METHOD FOR PROVIDING TUMOUR-SPECIFIC T CELLS

Abstract

The present invention relates to a method for providing a tumour specific T cell preparation, comprising the steps of: a. selecting tumour-specific T cell clones by: providing a tumour sample obtained from a patient; isolating a nucleic acid preparation from the tumour sample in a nucleic acid isolation step; obtaining a plurality of T cell receptor nucleic acid sequences from the nucleic acid preparation or a plurality of T cell receptor amino acid sequences encoded by the plurality of T cell receptor nucleic acid sequences; selecting a tumour-specific T cell receptor nucleic acid sequence from the plurality of T cell receptor nucleic acid sequences or a tumour-specific T cell receptor amino acid sequence from the plurality of T cell receptor amino acid sequences in a sequence selection step; b. sorting tumour-specific T cell clones by: providing a lymphocyte preparation obtained from the patient; isolating cells that comprise the selected tumour-specific T cell receptor nucleic acid sequence or the selected tumour-specific T cell receptor amino acid sequence from the lymphocyte preparation in an isolation step.

Claims

1. A method for providing a tumour specific T cell preparation, comprising the steps of: a. selecting tumour-specific T cell clones by: providing a tumour sample obtained from a patient; isolating a nucleic acid preparation from said tumour sample in a nucleic acid isolation step; obtaining a plurality of T cell receptor nucleic acid sequences from said nucleic acid preparation or a plurality of T cell receptor amino acid sequences encoded by said plurality of T cell receptor nucleic acid sequences; selecting a tumour-specific T cell receptor nucleic acid sequence from said plurality of T cell receptor nucleic acid sequences or a tumour-specific T cell receptor amino acid sequence from said plurality of T cell receptor amino acid sequences in a sequence selection step; b. sorting tumour-specific T cell clones by: providing a lymphocyte preparation obtained from said patient; isolating cells that comprise said selected tumour-specific T cell receptor nucleic acid sequence or said selected tumour-specific T cell receptor amino acid sequence from said lymphocyte preparation in an isolation step.

2. The method according to claim 1, wherein said isolation step comprises the steps of: contacting said lymphocyte preparation with a specifically reactive ligand being able to bind an amino acid sequence comprised within the V region of the T cell receptor that corresponds to said selected tumour-specific T cell receptor nucleic acid sequence or to said selected T cell receptor amino acid sequence, wherein said ligand is attached to a detectable label, and wherein particularly said ligand binds to said amino acid sequence with a dissociation constant of 10.sup.7, 10.sup.8 or 10.sup.9 mol/l or less, and isolating T cells carrying said detectable label from said lymphocytes preparation.

3. The method according to claim 1, wherein said isolation step comprises the steps of; contacting said lymphocyte preparation with a nucleic acid probe specifically binding to said selected tumour-specific T cell receptor nucleic acid sequence, wherein said nucleic acid probe is attached to a detectable label; isolating T cells carrying said detectable label from said lymphocyte preparation.

4. The method according to claim 1, wherein said isolation step comprises: a separating step, wherein said lymphocyte preparation is separated into a plurality of fractions, an expanding step, wherein cells comprised within said plurality of fractions are expanded under conditions of cell culture, and a selecting step, wherein at least one fraction of said plurality of fraction that comprises said selected tumour-specific T cell receptor nucleic acid sequence or said selected tumour-specific T cell receptor amino acid sequence is selected, and wherein said isolation step optionally further comprises repeating said separating step, said expanding step and said selecting step with said selected at least one fraction of said plurality.

5. The method according to claim 4, wherein said selecting step comprises, contacting said plurality of fractions with a nucleic acid probe specifically binding to said selected tumour-specific T cell receptor nucleic acid sequence, wherein said nucleic acid probe is attached to a detectable label and identifying fractions comprising said selected tumour-specific T cell receptor sequences, or obtaining T cell receptor nucleic acid sequences from said plurality of fraction and identifying fraction comprising said selected tumour-specific T cell receptor nucleic acid sequence.

6. The method according to claim 1, wherein said sequence selection step comprises the steps of aligning said plurality of T cell receptor nucleic acid sequences or said plurality of T cell receptor amino acid sequences; grouping T cell receptor nucleic acid sequences comprised in said plurality of T cell receptor nucleic acid sequences or T cell receptor amino acid sequences comprised in said plurality of T cell receptor amino acid sequence into a plurality of tumour sample clonotypes, wherein nucleic acid sequences or amino acid sequence comprised within a particular clonotype exhibit a virtually identical or an identical sequence; determining the number of T cell receptor nucleic acid sequences associated with each clonotype or determining the number of T cell receptor amino acid sequences associated with each clonotype, thereby yielding a clonotype frequency for each of said clonotypes; selecting a tumour-specific clonotype from said plurality of tumour sample clonotypes, wherein said tumour-specific clonotype is one of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes and/or is another clonotype of said plurality of tumour sample clonotypes that comprises a T cell receptor amino acid sequence being identical or virtually identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic sequences comprised within said one tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes, and selecting a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said selected tumour-specific clonotype as said tumour-specific receptor nucleic acid sequence or selecting a T cell receptor amino acid sequenced of said plurality of said T cell receptor amino acid sequences comprised within said selected tumour-specific clonotype as said tumour-specific amino acid sequence.

7. The method according to claim 6, further comprising providing a non-tumour sample obtained from said patient; isolating a nucleic acid preparation from said non-tumour sample in a nucleic acid isolation step; obtaining a plurality of T cell receptor nucleic acid sequences from said nucleic acid preparation or a plurality of T cell receptor amino acid sequences encoded by said plurality of T cell receptor nucleic acid sequences, yielding a plurality of non-tumour-specific T cell receptor nucleic acid sequences or a plurality of non-tumour-specific T cell receptor amino acid sequences; aligning said plurality of non-tumour-specific T cell receptor nucleic acid sequences or said plurality of non-tumour-specific T cell receptor amino acid sequences; grouping T cell receptor nucleic acid sequences comprised in said plurality of non-tumour-specific T cell receptor nucleic acid sequences or said plurality of non-tumour-specific T cell receptor amino acid sequences into a plurality of non-tumour-specific clonotypes, wherein T cell receptor nucleic acid sequences or T cell receptor amino acid sequences comprised within a particular clonotype exhibit a virtually identical or an identical sequence; selecting a tumour specific clonotype from said plurality of tumour sample clonotypes, wherein said tumour specific clonotype is one of the 100 most frequent clonotypes of said plurality of tumour sample or is another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being identical or virtually identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said one tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes, and said tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes is absent in said non-tumour sample or can be assigned to a non-tumour-specific clonotype that exhibits a frequency of not more than 20%, 15%, 10% or 5% of the frequency of said tumour-specific clonotype.

8. The method according to claim 7, further comprising: providing a blood sample obtained from said patient; isolating a nucleic acid preparation from said blood sample in a nucleic acid isolation step; obtaining a plurality of T cell receptor nucleic acid sequences from said nucleic acid preparation or a plurality of T cell receptor amino acid sequences encoded by said plurality of T cell receptor nucleic acid sequences; aligning said plurality of T cell receptor nucleic acid sequences or said plurality of T cell receptor amino acid sequences; grouping T cell receptor nucleic acid sequences comprised in said plurality of T cell receptor nucleic acid sequences or T cell receptor amino acids sequences into a plurality of blood sample clonotypes, wherein T cell receptor nucleic acid sequences or T cell receptor amino acids sequences comprised within a particular clonotype exhibit a virtually identical or an identical sequence; selecting a tumour specific clonotype from said plurality of tumour sample clonotypes, wherein said tumour specific clonotype is one of the 100 most frequent clonotypes of said plurality of tumour sample or is another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being identical or virtually identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said one tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes, and said tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample can be assigned to a blood sample clonotype that shows a frequency of less than the frequency of said tumour-specific clonotype.

9. The method according to claim 8, further comprising: providing a cell-free sample obtained from said patient; isolating a nucleic acid preparation from said cell-free sample in a nucleic acid isolation step; obtaining a plurality of T cell receptor nucleic acid sequences from said nucleic acid preparation or a plurality of T cell receptor amino acid sequences encoded by said plurality of T cell receptor nucleic acid sequences; aligning said plurality of T cell receptor nucleic acid sequences or said plurality of T cell receptor amino acid sequences; grouping T cell receptor nucleic acid sequences comprised in said plurality of T cell receptor nucleic acid sequences or T cell receptor amino acid sequences comprises in said plurality of T cell amino acid sequences into a plurality of cell-free sample clonotypes, wherein T cell receptor nucleic acid sequences or T cell receptor amino acid sequences comprised within a particular clonotype exhibit a virtually identical or an identical sequence; selecting a tumour specific clonotype from said plurality of tumour sample clonotypes, wherein said tumour specific clonotype is one of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes or is another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being identical or virtually identical to a T cell receptor amino acid sequence encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said one tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes, and said tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample can be assigned to a cell-free sample clonotype.

