CN117377684A

CN117377684A - CXCR3+ cells or cell preparations for use in cancer treatment

Info

Publication number: CN117377684A
Application number: CN202280019623.6A
Authority: CN
Inventors: 迪特尔·沃尔克; 迈克尔·施穆克-亨内塞; 蒂诺·沃尔默; 佩特拉·雷因克; 斯蒂芬·施利克塞尔
Original assignee: Charlotte Medical College Berlin
Current assignee: Charlotte Medical College Berlin
Priority date: 2021-01-12
Filing date: 2022-01-12
Publication date: 2024-01-09

Abstract

The present invention provides a modified T cell or isolated population of immune cells expressing a CXCR3 isoform selected from CXCR3A, CXCR B and CXCR3alt, and optionally further expressing a transgene comprising an artificial T cell receptor and/or a CXCR3 ligand, for use as a medicament. The invention also provides methods of obtaining the cell or population of cells from a plurality of immune cells derived from a human subject. The invention also relates to the assessment of CXCR3 splice variants and their ligands CXCL9, CXCL10 and CXCL11 in patients with myo-lamellar invasive bladder cancer (MIBC) to enable stratification of the patient according to the patient's predicted response or clinical outcome to treatment with a chemotherapeutic agent.

Description

CXCR3+ cells or cell preparations for use in cancer treatment

The present invention relates to transgenic expression of the chemokine receptor CXCR3 and its ligands in human T cells to enhance their efficacy and survival to provide an improved cell transfer agent in patients suffering from cancer, chronic viral infection or autoimmunity.

The present application claims priority from european patent applications EP21151233.0 and EP21151232.2, both filed on 1 month 12 of 2021, and from european patent applications EP21151438.5 and EP21151447.6, both filed on 1 month 13 of 2021, which are incorporated herein by reference in their entirety.

Background

Bladder Cancer (BC) is listed among the 10 most common malignancies in europe and the united states. T cell responses to eradication of tumors can be induced in restricted non-myogenic invasive bladder cancer (NMIBC) via BCG therapy, or sometimes in developing Myogenic Invasive Bladder Cancer (MIBC) via PD-1/PD-L1 blockade. In current medical-level therapies, MIBC patients received neoadjuvant chemotherapy (NAC) prior to Radical Cystectomy (RC), which improves Overall Survival (OS) compared to RC alone (Vale C. (Lancet) (2003) 361:1927-1934). 25% to 40% of MIBC patients respond to NAC as defined by the decline phase of the oncological grade. Non-responders MIBC patients did not show tumor reduction and remained in myometrial invasive disease status until RC began. Therefore, it is critical to effectively identify cancer patients who are most likely to benefit from neoadjuvant therapy. A new layered system for predicting response to NAC is needed to improve treatment regimens to gain clinical benefit to non-responder MIBC patients. The benefits of neoadjuvant therapy have also been demonstrated for locally advanced breast cancer (Eltahir A. Et al, J.Surg., U.S. J. surgical journal 1998:175 (2): 127-32), gastric cancer (Cunningham D. Et al, new England medical journal (N.Engl. J.Med.)), 2006:355 (1): 11-20), and esophageal cancer (van Hagen P., new England medical journal 2012:366 (22): 2074-84).

Powerful CD8 against malignant cells ⁺ T Cell responses can be induced by chemotherapy and other types of Cancer treatment (Galluzzi et al, cancer cells (2015), 28:690-714). Chemokine receptors CXCR3 (uniProt P49682) in CD8, which are bound by IFN-gamma inducible ligands CXCL9 (MIG, uniProt Q07325), CXCL10 (IP-10,UniProt P02778) and CXCL11 (I-TAC, uniProt O14625) ⁺ Heterogeneous expression in T cell compartments. CXCR3 mediated CD8 ⁺ T cells home and localize in secondary lymphoid compartments, and mediate cd8+ T cell home and localize to peripheral inflammation and malignant tissues. The activity of the CXCR3 chemokine system in tumors is directed to CD8 ⁺ T cell responses and efficacy against PD-1 inhibition in mice. The CXCR3 ligand axis can be activated via chemotherapeutic agents in preclinical models supporting anti-tumor efficacy.

In contrast to single transcripts of CXCR3 in the mouse genome, humans express three CXCR3 isoforms, the major isoform CXCR3A and two splice variants CXCR3B and CXCR3alt (Ehlert J. Et al J. Immunol.) (2004) 172:6234-6240). CXCR3 isoforms exhibit different affinities for CXCR3 ligands (CXCL 9, 10, 11) and selectively activate different signaling channels when binding to different ligands (metamaekers m. (front. Immunol.) (2018) 8:1970).

Disclosure of Invention

The immune compartments in tumors of patients treated with chemotherapy are studied primarily after treatment, and the identified immune markers rarely test their functional anti-tumor efficacy. In the examples presented herein, the inventors stratified the intratumoral activity of the CXCR3 chemokine system in a population of BC patients with respect to their response to NAC. The inventors point out that the chemokine CXCL11 and its specific receptors CXCR3alt and CXCR3A are powerful predictors of the response of MIBC patients to NAC and profile the functional relevance of CXCR3 ligand signals through each receptor in determining T cell efficacy in response to cancer or viral antigens.

A first aspect of the invention is a T cell expressing a CXCR3 variant from a transgene, the CXCR3 variant selected from CXCR3A, CXCR B and/or CXCR3alt, in particular when the CXCR3 variant, or one of the variants, is CXCR3A or CXCR3 alt. In a particular embodiment, the modified T cell is CD3 ⁺ CD8 ⁺ T cells. In certain embodiments, the CXCR3 variant transgene comprises the reverse complement of the pre-mRNA (reverse complement), or the encoding mRNA transcript of CXCR3A, CXCR3B or CXCR3alt, or a sequence encoding 95% of the amino acid sequence encoded by the above sequences, thereby preserving the biological function of the CXCR3 variant protein. In some embodiments, CXCR3A and/or CXCR3alt are expressed higher than CXCR3B, specifically an expression ratio of more than 1.

In optional embodiments, the modified T cells additionally express a recombinant Chimeric Antigen Receptor (CAR) or a transgenic T cell receptor (TgTCR), thereby recognizing a cancer, pathogen-derived, or tissue-specific antigen. In further embodiments, the modified T cells also express one or more CXCR3 ligand transgenes comprising the reverse complement of a pre-mRNA, or encoding transcripts of CXCL9, CXCL10, and/or CXCL 11.

