RU2808595C1 - Method of obtaining a culture of lymphocytes enriched in tumor-specific t-lymphocyte clones and cell cultures obtained using the specified method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of obtaining a culture of lymphocytes enriched with tumor-specific clones of T-lymphocytes. The method involves the steps of isolating autologous tumor-infiltrating lymphocytes (TILs) from a patient suffering from solid cancer, autologous TILs are cultured in a medium containing IL-2 and at least one of agents selected from IL-21 and a signal transduction blocking antibody PD-1:PD-L1, determine the repertoire of nucleotide sequences of T-cell receptors using high-throughput sequencing, assess the degree of enrichment of cultured TILs with tumor-specific clones using bioinformatics analysis, which determines the presence of clusters of convergent variants of T-cell receptors in the repertoire.

EFFECT: invention is effective for large-scale expansion using known ex vivo expansion protocols and administration to a cancer patient during adoptive cell therapy.

18 cl, 7 dwg, 1 tbl, 2 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of medicine, in particular to the field of immunotherapy of cancer, specifically to adoptive T-cell therapy. More specifically, the present invention provides a method for producing a culture of lymphocytes enriched in tumor-specific T lymphocyte clones, which can then be scaled up using known ex vivo expansion protocols and administered to a cancer patient through adoptive cell therapy (ACT). .

State of the art

Tumors develop various mechanisms to evade the immune response, including tissue hypoxia, inflammation, creation of an immunosuppressive microenvironment, suppression of antigen presentation, stimulation of regulatory T cells (Treg) infiltration and proliferation, and induction of T cell dysfunction. Administration of large numbers of ex vivo cultured autologous tumor-reactive T lymphocytes—usually following treatment regimens aimed at lymphodepletion—is a powerful therapeutic approach that can overcome the immunosuppressive mechanisms used by tumors. Clinical protocols for such ACT therapeutic strategies are currently being actively developed and used to treat patients (Dudley, M. E. et al. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J. Immunother. 26, 332-342 (2003); Kelderman, S. et al. Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy. Eur. J. Immunol. 46, 1351-1360 (2016); Rosenberg, S. A. & Restifo , N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62-68 (2015)).

ACT is a promising approach to cancer immunotherapy, but its effectiveness fundamentally depends on the degree of enrichment of the graft with tumor-specific T lymphocytes. Current methods for enriching a population of autologous cultured lymphocytes with tumor-reactive T lymphocytes are based on the identification of patient-specific peptide neoantigens (antigens resulting from mutations occurring in tumor cells), which are then used for functional characterization and selection of cultured tumor-infiltrating lymphocytes (TILs). ) (Yossef, R. et al. Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 3, (2018); Leko, V. et al. Identification of Neoantigen-Reactive Tumor-Infiltrating Lymphocytes in Primary Bladder Cancer J Immunol 202, 3458-3467 (2019). It is known that tumor cells in the early stages of carcinogenesis are practically indistinguishable in their molecular composition from the cells of the surrounding tissue; they cannot be recognized and destroyed by the immune system, being non-immunogenic. As the tumor grows, due to the accumulation of mutations in DNA, so-called neoantigens are formed, which can be presented by dendritic cells to T-helper cells, which are activators of specific immunity to tumors. The appearance of neoantigens occurs with a delay, which allows tumor cells to implement defense mechanisms against the immune system, in particular, to create a microenvironment that inhibits specific antitumor immunity.

For the purpose of identifying tumor-specific lymphocytes, known tumor-associated antigens (TAAs) can be used. OAAs are an affordable alternative to neoantigens, since the identification of unique neoantigens is expensive, labor-intensive, and functionally limited in terms of the spectra of identified antigens. Also, cultured autologous tumor tissue can be used as a source of antigen-specific stimulus (Seliktar-Ofir, S. et al. Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation. Front. Immunol. 8, 1211 ( 2017)), and both live and lysed tumor cells can be used for these purposes (Gonzalez FE et al. Tumor cell lysates as immunogenic sources for cancer vaccine design. Hum Vaccin Immunother. 2014;10(11):3261-9 ).

Certain cell surface markers, such as PD-1, CD39, CD69, CD103 or CD137, may also be useful in identifying a subset of T lymphocytes (usually CD8+) that are enriched in clonally expanded tumor-reactive T lymphocytes (Gros, A. et. al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J. Clin. Invest. 124, 2246-2259 (2014); Duhen, T. et al. Co-expression of CD39 and CD103 identifies tumor -reactive CD8 T cells in human solid tumors. Nat. Commun. 9,2724 (2018)), therefore culturing selected TIL subpopulations, such as PD-1 ⁺ T lymphocytes, is one of the possible options.

However, in all of the above scenarios, a reliable method is needed to assess the enrichment of transplanted cells with tumor-specific T lymphocytes. It is known that the developing adaptive immune response to various pathogens is often represented by groups of T-lymphocyte clones with highly homologous T-cell receptor (TCR) amino acid sequences (and their corresponding coding nucleotide sequences) that recognize the same epitopes (Dash, P Glanville, J. et al. Identifying specificity groups in the T cell receptor repertoire. Nature 547, 94-98 ( 2017)). Hypothetically, the identification of such convergent TCR clusters could be a highly effective approach to identifying clonotypes involved in the formation of an antitumor immune response.

Accordingly, in oncology and in particular in the field of ACT, there is a great need for both optimization of existing enrichment methods and methods for assessing the extent to which cultured TILs can be enriched with autologous T lymphocytes specific for tumor antigens for use in ACT protocols.

Such protocols, known as the Rapid Expansion Protocol (REP), are described in Dudley et al., Science 2002, 298, 850-854; Dudley et al., J. Clin. Oncol., 2005, 23, 2346-2357; J. Clin. Oncol., 2008, 26, 5233-5239, etc. The parameters of the composition of the TIL population (positive for CD28, CD8 or CD4), as well as numerical values characterizing the degree of population multiplication and its viability, were considered as parameters for the acceptability of TIL grown and propagated in ex vivo conditions for infusion into a patient for ACT purposes in these protocols.

These protocols suffer from a number of shortcomings. Thus, in particular, the possibility of their widespread commercialization is seriously limited by the duration of the process, high cost, and sterility problems. In this regard, protocols have been developed that are suitable for commercialization on an industrial scale and meet regulatory requirements for use in human patients. These protocols primarily include methods developed by Iovance Biotherapeutics and described in patents US 10420799, US 10639330, US 10918666, US 11273180.

Disclosure of the invention

The present invention is based on the unexpectedly discovered ability by the authors of the invention to effectively identify tumor-specific T lymphocytes by analyzing the repertoire of T-cell receptors of TIL using bioinformatics methods, such as, in particular, cluster analysis of the sequences of these receptors. This approach allows: 1) to detect the presence of tumor-specific T lymphocytes in the TIL pool, 2) to optimize the conditions for culturing TIL, and 3) to determine, on the basis of surface markers, the enrichment of freshly isolated or re-stimulated TIL with tumor-specific T lymphocytes.