10. The method according to claim 9, wherein said tumour-specific clonotype of the 100 most frequent clonotypes of said plurality of tumour sample clonotypes can be assigned to another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being identical or virtually identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said one tumour-specific clonotype of the 100 most frequent tumour-specific clonotypes or to a T cell receptor amino acid sequence of said plurality of T cell receptor amino acid sequences comprised within said one tumour-specific clonotype of the 100 most frequent tumour-specific clonotypes.

11. The method according to claim 10, wherein the most frequent clonotype of said tumour sample clonotypes or another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being virtually identical or identical to a T cell receptor amino acid encoded by a T cell receptor sequence of said plurality of T cell receptor nucleic acid sequences comprised within said most frequent tumour-specific clonotype is selected, wherein particularly said most frequent clonotype is absent in said non-tumour sample or can be assigned to a non-tumour clonotype that shows a frequency of not more than 20%, 15%, 10% or 5% of the frequency of said tumour-specific clonotype, and/or can be assigned to a blood sample clonotype that shows a frequency less than the frequency of the respective said tumour sample clonotypes, and/or can be assigned to a cell-free clonotype and/or can be assigned to another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being identical or virtually identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said most frequent tumour-specific clonotype.

12. The method according to claim 11, comprising selecting 5, 10, 15 or 20 tumour-specific clonotypes from said tumour sample, wherein said selected tumour-specific clonotypes are 5, 10, 15 or 20 of the 100 most frequent clonotypes, the 5 most frequent clonotypes, the 10 most frequent clonotypes, the 15 most frequent clonotypes, or the 20 most frequent clonotypes of said plurality of tumour sample clonotypes and/or are another clonotypes of said plurality of tumour sample clonotypes that comprise a T cell amino acid sequence being virtually identical or identical to a T cell receptor amino acid encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within any one of said selected 5, 10, 15 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes, and optionally said selected 5, 10, 15 or 20 tumour-specific clonotypes are absent in said non-tumour sample or can be assigned to a non-tumour-specific clonotype that exhibits a frequency of not more than 20% 15%, 10% or 5% of the frequency of said selected 5, 10, 15 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes, and/or said selected 5, 10, 15 or 20 tumour-specific clonotypes can be assigned to a blood sample clonotype that shows a frequency of less than the frequency of said selected 5, 10, 15 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes, and/or said selected 5, 10, 15 or 20 tumour-specific clonotypes can be assigned to a cell-free sample clonotype, and/or said selected 5, 10, 15 or 20 tumour-specific clonotypes can be assigned to another clonotype of said plurality of tumour sample clonotypes that comprises a T cell amino acid sequence being virtually identical or identical to a T cell receptor amino acid encoded by any one of said T cell receptor nucleic acid sequences of said plurality of T cell receptor sequences comprised within said selected 5, 10, 15 or 10 tumour-specific clonotypes of said plurality of tumour sample clonotypes.

13. The method according to claim 12, wherein any one of said one of the 100 most frequent clonotypes, said selected 5, 10, 15 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes is assigned to a non-tumour-specific clonotype, if a T cell receptor amino acid sequence encoded by a T cell nucleic acid receptor sequence of said plurality of T cell receptor nucleic acid sequences comprised within said tumour-specific clonotype or a T cell amino acid sequence of said plurality of amino acid sequences comprised with said tumour-specific clonotype is identical to a T cell receptor amino acid sequence encoded by a T cell receptor nucleic acid sequence comprised within said non-tumour sample clonotype, or if a T cell amino acid sequence of said plurality of T cell receptor amino acid sequences comprised with said tumour-specific clonotype is identical to a T cell receptor amino acid sequence comprised within said non-tumour sample clonotype, and/or, any one of said one of the 100 most frequent clonotypes, said selected 5, 10, 15 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes is assigned to a blood sample clonotype, if a T cell receptor amino acid sequence encoded by a T cell receptor sequence comprised within said tumour-specific clonotype is identical to a T cell receptor amino acid sequence encoded by a T cell receptor nucleic acid sequence comprised within said blood sample clonotype, or if a T cell amino acid sequence comprised with said tumour-specific clonotype is identical to a T cell receptor amino acid sequence comprised within said blood sample clonotype, and/or, any one of said one of the 100 most frequent clonotypes, said selected 5, 10 or 20 tumour-specific clonotypes of said plurality of tumour sample clonotypes is assigned to a cell-free sample clonotype, if a T cell receptor amino acid sequence encoded by a T cell receptor nucleic acid sequence of said plurality of T cell receptor nucleic acid sequences comprised within said tumour-specific clonotype is identical to a T cell receptor amino acid sequence encoded by a T cell receptor nucleic acid sequence comprised with said cell-free sample clonotype, or if a T cell amino acid sequence of said plurality of T cell amino acid sequences comprised with said tumour-specific clonotype is identical to a T cell receptor amino acid sequence comprised with said cell-free sample clonotype.

14. The method according to claim 3 wherein said nucleic acid probe is a double stranded oligonucleotide, wherein a first strand of said oligonucleotide is complementary to said selected tumour-specific nucleic acid sequence and connected to a nanogold particle, and wherein a second strand is complementary to said first strand and bears a fluorescent label, wherein said fluorescent label is quenched by said gold particle if said second strand is bound to said first strand, a peptide nucleic acid probe, wherein a nucleobase is replaced by a dye which luminesce upon probe binding to said selected tumour-specific T cell receptor nucleic acid sequence, or a nucleic acid probe, wherein a nucleobase is replaced by a dye which luminesce upon probe binding said selected tumour-specific T cell receptor nucleic acid sequence.

15. The method according claim 1, wherein said nucleic acid isolation step comprises the steps of a. isolating T cells from said tumour sample and isolating nucleic acid from said isolated T cells, and/or b. conducting a nucleic acid amplification reaction that specifically amplifies T cell receptor nucleic acid sequences.

16. The method according to claim 15, wherein said isolation step is followed by an expansion step, wherein said isolated T cells are expanded under conditions of cell culture.

17. The method according to claim 16, wherein said 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 said tumour-specific T cell receptor amino acid sequence is or 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.

18. The method according claim 17, wherein said 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.19.

19. (canceled)

20. A method for treating cancer in a patient having a tumour, comprising providing a tumour specific T cell preparation by a method according to claim 1 from said patient, administrating said tumour specific T cell preparation to said patient.

21. (canceled)

22. A method for manufacturing an artificial tumour-specific T cell receptor, comprising the steps of: providing a tumour specific T cell preparations by a method according to claim 1, isolating an individual tumour-specific T cell from said tumour-specific T cell preparation; determining the CDR3 regions of both subunits of said T cell receptor of said isolated individual tumour-specific T cell; preparing an artificial T cell receptor comprising said determined CDR3 regions of both subunits.

23.-28. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0358] FIG. 1 shows in A: scheme of the CDR3 region of the alpha-chain of the human T cell receptor. B: Same as A, but for the beta chain of the human T cell receptor. C: Principle of amplification of the genomic region containing CDR3. PCR primers are specific for the repertoires of V/Jsegments and amplify small regions of the V- and J-segments with the CDR3 region between them.

[0359] FIG. 2 shows a flow diagram depicting the principles of the method of the invention.

[0360] FIG. 3 shows how the ratio of TILs and non-tumour T cells separates TILs into highly tumour-reactive and minor tumour-reactive T cell clonotypes. The dashed line depicts the tumour vs non-tumour ratios (T/nT), the dotted and solid lines show the frequencies of T cell clones carrying/expressing specific activation markers (PD-1, IFNgamma). For a threshold ratio T/nT>5 almost all tumour-reactive clones are correctly predicted, for T/nT>20 2 clones are selected and correctly predicted as tumour-reactive.

EXAMPLES

Example 1: Identification of Tumour-Specific T Cells and Tumour-Specific Sequences by Comparative Sequence Analysis

[0361] Available Next-Generation-Sequencing (NGS) technology was used to sequence many thousand TCR beta CDR3 regions (one TCR corresponds to one T cell) per sample in high-throughput, whereby sequencing libraries for the CDR3-region of human TCR beta were generated. The resulting sequences were analysed by bioinformatics tools and the final result per sample is a table listing the respective clonotypes (types of T cells with the same TCR beta).

[0362] The CDR3 region of the T cell receptor is determined by the constant V- and J-segments (see FIG. 1) and the highly variable regions between them. Due to this structure one and the same CDR3 amino acid sequence can be encoded by multiple nucleotide sequences, which may be even composed of distinct V/J-segments. The occurrence of multiple (>1) nucleotide CDR3 sequences per one amino acid sequence among the set of tumour-specific T cells and potential tumour-reactive T cells (TRTC) is a strong hint that the T cells with the respective CDR3 amino acid sequence is reactive with respect to the tumour cells.

