Patent Application
20220143083
This application claims the benefit of priority of EP 19156745.2, filed 12. February 2019, which is incorporated herein by reference.
The present invention relates to a method for generating tumour specific T cells that counteract tumour immune escape.
Concurrent to the great immunotherapeutic advances of cancer treatment in recent years, treatment failures caused by tumour escape have become an increasingly important issue. It has been shown that a major mechanism of tumour escape can be explained by the activity of CD8+ T-cells (cytotoxic T-lymphocytes, CTLs). While the initial tumour-destructive activity of CTLs is beneficial to the patient, under circumstances of incomplete tumour elimination, the CTLs' activity may lead to selective loss of cognate antigen from tumour cells in a process named immune editing, thus promoting tumour escape.
Whilst CD8+ T-cells specifically eliminate their tumour target cells, CD4+ regulatory T-cells (Tregs) show the opposite behaviour. Through their immunosuppressive activity, Tregs provide a growth advantage to tumour cells carrying the cognate antigen of these Tregs. In fact, Tregs are present throughout the progressing tumour and their number is often correlated with tumour size. This argues for the continuous presence of specific tumour antigens recognized by Tregs.
Given the persistence of specific antigens for Tregs in the tumour and in tumour derived metastases, it follows that the cognate T-cell receptors of Tregs (Treg TCRs) constitute prime tumour-specific recognition elements that may be employed for the development of immunotherapies counteracting tumour escape.
Based on this background, the problem underlying the present invention to provide means for the treatment of cancer that overcome T cell mediated tumour escape. A solution to this problem is provided by the subject matter of the independent claims, with particular embodiments discussed in the dependent claims and the following description.
The fundamental concept underlying the present invention is the realization that regulatory T cells residing in the tumour, which mediate inhibition of cytotoxic T cells and other immune responses, may provide the key information, in form of their T cell receptor, to recognize tumour specific antigens. The potential of this concept can be turned into therapeutic tools in at least two ways:
The selection of tumour-specific Treg TCRs as provided by the invention represents a clear advantage for immunotherapeutic approaches. Two selection steps are employed in combination in order to achieve tumour specificity:
The preparations of tumour-specific T cells provided by the invention exhibit anti-tumour activity even in tumours with loss of function of Beta-2-Microglobulin through interactions with antigen presenting cells in the tumour microenvironment or more directly with malignant cells capable of MHC class II presentation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.
The term virtually identical in the context of the present specification relates to a sequence that shows ≥98% identity in relation to the sequence it is said to be virtually identical to. The term virtually identical sequences is used for, but not limited to, sequences that originate from a sequencing event of the same physical sequence or an identical copy thereof, where methodical errors or misreads during the sequencing process have introduced artifactual errors into the sequence.
The term translation of nucleic acid sequences in the context of the present specification relates to the assignment of amino acid (polypeptide) sequences to nucleic acid sequences based on the genetic code and the known reading frame of the nucleic acid sequence. It does not relate to the biological act of translation in the cell performed by ribosomes.
The term CDR3 in the context of the present specification relates to the variable complementarity determining region 3 of the TCR complex. The size of CDR3 is characterized by the total number of amino acids (AA) and respective nucleotides from the conserved cysteine in the Vβ, or Vα or Vγ or Vδ segment to the position of the conserved phenylalanine in the Jβ or Jα, Jγ or Jδ segment.
The term “tumour sample” in the context of the present specification refers to a sample or a pool of samples obtained from a tumour of a patient. The tumour sample may also include or consist of a metastasis or a collection of metastases.
The term “non-tumour tissue sample” in the context of the present specification refers to a sample or a pool of samples obtained from non-tumour tissue comparable or identical to the tumour's tissue of origin or its position in case of metastasis. An advantageous source for such non tumour tissue sample is tissue in close proximity to the tumour of the patient.
In the present specification, the term positive, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescently labelled antibody, wherein the label's fluorescence on the structure (for example, a cell) referred to as “positive” is at least 30% higher (≥30%), particularly ≥50% or ≥80%, in median fluorescence intensity in comparison to staining with an isotype-matched fluorescently labelled antibody which does not specifically bind to the same target. Such expression of a marker is indicated by a superscript “plus” (+), following the name of the marker, e.g. CD4+. If the word “expression” is used herein in the context of “gene expression” or “expression of a marker or biomolecule” and no further qualification of “expression” is mentioned, this implies “positive expression” as defined above.
In the present specification, the term negative, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescently labelled antibody, wherein the median fluorescence intensity is less than 30% higher, particularly less than 15% higher, than the median fluorescence intensity of an isotype-matched antibody which does not specifically bind the same target. Such expression of a marker is indicated by a superscript minus (−), following the name of the marker, e.g. CD127−.
