened human primary T-cells harboring CARs with different antigen recognition domains in the context of multiple CAR architectures in order to identify candidates displaying specific activity against cell lines expressing variable levels of the CD123 antigen. To provide proof of concept for the general applicability of the allogeneic approach we have manufactured a TCR-deficient CD123 CAR T-cell (UCART123) and demonstrated that this product maintains a potent anti-tumoral activity in vitro. Experiments in an orthotopic AML mouse model using UCART123 cells are currently ongoing, in order to establish the absence of alloreactivity and the anti-tumoral activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T Cells from a single healthy donor could thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients. DisclosuresGaletto:Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

Genomic analysis offers insights into the evolution of the bovine TRA/TRD locus.

BackgroundThe TRA/TRD locus contains the genes for V(D)J somatic rearrangement of TRA and TRD chains expressed by αβ and γδ T cells respectively. Previous studies have demonstrated that the bovine TRA/TRD locus contains an exceptionally large number of TRAV/TRDV genes. In this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine TRA/TRD locus and the remarkable TRAV/TRDV gene repertoire.ResultsAnnotation of the UMD3.1 assembly identified 371 TRAV/TRDV genes (distributed in 42 subgroups), 3 TRDJ, 6 TRDD, 62 TRAJ and single TRAC and TRDC genes, most of which were located within a 3.5 Mb region of chromosome 10. Most of the TRAV/TRDV subgroups have multiple members and several have undergone dramatic expansion, most notably TRDV1 (60 genes). Wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential ‘birth’ and ‘death’ rates have been used to form a functional bovine TRAV/TRDV repertoire which is phylogenetically distinct from that of humans and mice. The expansion of the bovine TRAV/TRDV gene repertoire has predominantly been achieved through a complex series of homology unit (regions of DNA containing multiple gene) replications. Frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a ‘collateral’ expansion of the latter. Transcript analysis was used to define the TRAV/TRDV subgroups available for recombination of TRA and TRD chains and demonstrated preferential usage of different subgroups by the expressed TRA and TRD repertoires, indicating that TRA and TRD selection have had distinct impacts on the evolution of the TRAV/TRDV repertoire.ConclusionBoth TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire. However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups. Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-994) contains supplementary material, which is available to authorized users.

TRAV gene expression in PBMCs and TILs in patients with breast cancer analyzed by a DNA melting curve (FQ-PCR) technique for TCR alpha chain CDR3 spectratyping

To explore the expression of the TRAV gene in peripheral blood mononuclear cells (PBMCs) and in tumor-infiltrating lymphocytes (TILs) in the patients with breast cancer using a DNA melting curve (FQ-PCR) technique for T cell receptor (TCR) alpha chain CDR3 spectratyping. Peripheral blood samples and tissue samples were obtained from thirty breast cancer patients. Total RNA was extracted from PBMCs and tumor tissues and then reverse transcribed into cDNA. FQ-PCR was used to amplify the human TCR alpha chain CDR3 region with the primers to the TRAV and TRAC genes. TCR alpha chain CDR3 spectratyping and partial CDR3 sequencing were used to determine use of TRAV gene product in T cell responses. TCR alpha CDR3 spectratyping showed preferential usage of certain TRAV genes in the PBMCs and TILs of all patients with breast cancer. The frequencies of TRAV1.1, TRAV9, and TRAV29 exceeded 30% in PBMCs and the frequencies of TRAV1.1 and TRAV22 exceeded 30% in TILs. More than three quarters of the patients (23/30) overexpressed the same gene in both PBMCs and TILs; for example, patient-1 highly expressed TRAV9 in the PBMCs and TILs. Patients with positive or negative tumor markers of estrogen receptor (ER), progesterone receptor (PR), pS2, C-erbB-2, nm23, P53, and Ki-67 showed no significant common TRAV gene expression, but some TRAV gene preferential usage frequencies exceeded 20%. For example, five of seven patients positive for ER had high levels of expression of TRAV1.1 and TRAV3. Finally, the amino acid sequence of TCR CDR3 region showed some common motifs in some of the patients. TRAV gene expression was complex and diverse in the patients with breast cancer. The TRAV gene usage may be closely related to the diversity of breast tumor antigens and the differential immune responses observed in individual patients. Research into the immunological mechanism of T cells may provide guidance for individual T cell-directed therapy for breast cancer.