[0363] CDR3 sequences, with this property are always added to the final selection of CDR3 sequences, if their score (see table 1 below) is greater or equal to 1000.

Scoring Schema for Identification of Tumour Specific T Cells (TSTCs) by Sequence Profiling and Bioinformatics Analysis.

[0364] The method is based on a scoring system given below (Table. 1), where one or several samples are taken and analysed in parallel, and the best scores are gained for clonotypes with respective ratios of frequencies per sample type. Generally, tumour infiltrating lymphocytes were identified by the following series of analysis steps: [0365] a.) Next-Generation-Sequencing (NGS) is performed starting from tumour samples. Tumour samples are either defined as one sample or a set of replicate samples taken from tumour tissue. In practice, the material to analyse the TCRs is obtained by either [0366] i.) Selecting distinct biopsies or different areas of one biopsy. This may be assisted by immunohistochemical staining, wherein particularly tumour reactive T cells (TRTCs) are immunohistologically stained with preferably T cell activation markers such as LAGS, OX40, CD107a or CD137 and stained regions are selected for DNA extraction. [0367] ii.) Lysis of tumour tissue e.g. by bead-based technologies for preparation of single cell suspensions as starting point for TCR analysis. Single cell suspensions may be separated in different T cell subsets, e.g. CD4+ and CD8+ subsets. [0368] In addition, tumour samples may be stored under cell-preserving conditions as resource for cell materials. [0369] b) Non-tumour samples from the same patient are selected from tissue/regions adjacent to tumour sample, if possible in replicates, where possible from distinct tissue spots and - and/or -TCR/CDR3 NGS sequence analysis was performed. [0370] c) Blood samples (cellular components) are taken from the same patient: By standard hematological fractionation cellular components were isolated from full blood, a and/or TCR/CDR3 NGS sequence analysis was performed and TCR-profiles were calculated. [0371] d) Serum, plasma or other cell-free biological fluids/tissues are taken from the same patient, optionally by additional removal of cellular components by standard hematological fractionation. The presence of TCR-specific DNA in cell-free samples can be a strong hint for apoptotic processes against T cells. If a significant amount of clonotypes (see below, Table.1) is found in cell-free sample and tumour, the score contributes to the scoring table (Table. 1). [0372] e) Optionally, 2 or more time points in the course of the patients treatment/diagnosis are used for screeningi.e. samples are taken at distinct time points from blood, etc. (see 2.a-d.). This will enable e.g. diagnosis of relapse or detection of new TSTCs directed against metastases etc.
Principles of Clonotype (Sequence Cluster) Calculation from NGS Data [0373] a. CDR3 regions of the TCR- and TCR-chain are sequenced with NGS technology. A 2-step PCR method (as disclosed in WO 2014/096394 A1) was used with TCR or TCR primers binding specifically to the V- and J-segments adjacent to the CDR3 region. DNA was used as starting material for the NGS process. [0374] b. Per sample a large (>10.sup.5) number of reads (nucleotide sequences) is commonly produced by NGS, the reads are merged into clusters of virtually identical nucleotide sequences, the number of reads per cluster determines the frequency of that cluster, where frequency of a cluster is measured in percentage of reads of this sample falling into this cluster. [0375] c. Clustering is very conservative and 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 [0376] i. not more than 1 bp mismatch and [0377] ii. where one cluster (cluster A) has at least 20 more reads than the other cluster (cluster B) [0378] are merged and regarded as identical to cluster A. The nucleotide sequence clusters are regarded as equivalent to clonotypes. [0379] d. 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 in (1.b). The frequency is a direct measure of the frequency of the respective T cell in the sample. [0380] e. Clusters (Clonotypes) sharing a virtually identical amino acid sequence are merged into clustertypes, the frequency of a clustertype is identical to the sum of frequencies of nucleotide sequence clusters being elements of said clustertype.

[0381] The ranking of TSTC (tumour-specific T cell) score is given in 4 digit numbers 1011,1010,1001,1000 (from best to lowest), all other cases are excluded.

[0382] Within the columns the scoring is defined as follows

TABLE-US-00001 TABLE 1 The scoring table for selection of best TSTC-clonotypes. T cell CDR3 B: non- nucleotide TSTC A: Tumour tumour C: blood D: cell- sequence score tissue tissue cellular free DNA Seq1 1011 1 0 1 1 Seq2 1110 1 1 1 0 Seq3 1001 1 0 0 1 . . .

[0383] Within each column (1 column per tissue type) simple binary scores are given per CDR3 nucleotide sequence (Seq1,2,3, . . . ): 1 means, that the respective CDR3 DNA sequence occurs, 0 means it is either absent or found in low levels. The precise definition is given below. The binary scores are combined to a 4-digit TSTC score as shown in Table 2. The ranking of accepted TSTC scores is given by their natural order: 1011, 1010, 1001, 1000 (from best to worst), all other scores are excluded. The TSTC scoring schema also includes cases, where e.g. no blood sample exists, i.e. columns C and D would be filled with 0, or where there are only tumour samples, i.e columns B, C and D would be filled with 0. The binary scores per column (=tissue type) is defined as follows:

[0384] A: score=1: The sequence (seq1,2, . . . ) 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

[0385] B: score=0: The sequence (Seq1,2,3, . . . ) is either absent in non-tumour sample or found identical in non-tumour sample, but with a ratio R=pepB/pepA less or equal to 0.2, 0.15, 0.1 or 0.05, if pepB is the frequency in non-tumour sample and pepA is the frequency in tumour sample. In all other cases score=1.

[0386] C: score=1: The frequency of sequence Seq1,2,3, . . . is lower than the frequency of the respective sequence in tumour tissue (A), otherwise score=0

[0387] D: score=1: The frequency of the sequence Seq1,2,3, . . . is higher than 0.001% of all sequences derived from cell-free DNA, otherwise score=0

[0388] E: For CDR3 sequences Seq1,2,3, . . . already selected by their TSTC score (see A-D above), optionally the following additional filter can be applied: if identical CDR3 amino acid sequences from A (tumour sample) are encoded by different CDR3 nucleotide sequences Seq1,2,3, . . . this is indicative of convergent recombination and highly immunogenic tumour antigens. Clonotypes with this property are given the highest TSTC score=1011.

[0389] F: For CDR3 sequences Seq1,2,3, . . . selected by their TSTC score (see A-D above), optionally the following additional filter is applied: CDR3 sequences translated into amino acid sequences from A (tumour sample) may be compared among each other by protein alignment (blast) using amino acid substitution matrices like BLOSUM80 or BLOSUM62. Amino acid sequences being highly similar with maximal 1 mismatch are grouped into similarity clusters and each member (Seq1,2,3 . . . ) of the similarity cluster is given the same TSTC-score as the best scoring CDR3 sequence in that similarity cluster.

[0390] Within each score group 1011, 1010, 1001, 1000 (from best to worst) the CDR3 nucleotide sequences are sorted by their frequency from highest to lowest and from the final sorted list the top 1-100 CDR3 nucleotide sequences are selected as candidate set for the next steps. In other embodiments the top 5, 10, 15, 20, 30, 40 or 50 CDR3 sequences are selected. But preferred are 20.

[0391] The best scoring clonotypes (up to 20) are stored as [0392] a. template for the synthesis of fluorescent tags [0393] b. template for the synthesis of novel tumour-specific T cells by gene transfer.

[0394] The above mentioned tumour sample may be a single sample or a set of samples from the patient. Therefore, a plurality of tumour samples from one patient may be analysed as described above. Clonotypes that occurred in different tumour samples are preferred over clonotypes that occur in the minority of tumour samples.

Example 2: Target Sequence Identification

[0395] Once the TCR nucleic acid sequences of the T cell clones of interest are identified, further steps are necessary to define the ideal target sequences that can be used for detection and enrichment of said T cell clones. At first, the specific genomic sequence is used to generate an at least partial mature mRNA sequence in order to discard any intronic parts that cannot serve as target for specific recognition by probes in living cells. Said clonal mature mRNA sequences are then compared with the complete transcriptome including the mature TCR mRNA of all other T cells not belonging to the clones of interest in order to identify only target-specific sequences. Particularly, mainly the CDR3 regions of the TCR mRNA are different on a clonotype basis and display difference to other transcripts in the cell as well. The target-clone specific sequences can be further analysed for structures that interfere with probe hybridisation. This can be performed either experimentally by checking the hybridisation efficiency, or by computational analysis using tools such as MFold or UNAFold (http://mfold.rna.albany.edu/). It is preferred that the region with the highest delta G (closer to zero) is chosen for probe design.