High expression of a marker, for example high expression of CD25, refers to the expression level of such marker in a clearly distinguishable cell population that is detected by FACS showing the highest fluorescence intensity per cell compared to the other populations characterized by a lower fluorescence intensity per cell. A high expression is indicated by superscript “high” or “hi” following the name of the marker, e.g. CD25high. The term “is expressed highly” refers to the same feature.
Low expression of a marker, for example low expression of CD127, refers to the expression level of such marker in a clearly distinguishable cell population that is detected by FACS showing the lowest fluorescence intensity per cell compared to the other populations characterized by higher fluorescence intensity per cell. A low expression is indicated by superscript “low” or “lo” following the name of the marker, e.g. CD127low. The term “is expressed lowly” refers to the same feature.
The expression of a marker may be assayed via techniques such as fluorescence microscopy, flow cytometry, FACS, ELISPOT, ELISA or multiplex analyses.
The term “clonotype” in the context of the present specification refers to a group of T cells that comprise T cell receptor nucleic acid sequences that exhibit a virtually identical nucleic acid sequence with respect to the variable region of the TCR, or that comprise T cell receptor amino acid sequences that exhibit a virtual identical amino acid sequence with respect to the variable region of the TCR. Clonotypes exhibiting a virtual identical amino acid sequence with respect to the variable region of the TCR may also referred to as clustertype.
The invention relates to a method for generating a preparation of tumour-specific T cells. This method comprises the following steps:
Obtaining a plurality of nucleic acid sequences encoding T cell receptors from the isolated T regulatory cells is equivalent to analysing the T cell receptor repertoire of the cells. In other words, an estimation is made as to which TCR sequences are present in the T cell population, and at what abundance (what is the relative incidence among the total number of TCR sequences).
Prior to the tumour T cell sequencing step, CD4+ cells are separated from the T cells isolated from the tumour sample, or the tumour infiltrating T cells separated from the tumour are restricted to CD4+ cells in the first place, for example by selection through CD4 antibodies. In the tumour T cell sequencing step, only or predominantly sequences encoding T cell receptors originating from CD4+ T cells are sequenced.
Non-limiting options for conducting the T cell sorting for analysing Treg include a) using cell surface marker CD4+CD25+CD127low/−, b) using CD4+ and intracellular FoxP3+ staining, c) using surface marker CD4+CTLA4high or CD4+CD39high. For cell sorting of non-Treg conventional CD4 T cells (Tconv) the surface marker CD4+CD25−CD127high can be used to determine potential overlaps in TCR-repertoires of Treg and Tconv. Separated T cell subsets are selected for DNA extraction and α- and/or β-TCR/CDR3 NGS sequence analysis is performed.
In particular embodiments, T cells that are characterized by expression of CD4 and CD25, and low expression of CD127 (CD4+CD25+CD127low) and/or by expression of both CD4 and FOXP3 (CD4+FOXP3+) are isolated in the tumour T cell isolation step, and subsequently sequenced exclusively. In certain embodiments, T cells with a T regulatory phenotype are characterized by markers known in the art including, without being limited to, CTLA-4, TIM3, GITR, LAG-3, CD69, TGF-beta, IL-10, particularly characterized by expression of CD4 and CD25 (Mason et al. J. Immunol. 2015, 195 (5) 2030-2037).
In particular embodiments, the sequence selection step comprises comparing said tumour TCR sequences among each other, thereby determining the relative incidence (frequency) of any individual sequence, and selecting sequences on account of their incidence in the tumour and in non-tumour tissue or blood. This information is valuable in determining the contribution of a particular sequence to the totality of the TCR mediated immunological equilibrium, and the likelihood of it being a representative of an important population of T cells, in the sense that they are likely to be specific for an antigen that is present within the local tumour tissues. The tumour-specific clonotype is absent or exhibits a frequency of <20%, <15%, <10% or <5% within the set of non-tumour tissue CD4+ TCR sequences. Conversely, if the ratio between the frequency of a particular clonotype in the tumour and the frequency of the same clonotype in non-tumour tissue is a 5 this is indicative of tumour specificity.