TIL lymphocytes are usually understood as a population of lymphocytes that migrated into the tumor tissue. TIL cells include, but are not limited to, CD8 ⁺ cytotoxic T lymphocytes, Th1 and Th17 CD4 ⁺ lymphocytes, natural killer cells, dendritic cells and M1 macrophages. T-lymphocytes infiltrating the tumor

enriched with polyclonal T cells with diverse antigen specificities. For example, it is known that melanoma is characterized by a high mutational load and a highly individualized set of neoantigens. Cultivation of TIL in ex vivo conditions after removal of a tumor fragment removes TIL from the aggressive microenvironment present in the tumor and reduces the immunosuppressive effects exerted by regulatory T cells ( _Treg ) intratumorally. Thus, culturing autologous TILs in ex vivo conditions allows not only to preserve the entire unique spectrum of the original tumor-reactivity of T lymphocytes, but also to increase them in many times increased numbers, which makes it possible to carry out ACT through subsequent administration to the patient.

TILs can be characterized biochemically or functionally by their ability to infiltrate the tumor and thereby exert a therapeutic effect. In terms of biochemical characteristics, TILs can generally be categorized based on the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, CD137, PD-1, CD39, CD69, CD103 (Duhen T et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun. 2018; 9(1):2724) and CD25 (Abd Hamid M et al. Self -Maintaining CD103+ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses. Cancer Immunol Res. 2020;8(2):203-216; Draghi A et al. Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor Antigens. Front Immunol. 2021;12:705422).

To identify groups of TCR clonotypes involved in the antitumor immune response, the present invention proposes to use cluster analysis. The proposed approach allows us to successfully identify known TAA-specific TCR clonotypes among the TCR repertoires in patients with melanoma and HLA-A*02. The number of clonotypes in clusters and the proportion of the total TIL repertoire they account for were found to increase significantly after immunotherapy using anti-PD-1 antibodies. A study of TCR content in sorted CD4 ⁺ and CD8 ⁺ CD39 ⁺ PD1 ⁺ TILs showed that these subpopulations are noticeably enriched in TCR clusters, a significant portion of which are formed by tumor-specific TCRs. These results provided the rationale to focus on the use of CD39 ⁺ PD1 ⁺ TILs in cancer ACT. Cluster analysis of the TCR repertoire facilitates the optimization of TIL culture conditions and allows one to assess the degree of enrichment of cultured donor cells and sorted TAA-activated T lymphocytes with tumor-specific T lymphocytes.

The use of TCR cluster analysis is a powerful tool with potential for practical application in clinical ACT.

The authors of the present invention have discovered that homology cluster analysis of T-cell receptors (TCRs) effectively identifies tumor-reactive TCRs, allowing: 1) to detect their presence in the TIL pool; 2) optimize TIL culture conditions, with the combination (low IL-2)/IL-21/anti-PD-1 demonstrating increased efficiency; 3) examine enrichment based on surface markers of tumor-targeting T cells in freshly isolated TIL (enrichment confirmed for CD4 ⁺ and CD8 ⁺ PD-1 ⁺ /CD39 ⁺ subsets) or restimulated TIL (indicates enrichment in 4-1BB- sorted cells (CD137 ⁺ cells)).

The antitumor immune response, like the immune response to any “foreign” antigen/pathogen, includes adaptive and innate components. The adaptive immune response that develops during vaccination or during an infectious disease is characterized by the expansion of many TCR variants, among which there is no clearly defined dominance of individual variants over the rest. To date, such an analysis has not been carried out for TIL. Accordingly, the discovery that using the present invention, including cluster analysis of the TCR sequence repertoire from TIL, it is possible to detect the antitumor immune response at the initial stages of its development; the fact that clusters of homologous sequences can be found precisely among TILs; and also the fact that the clusters are enriched specifically with tumor-reactive TCRs seems unexpected.

The technical result achieved by implementing the invention is to simplify the method for assessing the degree of enrichment of a TIL culture with tumor-reactive T-lymphocyte clones. Also, the technical result consists in obtaining a TIL culture enriched with tumor-reactive T-lymphocyte clones.

In its first aspect, the present invention relates to a method for producing a culture of TILs enriched in tumor-specific clones, comprising the steps of isolating autologous TILs from a patient suffering from solid cancer; autologous TILs are cultured in a medium containing IL-2 and at least one of agents selected from IL-21 and an antibody that blocks PD-1:PD-L1 signaling; determine repertoires of nucleotide sequences of T-cell receptors using high-throughput sequencing; assess the degree of enrichment of cultured TILs with tumor-specific clones using bioinformatics analysis that determines the presence of clusters of convergent T-cell receptor variants in the repertoire.

In one embodiment of the method, convergent variants of T-cell receptors are variants of T-cell receptors characterized by homologous amino acids

sequences containing no more than 3 differences, representing amino acid substitutions and/or insertions and/or deletions.

In yet another embodiment of the method, the degree of enrichment of tumor-specific T-lymphocyte clones is characterized by the proportion of the T-cell receptor repertoire occupied by T-cell receptor clones belonging to clusters of convergent T-cell receptor variants.

In the next embodiment of the method, the degree of enrichment of tumor-specific T-lymphocyte clones is characterized by the number of T-cell receptor clonotypes belonging to clusters of convergent T-cell receptor variants.

In yet another embodiment of the method, at the culture stage, autologous lymphocytes are cultured in a medium containing IL-2, IL-21 and an antibody that blocks PD-1:PD-L1 signaling. In a preferred embodiment of the method, the antibody that blocks PD-1:PD-L1 signaling is nivolumab.

In a further embodiment of the method, autologous lymphocytes are obtained from the patient's tumor material obtained during surgery.

In yet another embodiment of the method, autologous lymphocytes are obtained from the patient's blood.

In one embodiment of the method, the solid cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, ovarian cancer, breast cancer, pancreatic cancer.

In one embodiment, the method further includes the step of pre-isolating from autologous lymphocytes isolated from the patient a fraction enriched in lymphocytes bearing at least one surface marker selected from the group consisting of PD-1, CD39, CD137. In a preferred embodiment, said fraction is enriched in lymphocytes bearing the surface markers PD1 and/or CD39.

In one embodiment of the method, the medium in which the resulting lymphocytes are cultured additionally contains tumor cells and/or at least one product of their processing.

In one embodiment of the method, the medium in which the resulting lymphocytes are cultured additionally contains at least one tumor-associated antigen.

In yet another embodiment of the method, the medium in which the resulting lymphocytes are cultured additionally contains at least one tumor neoantigen. In a preferred embodiment, said at least one tumor neoantigen contains at least one driver mutation.

In yet another embodiment of the method, cultured lymphocytes are further enriched for tumor-specific variants by activation in the presence of at least one tumor antigen and subsequent enrichment using at least one T-lymphocyte activation marker.

Finally, in yet another embodiment of the method, the resulting culture is used in an ex-vivo expansion protocol. In a preferred embodiment, the optimal culture for subsequent use in the ex vivo expansion protocol is selected based on the results of assessing the degree of enrichment of cultured TILs with tumor-specific clones using bioinformatics analysis.

In its next aspect, the present invention relates to a culture of TIL enriched in tumor-specific clones obtained by the method of the present invention.