Example 3: Probes for In Vivo Detection

[0396] Having identified the target-specific DNA sequences of the clones of interest, probes for the detection in living T cells can be designed. Different probe formats can be used. However, depending on the length of the target-specific region multipartite probes or single oligonucleotide probes may be chosen. Molecular beacons can be designed to hybridise to target RNA at a temperature compatible with cell cultivation. Software packages such as Beacon Designer developed by PREMIER Biosoft International (www.premierbiosoft.com) are commercially available. Molecular probes can have a pair of mostly terminally conjugated dyes that are quenched due to formation of a stem while not hybridised to a target. Upon target hybridisation, the terminal stem is opened and the dyes are unquenched. However, in a complex environment such as the cytoplasm of living cell, unspecific interaction with proteins may open up the stem resulting in false positive signals. In order to enhance the specificity of a molecular beacon, a second molecular beacon can be designed to hybridise directly adjacent to the first molecular beacon as a bipartite probe. If the termini of both beacons are specifically hybridised within a distance of up to four nucleotides, a highly specific FRET signal between the adjacent dyes can be used to detect the hybridisation event. A multipartite recognition can also be achieved with unstructured probes other than in a molecular beacon format. The so-called SmartFlare is a new probe format that combines the properties of nanogold particles of enhancing cell transfection and quenching of fluorescent dyes which are immobilised in close proximity to the gold surface. Thus a simple probe complementary to a given target sequence bearing a single fluorescent dye is sufficient. The dye of the probe is effectively quenched when hybridised to another nucleic acid which is anchored to a gold nanoparticle. Upon transfection into a living cell, the probe is able to be displaced by specific hybridisation to its complementary target sequence, thus becoming fluorescent by detachment from the nanoparticle. Forced intercalation probes (FIT-probes, WO 2006/072368 A2) are a yet more desirable format. The intercalation of certain dyes between nucleobases of the formed probe-target duplex restricts the torsional flexibility of two heterocyclic ring systems of said dyes. As a result, FIT probes show strong enhancements of fluorescence upon hybridization. A FIT-probe with thiazol orange (TO) has been reported to yield a signal in the presence of complementary DNA or RNA with at least 25-fold enhancement of fluorescence intensity. More recently, it was discovered that dual fluorophore-labelled PNA FIT-probes are extremely responsive and bright hybridization probes for the sensitive detection of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. In contrast to existing DNA-based molecular beacons, this PNA-based probe form does not require a stem sequence to enforce dye-dye communication. Oxazole yellow (YO) containing FIT-probes have been shown to discriminate against single base mismatches by attenuation of fluorescence and may be used if single-nucleotide polymorphisms (SNPs) have to be detected specifically. Furthermore, it has been demonstrated that addition of C-terminal lysine residues enables uptake into living cells without the need for any further transfection reagent. Although FIT-probes have been originally published as PNA-based probes, FIT-probes based on DNA and LNA have been developed as well. DNA FIT probes with dual dye combinations such as TO and YO were found to be very specific in vivo exceeding the brightness of molecular beacons. In addition, so-called mixmers of PNA and DNA have become commercially available. Thus it is possible to optimise specificity, solubility and melting temperature to generate FIT-probes for the efficient fluorescent detection of living T cell clones.

[0397] Depending on their base composition and type of nucleotide, different lengths will be optimal for cytoplasmic recognition of target TCR mRNA. It is preferred that the target-specific hybridising part of standard PNA probes are shorter than 20 bases and standard DNA probes less than 35 bases. However, many non-standard modifications exist which can be used to elevate or decrease the specificity and/or melting temperature of nucleic acids. For example, abasic sites and unlocked nucleic acids may decrease melting temperature and increase specificity. LNA has a higher melting temperature than DNA and is protected from nuclease degradation. Even modified bases such as inosine which may pair to three of the four natural bases can be used to fine-tune intracellular recognition.

[0398] Due to the vast complexity of nucleic acid structures that may arise in vivo, it is preferred to choose monopartite probes that do not rely on structures for their functionality. Provided with the preferred specific target region previously identified by comparison to other cellular transcript and structural accessibility, the skilled person would know how to design an appropriate probe using respective bioinformatic design tools.

Example 4: Probe Uptake Mechanisms

[0399] Nucleic acids can be taken up into living cells by a multitude of mechanisms. The process is called transfection, when eukaryotic cells are targeted by a non-viral mechanism. Three general transfection methods are available called chemical-based transfection, non-chemical transfection and particle-based transfection. The chemical-based transfection methods make use of additional chemicals that facilitate cellular uptake. Such additives can be salts, polymers, liposomes and nanoparticles or a mixture thereof.

[0400] The efficiency of transfection methods is strongly dependent on the size and form of nucleotides as well as cell-type. Small nucleic acids can be efficiently transfected by pore-forming compounds. Streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small nucleic acids such as siRNA or molecular beacons and is compatible with T cells. In addition, T cells have been effectively transfected by gold nanoparticle conjugates with labelled probes such as SmartFlares. Also Lipofectamine was effectively used for transfection of small oligonucleotides such as siRNA or antisense RNA into T cells. Especially PNA can be simply elongated by a few lysine residues to achieve cellular uptake without any additional transfection reagents. Preferred non-chemical transfection methods are magnetofection and electroporation. More preferred is cell squeezing which was demonstrated to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to over 20 cell types, including embryonic stem cells and nave immune cells. The microfluidic platform of Sqz Biotechnologies Co. allows for the high throughput and efficient transfection of T cells without the need of transfection reagents.

Example 5: Increase of Specific Signals

[0401] The level of TCR mRNA transcripts in a cell can be increased in order to provide a higher signal to noise ratio for the specific detection by preferably monopartite probes. The inventors have discovered that a previous overnight treatment of T cells with 10 U/ml IL-2 can increase the transcript level of TCR mRNA. Alternatively, the TCR mRNA level can be increased with cycloheximide. The protein synthesis inhibitor cycloheximide (CHX) induces a 20-fold increase in mature TCR-alpha transcript accumulation without a concomitant increase in TCR-alpha gene transcription suggesting that CHX reverses the nuclear post-transcriptional events which prevent mature TCR-alpha mRNA accumulation. CHX also induces full length TCR-beta transcripts greater than 90-fold while TCR-beta gene transcription increases only 2- to 4-fold (Wilkinson & McLeod EMBO J. 1988 January; 7(1): 101-109.). Since the inhibition by CHX was found to be reversible, it is preferred to perform only a brief period of incubation sufficient to raise the mRNA level for detection by probes by a factor of 10.

[0402] Another alternative is to activate T cells and incubate activated cells for a period of 24 h, thereby doubling the amount of mRNA for specific detection.

Example 6: Array-Based Method for Sequence-Specific Isolation of T-Cell Clonotypes

[0403] T cell clonotypes, particularly the tumour-specific clonotypes of the invention may be isolated by the following iterative approach comprising diluting T-cells in clonotype-positive wells and repeating the method until a homogeneous T-cell population comprising the desired clonotype is generated.

[0404] The nucleic acid based assay may be performed by either direct probe hybridisation in cells or specific amplification of target sequences for detection. The direct probe hybridisation can be carried out using dead cells (analysis by Microscope, microtiter well scan, or FACS) or live cells (FIT-probes, etc. analysis by Microscope, microtiter well scan, or FACS). The amplification reaction is preferably a (RT-)PCR on array samples.

[0405] Suitable samples comprise, without being restricted to, extracellular nucleic acid (cell free), supernatant or array surface may comprise cell-free nucleic acid that can be used for specific and sensitive identification without killing valuable cells. This may allow a more rapid isolation of target cells without the need for cell division, crude lysate derived from an aliquot of the array (well or position), purified nuclear DNA, purified mRNA.

[0406] Different array formats that are compatible with the method comprise, without being restricted to. [0407] microtiter wells (at least 2 wells, preferably more than 6 wells, more preferably between 128 and 384 wells) [0408] embedded array. The cells are preferably embedded by a matrix that hinders free diffusion of cells and hence preserves the coordinates of an initially deposited clone. The matrix preferably comprises polymers such as agarose, gelatine or polyacrylamide. [0409] random array. The random array is not dependent on a preformed grid to contain samples. [0410] Microfluidic. A microfluidic array can be specifically formed by channels and other structures that may allow handling steps comprising initial cell distribution, washing, dilution, expansion and retrieval of cells and/or nucleic acids. A non-liming example of such microfluidic array is shown at http://www.biomemsrc.org/research/cell-tissue-microengineering/living-cell-array.

[0411] Depending on the frequency of the target clonotype in a sample, an appropriate limiting dilution may be performed in order to ensure that not more than one clonotype is present in a given diluted aliquot or well. Even single cells can be directly entrapped in an array with communicating microwells by dielectrophoresis (the process whereby dielectric particles, such as living cells, in a non-uniform electrical field, are prevented from leaving microwells).