In particular embodiments, the sequence selection step comprises comparing said tumour TCR sequences to a set of TCR sequences obtained from T cells (particularly to a set of TCR sequences obtained from CD4+ T cells, more particularly CD4+CD25+ T cells, even more particularly CD4+CD25+CD127low and/or CD4+FOXP3+ T cells) obtained from a non-tumour tissue sample of the same patient. By comparing the prevalence of particular TCR sequences between these two sites or samples, it is possible to determine whether a TCR sequence or a set of TCR sequences is specific for the tumour, or representative of TCR sequences residing outside of the tumour environment as it is the case in tissue specific autoimmunity and/or infection. The invention relies on a method of distinguishing tumour-specific from non-tumour regulatory TCR sequences, the latter being potentially harmful if isolated and used as transgene TCR sequences in an immune-stimulatory context as a cancer treatment, but the former bearing the potential to guide an anti-tumour response.
Additional information such as the existence of ubiquitous, systemically present clonotypes can be obtained by comparing the tumour derived Treg TCR sequences to those derived from blood CD4+ cells.
In particular embodiments, the sequence selection step comprises comparing said tumour TCR sequences to a set of reference TCR sequences. Conceivably, recurrent practice of the invention disclosed herein will lead to a wealth of TCR information enabling the creation of a database of tumour and non-tumour TCR sequences which are common between patients, allowing the user to identify tumour-specific TCR sequences without reference to a second biological sample.
Sequences are selected that are characterized by high incidence (frequent) in the tumour and/or unique to the set of tumour TCR sequences.
In particular embodiments, prior to the sequence selection step, nucleic acid sequences are translated to amino acid sequences and the comparison of tumour TCR sequences is performed on the basis of amino acid sequences. The skilled person recognizes that any of the sequence determination and comparison steps disclosed herein can be performed on the nucleic acid sequence level, but also-often in a second step-on the amino acid sequence level. While obtaining sequences from samples is almost invariably going to be performed on the nucleic acid level, sequence alignment and comparison may be performed on the amino acid level as certain differences in nucleic acid sequence may not lead to a difference in the polypeptide, which for the purposes of antigen or MHC recognition is the operative entity on the biological level.
In particular embodiments, the sequence selection step comprises the steps of
Particularly, a first T cell receptor nucleic acid sequence is virtually identical to a second T cell receptor nucleic acid sequence, if both sequences differ in not more than one base-pair position.
In certain embodiments, a first T cell receptor nucleic acid sequence is virtually identical to a second T cell receptor nucleic acid sequence, if both sequences differ in not more than one base pair, and the first T cell receptor nucleic acid sequence exhibits in the respective sample, particularly in the tumour sample, an at least twentyfold frequency compared to the second T cell receptor nucleic acid sequence. Consequently, both first and second T cell receptor nucleic acid sequences are assigned to the same clonotype.
Likewise, a first T cell receptor amino acid sequence is virtually identical to a second T cell receptor amino acid sequence if both amino acid sequences differ from each other in not more than at one or two positions. The above-mentioned T cell receptor amino acid sequences may be comprised within the alpha and/or beta chain of the TCRα/β or within the gamma or delta chain of the TCRγ/δ.
The clonotype frequency is a measure of the relative or absolute frequency of the T cell identified by the TCR nucleic acid sequence within the set of sequences for which the frequency is determined.
Particularly, the clonotype frequency in a given sample is a measure of the relative or absolute frequency of the T cell identified by the TCR nucleic acid sequence within said sample.
In certain embodiments, the T cell receptor nucleic acid sequences of the above-mentioned plurality are comprised within nucleic acid sequences encoding one of the polypeptide chains that form a human T cell receptor, particularly TCRα/β or TCRγ/δ. In certain embodiments, the T cell receptor nucleic acid sequences of the above-mentioned plurality do not comprise non-coding nucleic acid sequences. Non-coding sequences refer to clonotypes with stop-codons or frame shifts that lead to non-functional TCR protein sequences.
In certain embodiments, a tumour-specific T cell receptor nucleic acid sequence is characterized by a length of 30 nucleotides to 110 nucleotides.
In certain embodiments, the tumour-specific T cell receptor nucleic acid sequence encodes a unique amino acid sequence comprised within any one of the polypeptide chains (alpha, beta, gamma and delta) that form a human T cell receptor, wherein the unique amino acid sequence exclusively occurs in a particular clonotype or clustertype and not any other clonotype or clustertype.
In particular embodiments, comparison of the sequences is performed with respect to the CDR3 sequence tract and all members of a particular clonotype exhibit an identical CDR3 sequence.
In particular embodiments, the method further comprises the following steps that enable to obtain and identify a set of non-tumour sequences:
In particular embodiments, obtaining T cell receptor sequences comprises the steps of
Methods for obtaining TCR sequences are known in the art. WO2014096394, also published as US2015337368, both incorporated by reference herein in their entirety, show methods used in the examples. Another system is available under the tradename “ImmunoSEQ” from Adaptive Biotechnologies world-wide.