Brief description of drawings

Fig. 1. TCR clusters from lymphocytes infiltrating melanoma before and after immunotherapy. A, B. Normalized number of clonotypes in TKRβ clusters (normalized ALICE hits) from metastatic melanoma samples before and after anti-PD-1 therapy described in references Riaz, N. et al. Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab. Cell 171, 934-949.e16 (2017) (A) and Tumeh, P. C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568-571 (2014). (B). B. Proportion of OAA-specific TCRs matching those presented in the VDJdb database, within the entire repertoire (total) and within clonotypes in clusters (among hits) in patients with at least one cluster matching with VDJdb (N=8 patients) from the two data sets provided in the above references. N is the number of patients. Data were analyzed using paired Student's t test (A) or Wilcoxon test (B, C).

Figure 2. TCRβ clusters from a sample from patient pt44 (from the complete/partial response group, reference Riaz, N. et al., 2014 (see above)]). A. Number and size of clusters before and after therapy. One point on the graph corresponds to one cluster. B. TKRβ clusters Clusters of OAA-specific clonotypes matching VDJdb are shown in gray.

Fig. 3. Analysis of the TCRβ repertoire for CD8 ⁺ (A, C, E)) and CD4 ⁺ (B, D, F) subpopulations of DP and non-DP TIL obtained by sorting from metastatic lymph nodes of eight melanoma patients. A, B. Repertoire clonality calculated as [1-normalized Shannon-Wiener index]. B, D. Normalized number of clonotypes in clusters (normalized ALICE hits). D, E. Total frequency of clonotypes in clusters). Paired Student's t test (t-test). * - p<0.05, ** - p<0.01, **** - p<0.0001.

Fig. 4. TCRβ clusters identified in the repertoire obtained from fresh frozen tumor tissue (FTT), as well as from sorted CD8 ⁺ DP and non-DP TIL for patient mp26 (HLA-A*02). A. Distribution of TCRβ by clusters depending on tumor reactivity. OAA-specific clusters matching VDJdb are shown in gray; black - clusters with unidentified specificity. B. Distribution of TCRβ into clusters depending on double positivity for CD39 and PD1 (DP) markers. DP clusters are shown in gray, other clusters in black.

Fig. 5. TCR clusters in CD39 ⁺ PD1 ⁺ TIL. A. Cumulative frequency of OAA-specific clonotypes matching VDJdb in the CD8 ⁺ DP, CD8 ⁺ non-DP and COT TCRβ repertoires of patient mp26. B. Cumulative frequency of OAA-specific clonotypes matching VDJdb in clusters in the CD8 ⁺ DP, CD8 ⁺ non-DP and COT TKPJ3 repertoires of patient TP26. One-way analysis of variance with Bonferroni correction for multiple comparisons. B. Proportion of CD137 ⁺ cells (4-1BB ⁺ ) among CD8 ⁺ T lymphocytes in DP and non-DP TILs from patient mp26 that were cultured and restimulated with OAA-loaded (TAA) or control (control) dendritic cells. Bars shaded in gray refer to CD8 ⁺ DP. Open bars refer to CD8 ⁺ non-DP. D. Proportion of TAA-specific clonotypes matching VDJdb in sorted CD137 ⁺ CD8 ⁺ T cells in DP and non-DP TILs from patient mp26 that were cultured and restimulated with TAA-loaded (TAA-stimulated) or control (control ) dendritic cells. Paired Student's t test (t-test). * - p<0.05, ** - p<0.01, **** - p<0.0001.

Fig. 6. Influence of cultivation conditions on TIL and TCR clusters. Analysis of the TCRβ repertoire for patient mp26 TILs cultured under four different conditions. Clusters of clonotypes with TAA specificity determined based on VDJdb are shown in black. Gray color indicates clusters of clonotypes with unidentified or other specificity.

Fig. 7. Analysis of the TCRβ repertoire for TILs from patient tr26 cultured under four different conditions. A. Normalized number of clonotypes in TKRβ clusters (normalized ALICE hits). B. Cumulative frequency of Melan-A-specific TCRβ clusters. B. Number of clonotypes from the DP fraction. D. Number of cells, % of the number of cells obtained under culture conditions y1 (high IL-2). Data presented in panels A, B, and D were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons, with each culture condition compared with the IL-21/(low IL-2)/anti-PD-1 culture conditions. Data in panel (B) were analyzed using the Kruskal-Wallis test, Dunne's multiple comparison test, with each group compared to IL-21/(low IL-2)/anti-PD-1 culture conditions. * - р=1.2⋅10 ^-2 , ** -р=1.7⋅10 ^-3 , **** - р<10 ^-4 . Abbreviations: SOT - fresh frozen tumor tissue. Cultivation conditions: y1 - (high IL-2); y2 - IL-21, (low IL-2); y3 - Nivolumab, IL-21, (low IL-2); y4 - IFNγ, Nivolumab, IL-21, (low IL-2).

Carrying out the invention

Definitions

“Solid tumors” generally refer to abnormal tissue masses that usually do not contain cysts or fluid areas. Solid tumors can be either benign or malignant. The term "solid cancer" means malignant (cancerous) solid tumors. Solid cancers include, but are not limited to, sarcomas, carcinomas and lymphomas, including lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer, melanoma. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative cancer breast and non-small cell lung carcinoma, melanoma. What is common to the tissue structure of all solid cancers is that it contains interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, which can provide a supportive microenvironment among which the cancer cells are distributed.

“Programmed death protein 1 (PD-1)” is a member of the CD28 costimulatory gene family, is moderately expressed on naïve T, B and NKT cells and is up-regulated by T/B cell receptor signaling on lymphocytes, monocytes and myeloid cells. cells. PD-1 has two known ligands with different expression profiles, PD-L1 and PD-L2. The expression of PD-L2 is relatively limited, whereas PD-L1 is expressed more widely, including expression on naive lymphocytes, and its expression is induced on activated B and T cells, monocytes and dendritic cells. PD-1 is considered an important element in immune regulation and maintenance of peripheral tolerance. In the mouse, this has been shown to require PD-L1 expression in peripheral tissues and binding to PD-1 on potentially autoreactive T cells to negatively modulate T cell activation involving the ITIM sequence in the cytoplasmic domain of PD-1.

Human malignancies arising in various tissues have been found to overexpress PD-L1 or PD-L2. In large sample sets of, for example, ovarian, kidney, colorectal, pancreatic, liver, and melanoma cancers, PD-L1 expression has been shown to correlate with poor prognosis and reduced overall survival, regardless of subsequent treatment. Similarly, PD-1 expression on tumor-infiltrating lymphocytes has been found to be a hallmark of dysfunctional T cells in breast cancer and melanoma and correlates with poor prognosis in kidney cancer. Using primary patient samples, it was shown that blockade of PD-1 or PD-L1 in vitro leads to increased tumor-specific human T cell activation and cytokine production. Thus, in several syngeneic mouse tumor models, blockade of PD-1 or PD-L1 significantly inhibited tumor growth or induced complete regression. In general, the PD-1/PD-L1 pathway is a well-validated target for the development of antibody-based therapeutics for the treatment of malignancies.