A second aspect of the invention is an isolated immune cell preparation, in particular a T cell preparation, which is positive for CXCR3A, CXCR B and/or CXCR3alt expression by at least (.gtoreq.50%, in particular 70%, more in particular.gtoreq.80%. In some embodiments, the isolated cell preparation is derived from a cancer patient sample, or comprises cells expressing a CXCR3 variant according to the first aspect of the invention, or conversely, is free of transgenes.

In particular embodiments, modified T cells or isolated populations of immune cells expressing CXCR3 variants have a chemotactic index of greater than 1, have enhanced lytic potential, or produce more effector cytokines when stimulated or proliferated by CXCR3 ligands as compared to an unmodified or unfractionated control immune cell.

The third aspect of the modified T cells or isolated cell preparations according to the above aspects of the invention provides their use as a medicament, in particular for ameliorating T cell immunity, inhibiting infection or inflammation, or more particularly for treating cancer in solid form.

The fourth aspect of the present invention provides a method for isolating cxcr3+ cells from a human leukocyte sample. A related aspect is a method of obtaining a cxcr3+ cell preparation according to the invention, the method comprising providing a plurality of human immune cells, and inserting a transgene or transgenes encoding a sequence selected from SEQ ID NOs 001 to 006 and optionally encoding another sequence selected from SEQ ID NOs 007 to 015. A final embodiment of the invention is a method of expanding or activating an isolated cell preparation obtained according to the method provided above by culturing cells with CXCL9, CXCL10 and/or CXCL 11.

Detailed Description

Terminology and definitions

For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall govern.

As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof are intended to have an equivalent meaning and are open ended, as one or more items following any of these terms are not intended to be an exhaustive list of such one or more items, or are intended to be limited to only the listed one or more items. For example, an article "comprising" components A, B and C may consist of (i.e., contain only) components A, B and C, or may contain not only components A, B and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents include the disclosure of embodiments that "consist essentially of" or "consist of".

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure and subject to any specifically excluded limit in the stated range. If the stated range includes one or both of the limitations, ranges excluding either or both of those included limitations are also included in the disclosure.

Reference herein to "about" a value or parameter includes (and describes) a variation for that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, including in the appended claims, the singular forms "a," "or" and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), 4 th edition (2012), cold spring harbor (Cold Spring Harbor, N.Y.), cold spring harbor laboratory Press, N.Y.), and Ausubel et al, fine-compiled molecular biology laboratory Manual (Short Protocols in Molecular Biology), 5 th edition (2002), john Wiley father company (John Wiley & Sons, inc.), chemical methods.

The term gene expression or expression, or alternatively the term gene product, may refer to either or both of the process of producing a nucleic acid (RNA) or producing a peptide or polypeptide and its products, as well as transcription and translation, respectively, or any intermediate process that modulates the processing of genetic information to produce a polypeptide product. The term gene expression may also be applied to transcription and processing of RNA gene products, such as regulatory RNAs or structural (e.g., ribosomal) RNAs. If the expressed polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. Expression can be determined at both the transcriptional and translational levels, in other words, at the mRNA and/or protein product levels. The inventors indicate in the data provided in the examples that two methods of CXCL11, CXCL9 and CXCL10 measurement can be used to predict OS in MBIC receiving chemotherapy. For expression of CXCR3 ligand CXCL11 in a sample, 22.4pg/10mg tissue as measured by an ELISA system such as Luminex is a useful threshold for positive expression according to the invention. CXCR3 isoforms can be assessed at mRNA expression levels, but can be measured at surface protein expression levels with ligands that distinguish variants. Fig. 4f shows thresholds for CXCR3A, CXCR B and CXCR3alt expression as measured by real-time qPCR using the assay provided in the section entitled "intratumoral analysis of mRNA CXCR3 variants" (Intra-tumoural analysis of mRNA CXCR 3-variants), whereby a sample is considered positive if CXCR3 variant expression is > 0.1-fold, in particular > 0.2-fold as many as the housekeeping genes indicated.

The term splice variants or isoforms refers herein to three polypeptides derived from different splicing arrangements of the CXCR3 gene. CXCR3A is referred to as a classical receptor (Loetscher et al J.Endoc.J.Exp.Med.) (1996, 184:963-969) the NCBI reference DNA sequence encoding CXCR3A is provided in SEQ ID NO 001. Two naturally occurring CXCR3 splice variants have been identified in humans. An alternative splice to the CXCR3 gene is CXCR3B mRNA (NCBI reference DNA SEQ ID NO 002) yielding a polypeptide characterized by an additional N-terminal 51 amino acids (Lasagni et al J.En.Experimental medicine 2003, 197:1537-1549). Alternative or CXCR3alt splice variant mRNA products are truncated (NCBI reference DNA SEQ ID NO 003, ehlert et al 2004) resulting in polypeptides lacking the 6 th and 7 th transmembrane helices and the 3 rd intracellular loop, resulting in a short cytoplasmic C-terminus. The relative abundance of these splice variants can be measured by variant specific primers or molecular probes such as those used in the examples.

In the context of this specification, the terms sequence identity and percent sequence identity refer to a single quantitative parameter that represents the result of a sequence comparison determined by comparing two aligned sequences from position to position. Methods for alignment of sequences for comparison are well known in the art. Sequence alignment for comparison can be performed as follows: the search similarity method by Smith and Waterman, local homology algorithm of application mathematics progression (adv. Appl. Math.) "2:482 (1981), global alignment algorithm by Needleman and Wunsch, journal of molecular biology 48:443 (1970), proc. Nat. Acad. Sci.)" 85:2444 (1988); or by computerized implementation of these algorithms, including but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analysis is publicly available, for example, through the national center for Biotechnology information (http:// BLAST. Ncbi. Nlm. Nih. Gov /).

One example of amino acid sequence comparison is the BLASTP algorithm using default settings: the desired threshold: 10; word length: 3, a step of; maximum match within query range: 0; matrix: BLOSUM62; gap penalty: there is 11, extension 1; composition adjustment: conditional component score matrix adjustment. One such example for comparing nucleic acid sequences is the BLASTN algorithm using default settings: the desired threshold: 10; word length: 28; maximum match within query range: 0; match/mismatch score: 1.-2; gap penalty: linearity. Unless otherwise indicated, sequence identity values provided herein refer to values obtained using the BLAST program set (Altschul et al, J. Mol. Biol. 215:403-410 (1990)), which uses the default parameters described above for protein and nucleic acid comparisons, respectively. References to identical sequences without specifying a percentage value means a sequence that is 100% identical (i.e., the same sequence).

As used herein, the term treating (treating) or treating (treating) any disease or disorder (e.g., cancer) refers in one embodiment to alleviating the disease or disorder (e.g., slowing or preventing or reducing the progression of the disease or at least one clinical symptom thereof). In another embodiment, "treatment" or "treatment" refers to reducing or alleviating at least one physical parameter, including those that may not be discernable by the patient. In another embodiment, "treatment" or "treatment" refers to modulating a disease or disorder on the body (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art unless specifically described below.