In particular embodiments, the nucleic acid amplification reaction specifically amplifies a sequence encoding the CDR3 region of a chain of the human T cell receptor, particularly the CDR3 region of the alpha chain or the beta chain of the human T cell receptor.
While the step of selecting tiTreg sequences does not require knowledge about the composition of the associated full T cell receptor with respect to individual alpha chains and beta chains, the reconstruction of a functioning TCR encoding expression vector for transfer into the recipient cell does require this knowledge. Several strategies may be employed to achieve this.
The currently practiced methodology is to obtain TCR sequences twice in the process: firstly, a first portion of T cells obtained from the tumour sample are sequenced only with regard to the CRD3 sequence tract. This allows determination of clonotypes. The non-tumour sample is sequenced similarly.
Then, in a second sequencing step, a second portion of T cells obtained from the tumour sample are separated into single cells and sequenced individually. This sequencing round (step) affords information about the full TCR polypeptide chains expressed in any one single cell, and enables construction of the respective expression vectors for transfer into the transfer system employed in the gene transfer step. One technology that can be employed is 10× sequencing from 10× genomics Inc., described in U.S. Pat. Nos. 9,644,204, 9,975,122, 10,053,723 and 10,071,377, all of which are incorporated by reference herein.
In particular embodiments, recipient T cells are selected from the group comprising cytotoxic T cells and T helper cells. Herein, the method comprises a step of selecting T helper cells from a sample obtained from the patient, yielding an enriched T helper cell preparation. The recipient T cells are prepared by depletion of CD8+ T cells and CD4+ regulatory T cells, particularly from a lymphocyte preparation of the patient.
In certain embodiments, the recipient T cells (which are not regulatory cells) belong to an enriched fraction of T helper cells described as CD3+CD4+CD8−CD25− that is isolated from PBMCs of the same patient by e.g. CD8 and CD25 negative selection.
In certain embodiments, the recipient T cells are part of an enriched fraction of T helper cells described as CD3+CD4+CD8−CD127+ that is isolated from PBMCs of the same patient by CD8 negative and CD127 positive selection.
The enriched T cell preparation is subjected to the gene transfer step.
Alternatively, cytotoxic T cells are selected from a sample obtained from the patient, yielding an enriched cytotoxic T cell preparation.
The term “non-tumour tissue sample” in the context of the present specification refers to a sample or a pool of samples obtained from tissue of the same type as the tissue in which the tumour originates; one non-limiting example for a source of non-tumour tissue is tissue in close proximity to the tumour of a patient.
Particularly, the non-tumour tissue T cell receptor amino acid sequences are grouped into the plurality of non-tumour-tissue clonotypes in the same manner as the T cell receptor amino acid sequences obtained from the tumour into the plurality of tumour sample clonotypes, particularly to allow a re-assignment of a clonotype identified in a tumour sample to a non-tumour tissue clonotype.
Particularly, a clonotype can be assigned to a non-tumour clonotype, if any one of the T cell receptor amino acid sequences of the plurality of T cell receptor amino acid sequences comprised within this clonotype is virtually identical or identical to a T cell receptor amino acid sequence comprised within a non-tumour clonotype.
Particularly, a clonotype identified in a tumour can be assigned to a non-tumour clonotype, if
In certain embodiments, the non-tumour sample is a sample of non-tumour tissue of the same type of tissue as the tumour, particularly a sample of non-tumour tissue adjacent to the tumour. Such non-tumour tissue can be identified by common techniques such as ultra sound examination, radiography, CT or immunostaining.
The term “blood sample” or “sample from blood” in the context of the present specification refers to a sample from blood or a pool of samples obtained from blood of a patient.
In certain embodiments, the T cell receptor amino acid sequences obtained from the blood sample are grouped into the plurality of blood sample clonotypes in the same manner as the T cell receptor amino acid sequences obtained from the tumour into the plurality of tumour sample clonotypes, particularly to allow an assignment of a tumour sample clonotype to a blood sample clonotype.
Particularly, a tumour-specific clonotype can be assigned to a blood sample clonotype if any one of the T cell receptor amino acid sequences of the plurality of T cell receptor amino acid sequences comprised within this clonotype exhibits a virtually identical or identical sequence to a T cell receptor amino acid sequence comprised within a blood sample clonotype.
In certain embodiments, the selected tumour-specific clonotype cannot be assigned to a known clonotype being reactive to viruses such as the human cytomegalovirus or the Epstein-Barr-virus.