For the purposes of the present invention, various agents can be used as agents that block the PD-1/PD-L1 pathway. In particular, PD-1 blocking monoclonal antibodies such as nivolumab (Bristol-Myers Squibb, USA) or pembrolizumab (Merck Sharp and Dohme, USA) marketed as Keytruda are preferred. The PD-L1 blocking drug atezolizumab, marketed as Tecentriq, may also be used.

In this invention, the expression “convergent T cell receptor variants” can be used to refer to T cell receptor variants characterized by homologous amino acid sequences containing no more than three differences in amino acid substitutions, insertions or deletions.

The term "driver mutation" as used herein means a change in DNA sequence (mutation) that turns a normal cell into a cancer cell and allows such cell to grow and spread in the body.

Interleukin-2 (IL-2) is a T-lymphocyte growth factor and includes all forms of interleukin-2, including human and animal origin, variants with conservative amino acid substitutions, glycoforms, and biosimilars of commercially available IL drugs -2. In particular, recombinant forms of human interleukin-2 may be useful for the purposes of the present invention, such as aldesleukin, commercially available from a number of manufacturers under the name Proleukin. Aldesleukin (des-alanyl-1, series-125 human IL-2) is a non-glycosylated form of recombinant human IL-2 with a molecular weight of approximately 12 kDa. Also useful for the purposes of the present invention is a recombinant form of IL-2 available from CellGenics, Inc. (Portsmouth, NH, USA) (CELLGRO GMP) or ProSpec-tany TechnoGene Ltd. (East Brunswick, NJ, USA). The term IL-2 also includes various PEGylated forms of IL-2, for example, those commercially available from Nektar Therapeutics (South San Francisco, CA, USA) or described in US 2014/0328791 A1 or WO 2012/065086 A1. In particular, for the purposes of the present invention, the drug Roncoleukin, produced by LLC NPK Biotech, Russia, can be used.

Interleukin-21 (IL-21) is a pleiotropic cytokine protein and includes all forms of interleukin-21, including human and animal origin, variants with conservative amino acid substitutions, glycoforms, as well as biosimilars of commercially available IL drugs -21. IL-21 is produced primarily by natural killer T cells and activated CD4 ⁺ T cells. Recombinant human IL-21 consists of a single non-glycosylated polypeptide chain containing 132 amino acid residues with a molecular weight of approximately 15.4 kDa. Recombinant human IL-21 is available from a number of manufacturers, including ProSpec-tany TechnoGene Ltd. (East Brunswick, NJ, USA) and Thermo Fisher Scientific Inc. (Waltham, MA, USA). In particular, for the purposes of the present invention, the drug IL-21, produced by CyStorLab LLC, Russia, can be used.

Any methods well known to one skilled in the art can be used to isolate genomic DNA or RNA. In particular, the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd edition can be used. Cold Spring Harbor Laboratory Press, NY, (1989), as well as commercially available nucleic acid isolation kits such as DNeasy or RNeasy (Qiagen, Germany) or equivalents.

RNA samples can be used to generate cDNA and gene libraries using reverse transcription and polymerase chain reaction (PCR), as is well known in the art, for example, using the Gubler-Hoffman method (Gubler U., Hoffman B.J. Gene, 1983 , v. 25, p.263-269). Methods using T4 phage DNA ligase can also be used (Akowitz, Manuelidis. Gene, 1989, vol. 81, p. 295-306; Lukyanov et al. Biophys. Biochem. Res. Comm., 1997, vol. 230, p.285-288), methods using other ligases, template-switching cDNA synthesis methods (Chenchik et al., (1998) Gene Cloning and Analysis by RT-PCR, ed. by P. Siebert and J. Larrick, BioTechniques Books , Natick, Mass., p.315-319); Zhu et al., Biotechniques, 2001, vol. 30, p.892-897), as well as other methods.

DNA or cDNA samples are subjected to a sample preparation procedure, which typically involves PCR amplification and insertion of adapters for subsequent sequencing. During preparation, samples can be enriched for nucleotide sequences encoding TCRs.

Methods useful for analyzing TCR repertoires by sequencing, in particular high-throughput sequencing, are well known in the art. These methods allow the identification of genomic sequences from tens to millions of TCR variants in blood samples, cell subpopulations, or tissue samples obtained from human donors or animal models of interest. In these methods, when creating libraries of TCR α- or β-chain genes, or gene libraries of paired TCR α- and β-chains, genomic DNA can be used as starting material (see, for example, Robins H.S., et al. Blood, 2009, p.4099-4107) or RNA. The generation of TCR gene libraries can be accomplished by PCR amplification using a set of gene-specific primers, or using an approach known as 5'RACE, or using other methods, either in the volume of the entire sample or for cells separated into individual microvolumes using multiwell plates or water-in-oil emulsions.

For example, a TCR gene library can be obtained using 5'RACE, as described in a number of publications (Britanova et al., J. Immunol., 2016, vol. 196(12), p.5005-5013). Total RNA from peripheral blood monocytes or sorted T cells is extracted using column-based methods, such as, for example, the RNeasy Micro Kit (Qiagen) or the like, or using Trizol reagent-based protocols. Alternatively, mRNA can be extracted using column-based methods, such as the Oligotex mRNA Mini Kit (Qiagen), or other methods. cDNA synthesis is carried out using primers complementary to TCR constant regions, or using primers containing oligo-dT regions, using reverse transcriptase capable of template switching. Examples of the latter approach include SmartScribe reverse transcriptase (Clontech, USA) or Mint (Evrogen, Russia) using oligonucleotides for template switching. Further amplification of TCR gene libraries is carried out using primers specific to the template switch oligonucleotide and the constant regions or J regions of the TCR genes.

Alternatively, PCR amplification using cDNA as a template can be performed using a multiplex set of primers specific for TCR genes.

Another alternative is to extract genomic DNA using any method known in the art and then use it as a template for PCR in the presence of a multiplex set of primers specific for TCR genes. In this case, one or several stages of PCR amplification can be carried out using nested and/or step-out primers. Adapters for sequencing can be included both during the PCR itself and subsequently through ligation. Examples of obtaining experimental data using the described approach are presented in a number of publications (Robins H.S., et al. Blood, 2009, p.4099-4107; Freeman J.D., et al. Genome Res. (2009), p.1817-1824; Mamedov I.Z. , et al. Front. Immunol., 2013, 456).

As another alternative for the purposes of the present invention, sequencing data obtained from transcriptome analysis (RNA-seq) or exome analysis (Exome-seq), or other sequencing data that contains information at least at least about partial TCR sequences. To extract information about the actual TCR sequences, software that is appropriate in its functionality can be used, such as, for example, MiXCR in RNA-Seq mode (Bolotin D.A. et al. Nat. Biotechnol., 2017, vol. 35, No. 10, p.908-911).

Sequencing adapters and methods for introducing them into nucleic acids are well known to those skilled in the art; their practical implementation is carried out in the form of commercially available kits for high-throughput sequencing.