Human T cells according to the invention encompass both αβ and γδ T Cell Receptors (TCRs) that express cells (expressing CD3, in particular CD4 or CD 8), as well as Natural Killer (NK) cells and NK T cells (expressing CD 56). These cells can also be characterized by the absence of cell surface markers, neurons, erythrocytes or fibroblasts, which characterize myeloid cells, B cells, innate lymphoid cells, endothelial cells, stromal cells or epithelial cells.

In the context of the present specification, the term neoadjuvant treatment relates to pharmaceutical formulations comprising one or more antitumor drugs. In the case of bladder cancer, antineoplastic agents are the most common chemotherapies treated with platinum-based drugs, but may also include bcg (Bacillus Calmette-Guerin) or cancer immunomodulation therapies. The neoadjuvant treatment regimen may additionally include radiation treatment of the tumor.

The standard treatment regimen for neoadjuvant chemotherapy is a formulation of the drugs methotrexate, vinblastine, doxorubicin or epirubicin and cisplatin, but may include similar drugs, such as paclitaxel, carboplatin, doxorubicin, gemcitabine, fegrid (filgrastim), pemetrexed, vinorelbine, oxaliplatin, vinflunine or docetaxel.

In the context of the present specification, the term cancer immunotherapy, biologic or immunomodulatory therapy is intended to cover the types of cancer treatments that help the immune system fight cancer. Non-limiting examples of cancer immunotherapy include immune checkpoint inhibitors and agonists, T cell metastasis therapies, cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells.

In the context of the present specification, the term checkpoint inhibitor or checkpoint inhibitory antibody is intended to encompass agents, in particular antibodies (or antibody-like molecules), capable of disrupting an inhibitory signalling cascade that limits immune cell activation, known in the art as immune checkpoint mechanisms. In certain embodiments, the checkpoint inhibitor or checkpoint inhibitory antibody is an antibody directed against CTLA-4, PD-1, PD-L1, B7H3, VISTA, TIGIT, TIM-3, CD158, or TGF- β.

In certain embodiments, the immune checkpoint inhibitor is selected from the clinically available antibody drugs ipilimumab (Bristol-Myers Squibb), CAS number 477202-00-9, na Wu Liyou mab (Bristol-Mischnobel; CAS number 946414-94-4), pamglizumab (Merck Inc.), CAS number 1374853-91-4), pidrizumab (CAS number 1036730-42-3), atilizumab (Roche AG), CAS number 1380723-44-3, avuzumab (Merck group (Merck KGaA), CAS number 1537032-82-8), devaluzumab (Zestinac (Astra Naca), CAS number 1428935-60-7) and Similant Li Shan mab (Sanofi Aventis), CAS number 1801342-60-8.

In the context of the present specification, the term checkpoint agonist agent or checkpoint agonist antibody is intended to encompass agents, in particular but not limited to antibodies (or antibody-like molecules), capable of enhancing the immune cell activation signaling cascade. The term checkpoint agonist agent also encompasses cytokines, vaccines, adjuvants and agonist antibodies that promote immune activation. Non-limiting examples of cytokines known to stimulate immune cell activation include IL-12, IL-2, IL-15, IL-21, and interferon-alpha. In certain embodiments, the checkpoint agonist agent or checkpoint agonist antibody is an antibody against CD122, CD137, ICOS, OX40, or CD 40.

In certain embodiments, the immune checkpoint agonist agent is selected from the clinically available drugs aldesleukin (Novartis, inc., cas No. 110942-02-4), interferon alpha-2 b (Merck, inc., CAS No. 215647-85-1), imiquimod (O Bei Taike (Apotex, CAS No. 99011-02-6), PF-8600 (Pfizer)), poly ICLC (Oncovir, CAS No. 59789-29-6), capeizumab (Apexigen, 1613144-80-1), or Wutu Mi Shan antibody (CAS No. 1417318-27-4).

In the context of the present specification, the term cycle threshold or CT relates to quantitative nucleic acid measurements, for example measurements with quantitative polymerase chain reaction (qPCR). The method involves repeated cycles of nucleic acid amplification using nucleic acid probes that hybridize to a target biomarker to generate a product that emits a fluorescent signal, which can be measured to determine the amount of starting genetic material. The cycle threshold may be an average, or an average of multiple duplicate samples. Other quantitative measurements may be substituted for the cycle threshold, such as crossover points or tuned inflection points.

As used herein, the term pharmaceutical composition or pharmaceutical formulation refers to a compound of the invention or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

The term response or drug responder (drug responder), as used herein, particularly with respect to anti-tumor treatment of BC, refers to tumor regression as measured by pathological analysis. The term also encompasses positive clinical results and the presence of immune infiltration in the tumor, in which case the patient's response to treatment can also be correlated with overall survival and the number of T cells present in the tumor.

The T cell activity status of a sample is a measure of the efficacy of T cells in a tissue sample reflecting their local numbers, activation phenotype and chemokine environment. The data presented in the examples indicate that the expression levels of CXCR3 isoforms CXCR3A and CXCR3alt and their ligands CXCL9, CXCL19 and CXCL11 are directly related to the T cell markers CD3 and CD8 and better OS. However, the presence of CXCR3B and CXCL4 is associated with poor OS. Phenotypic analysis shows that cd8+ cxcr3+ stem cell memory cells in healthy and patient lymphocyte samples respond to antigen and CXCR3 ligand cues (ligands cues) by migration, proliferation and production of cytokines. These correlations can be used to quantitatively assess the likelihood that cancer patients respond favorably to anti-tumor therapy, which relies on T cell activation as one of its mechanisms of action, and thus correlates with overall survival after treatment. Thus, the T cell activity status is a combined measure of the status of T cells present in a sample in terms of expression of cxcr3+ isoforms and their ligands, in other words, the presence of cxcr3a+ and cxcr3alt+ antigen-experienced T cells, together with the rich environment of CXCL9, CXCL10, CXCL11, confers high potential for local T proliferation and cytokine production. CXCR3 expression was observed on a variety of immune subpopulations that caused tumor infection and immunity, including natural killer cells, B cells, and macrophages, and autoimmunity. Those skilled in the art will appreciate that the presence of other immune cells may additionally contribute to the T cell activity status.

CXCR3+ cells or isolated CXCR3+ cell populations for medical use

A first aspect of the invention is a T cell expressing a CXCR3 variant from an artificially inserted transgene, the CXCR3 variant selected from CXCR3A, CXCR3B and/or CXCR3alt, particularly when the CXCR3 variant comprises CXCR3A or CXCR3 alt. This may be cells naturally expressing or not CXCR3, in particular CD4 ⁺ Or CD8 ⁺ T cells, or NK or NKT cells.