Such assignment may be performed by bioinformatics methods, wherein a tumour-specific T cell receptor nucleic acid sequence comprised within the selected tumour-specific clonotype is compared to nucleic acid sequences of known clonotypes being reactive to viruses such as the human cytomegalovirus or the Epstein-Barr-virus.
Particularly, the selected tumour-specific clonotype cannot be assigned to a clonotype known to be reactive to viruses such as the human cytomegalovirus or the Epstein-Barr-virus, if
In certain embodiments, the nucleic acid isolation step comprises the steps of
In certain embodiments, the tumour-specific T cell receptor nucleic acid sequence encodes the CDR3 region of a chain of the human T cell receptor, particularly the CDR3 region of the alpha chain or the beta chain of the human T cell receptor or the tumour-specific T cell receptor amino acid sequence is comprised within the CDR3 region of a chain of the human T cell receptor, particularly the CDR3 region of the alpha chain or the beta chain of the human T cell receptor.
In certain embodiments, the tumour-specific nucleic acid sequence is comprised within an RNA, particularly encoding an amino acid sequence comprised within the CDR3 region of the alpha chain or the beta chain of the human T cell receptor.
In certain embodiments, the tumour-specific nucleic acid sequence is comprised within an RNA, particularly encoding amino acid sequences comprised within the CDR3 regions of both alpha chain and beta chain of the human T cell receptor.
In certain embodiments, the tumour-specific nucleic acid sequence is comprised within an RNA, particularly encoding long stretches of amino acid sequences of the alpha chain or the beta chain of the human T cell receptor.
Particularly, the tumour reactivity of the tumour-specific Treg TCR-transduced T cell preparation of the invention may be confirmed by:
In one embodiment, the method of the invention provides a preparation of tumour-specific T cells by the following steps:
In certain embodiments, the gene transfer step is preceded by a transgene generating step, wherein the nucleic acid encoding said selected tiTreg TCR under control of a promoter sequence is generated by
Another aspect of the invention relates to a preparation of T cells obtained by the method of the invention, particularly for use in treatment of cancer, or in prevention of its recurrence.
Yet another aspect of the invention relates to a method to identify tumour-specific neoantigens by employing tumour-specific Treg TCRs identified and isolated as described in the above aspects and embodiments of the invention. These neoantigens may be employed as tumour vaccines or in the development of bispecific antibody or bispecific T cell Engager (BiTE® (Amgen Corp.)) related immunotherapies to counteract tumour escape.
Major targets of anti-tumour T cell responses are so-called neoantigens resulting from tumour-specific somatic mutations. They are identified by whole-exome- and transcriptome sequencing using genomic DNA and RNA of tumour cells derived from biopsies or surgical resection material. For comparison, corresponding nucleic acids from tumour-adjacent normal tissue or peripheral blood lymphocytes are sequenced as well. Neoantigen candidates, especially those with mutations in common oncogenic driver genes like KRAS, NRAS, TP53, PIK3CA, EGFR, BRAF, and/or the like are then tested for recognition by Treg TCR-transduced T cells in vitro. In addition to mutated neoantigens, candidate tumour-associated antigens (TAA) such as tumour type-specific cancer-germline antigens or antigens overexpressed or aberrantly (in alternative reading frames) expressed in tumour cells can be identified by transcriptome sequencing as well and tested for recognition by Treg TCR-transduced T cells. Both, peptides derived from neoantigens containing the amino acids exchanged by mutations and long-overlapping peptide libraries spanning the complete amino acid sequences of candidate TAA, are synthesized, pulsed onto autologous or HLA-matched antigen-presenting cells and tested for recognition by Treg TCR-transduced T cells via functional assays like IFN-γ-Elispot assays, ELISA or comparable tests.
Thus, in one embodiment the invention relates to a method for determining the ability of a neoantigen to elicit tumour-specific T cell responses. This method comprises the steps of:
Likewise, unmutated antigen candidates with tumour-restricted expression recognized by the tiTreg-transgenic T cells are assigned tumor-associated antigens.
The invention further provides the use of the tumour-specific regulatory TCR sequences as a biomarker.