TRAC gene expression analysis in in vitro drug metabolism screening

TRAC gene expression analysis in in vitro drug metabolism screening

Conjugative Transferability of the A/C Plasmids fromSalmonella entericaIsolates That Possess or LackblaCMYin the A/C Plasmid Backbone

The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)-based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30 degrees C and 37 degrees C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3-12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a bla(CMY) gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugants, and most of these donors carried multiple replicons. A/C cotransferred with B/O, N, and I1 at both 30 degrees C and 37 degrees C and with HI2 at 30 degrees C. Seven Salmonella Newport isolates that produced transconjugants possessed only the single A/C replicon and lacked bla(CMY). PCR mapping of the A/C transfer gene operon in ten Salmonella Newport isolates that carried bla(CMY) revealed a bla(CMY) inverted repeat element integrated between the traA and traC genes. These results suggest that A/C may have been a conjugative plasmid before the integration of bla(CMY) into the transfer gene operon. Additionally, transfer deficient A/C replicons may be mobilized in the presence of certain compatible conjugative plasmids.

Identification and analysis of rhizobial plasmid origins of transfer

Twelve Rhizobium leguminosarum isolates from France, Germany and the UK, each carrying between four and seven plasmids, were screened by PCR using primers designed to amplify partial traA and traC genes and the intergenic spacer (igs) between them, which is expected to contain oriT (the nick site for conjugal transfer). Five strains, 1062, RES-2, RES-6, RES-7 and RES-9, generated oriT-containing PCR fragments. Sequencing identified three types that are related to but different from other rhizobial plasmid oriT sequences in the database. Sequence comparisons revealed conserved motifs in the igs, including a 14-bp putative nic site, a stem-loop and a tra box. The RES-2, RES-6 and RES-9 PCR products were used as probes in Southern hybridisation studies to screen the 12 strains for related sequences. Eleven strains contain at least one homologous sequence, but of the 64 plasmids present among the 12 strains only 17 hybridised to the oriT probes. Four sequence variants of the repC plasmid replication initiation gene have previously been described in these strains, but there is no correlation between repC and oriT sequence distributions, and there is evidence for recombination to generate different repC-oriT combinations. Three plasmids, pYK32, pYK36 and pYK39, containing the oriT amplified from RES-2, RES-6 and RES-9, respectively, were constructed for functional analysis of the oriT sequence variation. Each plasmid was transformed into R. leguminosarum strains 1062, RES-2 and RES-9 to generate nine donor-plasmid combinations, and their mobilisation frequencies into Escherichia coli and Agrobacterium tumefaciens measured following biparental matings. All three plasmid constructs were mobilised at a similar frequency (10(-7) to 10(-8) per recipient) by each donor strain, suggesting that there is no discrimination by the transfer proteins between the different oriT sequences.

Identification and analysis of rhizobial plasmid origins of transfer

Twelve Rhizobium leguminosarum isolates from France, Germany and the UK, each carrying between four and seven plasmids, were screened by PCR using primers designed to amplify partial traA and traC genes and the intergenic spacer ( igs) between them, which is expected to contain oriT (the nick site for conjugal transfer). Five strains, 1062, RES-2, RES-6, RES-7 and RES-9, generated oriT-containing PCR fragments. Sequencing identified three types that are related to but different from other rhizobial plasmid oriT sequences in the database. Sequence comparisons revealed conserved motifs in the igs, including a 14-bp putative nic site, a stem-loop and a tra box. The RES-2, RES-6 and RES-9 PCR products were used as probes in Southern hybridisation studies to screen the 12 strains for related sequences. Eleven strains contain at least one homologous sequence, but of the 64 plasmids present among the 12 strains only 17 hybridised to the oriT probes. Four sequence variants of the repC plasmid replication initiation gene have previously been described in these strains, but there is no correlation between repC and oriT sequence distributions, and there is evidence for recombination to generate different repC-oriT combinations. Three plasmids, pYK32, pYK36 and pYK39, containing the oriT amplified from RES-2, RES-6 and RES-9, respectively, were constructed for functional analysis of the oriT sequence variation. Each plasmid was transformed into R. leguminosarum strains 1062, RES-2 and RES-9 to generate nine donor–plasmid combinations, and their mobilisation frequencies into Escherichia coli and Agrobacterium tumefaciens measured following biparental matings. All three plasmid constructs were mobilised at a similar frequency (10 −7 to 10 −8 per recipient) by each donor strain, suggesting that there is no discrimination by the transfer proteins between the different oriT sequences.