In a particular embodiment, the modified immune cell is CD3, depending on the desired immunomodulation in the final product ⁺ T cells selected from CD4 ⁺ Helper T cells or CD8 ⁺ T cells (expressing CD 3) ⁺ Alpha beta or gamma delta CR+, and/or lineage specific transcriptional markers such as GATA3, tbet or degermination proteins (Eomes)), or regulatory T (Treg) cells (expressing CD4, CD25 and transcriptional marker fork-box P3 (foxp 3)), more particularly CD3 ⁺ CD8 ⁺ T cells. The present invention provides potent T cell products that can be produced by conferring on, for example, suppressor cells (e.g., GATA3 ⁺ CD4 ⁺ T cells, or foxp3+ Treg), or inflammatory cells CD8 ⁺ CD45RO ⁺ Memory cells or CD56 ⁺ NKT cells deliver different immunomodulatory signals with enhanced CXCR 3-dependent activation and proliferation.

The data in the examples show that CXCR3 variants CXCR3A and CXCR3alt are directly related to the increased number and/or greater proportion of T cell markers in tumors, which in turn are in question for BC patients Better clinical outcome after neoadjuvant therapy for broad antigen stimulation of tumor-specific T cells is relevant. Different relationships between each CXCR3 variant, CD 3T cells, and patient outcome indicate that isoforms have specific downstream effects, which can be used for medical treatment in different environments (settinguses) by transgenic manipulation of CXCR3 expression profiles. In particular, the stimulation of CXCR3 with CXCR3 ligands ^{High height} Stem cells and central memory cells lead to improved proliferation, migration and cytokine secretion of tumor-specific and virus-specific cd8+ T cells in vitro. The inventors propose that the transgenic expression of CXCR3 variants, in particular CXCR3A and CXCR3alt, strongly correlated with the presence and efficacy of a T cell response will confer immune cells, in particular CD3, on the receptor ⁺ T cells improve proliferation, migration and cytokine secretion.

In certain embodiments, the modified cell expresses a CXCR3 variant transgene, the CXCR3 variant transgene comprising:

CXCR3alt, CXCR3B, or specifically CXCR3A only.

In certain embodiments, the modified cell expresses a transgene or two transgenes comprising:

CXCR3A and CXCR3B, CXCR3alt and CXCR3B, or specifically CXCR3A and CXCR3alt together.

In certain embodiments, the modified cells express a transgene or transgenes encoding CXCR3alt, CXCR3A, and CXCR3B proteins.

To confer a desired functional phenotype, it is understood that these CXCR3 variants should be expressed as proteins present on the cell surface. Based on prior knowledge of these newly described isoforms, the CXCR3 variant gene sequences provided herein are transient and specific amino acids or nucleic acids may be altered in the future when data from more human subjects reveals new alleles. Transgenes typically encompass different isoforms of CXCR3 protein of UniProt P49682.

The following embodiments of the invention relate to compositions of CXCR3 transgenes that can be transiently or stably incorporated into cells. In some embodiments, the one or more CXCR3 variant transgenes comprise a reverse complement of a pre-mRNA transcript containing both an intron and an exon of the CXCR3 variant CXCR3A, CXCR B or CXCR3alt, specifically a sequence selected from SEQ ID NO 001, SEQ ID NO 002, and/or SEQ ID NO 003. In alternative embodiments, the one or more CXCR3 transgenes comprise a reverse complement sequence encoding an mRNA transcript having only the exons of the CXCR3 variant gene, specifically a sequence selected from SEQ ID NO 004, SEQ ID NO 005, and/or SEQ ID NO 006. In another embodiment, a CXCR3 transgene comprises a sequence that is not less than 95% identical to the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006, wherein the encoded protein has the same biological activity as SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006, respectively, particularly if the CXCR3 transgene encodes an amino acid sequence that has not less than 96%, not less than 97%, not less than 98% or even not less than 99% sequence identity to the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006.

The biological activity conferred by functional CXCR3 transgene expression can be assessed by several different methods by comparing cells or cell populations carrying one or more transgenes to unmodified control cells or populations. The first functional assay provided for CXCR3 is the ability to migrate towards a stimulus of 100ng/ml CXCL9, CXCL10 or CXCL11, as shown in fig. 3a and 3b. CXCR3 also confers enhanced proliferation in response to combined stimulation with antigen and CXCL11, for example using an assay that measures CFSE dilution, as used in fig. 4 e.

The data in the examples show that the functionality of CXCR3 expression from human T cells from healthy volunteers or cancer patients mediates migration towards CXCL9, CXCL10 or CXCL11 stimulation with a chemotactic index exceeding 2 (fig. 3a and 3 b), or up to 10 of cxcr3+ cells after 21 days of simulation with CMV epitopes and CXCL11 ⁴ Double enrichment, and increased intracellular production of ifnγ and tnfα in response to tumor antigens compared to unstimulated cells (fig. 4a and 4 c). The listed assays reflect enhanced effector efficacy and post-transplant survivalThe desired quality, which is ideal for modified cells used as immune status modulating drugs.

The genetic engineering method employed to insert one or more transgenes according to the invention into a cell is not particularly limited and may be selected from, but is not limited to, the following technical methods:

1. Random integration, wherein the transgene is delivered into the cell by:

a. lentiviruses, in particular 3 rd generation lentiviruses (Dull et al J.Virol.) (1998, 72 (11): 8463);

b. gamma-retroviruses, in particular T-cell transduction with gamma-retroviruses (Rossig et al Blood 2002, 99 (6): 2009);

c. transposon-transposase based methods, in particular the PiggyBac transposon (Nakazawa et al J. Immunotherapy journal (J. Immunother.)) (2009, 32 (8): 828), or Sleeping Beauty (sleep beautyy) (TC 1-like) transposon system (Ivics et al Cell (Cell)) (1997, 91 (4): 501, (Huang et al blood 2006, 107 (2): 483);

2. targeted integration, including targeted transgene integration using a Homology Directed Repair (HDR) -mediated or nuclease-assisted system comprising:

a. template formats, such as those selected from single-or double-stranded DNA systems (Roth et al Nature) 2018, 559 (7714): 405), or adeno-associated virus (AAV) integration sites, such as AAV serotype 6 (Dever et al Nature 2016, 539 (7629): 384; eyquem et al Nature 2017, 543 (7643): 113), or non-integrating lentiviruses (Lombard et al Nature Biotechnology (Nature Biotechnol.) (2007, 25 (11): 1298);

b. Nucleases, e.g., zinc finger nucleases, transcription activator-like (TAL) effector nucleases, mega-TAL, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -Cas;

c. a nuclease-free system;

transient transgene delivery of mrna using:

a. in vitro electroporation (Schaft et al Cancer immunology (Cancer immunol.)) (2005, 55:1132);

b. a nanoparticle;

c. and (3) viruses.