The nucleotide sequences of Treg TCRs may be synthesized and transferred into expression vectors (by way of non-limiting example, retroviral expression vectors) for the purpose of transducing fresh effector T cells. The sequences of the α- and β-chains may be designed as codon-optimized sequences (or as a nucleic acid sequence significantly differing from the TCR sequence found in the patient without being different on the amino acid level), giving them unique sequence features that can easily be recognized and quantified by high throughput sequencing of ex vivo material such as peripheral blood lymphocytes. After adoptive transfer of Treg TCR-transduced T cells into the patient, the Treg TCR-sequences can be monitored at different time points after therapy and correlated with the clinical course of the treated patient.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Handling and processing of different tissues and cell sorting
For cell sorting of non-Treg conventional CD4 T cells (Tconv) the surface marker CD4+CD25− CD127high and/or CD4+FOXP3− can be used to determine potential overlaps in TCR-repertoires of Treg and Tconv. Such an overlap of a unique clonotype in the Treg and Tconv fraction is an indication of induced Treg formation. When using Treg TCRs for immunotherapeutic approaches, it can make a difference whether the TCR is derived from a natural or an induced regulatory T-cell. The TCR of an induced Treg originates from a tumour-specific T helper cell. Thus its use should be tumor-specific and safe. On the other hand, TCRs of natural regulatory T-cells may recognize cognate self-antigens at other sites in the organism, causing adverse effects by induction of autoimmunity. Such side effects may be of inflammatory rather than cytotoxic nature and may require anti-inflammatory treatment Moreover, peripherally induced Treg cells can be distinguished from thymic natural Treg cells by lack of expression of the surface marker neuropilin1 and the intracellular marker Helios. Separated T cell subsets are selected for DNA extraction and α- and/or β-TCR/CDR3 NGS sequence analysis is performed.
In addition, tumour samples may be stored under cell-preserving conditions as resource for cell material.
Blood samples are taken from the same patient: By standard hematologic fractionation cellular components are isolated from full blood. Cellular components may be separated in different T cell subsets. After DNA extraction, α- and/or β-TCR/CDR3 NGS sequence analysis is performed.
Providing Anti-Tumour T-Cells by Gene Transfer of Tumour-Specific Treg TCRs
Preparation of Cell Suspensions of T-Lymphocytes from Tumour (NSCLC) and Lung Tissue:
Each tumour specimen is dissected free of surrounding normal tissue and necrotic areas. Approximately 0.8 g cubes from tumour and normal lung tissue are cut into small chunks measuring about 2-3 mm in each dimension. Sliced tumour (and also non-tumour) biopsies are subjected to a commercial mechanical/enzymatic tissue dissociation system (gentleMACS, Miltenyi Biotec), using the Tumour Dissociation Kit (Miltenyi Biotec) and following the manufacturers instructions.
After gentleMACS disaggregation, cell suspensions are passed through 70-μm strainers. Aliquots of tumour cells are taken at this point and cryopreserved in 10% DMSO (Sigma-Aldrich) and 90% FCS (Life Technologies) for later use. The remaining cell suspension is subjected to density gradient centrifugation using a 40%/80% step gradient of Percoll® (GE Healthcare Europe GmbH) in PBS/RPMI 1640. T-lymphocytes are harvested from the interphase and washed in complete medium (RPMI 1640, Lonza). Subsequently, T-lymphocytes are cultured by placing cells at a concentration of 0.5×106 cells/ml in each well of a 24-well tissue culture plate with 2 mL of TexMACS medium (=recovery medium, Miltenyi Biotec) supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin. Plates are placed in a humidified 37° C. incubator with 5% CO2 and cultured overnight.
Sorting of T-Lymphocytes
The next day, cells are harvested and pooled from the wells. For isolation of the Treg fraction (CD4+, CD25high, CD127low/neg) and the corresponding Tconv fraction (CD4+, CD25low/neg, CD127+) cells are stained for FACS sorting with the following antibody panel (Miltenyi Biotec): human CD3-APC-Vio770, CD4-PE-Vio770, CD25-PE, CD127-APC. Additionally cells are stained to test for viability using 7-Aminoactinomycin D (7-AAD, BioLegend). DNA is extracted from each separated fraction and utilized for library preparation.
Genomic DNA Isolation and TCRsafe® Analysis
Genomic DNA (gDNA) is extracted from tissue materials using the NucleoSpin® Tissue Kit from Macherey-Nagel (Düren, Germany). Blood gDNA is isolated from 2-3 ml fresh blood with either QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) or AllPrep® DNA/RNA/miRNA Universal Kit (Qiagen) following the manufacturer's protocols.
CDR3 regions of the TCRβ-chain are sequenced with NGS (Illumina MiSEQ) technology following the TCRsafe® method, a proprietary 2-step PCR amplification procedure (as disclosed in WO 2014/096394 A1) which uses TCRβ primers binding specifically to the V- and J-segments adjacent to the CDR3 region. Genomic DNA is used as starting material for the NGS process.