A number of platform solutions are currently available commercially for sequencing. In particular, sequencing systems such as HiSeg™, MiSec™, and/or Genome Anlyzer™ available from Illumina® can be used for the purposes of the present invention; Ion PGM™ or Ion Proton™ sequencing systems available from Ion Torrent™; PACBIO RS II sequencing system from Pacific Biosciences; Life Technologies™ SOLID sequencing system; 454 GS FLX+ or GS Junior sequencing systems from Roche or any other sequencing platforms.

The culture of lymphocytes enriched with tumor-specific T-lymphocyte clones obtained by the method of the present invention can then be used for large-scale expansion using known ex vivo expansion protocols.

The invention presented herein will be illustrated below with reference to specific examples. It should be understood that the given examples serve solely for the purpose of illustrating the most preferred embodiments of the invention and are not intended to limit its scope. The scope of the claims is determined by the claims presented below.

Materials and methods

Patients and samples

All clinical samples were obtained from the National Medical Research Center (NMRC) of Oncology named after. N.N.

Blokhin in accordance with protocol MoleMed-0921, approved by the ethical committee. All patients in the study were diagnosed with metastatic melanoma. Patients signed informed consent before collecting their biomaterial. Most experiments were performed on resected metastatic lymph nodes obtained during surgery. Tumor samples were genotyped for the BRAF ^V600E mutation. 20–30 ml of peripheral blood was also obtained from each patient included in this study before surgery. Patient information is presented below in Table 1.

Short-term cultivation of TIL

Freshly resected tumor samples were divided into fragments measuring 1–3 mm in each dimension. Several fragments were frozen in liquid nitrogen for further preparation of cDNA libraries and HLA typing. Individual fragments were seeded into the wells of a 24-well tissue culture plate containing 2 ml of complete T-lymphocyte (SM) culture medium supplemented with 5% heat-inactivated human AB serum (PanBiotech, Germany) and 1000 IU/ml IL-2 (Roncoleukin , Biotechnology LLC, St. Petersburg, Russia). The SM medium contained RPMI-1640 (PanEco, Russia), 25 mM HEPES, pH 7.2 (PanEco, Russia), penicillin 100 IU/ml, streptomycin 100 μg/ml, gentamicin 10 μg/ml, 1x mixture of non-essential amino acids, lx GlutaMAX, β-mercaptoethanol (0.55 μM) (all from Gibco, Thermo Fisher Scientific, USA) and 110 μg/ml sodium pyruvate. On the fourth day of culture, TILs were collected, filtered through a 70-μm sieve, stained with fluorophore-labeled antibodies, and subjected to flow cytometry sorting (FACS). To generate TCR repertoire libraries, T cells were sorted directly into RLT cell lysis buffer (QIAGEN, The Netherlands) and stored at -80°C until used for RNA isolation. For

functional assays TILs were sorted into 1.5 ml Eppendorf tubes containing 0.5 ml RPMI-1640. Live sorted T lymphocytes were seeded into the wells of a 96-well cell culture plate at a concentration of 10 ⁶ cells/ml in SM medium and cultured for at least five days. Then 1000 IU/ml IL-2, 50 ng/ml IL-21 (SCI-STORE, Russia), 20 μg/ml nivolumab (Bristol-Myers Squibb, USA) and 10% autologous serum obtained from patients were added to the SM. Half of the medium was replaced three times a week. One day before functional assays, all media were replaced with fresh SM medium that did not contain interleukins or nivolumab.

Cultivation of OIL

Tumor specimen fragments from each of four melanoma patients (mp26, mp32, mp34, and mp42) were seeded into separate wells of a 24-well tissue culture plate (1 fragment per well, 1 plate per patient) with basal culture medium based on RPMI-1640 and 5% heat-inactivated human AB serum. TIL bulk was grown from tumor fragments under the following different conditions (6 wells/condition) for 9 days: 1) high IL-2 (3000 IU/ml), 2) (low IL-2) (100 IU/ml), 3 ) IL-21 (25 ng/ml) + low IL-2, 4) nivolumab (20 μg/ml) + IL-21 + low IL-2, and 5) IFNγ (100 μg/ml) + nivolumab + IL- 21 + low IL-2. TILs from patient tr26 were cultured under conditions 1, 3, 4, and 5; TILs from patients mp32, mp34 and mp42 were cultured under conditions 1-4. Half of the medium was replaced on days 3 and 5 with the same medium that was originally used for inoculation. On day 9 of culture, TILs from each well were counted and lysed at a density of 5×10 ⁵ cells/ml in RLT buffer to prepare TCR repertoire libraries.

Cultivation of sorted T-lymphocyte populations FACS-sorted T-lymphocytes were expanded by nonspecific TCR-dependent stimulation using anti-CD3/CD28 antibody-coupled beads (anti-CD3/CD28 Dynabeads) (Thermofisher Scientific, USA). Beads were added to the culture medium the next day after cell seeding at the rate of 2 μl of bead solution per 10 ⁵ cells. FACS-sorted TILs were expanded over a period of 2 weeks. The beads were removed using a magnet once the desired cell number was reached. Before co-culture experiments, cells were allowed to rest for 24 hours in IL-free SM medium supplemented with 10% autologous patient serum (i.e., “resting medium”).

Cultivation of Monocyte-Derived Dendritic Cells Autologous dendritic cells were prepared as described below. CD14 ⁺ cells (monocytes) were isolated from patient PBMCs using a magnetic enrichment procedure using anti-CD14 MicroBeads (Miltenyi Biotec, Germany). Monocytes were then seeded into the wells of a 24-well tissue culture plate at a concentration of 5×10 ⁵ cells/well. For monocyte differentiation, X-Vivo-15 medium (Lonza, Switzerland) with 400 U/ml IL-4 and 800 U/ml GM-CSF was used. On the fourth day of culture, the medium was refreshed, and dendritic cells (DCs) were loaded with a mixture of melanoma OAA peptides (PepTivator Melan-A/MART-1, gp100/Pmel and human MAGE-A3; Miltenyi Biotec) at a concentration of 600 nM each. The next day, loaded dendritic cells were subjected to maturation using 1 μg/ml PGE, 10 ng/ml IL-1β, and 25 ng/ml TNF-α. After maturation for 24 hours, DCs were collected and used for co-culture with T lymphocytes.

Antigen-specific activation assay of CD-137

FACS-sorted PD1 ⁺ CD39 ⁺ (double positive, DP) and non-DP populations were co-cultured with antigen-loaded autologous DCs in a 10:1 ratio (T cells: DK). The co-culture medium consisted of SM and serum-free AIM-V medium (Gibco, USA), taken in a 1:1 ratio, supplemented with 50 ng/ml IL-21. For further RNA isolation and TCR library preparation, both CD137 ^high and CD137 ^low T cells were lysed using RLT buffer. The proportion of cells with increased expression of CD137 was measured for both CD4 ⁺ and CD8 ⁺ TILs. Antigen-specific activation was measured as the ratio of the proportion of CD137 ^high T cells in TILs that were cocultured with antigen-loaded DCs compared with unloaded DCs.