[0412] Cultivation conditions may be chosen to optimise the proliferation of cells. This may comprise the co-cultivation with feeder cells that prevent the cell death or lack of growth of single cells that were diluted from a sample. In addition, cytokines and nutrients can be included in the media to further enhance cell division. Depending on the desired T-cell type different optimal conditions may be applied. In some cases it may be advantageous to trigger or enhance the production of exosomes by target T-cells as a source of cell-free nucleic acid for testing, wherein the aforementioned production of exosomes may be triggered by activation of said T-cells by antigen presenting cells or contacting with IL-2.

[0413] Given that a population of 10.sup.6 T-cells isolated from a blood sample contains 1 T-cell of interest with a previously identified CDR3, an array-based screening procedure can be employed. A typical RT-PCR machine can handle 384-well microtiter plates. The 10.sup.6 T-cells can be equally diluted into 384 wells, amounting to about 310.sup.3 cells per well, one of which harbours the clonotype of interest. After 4 divisions each cell would be present in 8 copies, whereby the clone of interest is ideally still present in the same 1:310.sup.3 ratio as before. One half of the supernatant is withdrawn and the DNA (or mRNA) is purified while keeping the coordinates in the aliquot 384 microtiter plate. (For automated DNA or RNA purification methods see here: https://www.promega.de/resources/tools/automated-methods/). The samples are subjected to RT-PCR to detect the coordinates of the target clonotype. Once the coordinates are known, the aliquot of living cells (4 in 10.sup.4 cells) from the coordinate is diluted into another 384 well plate. Now up to 4 wells may contain the target clonotype with a ratio of about 1:30. After 4 cell divisions, the wells are screened again by PCR and the aliquot of positive wells (4 in 120) may be diluted again into a microtiter plate with appropriate dimensions to yield clonal cultures. All positive wells may be diluted into one 384 plate, even at the potential loss of some target cells. After further 4 cell divisions, the positive clones can be quickly identified in an aliquot by RT-PCR or other probe-based methods. In order to optimise growth conditions appropriate media with cytokines and feeder cells (which can be easily distinguished by surface antigens) can be used.

[0414] In the case that more positive clones are present in the original sample of 1 million cells, the procedure can process more of these to have a higher chance of obtaining proliferating clonotypes for expansion.

[0415] The cell division rate and the capacity to expand of target clonotypes is limiting for this procedure. It may take 24-48 h for a CD4+ T-cell to divide for the first time in vitro whereas subsequent divisions typically occur much faster. If one T-cell division takes 1 day, then the procedure with 3 arrays will take at least 2 weeks. However, for effective treatment prior expansion of clonotypes is imperative. The above method intrinsically favours the isolation of proliferative T-cells. If the cell-free supernatant contains TCR-beta mRNA, then isolation may proceed faster by non-destructive analysis of the supernatant.

Example 7: Efficiency of the Sequence Based Prediction of Tumour Reactive T Cell Clonotypes

[0416] In table 2 the 100 most frequent clonotypes are exemplary depicted (NN: clonotype could not be measured in non-tumor tissue). The shown 100 most clonotypes equate to SEQ ID 01 to 100. In Table 3, 4 and 5 the most frequent 5, 10 and 15 clonotypes, respectively, of freshly isolated TILs from NSCLC-tumour samples are shown (column E), characterized by unique CDR3-beta peptides (column A) and their flanking V- and J-segments (columns B and C). IFNgamma secretion assay after co-incubation of expanded CD4.sup. TILs with autologous tumour cells reveals the presence of a significant number of clearly tumour-reactive CD8+ clones within the TOP 5, 10 and 15 (column H in Table 3-5, IFNgamma>0.25).

[0417] In table 3 the CDR3 region (peptide) of the beta T cell receptor is shown as found identical in different samples of the same tumour patient (NSCLC) for the top 5 TILs CD8+ clonotypes. V-segments and J-segments are denoted according to IMGT nomenclature. The CDR3 frequencies as percent of sequence reads are given for the following samples: BLOOD: T cells were extracted from blood (PBMCs). TILs CD8.sup.+: T cells from tumour (TILs) were extracted and sorted with respect to CD8.sup.+. non-TUMOUR CD8.sup.+: lung tissue samples were taken distal from tumour and T cells extracted and sorted (CD8.sup.+). TILs CD4.sup.PD1.sup.+: T cells were extracted from tumour, depleted with respect to CD4 and sorted by a PD1 specific antibody, which results in the fraction of activated cytotoxic T cell. IFNgamma CD4.sup.: T cells originally extracted from tumour were kept in culture for 20 days, co-cultured with tumour cells and measured for secretion of IFNgamma by a commercial assay, which shows the activation of T cells as a direct measure of tumour reactivity. TILs CD8.sup.+/non-TUMOUR CD8.sup.+: Ratio of frequencies found in TILs and non-tumour samples (CD8.sup.+). For ratios>5 (>20) there is a clear prevalence of highly tumour reactive clonotypes as shown simultaneously by the IFNgamma and PD1+ frequencies.

[0418] Table 4 shows the same as in Table 3, but for the top 10 TILs CD8.sup.+ clonotypes. Again, for TILs CD8.sup.+/non-TUMOUR CD8.sup.+ ratios>5 (>20) there is a clear prevalence of highly tumour reactive clonotypes as shown simultaneously by the IFNgamma and PD1+ frequencies.

[0419] Table 5 shows the same as in Table 3, but for the top 15 TILs CD8.sup.+ clonotypes. Again, for TILs CD8.sup.+/non-TUMOUR CD8.sup.+ ratios>5 (>20) there is a clear prevalence of highly tumour reactive clonotypes as shown simultaneously by the IFNgamma and PD1+ frequencies.

[0420] These high frequency, tumour-reactive clones can be predicted and identified applying the ratio of frequencies between tumour and non-tumour CD8+ T-cells (T/nT ratio, column I).

[0421] In table 3, within the Top 5, the T/nT ratio of >20 identifies clone 2, the ratio of >5 the clones 1, 2 and 4. Thus, all tumour-reactive clones within the Top 5 are identified using the T/nT ratio.

[0422] In table 4, within the TOP 10, the ratio of >20 identifies the clones 2 and 7, the ratio of >5 the clones 1, 2, 4, and 7 as tumour-reactive.

[0423] In table 5, within the TOP 15, the ratio of >20 identifies the clones 2 and 7, the ratio of >5 the clones 1, 2, 4, 11 and 12, comprising all tumour-reactive clones within the 15 most frequent CD8+ TILs.

[0424] In table 6 the comparison of 3 methods of identifying tumour specific T cells is shown for IFNgamma frequencies>0.25: a) only tumour tissue is used, i.e. all statistics refer to TILs alone. b) TIL (CD8+) frequencies are compared to T cells (CD8+) from non-tumour tissue and only TILs with a tumour/non-tumour ratio of >20 are used. c) TIL (CD8+)frequencies are compared to T cells (CD8+) from non-tumour tissue and only TILs with a tumour/non-tumour ratio of >5 are used. It is obvious that the best results in terms of number of tumour reactive T cells and strength of measured IFN signal are reached by the tissue comparisons, preferably with a ratio>5.

[0425] For a selection of TOP 15 clonotypes, the prediction of tumour-reactivity is shown to be quite accurate in FIG. 1: The rule ratio T/nT>5 separates the T cell clonotypes efficiently into highly tumour-reactive and minor tumour-reactive ones. For a ratio T/nT>20 the prediction of tumour-reactivity is 100% correct, with the price to miss a number of truly tumour-reactive clonotypes.

Example 8: TCR-Sequence-Specific Isolation of Tumour-Reactive Clonotypes

[0426] The identification of tumour-reactive clonotypes characterized by specific sequences (CDR3beta, Vbeta segment) opens the way for sequence specific strategies for enrichment of tumour-reactive clones.

[0427] 6 weeks after resection of the tumour (NSCLC), blood was taken from the patient and PBMCs prepared. Part of the PBMCs were sequenced for TCRbeta. An aliquot of the PBMC preparation was incubated with a Vbeta-30 antibody specific for clone 2 of the patient.

[0428] Result are given in Table 7 showing the enrichment of desired T cell clones by sequence specific sorting with respective Vbeta-segment specific antibodies. CDR3 peptide: The CDR3 region (peptide) of the beta T cell receptor. V-segments and J-segments are denoted according to IMGT nomenclature. TILs CD8.sup.+: T cells from tumour (TILs) were extracted and sorted with respect to CD8.sup.+. IFNgamma CD4.sup.: T cells originally extracted from tumour were kept in culture for 20 days, co-cultured with tumour cells and measured for secretion of IFNgamma by a commercial assay, which shows the activation of T cells stimulated by the respective antigens. TILs CD4.sup.PD1.sup.+: T cells were extracted from tumour, depleted with respect to CD4 and sorted by a PD1 specific antibody, which results in the fraction of activated cytotoxic T cell. TILs CD8.sup.+/non TUMOUR CD8.sup.+: Ratio of frequencies found in TILs and non-tumour samples (CD8.sup.+). Vbeta-AB: The respective Vbeta-segment specific antibody used for capturing of dedicated clonotypes. Freq. in Vbeta AB selection: Frequency of respective clonotype after using bead separation with a Vbeta-specific antibody. Freq. in PBMC: Frequency of respective clonotype in peripheral blood. Enrichment factor: The ratio of clonotype frequencies after separation by Vbeta-antibody versus frequency in peripheral blood. For the second clonotype there was no detectable frequency in peripheral blood, so that the enrichment factor could only be guessed by employing the lower threshold of 0.001% as the highest possible value.