Calculation of Clonotype (Sequence Cluster) Frequencies from NGS Data
Per sample a large (>105) number of paired reads (nucleotide sequences) is commonly produced by NGS. The read-pairs typically overlap by 40 to 80 bases and are merged read-pair by read-pair to contiguous sequences. These sequences are then assembled into clusters of virtually identical nucleotide sequences with the number of reads per cluster determining the frequency of that cluster. Frequency of a cluster is a measure of the percentage of reads within this sample falling into this cluster.
Clustering works in two rounds: In a first step all reads with 100% nucleotide sequence identity are counted as 1 cluster with the cluster sequence being identical to the read sequence. In the second step clusters are compared among each other and those with
The nucleotide sequence clusters are translated to amino acid sequences (peptides) and tabulated. Each cluster is regarded as one clonotype with a frequency as defined above. The frequency is a direct measure of the frequency of the respective T cell in the sample.
Comparison of TCR Sequence Profiles Between Samples
CDR3 amino acid sequences of clonotypes were compared between samples by an identity test procedure, where only sequences without mismatches are accepted as one and the same CDR3 amino acid sequence. The result of a multi-sample comparison is a table with one TCRβ CDR3 amino acid sequence shared by one or more samples per row, each sample is represented by one column containing the respective CDR3 frequencies in that sample (see
Identification of Tumour-Specific Treg Clonotypes by Comparative Sequence Analysis
In one exemplary embodiment, tumour-specific Treg clonotypes are identified by the 4-digit score 1100. The score is combined by comparative sequence analysis according to Table 1.
For each CDR3 peptide sequence (Seq1,2,3, . . . ) simple binary scores are given in each sample (in each column). ‘1’ means, that the respective CDR3 peptide sequence is present in the sample, ‘0’ means it is either absent or found at low levels. The precise definition is given below. The binary scores are combined to a 4-digit score as shown in Table 1. The scoring schema also includes cases, where no blood sample exists, i.e. the column D would be filled with ‘0’, or where no non-tumour sample exist, i.e. the column C would be filled with ‘0’. The binary scores per column (sample type) is defined as follows:
A: score=1: The sequence (Seq1,2,3, . . . ) is among top 100 clonotypes (sorted by their frequency from highest to lowest) and shows an intact open reading frame, i.e. no stop codons or frame shifts are found, otherwise score=0
B: score=1: The sequence (Seq1,2,3, . . . ) is present in the sample. The ratio B/A is greater than 0,5, if B is the frequency in the tumour for TCRs selected as CD4+CD25+CD127low/− and A is the frequency in the tumour CD4+ sample. In all other cases score=0.
C score=0: The sequence (Seq1,2,3, . . . ) is either absent in the non-tumour sample or found identical in the tumour sample, but with a ratio C/A ≤0.2, if C is the frequency in the non-tumour CD4+ sample and A is the frequency in the tumour CD4+ sample. In all other cases score=1.
D: score=0: The frequency of the sequence (Seq1,2,3, . . . ) is lower than the frequency of the respective sequence in tumour tissue (A), otherwise score=1.
A score of 1100 is needed to identify the respective TCR sequence as tumour specific. Seq2 in Table 1 has a score of 1100 and is identified as a tumour-specific T-reg clonotype.
TCR Alpha/Beta Coupled Sequencing of Tumour-Specific Treg Clonotypes and Synthesis and Cloning of Complete Alpha/Beta Treg TCRs
Paired sequencing of both chains of the T-cell receptor is an essential prerequisite for tailored production of T-cells with target-specific antigen receptors—where the target can be one or several tumour antigens, antigens of viral or microbial origin, antigens related to autoimmune diseases et cetera. As the two chains of any T-cell receptor are encoded on different chromosomes, a sophisticated paired-sequencing approach is required to correlate respective RNAs transcribed form different chromosomes on the single-cell level. There are established technologies to perform paired-sequencing of both chains of the T-cell receptor, with e.g. the technology offered by the company 10×genomics (https://www.10xgenomics.com/solutions/vdj/) being the one which can be implemented in any molecular biological laboratory by using the system's chromium controller for single cell sequencing. Another approach is based on well-plated cell-arrays and deep parallel sequencing followed by statistical analysis (Howie B. et al., High-throughput pairing of T cell receptor α and β sequences. Science Translational Medicine 7, 301 (2015)).
TCRβ CDR3 parts of T-cell receptors identified as tumour specific by the method described above will be paired with their respective α-chains in a separate step by performing single-cell sequencing, usually with a sample of up to 10000 T-cells and selection of the respective paired TCR sequences containing the tumour specific TCRβ CDR3 subsequence. Based on the paired TCR sequence the receptor can be synthesized.