Flow cytometry

For FACS sorting of PD1 ⁺ CD39 ⁺ TIL subpopulations, cells were stained with the following antibodies: CD4-BV510 (RPA-T4), CD8-Alexa-647 (SK1), CD127-APC-Cy7 (IL-7Rα, A019D5), PD-1- BV421 (CD279, EH12.2H7) (BioLegend, Germany), CD25-PE (IL-2Rα, Beckman Coulter), CD39-FITC (eBioAl) (eBioscience, USA). Cells were pelleted, resuspended in a staining solution containing fluorescent antibodies, and incubated for 1 h at 4°C in the dark. The cells were then washed and resuspended in PBS at a density of approximately 5×10 ⁶ cells/ml. T lymphocytes were sorted using an Aria III flow cytometer (BD Biosciences, USA). Data analysis was performed using FlowJo software (FlowJo, LLC). In order to avoid antigen-specific Treg-dependent suppression of T cells in further functional analyses, Treg cells were identified as CD4 ⁺ CD25 ⁺ CD127 ^- cells and sorted separately. For functional assays, T cells were divided into CD39 ⁺ PD-1 ⁺ (double positive, DP) and non-DP (positive for one of the CD39 and PD-1 antigens or double negative) subsets, without dividing into CD4 ⁺ and CD8 ⁺ cells . To obtain the TCR library, CD4 ⁺ DP and CD8 ⁺ DP cells, as well as corresponding populations of non-DP cells, were sorted individually to assess the individual properties of their TCR repertoires. To analyze CD137 activation, T lymphocytes were stained with fluorescently labeled antibodies CD4-BV510 (RPA-T4), CD8-Alexa-647 (SK1), CD137-PE (4 B4-1) (BioLegend, Germany).

HLA typing

In patients mp39, mp41, mp42 and mp44, only the presence of HLA-A*02 was tested. Aliquots of PBMC or TIL from these patients were stained with anti-HLA-A*02-PE (BD7.2) (BD Biosciences, USA) and analyzed using a Navios flow cytometer. HLA typing of the remaining patients (Table 1) was performed using next generation sequencing (NGS).

RNA isolation and preparation of TCR libraries RNA from fresh frozen tumor fragments was isolated using TRIzol reagent (Invitrogen). RNA from RLT-lysed cells was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. RNA concentration was measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, USA). For cDNA synthesis, no more than 500 ng of total RNA was used.

cDNA libraries were generated using the Human RNA TCR Multiplex kit (MiLaboratories) according to the manufacturer's protocol. Achievable sequencing depth can be 20 paired-end reads per cell, or more, for sorted and cultured TIL populations. Sequencing was performed using the Illumina NextSeq platform (read length 2×150 bp).

Analysis of TCR repertoire sequencing data

Sequencing data of TCR repertoires were analyzed using MiXCR software (MiLaboratories) to identify TCRβ CDR3 clonotypes. VDJtools software (Shugay, M. et al.) was used to process the output data on the TCR repertoire obtained using MiXCR, to calculate the diversity of the TCR repertoire, and to preprocess the TCR repertoires (i.e., combining the TCR repertoires and reducing the discreteness of the TCR repertoires). VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires. PLoS Comput. Biol. 11, el004503 (2015).

Cluster analysis of TKPβ TIL repertoires

Cluster analysis was performed using the ALICE algorithm (Pogorelyy, M. V. et al. Detecting T cell receptors involved in immune responses from single repertoire snapshots. PLoS Biol. 17, e3000314 (2019)). In order to exclude insufficiently corrected erroneous TCR variants, which could potentially be a source of false neighbors for the most frequently occurring clonotypes in cluster analysis, thereby distorting the process of identifying convergent clonotypes (hereinafter referred to as ALICE hits), only those clonotypes that were read >1 time. P _gen of amino acid sequences was calculated using Monte Carlo simulation. For each pair of V- and J-segments, 5 million TCR sequences were simulated and 20 iterations of the ALICE algorithm were performed. Only clonotypes with a p value <0.001, calculated with the Benjamini-Hochberg (BH) correction, were considered significant ALICE hits. Because the number of ALICE hits is highly dependent on the initial variability of the TCR repertoire, the number of ALICE hits from sample to sample was normalized based on the initial number of clonotypes with read counts greater than 1 in each sample.

The igraph function was used to visualize clusters resulting from convergent selection of TCRs (Csardi, G., Nepusz, T. & Others. The igraph software package for complex network research. InterJournal, complex systems 1695, 1-9 (2006)). This function is based on the de Bruijn graph method to calculate the distance between amino acid sequences of CDR3 hypervariable regions. In the resulting graph in GML format, each node represents an individual TCR clonotype, and the distance between nodes is proportional to the difference between the CDR3 amino acid sequences. Edges connect nodes representing TCR clonotypes characterized by a Hamming distance <2. Node size represents the frequency of the corresponding TCR clonotype. After constructing composite graphs including clonotypes from multiple TCR repertoires, identical clonotypes from different TCR repertoires are displayed as separate nodes.

Although other hypervariable regions (CDR1, CDR2) are also involved in antigen binding, it is the CDR3 region that, as is well known to one skilled in the art, makes a major contribution to the interaction with the antigen. Thus, due to the greater information content of CDR3 sequences, it was the TCR CDR3 sequences that were chosen for cluster analysis.

Correlation of clonotypes in TCR clusters with the VDJdb database TCR repertoire data were annotated using the VDJdb database for T-cell receptors with known specificity (Shugay, M. et al. VDJdb: a curated database of T-cell receptor sequences with known antigen specificity. Nucleic Acids Res. 46, D419-D427 (2018); Bagaev, D. V. et al. VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium. Nucleic Acids Res. 48, D1057-D1062 (2020) ). Annotation was based on the assumption that the TCRs under study have the same specificity as the TCRs from the database if: i) the CDR3 regions of the compared TCRs differ by no more than one central amino acid substitution, i) the substituted amino acids belong to the same group based on the properties of their R side chains (polar, aliphatic, aromatic, positively/negatively charged), and iii) HLA restriction TCR clonotypes from the VDJdb database match one of the patient's HLA alleles, if known.

TCR clusters consisting predominantly of OAA-specific clonotypes, but not clonotypes with other specificities, were considered OAA-specific overall. Members of OAA-specific clusters for which no matches were found in VDJdb were assumed to have the same specificity as the entire cluster, based on structural similarity to clonotypes that had matches in VDJdb, and were included in subsequent analyses.

Analysis of differentially proliferating clonotypes To identify TCRβ clonotypes that were present in significant quantities in the total TIL of patients mp26 and mp34, we used a statistical approach implemented in the edgeR package (Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics vol. 26 139-140 (2010). The TCR repertoires of TILs cultured under experimental conditions 2-4 (as described above) and TCRs cultured under “high IL-2” conditions were compared. Biological samples taken in six and four replicates, obtained under each of the specified experimental culture conditions, were used to analyze the TCR repertoires of patients mp26 and mp24, respectively. TCR clonotypes were considered proliferating if the false-positive rate-corrected p value was <0.01 and the log2 fold change was >1.