[0429] Clone 2 was measured in the PBMCs of the patient with a frequency of 0.097% (column J). Using the Vbeta-30 antibody and beads separation the frequency was increased to 5.52% (column I). This is an enrichment factor of 57.0, setting the stage for full isolation of the clone with standard procedures from peripheral blood of the patient.

Methods and Materials

[0430] The following experiments were approved by the Berlin chamber of physicians ethics committee (Nr. Eth-62-15).

Initiation and Expansion of T-Lymphocyte Microcultures from Tumour and Lung Tissue Fragments

[0431] Each tumour specimen was dissected free of surrounding normal tissue and necrotic areas. Approx. 1 g cubes from tumour and normal lung tissue were cut into small chunks measuring about 2-3 mm in each dimension. Sliced tumour (and also non-tumour) biopsies were subjected to a commercial mechanical/enzymatic tissue dissociation system (GentleMACS, Miltenyi Biotec, Bergisch-Gladbach, Germany), using the Tumour Dissociation Kit (Miltenyi Biotech) and following the manufacturer's instructions.

[0432] After GentleMACS disaggregation, cell suspensions were passed through 70-m strainers. Aliquots of tumour cells were taken and cryopreserved in 10% DMSO (Sigma-Aldrich) and 90% FCS (Life Technologies) for later use. The remaining cell suspension was subjected to density gradient centrifugation using a 40%/80% step gradient of Percoll (GE Healthcare Europe GmbH) in PBS/RPMI 1640. T-lymphocytes were harvested from the interphase and washed in complete medium (RPMI 1640, Lonza). Subsequently, T-lymphocyte were placed in a 24-well tissue culture plate with 2 mL of recovery medium (RM) at a concentration of 0.510.sup.6cells/ml. RM consisted of RPMI 1640 supplemented with 25 mM HEPES pH 7.2 and L-glutamine (Lonza), 100 IU/mL penicillin, 100 g/mL streptomycin, and 50 M -mercaptoethanol (ThermoFisher Scientific, Waltham, Mass., USA), supplemented with 10% autologous human serum. Plates were placed in a humidified 37 C. incubator with 5% CO.sub.2 and cultured overnight.

[0433] The next day, cells were harvested and pooled from the wells and separated by the magnetic beads-based MidiMACS system, using CD4 and CD8 MicroBeads and LS columns (Miltenyi Biotech), according to the manufacturer's protocol. The flow-through of the CD4MicroBeads experiments, i.e. the CD4-depleted cell fractions were further used for tracking CD8 TIL clnotypes in PD1 and INFgamma experiments. For separation of PD1+ clonotypes PD-1 Microbeads (Miltenyi Biotech, positive selection) were used. All cell fractions were cultured in complete medium (CM) at a density of 0.5-110.sup.6 cells/ml. CM consisted of RPMI 1640 supplemented with 25 mM HEPES pH 7.2 and L-glutamine (Lonza), 100 IU/mL penicillin, 100 g/mL streptomycin, 2.5 mg/L amphotericin B (Sigma-Aldrich, St Louis, USA), and 50 M -mercaptoethanol (ThermoFisher Scientic), supplemented with 10% fetal calf serum (FCS), plus 3000 IU/mL of recombinant human IL-2 (Miltenyi Biotec) and Dynabeads Human T-Activator CD3/CD28 (Life Technologies) at a bead:T-cell ratio of 1:1. The plates were placed in a humidified 37 C. incubator with 5% CO.sub.2 and cultured until day 22. Every second or third day, half of the medium was removed and replaced with fresh medium supplemented with fresh IL-2. Whenever necessary, cells were split in doubled wells by the addition of fresh medium supplemented with IL-2 to maintain a cell density of 10.sup.6 cells/ml. Within the first week, the cell cultures were harvested for DNA extraction and NGS library preparation, residual TILs were further expanded. Between day 14 and 18 Dynabeads were removed, IL-2 concentration in the medium was reduced to 1500 IU/ml and 10% FCS was replaced by 6% autologous human serum.

Interferon-Gamma Secretion AssayCell Enrichment and Detection

[0434] For the tumour co-culture assay on day 22, the IL-2 was omitted from the medium. The co-culture was established with a 1:1 ratio of expanded TILs and autologous tumour cells (10.sup.5 TILs and 10.sup.5 autologous tumour cells per well). Tumour cells were derived from the initial tumour digest that was cryopreserved in 10% DMSO (Sigma-Aldrich) and 90% FCS (Life Technologies) and were washed in RPMI 1640 before addition. The co-culture was incubated in a humidified incubator for 20 h at 37 C. before the cells were harvested and analysed for interferon gamma (IFN) production in an IFN Secretion Assay and Detection Kit (Miltenyi Biotec) according to the manufacturer's instructions. Beads bound cells were eluted and pelleted for genomic DNA isolation and NGS library preparation.

V-Beta Antibody-Based Cell Enrichment

[0435] For isolation of T cells from blood 3 ml of freshly drawn blood were incubated with 5 volumes of erythrocyte lysis buffer (EL buffer, Qiagen) for 15 minutes at 4 C. Mononuclear cells were pelleted in a refrigerated centrifuge at 400 g. Cells were washed several times with EL buffer and PBS and finally labeled with anti-Vbeta 30 antibody PE-conjugate (Beckman Coulter, Brea, Calif., USA). Cells were indirectly magnetically labeled with anti-PE MicroBeads (Miltenyi Biotec) and separated on MS columns using the MiniMACS magnetic separation system following the manufacturer's instructions (Miltenyi Biotec). Beads bound cells were eluted and pelleted for genomic DNA isolation and NGS library preparation.

Genomic DNA Isolation

[0436] Genomic DNA (gDNA) was extracted from tissue materials using the NucleoSpin Tissue Kit from Macherey-Nagel (Duren, Germany). Blood gDNA was 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.

Calculation of Clonotype (Sequence Cluster) Frequencies from NGS Data

[0437] CDR3 regions of the TCR-chain were sequenced with NGS (Illumina MiSEQ) technology following a proprietary 2-step PCR amplification method (as disclosed in WO 2014/096394 A1) which is using TCR primers binding specifically to the V- and J-segments adjacent to the CDR3 region. Genomic DNA was used as starting material for the NGS process.

[0438] Per sample a large (>10.sup.5) number of paired reads (nucleotide sequences) is commonly produced by NGS. The read-pairs are overlapping by typically 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, the number of reads per cluster determines the frequency of that cluster, where frequency of a cluster is measured in percentage of reads of this sample falling into this cluster.

[0439] Clustering is very conservative and 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 [0440] not more than 1 bp mismatch and [0441] where one cluster (cluster A) has at least 20 more reads than the other cluster (cluster B)
are merged and regarded as identical to cluster A. The nucleotide sequence clusters are regarded as equivalent to clonotypes.

[0442] 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

[0443] 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 TCR 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. Ratios between distinct samples (sharing the same CDR3 amino acid sequence) are calculated by ratio of the respective frequencies.