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.

Nucleotide sequence of the F plasmid gene, traC, and identification of its product

The traC gene of the F plasmid tra operon is required for the assembly of mature F-pilin subunits into extended F pili. The nucleotide sequence of traC was determined with a deduced coding region of 875 amino acids (aa) and 99066 Da. The traC1044 mutant allele, which allows filamentous phage infection in the absence of pilation, contains a C-to-T transition leading to an Arg → Cys substitution. Confirmation of the translational start came from the direct N-terminal aa sequencing of a TraC-alkaline phosphatase fusion protein.

Location of F plasmid transfer operon genes traC and traW and identification of the traW product.

As part of an analysis of the conjugative transfer genes associated with the expression of F pili by plasmid F, we have investigated the physical location of the traC and traW genes. We found that plasmid clones carrying a 2.95-kilobase EcoRI-EcoRV F transfer operon fragment were able to complement transfer of F lac traC mutants and expressed an approximately 92,000-dalton product that comigrates with TraC. We also found that traW-complementing activity was expressed from plasmids carrying a 900-base-pair SmaI-HincII fragment. The traW product was identified as an approximately 23,000-dalton protein. The two different F DNA fragments that expressed traC and traW activities do not overlap. Our data indicate that the traC gene is located in a more-tra operon promoter-proximal position than suggested on earlier maps and that traW is distal to traC. These results resolve a long-standing question concerning the relationship of traW to traC. The clones we have constructed are expected to be useful in elucidating the role of proteins TraC and TraW in F-pilus assembly.

A traC mutant that retains sensitivity to f1 bacteriophage but lacks F pili.

An F lac pro mutant which was temperature sensitive for infection by the filamentous bacteriophage f1 but resistant to the F-specific icosahedral RNA phage f2 was isolated. Cells carrying the F' mutation failed to elaborate F pili at all temperatures. Mutant cells were able to pair with recipient cells during bacterial conjugation, but transfer of conjugal DNA occurred at a greatly reduced frequency. Complementation analyses showed the F' mutation to be in the traC gene. When a plasmid carrying traC was introduced into hosts harboring the F' mutation, phage sensitivity, the ability to elaborate F pili, and conjugation efficiency were restored. The mutation was named traC1044. The F lac pro traC1044 mutant appears to be unique among traC mutants in retaining host sensitivity to the filamentous phage f1 in the absence of expression of extended F pili. Phage f1 attachment sites appeared to be present at the cell surface in traC1044 mutants. The reduced accessibility of these sites may account for the reduced efficiency of phage f1 infection of traC1044 hosts, although the possibility that a defect was present in the receptor site itself was not eliminated. Membranes of hosts carrying the F' mutation contained a full complement of mature F-pilin subunits, so the product of traC is presumably required for pilus assembly but not for pilin processing. This, together with the deficiency in conjugal DNA transfer, suggests that traC may be part of a membrane-spanning tra protein complex responsible for pilus assembly and disassembly and conjugal DNA transmission.

Conjugal transfer system of the IncN plasmid pKM101.

The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically. Its organization differed significantly from that of the F plasmid. The tra genes are located in three regions, each between 3 and 4 kilobases in length. All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus. The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region. Using two different methods, we have identified 11 complementation groups required for transfer. One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids. One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.

Genetic analysis of deletions of R100-1 that are both transfer-deficient and tetracycline-sensitive.

The extent of the deletions of five Tets Tra- mutants of R100-1 was determined by complementation experiments with wild-type and tra mutants of Flac. The presence or absence of the origin of transfer on the mutants was also investigated. Using the results, a tentative map of this region of the R factor was drawn: it was essentially similar to the analogous region of the E. coli K12 F factor, except that tet was located between traJ and traA. Some of the deletions had removed the promoter for the transfer operon. This allowed detection of the transcription of traC and distal genes from a weak, traJ-independent promoter. This is probably the Is2 promoter, since R100-1 carries and Is2 insertion sequence located immediately to the left of traC in the correct orientation. Since neither the transfer operon promoter nor the Is2 promoter seemed to be required for transcription of traI, it was concluded that, unlike the F factor, this was located in a separate operon.