In certain embodiments, the modified immune cells according to the invention express more CXCR3A and/or CXCR3alt than CXCR3B, particularly wherein the ratio of CXCR3A, CXCR alt or combined CXCR3A and CXCR3alt expression to CXCR3B expression is greater than 1.CXCR3 variants can be measured at the protein level or the nucleic acid level, as shown in fig. 4 f. The data in the examples show that on stem cell memory cd8+ T cells, the expression ratio of CXCR3A and CXCR3alt expression compared to CXCR3B expression is greater than 1, which shows enhanced responsiveness to CXCR3 ligand stimulation in vitro.

In a further embodiment of a modified T cell according to the first aspect of the invention expressing one or more CXCR3 variant transgenes, the cell expresses an additional transgenic protein. In some embodiments, the additional transgenic protein is a recombinant chimeric antigen receptor protein comprising the following essential structural components:

a. A signal peptide, wherein the signal peptide,

b. a target-specific recognition domain, in particular wherein the target is selected from a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen or a virus-specific surface antigen,

c. an effector domain comprising a transmembrane region and one or more intracellular signaling domains (signalling domain), in particular a CD3 zeta signaling domain, and

d. a linker region connecting domain (b) and domain (c).

In another embodiment, the additional transgene expressed by the modified T cell is a transgenic T cell receptor (TgTCR), wherein the TgTCR recognizes an immunotherapeutic target selected from a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen.

The CAR target specific recognition domain or TgTCR from the above embodiment can recognize, in other words, exhibit specific binding to an epitope from a molecule selected from, but not limited to, LMP1 (epstein-barr virus), CMV (cytomegalovirus), GD2, L1CAM (neuroblastoma), her2 (colon, sarcoma, glioblastoma, bladder), IL13Ra2, egfrvlll (glioblastoma), CD133 (HCC, pancreas and colorectal), mesothelin (pancreas), CAIX (kidney), CEACAM5 (gastrointestinal), TAG-72, CEA (colon), COA-1 (colorectal), PSMA (prostate) or c-MET (breast). These antigens are viral, or tissue antigens, or antigens that are up-regulated in tumor cells, conferring tissue or disease specific activation signals, which together with increased CXCR 3-mediated migration towards inflammation characterized by CXCR3 ligands, target T cell activity to defined physiological sites (Li et al Signal Trans. And Targeted therapy 2019, 4:35).

In another embodiment of the modified T cell according to the invention, the cell expresses a CXCR3 ligand transgene encoding a recombinant protein comprising a human CXCR3 ligand, so as to confer an autocrine activation signal on the transgenic CXCR3 receptor. In one embodiment, the CXCR3 ligand transgene comprises a CXCR3 ligand transgene promoter sequence and one of the following CXCR3 ligand sequence variants:

reverse complement of the pre-mRNA transcript (both introns and exons) of CXCL9, CXCL10 and/or CXCL11, in particular a sequence selected from SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010 and/or SEQ ID NO 011, or

Reverse complement of mRNA transcripts encoding CXCL9, CXCL10 and/or CXCL11 (exons only), in particular a sequence selected from the group consisting of SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, or

c. Nucleic acid encoding an amino acid sequence having at least (gtoreq) 95% sequence identity to the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, in particular wherein the encoded protein has the same biological activity as the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, respectively. In more specific embodiments, the CXCR3 transgene encodes an amino acid sequence having greater than or equal to 96%, > or equal to 97%, > or equal to 98%, or even greater than or equal to 99% sequence identity to the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, and/or SEQ ID NO 015.

In certain embodiments, the CXCR3 ligand transgene promoter is a constitutive promoter, such as the CMV immediate early promoter. In other embodiments, the promoter is conditionally expressed, e.g., activated by a T cell receptor linkage, to allow inducible expression of the CXCR3 ligand transgene downstream of the native or transgenic antigen receptor linkage, so as to limit non-specific activation and deleterious inflammatory side effects. This may be achieved by including an antigen responsive element such as ARRE-1 or ARRE-2 or CD28 responsive region. It will be appreciated that in order for a CXCR3 ligand to confer a functional advantage to a modified T cell, the ligand must be secreted for signaling through CXCR 3.

SEQ NO ID 007 through SEQ NO ID 015 provide splice variants of the CXCL9, CXCL10 and CXCL11 genes currently known in the art. In the examples, commercial CXCL9, CXCL10 and CXCL11 at a concentration of 10ng/ml are shown to be applied to CXCR 3-expressing CD8+ T cells from healthy donors or patients, enhancing T cell proliferation and cytokine secretion, indicating that autocrine production can enhance CXCR3 ⁺ Functionality of modified T cells. The biological function of CXCL9, CXCL10 and CXCL11 transgenes can be assessed by whether they cause the same increase in cytokine production as compared to the CXCR3 ligand transgene sequences provided, using CFSE dilution of cxcr3+ cells as assayed or antigen stimulated in fig. 4.

The second aspect of the invention provides an isolated immune cell preparation, in particular a T cell preparation, wherein the isolated immune cell preparation comprises at least (> 50%, in particular 70%, more in particular > 80%, even more in particular > 90% of immune cells, in particular T cells, which express a human CXCR3 variant selected from CXCR3A, CXCR alt+ and/or CXCR3B, in particular when one of the human CXCR3 variants or the human CXCR3 variant is CXCR3A and/or CXCR3alt. In some embodiments, the population of T cells expressing a CXCR3 variant is a direct ex vivo sample; in other embodiments, the isolated population has undergone an enrichment, amplification, or transgene insertion procedure to increase the cxcr3+ population from the starting sample of immune cell precursors.

In certain embodiments, most immune cell formulations express CXCR3alt only, CXCR3B only, or CXCR3A only.

In certain embodiments, a majority of the immune cell formulations express CXCR3A and CXCR3B, or CXCR3alt and CXCR3B. In particular embodiments, a majority of immune cells in an immune cell preparation together express CXCR3A and CXCR3alt.

In certain embodiments, a majority of the immune cell formulations express all three CXCR3alt, CXCR3A, and CXCR3B proteins.