TABLE-US-00002 TABLE2 F G E non- TILsCD8.sup.+/ A B C D TILs TUMOR non_TUMOR CDR3peptide Vsegm Jsegm BLOOD CD8.sup.+ CD8.sup.+ CD8.sup.+ CASSVDRGAEAFF V19*01/*02/*03 J1-1*01 0,000 3,660 0,407 8,993 CAWNKQVDGYTF V30*01/**03/*05 J1-2*01 0,026 2,394 0,055 43,527 CASSFGVMNTEAFF V5-5*01/*02/*03 J1-1*01 0,000 2,330 1,461 1,595 CASSPDGETQYF V4-2*01/*02 J2-5*01 0,000 2,256 0,201 11,224 CASSLGQAYEQYF V7-8*01/*02/*03 J2-7*01 0,608 1,786 1,016 1,758 CASSPVAGMNTEAFF V7-3*01/*05 J1-1*01 0,035 1,417 3,386 0,418 CAISDWIGSNYGYTF V10-3*01/*02/*03/*04 J1-2*01 0,000 1,162 0,058 20,034 CASSGRGDLLEQYF V5-6*01 J2-7*01 0,326 1,121 0,596 1,881 CASSETGAAETQYF V18*01 J2-5*01 0,000 1,095 4,883 0,224 CASSRLAGGTDTQYF V7-3*01/*05 J2-3*01 0,564 0,950 2,477 0,384 CASSSGLVYEQYF V19*01/*02/*03 J2-7*01 0,000 0,898 0,128 7,016 CASSTGTGGLGELFF V28*01 J2-2*01 0,000 0,875 0,102 8,578 CASSEAPPLYYEQYF V6-1*01/V6--5*01/ J2-7*01 0,051 0,855 0,211 4,052 -6*01/-6*02/-6*03/ -6*04/-6*05 CASSNDRAGLNEQFF V6-1*01/V6--5*01/ J2-1*01 0,352 0,846 0,739 1,145 -6*01/-6*02/-6*03/ -6*04/-6*05 CATSDGRLEQFF V24-1*01 J2-1*01 0,127 0,822 0,113 7,274 CASSLGYRYGTEAFF V5-4*01/*02/*03/*04 J1-1*01 0,752 0,810 3,512 0,231 CASSQDNGGYGYTF V4-1*01/*02 J1-2*01 0,000 0,803 0,148 5,426 CASSQGDSFYGYTF V4-1*01/*02 J1-2*01 0,232 0,800 0,195 4,103 CASSADLGDRVNGYTF V5-1*01/*02 J1-2*01 0,000 0,782 0,807 0,969 CASSLDRGGYEQYF V4-1*01/*02 J2-7*01 0,000 0,754 0,140 5,386 CARPPAGIPDTQYF V28*01 J2-3*01 0,000 0,731 0,000 NN CASSDQGHSNQPQHF V4-1*01/*02 J1-5*01 0,160 0,713 0,130 5,485 CASSRPSFRVSEQFF V4-1*01/*02 J2-1*01 0,464 0,704 1,214 0,580 CASSLLLAGASYEQYF V5-5*01/*02/*03 J2-7*01 0,343 0,665 0,392 1,696 CASSSFQGGNEQFF V28*01 J2-1*01 0,874 0,614 3,060 0,201 CASSLVRGNEQFF V27*01 J2-1*01 0,068 0,609 0,189 3,222 CASSLERSERPYEQYF V7-9*01-*07 J2-7*01 0,801 0,596 4,943 0,121 CASTPRGNTGELFF V6-1*01/V6--5*01/ J2-2*01 0,145 0,571 0,280 2,039 -6*01/-6*02/-6*03/ -6*04/-6*05 CASNPGRGTREQYF V5-6*01 J2-7*01 0,020 0,564 0,098 5,755 CASSLRINYEQYF V5-5*01/*02/*03 J2-7*01 0,000 0,557 0,307 1,814 CASSRPEATNEKLFF V4-1*01/*02 J1-4*01 0,000 0,556 0,012 46,333 CASSWGTDTEAFF V27*01 J1-1*01 0,041 0,472 0,098 4,816 CAWAKGTEAFF V30*01/**03/*05 J1-1*01 0,000 0,471 0,015 31,400 CASSQVTGITEAFF V14*01/*02 J1-1*01 0,302 0,457 0,668 0,684 CASSPGGRPYEQYF V5-4*01/*02/*03/*04 J2-7*01 0,000 0,417 0,010 41,700 CASSPGQGEGYEQYF V4-1*01/*02 J2-7*01 0,070 0,387 0,104 3,721 CASSQVGSSVAGGRSEA V4-1*01/*02 J1-1*01 0,000 0,351 0,288 1,219 CASSSTGTGGSSWNEQF V6-1*01/V6--5*01/ J2-1*01 0,000 0,350 0,018 19,444 -6*01/-6*02/-6*03/ -6*04/-6*05 CATGTGSYEQYF V19*01/*02/*03 J2-7*01 0,000 0,284 0,000 NN CASSLWEASYGYTF V5-6*01 J1-2*01 0,012 0,282 0,174 1,621 CASSQTGTGSYEQYF V4-1*01/*02 J2-7*01 0,000 0,280 0,130 2,154 CASSIAQGVYEQYF V27*01 J2-7*01 0,000 0,278 0,866 0,321 CASSQRRLNTEAFF V16*01/**02/*03 J1-1*01 0,000 0,273 0,000 NN CASSLGTAKETQYF V7-9*01-*07 J2-5*01 0,214 0,262 1,014 0,258 CASSFEAPAYEQYF V5-8*01/*02 J2-7*01 0,000 0,252 0,258 0,977 CASSLAGGLVEQYF V19*01/*02/*03 J2-7*01 0,267 0,249 0,188 1,324 CATTQAGTENTEAFF V19*01/*02/*03 J1-1*01 0,000 0,246 0,055 4,473 CASSPGQGEGYEQYF V4-1*01/*02 J2-7*01 0,030 0,242 0,022 11,000 CASSQEGEGETQYF V4-1*01/*02 J2-5*01 0,026 0,237 0,019 12,474 CASSVGPGLNMQVTDTQ V7-6*01/*02 J2-3*01 0,000 0,236 0,027 8,741 CASSYRDSSSYEQYF V9*01/*02/*03 J2-7*01 0,000 0,229 0,000 NN CASSYLAEPPGNEQFF V6-2*01/**02/**03/ J2-1*01 0,078 0,229 0,199 1,151 -3*01 CASSSYSETANYGYTF V5-1*01/*02 J1-2*01 0,014 0,223 0,097 2,299 CASSQERSTGELFF V4-2*01/*02 J2-2*01 0,000 0,223 0,132 1,689 CASSYWGGTNTEAFF V6-1*01/V6--5*01/ J1-1*01 0,000 0,219 0,189 1,159 -6*01/-6*02/-6*03/ -6*04/-6*05 CASSIDRGSEAFF V19*01/*02/*03 J1-1*01 0,000 0,217 0,144 1,507 CASSQVLSGGFYEQYF V4-1*01/*02 J2-7*01 0,000 0,216 0,154 1,403 CAWSKEYGYTF V30*01/**03/*05 J1-2*01 0,000 0,213 0,000 NN CAWTWGGGNEQYF V30*01/**03/*05 J2-7*01 0,194 0,213 0,084 2,536 CATSDLHRTPDLNTEAF V24-1*01 J1-1*01 0,038 0,207 0,111 1,865 CASSSQGDGTDTQYF V7-9*01-*07 J2-3*01 0,000 0,202 0,135 1,496 CASSPGPNYEQYF V7-6*01/*02 J2-7*01 0,021 0,201 0,012 16,750 CASSLEEYGYTF V7-2*01/*02/*03/*04 J1-2*01 0,605 0,199 1,816 0,110 CASSQDRSVAYEQYF V4-3*01/*02/*03/*04 J2-7*01 0,000 0,198 0,000 NN CASSLRGKTSTYEQYF V7-8*01/*02/*03 J2-7*01 0,017 0,191 0,194 0,985 CASSLSSKNEQFF V27*01 J2-1*01 0,000 0,188 0,085 2,212 CAVNQAGWGGTQYF V27*01 J2-3*01 0,108 0,180 0,060 3,000 CAWSFPGASGG*ETQYF V30*01/**03/*05 J2-5*01 0,000 0,180 0,132 1,364 CASSQRAAPYGYTF V4-1*01/*02 J1-2*01 0,000 0,177 0,039 4,538 CASSSGHGYNEQFF V3-1*01/*02 J2-1*01 0,000 0,169 0,129 1,310 CASSLLLSGGAADTQYF V27*01 J2-3*01 0,011 0,166 0,560 0,296 CASSRGPNYEQYF V7-6*01/*02 J2-7*01 0,043 0,158 0,172 0,919 CASSIDSNNEQFF V19*01/*02/*03 J2-1*01 0,077 0,155 0,163 0,951 CATSDLIDFDRVDGYTF V24-1*01 J1-2*01 0,000 0,153 0,000 NN CASSPLTGMQFF V7-6*01/*02 J2-1*01 0,000 0,147 0,098 1,500 CASIWRLGMNTEAFF V19*01/*02/*03 J1-1*01 0,000 0,145 0,260 0,558 CASSSTVAGEQYF V27*01 J2-7*01 0,444 0,145 1,494 0,097 CASSPRTGNTGELFF V4-2*01/*02 J2-2*01 0,065 0,143 0,164 0,872 CASTRSVGAGTEAFF V27*01 J1-1*01 0,000 0,140 0,085 1,647 CASSPGTDGSSLGSPLH V27*01 J1-6*01 0,000 0,137 0,015 9,133 CASSWDSSYEQYF V6-2*01/**02/**03/ J2-7*01 0,000 0,134 0,029 4,621 -3*01 CASSPLGGEKLFF V6-1*01/V6--5*01/ J1-4*01 0,000 0,134 0,271 0,494 -6*01/-6*02/-6*03/ -6*04/-6*05 CASSQAGIHGYTF V14*01/*02 J1-2*01 0,000 0,130 0,079 1,646 CASSIAGGPGETQYF V19*01/*02/*03 J2-5*01 0,000 0,126 0,088 1,432 CASSQVPDRDGYTF V4-3*01/*02/*03/*04 J1-2*01 0,000 0,123 0,194 0,634 CASSQGAALGYEQYF V4-1*01/*02 J2-7*01 0,000 0,122 0,000 NN CASSEYLEVQETQYF V25-1*01 J2-5*01 0,027 0,120 0,090 1,333 CASSLEANNEQFF V5-6*01 J2-1*01 0,000 0,120 0,101 1,188 CAISESKDRPSSYNEQF V10-3*01/*02/*03/*04 J2-1*01 0,000 0,119 0,129 0,922 CASSPGAGLYEQYF V5-4*01/*02/*03/*04 J2-7*01 0,000 0,119 0,149 0,799 CASSQKWGNIQYF V14*01/*02 J2-4*01 0,017 0,118 0,046 2,565 CATGLAGGQEQYF V24-1*01 J2-7*01 0,099 0,118 0,070 1,686 CASSLTDYGYTF V7-2*01/*02/*03/*04 J1-2*01 0,306 0,118 1,023 0,115 CASSLTDYGYTF V7-2*01/*02/*03/*04 J1-2*01 0,083 0,117 0,176 0,665 CASTPGSYRETQYF V5-1*01/*02 J2-5*01 0,055 0,116 0,491 0,236 CASGTDFPSYEQYF V19*01/*02/*03 J2-7*01 0,000 0,115 0,017 6,765 CAIPSSSGANVLTF V10-3*01/*02/*03/*04 J2-6*01 0,000 0,112 0,000 NN CASSLVGGPHEQYF V7-9*01-*07 J2-7*01 0,000 0,111 0,000 NN CASSSAGTGHNEQFF V6-1*01/V6--5*01/ J2-1*01 0,000 0,111 0,054 2,056 -6*01/-6*02/-6*03/ -6*04/-6*05 CASSQKDRYGYTF V4-2*01/*02 J1-2*01 0,000 0,109 0,010 10,900