Statistical analysis

Statistical analysis was performed using the Graph Pad Prism 8.0 software package (GraphPad Software Inc., USA). All data are presented as mean ± standard deviation. The Shapiro-Wilk test was used to assess normality in all cases. The names of the statistical tests and the number of replicates in each comparison group are indicated in the figure legends.

Example 1: Retrospective data analysis.

The primary analysis used published repertoires of TCR beta chain (TCRR) sequences of TIL obtained before and after immunotherapy using anti-PD-1 antibodies for two cohorts consisting of 21 and 8 patients with cutaneous melanoma (Riaz, N. et al. Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab. Cell 171, 934-949.e16 (2017); Tumeh, PC et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568-571 (2014). Using the ALICE algorithm, clusters of convergent TKRβ clonotypes were identified in all patients. The number of clonotypes in clusters increased significantly after treatment in both cohorts (p = 0.019 and 0.038, respectively; Fig. 1A, B), which may reflect treatment-dependent proliferation of convergent tumor-specific TILs. A search of the VDJdb database revealed clonotypes in clusters that were extremely similar or identical to known TKRβ variants specific for melanoma-associated HLA A*02-restricted antigens, such as Melan-A (Melan-A _aa26-35 -ELAGIGILTV) and NY-ESO-1 (NY-ESO-1 _aa157-165 - SLLMWITQC), in 20% and 50% of patients from the cohorts described in the above references, respectively. Clonotypes in the clusters were enriched for OAA-specific TCRβ variants compared to the TCR repertoire as a whole, indicating that cluster analysis can identify clonotypes involved in the developing antitumor immune response ( Fig. 1B ). In FIG. Figure 2 shows the identified TCR clusters for one of the patients (from the complete/partial response group) after immunotherapy.

Example 2. CD39 ⁺ PD-1 ⁺ subpopulation of freshly isolated TIL is enriched in clonal, convergent and tumor-specific TCRs

To test whether the enrichment of convergent TCR clusters in different TIL subpopulations is associated with the CD39/PD-1 phenotype, fluorescence-activated cell sorting (FACS) was performed on CD39 ⁺ PD-1 ⁺ (double positive, DP) and non-DP CD4 ⁺ and CD8 ⁺ T cells from TILs freshly isolated from lymph node metastases in eight melanoma patients. Overall, TIL composition was biased toward a greater predominance of CD4 ⁺ T lymphocytes, although cells with the DP phenotype were more abundant among CD8 ⁺ TIL compared with CD4 ⁺ cells. Analysis of the TCRβ repertoire revealed increased clonality and cluster enrichment for both CD4 ⁺ and CD8 ⁺ DP TILs compared with the corresponding non-DP subpopulations ( Fig. 3A–E ), with greater clonality among CD8 + TILs than CD4 + TILs independently on immune checkpoint expression status.

For TILs obtained from an HLA-A*02-positive mp26 patient with BRAFwt melanoma, a search of the VDJdb database identified three TCR clusters that included A*02-Melan-A _aa26-35 -specific clonotypes (Fig. 4). The CD8 ⁺ DP subpopulation, compared with the total TCR repertoire obtained using fresh frozen tumor tissue (FTT), and compared with the CD8 ⁺ non-DP subpopulation (Fig. 5 A, B), was significantly enriched in both Melan-A-specific clonotypes, and TCR clusters that include such clonotypes.

Similar results were obtained for another BRAFwt HLA-A*02-positive patient, pt41. Thus, the CD39 ⁺ PD1 ⁺ subpopulation is enriched in large and convergent T cell clones that participate in the developing tumor-specific immune response, a significant portion of which can be detected using TCR cluster analysis.

In order to confirm the obtained results at the functional level, an analysis of the surface expression of CD137 (4-1BB) was used. Sorted DP and non-DP TIL subpopulations from patient mp26 were cultured for two weeks and stimulated with monocyte-derived autologous dendritic cells that were loaded with a mixture of TAA peptides. CD8 ⁺ CD137 ^high subpopulations were quantified by flow cytometry and sorted to obtain a TCRβ library. As shown in FIG. 5B, the proportion of CD137 ^high cells was higher in cultured DP cells, but no differences were found between T cells stimulated with TAA-loaded dendritic cells or control dendritic cells. Analysis of the TCRβ repertoire revealed that a fraction of OAA-stimulated CD8 ⁺ DPs with high CD137 expression, but not (non-DP) and non-control DP cells, were enriched for known Melan-A-specific clonotypes ( Fig. 5D ). These clonotypes included an OAA-reactive TKRβ variant, CSARVGNQPQHF-TRBV20-TRBJ1-5, which was previously identified in a cluster analysis of uncultured CD8 ⁺ DP cells, and a variant CASSGGMGQPQHF-TRBV19-TRBJ1-5, homologous to another cluster. TAA-specific clonotypes totaled approximately 13% of the fraction of cultured and activated TAA DP TILs, characterized by a high level of CD137 expression. These results highlight the importance of analyzing the TCR repertoire of antigen-reactive cells, and demonstrate that analysis of the CD137 marker alone is not sufficiently informative. Example 3.

TILs were cultured under four different conditions: (high IL2), (low IL-2)/IL-21, (low IL-2)/IL-21/anti-PD-1 and (low IL-2)/IL- 21/anti-PD-1/IFNγ, where nivolumab was used as the anti-PD-1 agent and the IL-2 concentration was 100 IU/ml (low) and 3000 IU/ml (high). For each condition, the TCRβ repertoires of TILs independently cultured from 12 tumor fragments obtained from patient mp26 were analyzed. Identified TCR clusters from all samples were combined (Fig. 6). The following indicators were used:

i) normalized number of clonotypes in clusters (Fig. 7A),

ii) the cumulative proportion of the repertoire accounted for by Melan-A-specific TKRβ clusters (clusters predominantly comprising Melan-A-specific clonotypes defined in VDJdb) (Fig. 7B).

Among all culture conditions, the combination (low IL-2)/IL-21/anti-PD-1 yielded the highest number of clonotypes in clusters and the highest cumulative proportion of Melan-A-specific clusters (Fig. 7A, B). The addition of IFNγ did not further enhance the proliferation of potentially tumor-specific clones.

To identify clonotypes that proliferated significantly in tumor fragment cultures in the presence of IL-21 and (low IL-2) (both in the presence and absence of nivolumab and IFNγ) compared to pan-activating IL-2 culture conditions _high , used edgeR software. The (low IL-2)/IL-21/anti-PD-1 combination produced the highest number of reproducibly proliferating clonotypes, 60% of which were detected among CD8 ⁺ DP TILs sorted after a brief pre-culture. The total number of clonotypes matching the repertoire of the CD8 ⁺ DP fraction was also highest for this combination (Figure 7B). These results demonstrate the beneficial effects of PD-1 inhibition on the proliferative potential of CD8 ⁺ CD39 ⁺ PD-1 ⁺ T cells. There was also a slight increase in the number of CD8 ⁺ clonotypes from the non-DP fraction repertoire that proliferated under the same conditions - 10 clonotypes compared to three clonotypes in culture conditions (low IL-2)/IL-21 without nivolumab, which may be explained by nivolumab -dependent proliferation of PD-1 ⁺ CD39 ^- T lymphocytes.