This aspect of the invention encompasses native CXCR3 variant expression by immune cell preparations, or CXCR3 variant expression from transgenes. CXCR3 expression can be confirmed by real-time qPCR, for example using the probes provided herein, wherein expression of a signal > 0.1-fold, specifically > 0.2-fold housekeeping gene is considered positive. To confer a desired functional phenotype, it is understood that these CXCR3 variants should be expressed as proteins present on the cell surface. These cells may be isolated from peripheral blood or peripheral blood mononuclear cells, tissue samples, and/or tumor samples, and may include additional transgenic proteins. In certain embodiments of the invention related to cancer immunotherapy, the isolated immune cell preparation according to the above aspects of the invention has been isolated or expanded from a variety of immune cells isolated from a cancer patient sample such as peripheral blood or tumor-infiltrating lymphocytes (TILs) and/or lymph nodes derived from draining tumor tissue (Poch et al tumor immunology (oncoimmunol.) 2018,7 (9): e 1476816); dudley et al J.Immunotherapy 2003, 26:332; sakelariou et al 2018, 36:95).

The data presented in the examples demonstrate that CXCR3 derived from blood ⁺ The ability of T cells to respond to CXCR3 ligand migration signals (fig. 3 and 4), and the ability to produce inflammatory cytokines ifnγ and tnfα after stimulation with autologous tumor lysate or viral antigen (fig. 2, 4). These cells can be used asThe starting point for enriched preparations of natural antigen-specific cxcr3+ cells, as they are early effector cells associated with a stronger immune response and better clinical outcome of cancer. Alternatively, they may be useful starting populations for introducing additional transgenes such as CXCR3 variants, CXCR3 ligands, tgtcrs, CAR T cell receptors in transgenic approaches.

In another embodiment, the invention provides an isolated population of immune cells comprising ≡50%, specifically ≡70%, more specifically ≡80% of CXCR3 according to any embodiment of the first aspect of the invention ⁺ Genetically modified immune cells. In other words, the primary cells express CXCR3A, CXCR3B and/or CXCR3alt, and optionally an artificial antigen receptor and/or CXCR 3-binding chemokine from the transgene.

In certain embodiments, an isolated CXCR3 according to this aspect of the invention ⁺ The cell preparation is enriched by 50%, specifically 70%, more specifically 80% in one of the following functionally different T cell subsets:

a.CD8 ⁺ T cells, in particular CD8 ⁺ CCR7 ⁺ CD45RA ⁺ CD95 ⁺ Stem cell-like memory T cells and/or CD8 ⁺ CCR7 ⁺ CD45RA ^- A T cell for central memory, wherein the T cell,

b.CD4 ⁺ memory T cells, in particular helper T cell type 1, T-bet ⁺ CD4 ⁺ A memory T-cell, a cell line,

c.CD4 ⁺ treg cells, in particular CD4 ⁺ CD25 ⁺ Treg cells expressing foxp3, or

NK or NKT cells, in particular CD56 ⁺ NK or NKT cells.

The data presented in the examples demonstrate that the CXCR3 variant is CD8 in healthy blood and in LN of BC patients ⁺ CCR7 ⁺ CD45RA ⁺ CD95 ⁺ Stem cell-like memory T cells and CD8 ⁺ CCR7 ⁺ CD45 RA-central memory T cells are highly expressed. These natural cxcr3+ cells can be purified to provide an isolated cxcr3+ cell preparation according to the invention. In which CD4 assists, cytotoxic NKT cell activity or inhibitsIn embodiments where sex Treg cytokines are desirable in the isolated cell preparation, alternative embodiments b to c may be used. Th1 and NKT cells are useful against cancer, bacterial or viral infections, whereas Treg can suppress inflammation caused by, for example, autoimmune or graft-specific immune activation. Expression of surface molecules that recognize the subpopulations may be determined, for example, by flow cytometry, compared to isotype control or cells known to lack the marker in question, or by real-time qPCR.

In another embodiment, the modified T cell or isolated cxcr3+ cell preparation according to the first or second aspects of the invention, respectively, is defined by their properties in at least one assay that demonstrates the functionality of the expression of a transgenic CXCR3 variant. The concentration of CXCL9, CXCL10 or CXCL11 used in any of the following assays may be in the range of 1 to 1000ng/ml of CXCL11, specifically between 10 and 100 ng/ml. Titration at concentrations of 1, 10, 100 and 1000ng/ml are particularly useful, with unstimulated controls to identify kinetic or functional responses to stimulation.

According to this embodiment, the cell or cell population can have a chemotactic index of > 2 following in vitro stimulation with CXCL9, CXCL10, and/or CXCL11 using assays as provided on page 23, line 30 of the examples.

Alternatively, cells or cell populations may have a higher frequency of proliferating cells after stimulation with antigen and CXCL11 than unstimulated cells, in other words, the ratio of proliferation measurements of isolated cxcr3+ populations must be > 1 compared to pre-or non-isolated samples. This can be measured in an assay using in vitro cell division assays such as flow cytometry to measure the percentage of cells diluted with a labeling dye, such as those provided in the methods of page 24, line 23 (surrogate dyes, see Parish "immunoand cell biology (immunol.)) (1999, 77 (6): 499), or by thymidine incorporation, for example. Alternatively, or in addition to proliferation, assays that measure enrichment of cxcr3+ antigen-specific populations, such as those provided at page 24, line 6 that measure upregulation of CD137 after antigen incubation, can be used. For the present invention, the fold enrichment of cxcr3+ antigen-specific populations in response to antigen and CXCL11 stimulation must be > 1 fold, specifically > 1.2 fold, greater than that of the unfractionated control sample.

In other options provided by this embodiment, the T cells or isolated CXCR3 are compared to unmodified T cells lacking one or more CXCR3 transgenes or to cell preparations prior to unfractionation or isolation ⁺ Cell populations are characterized by having enhanced lytic potential. The lytic potential can be measured by% killing of target cell populations such as tumors or virus-infected cells marked with dyes or radiolabels, or by intracellular measurement of lyase granzyme B and/or perforin in a flow cytometry assay (clinic et al immunology front 2019, 10:1148; wagner et al natural medicine (nat. Med.) 2019, 25 (2): 242). The ratio of the% of target cells killed by the cxcr3+ cell population must be > 1, in particular > 1.5 times the ratio of the sample before fractionation or separation.

Finally, cxcr3+ cells or cell populations, upon stimulation with CXCL11, can produce more effector cytokines, specifically selected from ifnγ, tnfα, IL-10 and IL-2, and optionally antigen pulsed antigen presenting cells, TCR cross-linking agents and soluble antigens or calcium flux inducers. Exemplary protocols for intracellular flow cytometry of ifnγ and tnfα in the presence of brefeldin a, stimulated with antigen expressing cells and CXCL11 for 12 hours, are provided at page 24, line 20. For the purposes of the present invention, the% of cytokine+ cells in an expanded or enriched cxcr3+ cell population should be > 1-fold, specifically > 1.1-fold greater than the% of cytokine+ cells in an unaddressed or pre-enriched control sample.