Coupled T Cell Receptor Profiling and FOXP3 Gene Expression Analysis of Treg Cells Using 10×Genomics Technology
Paired TCR single-cell sequencing can be coupled to gene expression analysis to characterize subtypes of T cells and analyze their functional states. FOXP3 is the lineage-defining transcription factor of regulatory T cells. CD4+ Treg cells can be differentiated from effector type T cells by high and stable cell surface expression of CD25, absence of CD127 expression and constitutive expression of FOXP3 (CD25high/CD127−/FOXP3+). To identify Treg among TILs, CD4+/CD25+/CD127− T cells are sorted by FACS and subjected to combined paired TCR single-cell sequencing and FOXP3 expression analysis using 10×genomic's Chromium™ Single Cell V(D)J Reagent Kit according to the manufacturer's instructions. PCR-amplification of FOXP3 (Genbank Accession AF277993) for sequencing-library generation and subsequent sequencing is achieved by use of in-house designed primers (CTTCTCCTTCTCCAGCACCA (SED ID NO 101); GACACCATTTGCCAGCAGTG (SEQ ID NO 102); TTGAGGGAGAAGACCCCAGT (SEQ ID NO 103)). The 10×technology can detect both natural and induced regulatory T cells. Induced Treg are identified as cells of a given clonotype with identical α/β-TCRs, consisting of FOXP3-expressing and FOXP3-negative T cells.
Alpha- and β-chains of Treg cells are synthesized and cloned into retroviral expression vectors for the stable transduction of fresh effector T cells from autologous tumour patients (see next paragraph).
Gene Transfer of the Isolated and Cloned Tumour-Specific Treg TCRs
This is achieved using a retroviral transduction system (adapted from Voss et al., Adoptive Immunotherapy: Methods and Protocols, Springer Science and Business Media, 2005). Recombinant TCR-transgenic retroviruses are produced using the Phoenix-Ampho packaging cell line. They are transiently transfected with the expression vector encoding the bicistronic construct TCR-beta chain-p2A-TCR-alpha chain, called pMX-puro/βTCR-p2A-αTCR. Co-transfected are the plasmids pHIT60 (from Murine Moloney Leukemia Virus) encoding viral structure proteins and polymerase (reverse transcriptase) as well as pCOLT-GALV (from Gibbon Ape Leukemia Virus) coding for amphotropic envelope proteins. Supernatants containing infectious virus particles can be harvested after 40 to 48 hours. Enriched CD4+ Thelper cells derived from PBMCs of the same patient are infected by incubation with viral supernatant, seeded in 24-well plates and cultured in the incubator (37° C., 5% CO2) for 24 h. Subsequently, the cells are stimulated with anti-CD3/anti-CD28 in medium supplemented with puromycin in order to expand and enrich TCR-transgenic T-cells. Enrichment is monitored by flow cytometry using TCR-s-chain-specific antibodies. After sufficient expansion, T cells are frozen in aliquots forming a stock of effector T-cells for functional assays.
Detection of Tumor Reactivity of Treg TCR-Transduced Th1-Cells
Treg TCR-transduced Th1-cells (1×105 per well) and tumour cells (1×104 per well), either untreated or pre-treated with 200 IU ml−1 IFN gamma to stimulate expression of MHC class II molecules, are co-cultured in 200 μl RPMI 1640 (Lonza), supplemented with 10% autologous human serum, penicillin and streptomycin (ThermoFisher Scientific). After 48 h, culture supernatants are harvested and analyzed for IFN gamma using the cytometric bead array (BD Biosciences). For flow cytometric detection of intracellular levels of IFN gamma, transduced T cells are co-cultured with tumour cells for 24 h in the presence of Golgi-Plug (BD Biosciences; 1:1,000). Subsequently, cells are stained using surface antibodies to exclude dead cells, and intracellular IFNgamma is detected using the Cytofix/Cytoperm Kit and anti-IFN-γ staining (BD Biosciences), according to the manufacturer's protocol.
Identification of Induced Tumour Infiltrating Treg Sequences as Shown in
All data shown in
The table of
Each column is from sample preparations of a specific tissue and/or made from cell samples sorted with respect to specific markers.
10×Genomics' single-cell-sequencing technology allows to identify VDJ-pairing (T-cell receptor, both chains) and gene expression of dedicated genes (here FOXP3) from one cDNA library. The results are single-cell specific, i.e. we can compare sequences cell by cell. In this way distinct cells for one clonotype (same TCRs/VDJ) can be identified which differ by their FOXP3 expression pattern:
The shaded rows shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 19156745.2 | Feb 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/053648 | 2/12/2020 | WO | 00 |