In terms of total proliferative potential, the highest numbers of T cells were observed in the (high IL-2) condition, although roughly comparable numbers of cells were also observed in the (low IL-2) condition. In the presence of IL-21, suppression of IL-2-dependent TIL proliferation was observed, as previously reported for human CD4 ⁺ T lymphocytes.

Rational clustering of TCRs based on structural homology can be used to identify tumor-specific T-lymphocyte clones and assess the relative enrichment of TILs in them. Since the published data on the TCR repertoire in melanoma, clusters of convergent TCR clonotypes have been identified, the number and overall frequency of which increased after immunotherapy with anti-PD-1 antibodies. There was also a significant enrichment of convergent TCR clusters in PD1 ⁺ CD39 ⁺ subpopulations, related to both CD4 ⁺ and CD8 ⁺ TILs, which were previously shown to be enriched in terms of tumor reactivity. These conclusions are further supported by data obtained for cases in which the HLA context as well as certain TCR clonotypes and related antigens of interest were known. A search of the VDJdb database successfully identified OAA-specific TCR variants in HLA-A*02-positive patients, with approximately half of the clusters being correlated with known Melan-A-specific sequences. The overall frequency of these Melan-A-specific clusters in the CD8+ DP population was only slightly lower than the overall frequency of all identified Melan-A-specific TCRs. This observation is evidence that the proposed approach allows us to identify the vast majority of tumor-specific clonotypes, which are present at high frequency in the CD8+ DP subpopulation. TCR clusters are an important feature of a convergent immune response that includes multiple homologous clonotypes. The data presented demonstrate the effectiveness of this approach, even in situations where specific antigens are unknown.

In one embodiment, the proposed approach was used to optimize cultural conditions when growing TIL under ex vivo conditions. In particular, TILs cultured under IL-21 ⁺ conditions showed the highest amounts of TCRs in clusters, indicating a more pronounced influence of antigen-dependent TCR selection. Without intending to be limited by any scientific theory, the inventors of the present invention believe that IL-21 is capable of influencing the described cell culture system both at the stage of antigen presentation (since MHC-I presentation and, to a lesser extent, MHC-II presentation restriction of antigen by tumor cells occurs during the cultivation of tumor fragments ex vivo), and at the proliferation stage, at which IL-21 selectively promotes the proliferation of populations of non-regulatory (effector) T lymphocytes. Also, it was previously shown that the use of an antibody to PD1 induces the secretion of IFNγ and TNFα by CD8 ⁺ TIL cells expressing PD-1 (Park, J. et al. Immune Checkpoint Inhibitor-induced Reinvigoration of Tumor-infiltrating CD8+ T Cells is Determined by Their Differentiation Status in Glioblastoma Clin Cancer Res 25, 2549-2559 (2019) We found a greater increase in clustered and tumor-specific TCRs for cells cultured under IL-21 ⁺ /anti-PD- conditions. 1, compared to IL-21 ⁺ alone.

It is expected that the proposed approach to analyzing the degree of enrichment of TIL with tumor-reactive clones, based on an assessment of the TCR repertoire of TIL, will accelerate the clinical development of adoptive T-cell therapy. This approach can significantly facilitate the selection of optimal TIL subpopulations and optimization of TIL cultivation conditions followed by the enrichment procedure. In a clinical setting, the proposed approach will allow us to evaluate the abundance of tumor-reactive TILs for individual tumor samples both before and after cultivation and/or enrichment. As T cell therapies mature in their ability to identify lineage specificities and phenotypes that can effectively target each individual patient's tumor, ways to group convergent TCRs that react with the same tumor antigens will become an important part of the toolkit to develop rational T-cell therapies.

Claims (22)

1. A method for obtaining a culture of tumor-infiltrating lymphocytes (TILs) enriched with tumor-specific clones, including the stages at which: - isolate autologous TIL from a patient suffering from solid cancer; - autologous TILs are cultured in a medium containing IL-2 at a concentration of 100 IU/ml and IL-21; - determine repertoires of nucleotide sequences of T-cell receptors of cultured TIL using high-throughput sequencing; - assess the degree of enrichment of cultured TILs with tumor-specific clones using bioinformatics analysis, which determines the presence in the repertoire of nucleotide sequences of T-cell receptors of at least one cluster of convergent variants of T-cell receptors. 2. The method according to claim 1, in which convergent variants of T-cell receptors are variants of T-cell receptors characterized by homologous amino acid sequences containing no more than three differences, representing amino acid substitutions, and/or insertions, and/or deletions. 3. The method according to claim 1, in which the degree of enrichment of tumor-specific T-lymphocyte clones is characterized by the proportion of the T-cell receptor repertoire occupied by T-cell receptor clones belonging to clusters of convergent T-cell receptor variants. 4. The method according to claim 1, in which the degree of enrichment of tumor-specific T-lymphocyte clones is characterized by the number of T-cell receptor clonotypes belonging to clusters of convergent T-cell receptor variants. 5. The method according to claim 1, in which at the stage of cultivation, autologous lymphocytes are cultured in a medium additionally containing an antibody that blocks PD-1:PD-L1 signal transmission. 6. The method of claim 5, wherein the antibody that blocks PD-1:PD-L1 signaling is nivolumab. 7. The method according to claim 1, in which autologous lymphocytes are obtained from tumor material of the patient obtained during surgery. 8. The method according to claim 1, in which autologous lymphocytes are obtained from the patient’s blood. 9. The method of claim 1, wherein the solid cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, ovarian cancer, breast cancer, pancreatic cancer. 10. The method according to claim 1, further comprising the step of pre-isolating from autologous lymphocytes isolated from the patient a fraction enriched in lymphocytes bearing at least one surface marker selected from the group consisting of PD-1, CD39, CD137. 11. The method according to claim 10, in which the specified fraction is enriched with lymphocytes carrying surface markers PD-1 and/or CD39. 12. Method according to any one of paragraphs. 1-11, in which the medium in which the resulting lymphocytes are cultured additionally contains tumor cells and/or at least one product of their lysis. 13. Method according to any one of paragraphs. 1-11, in which the medium in which the resulting lymphocytes are cultured additionally contains at least one tumor-associated antigen. 14. Method according to any one of paragraphs. 1-11, in which the medium in which the resulting lymphocytes are cultured additionally contains at least one tumor neoantigen. 15. The method of claim 14, wherein said tumor neoantigen contains at least one driver mutation. 16. Method according to any one of paragraphs. 1-15, wherein the cultured lymphocytes are further enriched for tumor-specific variants by activation in the presence of at least one tumor antigen and subsequent enrichment using at least one T cell activation marker. 17. Method according to any one of paragraphs. 1-16, in which the resulting culture is used in an ex vivo expansion protocol. 18. The method according to claim 17, in which the culture that is optimal for subsequent use in the ex vivo expansion protocol is selected based on the results of assessing the degree of enrichment of cultured TILs with tumor-specific clones using bioinformatics analysis.

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