TABLE-US-00003 TABLE3 F G I E non- TILs H TILsCD8.sup.+/ J A B C D TILs TUMOR CD4.sup.- IFNgamma non_TUMOR TSTC CDR3peptide Vsegm Jsegm BLOOD CD8.sup.+ CD8.sup.+ PD1.sup.+ CD4.sup.- CD8.sup.+ score CASSVDRGAEAFF V19*01/*02/*03 J1-1*01 0,000 3,660 0,407 1,066 2,742 8,993 1010 CAWNKQVDGYTF V30*01/**03/*05 J1-2*01 0,026 2,394 0,055 1,231 0,740 43,527 1010 CASSFGVMNTEAFF V5-5*01/*02/*03 J1-1*01 0,000 2,330 1,461 0,533 0,089 1,595 1110 CASSPDGETQYF V4-2*01/*02 J2-5*01 0,000 2,256 0,201 0,971 1,127 11,224 1010 CASSLGQAYEQYF V7-8*01/*02/*03 J2-7*01 0,608 1,786 1,016 0,115 0,045 1,758 1110

TABLE-US-00004 TABLE5 F G I E non- TILs H TILsCD8.sup.+/ J A B C D TILs TUMOR CD4.sup.- IFNgamma non_TUMOR TSTC CDR3peptide Vsegm Jsegm BLOOD CD8.sup.+ CD8.sup.+ PD1.sup.+ CD4.sup.- CD8.sup.+ score CASSVDRGAEAFF V19*01/*02/*03 J1-1*01 0,000 3,660 0,407 1,066 2,742 8,993 1010 CAWNKQVDGYTF V30*01/**03/*05 J1-2*01 0,026 2,394 0,055 1,231 0,740 43,527 1010 CASSFGVMNTEAFF V5-5*01/*02/*03 J1-1*01 0,000 2,330 1,461 0,533 0,089 1,595 1110 CASSPDGETQYF V4-2*01/*02 J2-5*01 0,000 2,256 0,201 0,971 1,127 11,224 1010 CASSLGQAYEQYF V7-8*01/*02/*03 J2-7*01 0,608 1,786 1,016 0,115 0,045 1,758 1110 CASSPVAGMNTEAFF V7-3*01/*05 J1-1*01 0,035 1,417 3,386 0,212 0,244 0,418 1110 CAISDWTGSNYGYTF V10-3*01/*02/ J1-2*01 0,000 1,162 0,058 0,393 2,382 20,234 1010 *03/*04 CASSGRGDLLEQYF V5-6*01 J2-7*01 0,326 1,121 0,596 0,092 0,000 1,881 1110 CASSETGAAETQYF V18*01 J2-5*01 0,000 1,095 4,883 0,246 0,097 0,224 1110 CASSRLAGGTDTQYF V7-3*01/*05 J2-3*01 0,564 0,950 2,477 0,052 0,040 0,384 1110 CASSSGLVYEQYF V19*01/*02/*03 J2-7*01 0,000 0,898 0,128 0,217 0,888 7,016 1010 CASSTGTGGLGELFF V28*01 J2-2*01 0,000 0,875 0,102 1,050 0,651 8,578 1010 CASSEAPPLYYEQYF V6-1*01/ J2-7*01 0,051 0,855 0,211 0,000 0,000 4,052 1110 V6--5*01/-6*01/ -6*02/-6*03/ -6*04/-6*05 CASSNDRAGLNEQFF V6-1*01/ J2-1*01 0,352 0,846 0,739 0,017 0,000 1,145 1110 V6--5*01/-6*01/ -6*02/-6*03/ -6*04/-6*05 CATSDGRLEQFF V24-1*01 J2-1*01 0,127 0,822 0,113 0,725 0,000 7,274 1010

TABLE-US-00005 TABLE4 F G I E non- TILs H TILsCD8.sup.+/ J A B C D TILs TUMOR CD4.sup.- IFNgamma non_TUMOR TSTC CDR3peptide Vsegm Jsegm BLOOD CD8.sup.+ CD8.sup.+ PD1.sup.+ CD4.sup.- CD8.sup.+ score CASSVDRGAEAFF V19*01/*02/*03 J1-1*01 0,000 3,660 0,407 1,066 2,742 8,993 1010 CAWNKQVDGYTF V30*01/**03/*05 J1-2*01 0,026 2,394 0,055 1,231 0,740 43,527 1010 CASSFGVMNTEAFF V5-5*01/*02/*03 J1-1*01 0,000 2,330 1,461 0,533 0,089 1,595 1110 CASSPDGETQYF V4-2*01/*02 J2-5*01 0,000 2,256 0,201 0,971 1,127 11,224 1010 CASSLGQAYEQYF V7-8*01/*02/*03 J2-7*01 0,608 1,786 1,016 0,115 0,045 1,758 1110 CASSPVAGMNTEAFF V7-3*01/*05 J1-1*01 0,035 1,417 3,386 0,212 0,244 0,418 1110 CAISDWTGSNYGYTF V10-3*01/*02/ J1-2*01 0,000 1,162 0,058 0,393 2,382 20,234 1010 *03/*04 CASSGRGDLLEQYF V5-6*01 J2-7*01 0,326 1,121 0,596 0,092 0,000 1,881 1110 CASSETGAAETQYF V18*01 J2-5*01 0,000 1,095 4,883 0,246 0,097 0,224 1110 CASSRLAGGTDTQYF V7-3*01/*05 J2-3*01 0,564 0,950 2,477 0,052 0,040 0,384 1110

TABLE-US-00006 TABLE 6 percentage tumor non- tumor median reactive reactive reactive IFNgamma clones clones clones top 5TILs a. no non-tumor 0.74 3 2 60% tissue used b. ratio tumor/ 0.74 1 0 100% non-tumor >20 c. ratio tumor/ 1.13 3 0 100% non-tumor >5 top 10TILs a. no non-tumor 0.17 4 6 40% tissue used b. ratio tumor/ 1.56 2 0 100% non-turner >20 c. ratio tumor/ 1.76 4 0 100% non-tumor >5 top 15TILs a. no non-tumor 0.10 6 9 40% tissue used b. ratio tumor/ 1.56 2 0 100% non-tumor >20 c. ratio tumor/ 0.89 7 1 88% non-tumor >5

TABLE-US-00007 TABLE7 Freq. TILs in Freq. enrich- TILs IFNgamma CD4.sup.- TILS_CD8/ Vbeta- Vbeta in ment CDR3peptide Vsegm Jsegm CD8+ CD4.sup.-PD1.sup.+ PD1.sup.+ non_TUMOR AB AB PBMC factor CAWNKQVDGYTF V30*01/**03/*05 J1-2*01 2,394 0,740 1,231 43,527 V30-AB 5,525 0,097 57,019 CAWAKGTEAFF V30*01/**03/*05 J1-1*01 0,471 0,703 0,254 31,400 V30-AB 1,294 no >1000 value