The data presented in the examples demonstrate that in cancer patients, inflamed lymph nodes enriched for CXCR3 ligands are more highly infiltrated with cxcr3+ T cells, and that tumors with more CXCR3 biomarker expression, in particular CXCR3A and CXCR3alt, show signs of containing more T cells. Furthermore, in vitro assays demonstrate that cxcr3+ stem cell memory cells or central memory cells have a chemotactic index of more than 2 when stimulated by CXCL9, CXCL10 or CXCL11, and proliferate and produce more cytokines than other subpopulations in response to viral or tumor antigens when stimulated by CXCL 11. These assays form the basis for the functional characterization presented in this embodiment of the invention.

A third aspect of the invention provides a modified CXCR3 for use as a medicament ⁺ T cells or isolated CXCR3 ⁺ Cell preparation. In some embodiments, the medicament is used to enhance aspects of T cell immunity, covering the pro-inflammatory and anti-inflammatory T cell functions provided by the different subpopulations provided above that carry the transgene.

In certain particular embodiments, modified CXCR3 ⁺ T cells or isolated CXCR3 ⁺ Cell preparation for enhancing CD8 ⁺ T cell immunity, in particular for increasing immunity against a solid tumor selected from squamous cell carcinoma or adenocarcinoma, more in particular against a cancer selected from breast cancer, colorectal cancer, neuroblastoma, sarcoma, bladder cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, renal cancer, gastrointestinal cancer or prostate cancer. The data in the examples show that these cells are particularly useful for the treatment of solid cancers, rather than systemic cancers derived from lymphocytes, because CXCR3 transgenes or enriched CXCR3 expression confers the ability to home to inflamed tumor tissue expressing CXCL11, CXCL9 or CXCL 10.

In CXCR3 ⁺ T cells or isolated CXCR3 for use as a medicament ⁺ In an alternative embodiment of the cell preparation, the one or more cells are used for the treatment of an infectious disease. Since CXCR3 has been shown to enhance the inflammatory capacity of cd8+ T cells, this treatment can be particularly useful for treating diseases caused by intracellular pathogens, such as chronic viral infections, where the infected cells are susceptible to killing by cytotoxic CD 8T cells. Related viral diseases include, but are not limited to, EBV, CMV, human immunodeficiency virus, coronavirus, or hepatitis. The data presented in the examples show that in vitro stimulation of CXCL11 on CXCR3 variant expressing cells is able to activate CMV and EBV specific CD 8T cells in human samples (fig. 4).

A fourth aspect of the invention provides obtaining an isolated CXCR3 ⁺ A method of preparing a cell preparation, the method comprising providing a first sample of a human, in particular a peripheral blood sample, a lymph node or a tumour tissue sample, comprising immune cellsAnd (3) step (c). The data in the examples demonstrate that tumor draining lymph nodes enrich cxcr3+, tumor-specific cells. Tumor tissue of neoadjuvant therapy responsive BC patients also showed to be enriched for CXCR3 variant biomarkers. Likewise, any target tissue or lymph node draining the target tissue, such as an organ afflicted with a deleterious immune infiltration or viral infection, may be used as a suitable starting sample in which the desired antigen specificity of the CXCR3 expression profile is present.

The next step is to select or enrich immune cells, in particular T cells, expressing CXCR3 variants cxcr3alt+, cxcr3b and/or cxcr3a+ from the sample and/or to remove cells not expressing CXCR3 variants CXCR3A, CXCR3B and/or CXCR3 alt. This can be achieved, for example, by magnetic sorting with cxcr3alt+ specific antibodies carrying magnetic beads and subsequent retention in a magnetic field, or by flow cytometry sorting of fluorescently labeled cells. The method encompasses expression of total CXCR3 or individual variant combinations.

In some embodiments, the method of obtaining an isolated cxcr3+ cell preparation comprises an additional gene transfer step, wherein the transgene is inserted into a plurality of cells. The transgene may comprise a CXCR3 variant nucleic acid sequence selected from SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, optionally a CXCR3 ligand sequence selected from SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015. Alternatively, the transgenic nucleic acid sequence may encode an amino acid sequence having greater than or equal to 95% sequence identity to an amino acid sequence encoded by a sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, in particular wherein the transgene encodes an amino acid sequence having greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98% or even greater than or equal to 99% sequence identity to an amino acid sequence encoded by a sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, respectively. In this case, the protein encoded by the sequence has substantially the same biological activity as one of the sequences provided above, in particular in an assay of T cell migration, cytokine production or proliferation as outlined above and in the examples.

It may also be desirable to add a transgene of TgTCR or CAR at this step as detailed in the first aspect of the invention to provide a modified T cell.

The final embodiment of the method for preparing an isolated CXCR3 variant expressing cell preparation provides an expansion step wherein the cells are cultured with CXCL9, CXCL10 and/or CXCL11 at a concentration of 1 to 1000ng/ml, or specifically with CXCL11 at a concentration of 10 to 100 ng/ml. This may optionally be in the presence of antigens and/or gamma chain cytokines, in particular IL-2.

The data in the examples, in particular CXCR3 ligand titration migration assays, show that CXCL9, CXCL10 and/or CXCL11 of 10 to 100ng/ml can enhance proliferation, migration and cytokine production of CXCR3 expressing T cell subsets. The functional importance of in vitro assays is demonstrated in biomarker analysis that captures the direct correlation between the presence of CXCR3 chemokine family molecules in BC tumors and the positive outcome and overall survival of neoadjuvant cancer treatment.

The invention also relates to the following;

A. modified CD3 expressing CXCR3 transgene ⁺ A T cell, wherein the transgene encodes a recombinant protein comprising a human CXCR3 variant selected from the group consisting of:

CXCR3A, CXCR alt+ and/or CXCR3B,

in particular, wherein the human CXCR3 variant or one of the human CXCR3 variants is CXCR3A and/or CXCR3alt.

B. A modified T cell according to item A, wherein the T cell is a CD3+CD8+ memory T cell.

C. The modified T cell according to item a or B, wherein the CXCR3 transgene

a. Reverse complement of a pre-mRNA transcript comprising CXCR3A, CXCR alt and/or CXCR3B, in particular a sequence selected from SEQ ID NO 001, SEQ ID NO 002 and/or SEQ ID NO 003, or

b. Reverse complement of an mRNA transcript encoding comprising CXCR3A, CXCR alt and/or CXCR3B, in particular a sequence selected from SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006, or

c. Encodes an amino acid sequence having at least (. Gtoreq.95%) sequence identity to the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006, and wherein the encoded protein has the same biological activity as SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006,

in particular, wherein the CXCR3 transgene encodes an amino acid sequence having greater than or equal to 96%, greaterthan or equal to 97%, greaterthan or equal to 98%, or even greater than or equal to 99% sequence identity to the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, and/or SEQ ID NO 006.

D. The modified T cell according to any one of items a to C, wherein the expression level of CXCR3A and/or CXCR3alt is higher than the expression level of CXCR3B, in particular wherein the ratio of the expression level of CXCR3A and/or CXCR3alt compared to CXCR3B is greater than 1.

E. A modified T cell according to any one of a to D, which further expresses a Chimeric Antigen Receptor (CAR) comprising:

a. a signal peptide, wherein the signal peptide,

c. an effector domain comprising a transmembrane region and one or more intracellular signaling domains,

d. a linker region connecting domain (b) and domain (c).

F. A modified T cell according to any one of items a to D, which further expresses a transgenic T cell receptor (TgTCR) protein, wherein the TgTCR recognizes a target selected from a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen.

G. The modified T cell according to item E or F, wherein the target-specific recognition domain or TgTCR recognizes a target selected from a transgenic T cell receptor specific for an antigen selected from LMPA, CMV _pp65 GD2, L1CAM, her2, IL13Ra2, EGFRvIII, CD133, mesothelin, CAIX, CEACAM5, TAG-72, CEA, COA-1, PSMA or c-MET.

H. The modified T cell of any one of claims a to G, wherein the cell further expresses a CXCR3 ligand transgene comprising a CXCR3 ligand transgene promoter sequence and a recombinant human CXCR3 ligand, and wherein the transgene comprises:

reverse complement of a pre-mRNA transcript of CXCL9, CXCL10 and/or CXCL11, in particular a sequence selected from the group consisting of SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010 and/or SEQ ID NO 011, or

Reverse complement of mRNA transcripts encoding CXCL9, CXCL10 and/or CXCL11, in particular a sequence selected from the group consisting of SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, or

c. A nucleic acid sequence encoding an amino acid sequence having at least (. Gtoreq.) 95% sequence identity to the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, and wherein the encoded protein has the same biological activity as the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015,

Specifically, wherein the CXCR3 transgene encodes an amino acid sequence having greater than or equal to 96%, greaterthan or equal to 97%, greaterthan or equal to 98%, or even greater than or equal to 99% sequence identity to the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, and/or SEQ ID NO 015.

I. An isolated immune cell preparation, particularly a T cell preparation, wherein the isolated immune cell preparation comprises at least (No. 50), particularly 70%, more particularly No. 80%, even more particularly No. 90% of immune cells, particularly T cells, expressing a human CXCR3 variant selected from CXCR3A, CXCR alt+ and/or CXCR3B, particularly wherein one of the human CXCR3 variant or human CXCR3 variant is CXCR3A and/or CXCR3alt.

J. The isolated cell preparation according to item I, wherein the cells are derived from a cancer patient sample, in particular a cancer patient sample selected from peripheral blood, tumor tissue and/or tumor draining lymph node tissue.

K. The isolated cell preparation according to item I or J, comprising at least (. Gtoreq.) 50%, specifically.gtoreq.70%, more specifically.gtoreq.80% of any one of the modified immune cells as detailed in items A to H.

An isolated cell preparation according to item I or J, wherein the cell does not express any transgene.

The isolated cell preparation according to any one of claims I to L, wherein within immune cells expressing CXCR3 variants, 50% or more, specifically 70% or more, more specifically 80% or more are:

a.CD8 ⁺ memory cell, in particular CD8 ⁺ CCR7 ⁺ CD45RA ⁺ CD95 ⁺ And/or CD8 ⁺ CCR7 ⁺ CD45RA ^- CD95 ⁺ A memory T-cell, a cell line,

b.CD4 ⁺ memory T cells, in particular helper T cell type I, T-bet ⁺ CD4 ⁺ A memory T-cell, a cell line,

c.CD4 ⁺ regulatory T (Treg) cells, in particular CD4 ⁺ CD25 ⁺ Treg cells, or

NK or NKT cells, in particular CD56 ⁺ NK or NKT cells.

N. a modified T cell according to any one of a to H, or an isolated cell preparation according to any one of I to M, wherein the cell or cell population is characterized by:

a. chemotactic index > 2 after in vitro stimulation with CXCL9, CXCL10 and/or CXCL11,

b. the frequency of proliferating cells after stimulation with antigen and CXCL11 is higher than that of unstimulated cells,

and/or wherein as detailed in item C, the T cell or isolated cell population is characterized as compared to an unmodified T cell or an unseparated cell preparation lacking one or more CXCR3 transgenes:

c. enhanced cleavage potential, and/or

d. More effector cytokines, in particular selected from ifnγ, tnfα, IL-10 and/or IL-2, are produced after stimulation with CXCL 11.

O. a modified immune cell according to any one of a to H, or an isolated cell preparation according to any one of I to N, for use as a medicament.

P. a modified T cell according to any one of A to H, or an isolated cell preparation according to any one of I to O, for use in enhancing T cell immunity, in particular CD8 ⁺ T cell immunity.

A modified immune cell according to any one of a to H, or an isolated cell preparation according to any one of I to P, for use in the treatment of cancer, in particular a solid cancer such as squamous cell carcinoma or adenocarcinoma, more particularly a cancer selected from breast cancer, colorectal cancer, neuroblastoma, sarcoma, bladder cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, renal cancer, gastrointestinal cancer or prostate cancer.

A modified immune cell according to any one of a to H, or an isolated cell preparation according to any one of I to P, for use in the treatment of an infectious disease.

S. a method of obtaining an isolated cell preparation according to any one of the claims I to N, the method comprising the steps of:

-providing a sample comprising immune cells;

-selecting immune cells, in particular T cells, which express CXCR3 variants cxcr3alt+, cxcr3b and/or cxcr3a+ present in the sample and/or removing cells from the sample which do not express CXCR3 variants CXCR3A, CXCR3B and/or CXCR3 alt.

A method of obtaining an isolated cell preparation, the method comprising the steps of:

-providing a sample comprising a plurality of immune cells;

-inserting a transgene into each of the plurality of immune cells in a gene transfer step, wherein the transgene comprises

a. A nucleic acid sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, and optionally SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, or

b. A nucleic acid sequence encoding an amino acid sequence having at least (gtoreq) 95% sequence identity to an amino acid encoded by a sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, and optionally SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, and wherein the encoded amino acid sequence has the same biological activity as SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, in particular, wherein the transgene encodes an amino acid sequence having greater than or equal to 96%, greaterthan or equal to 97%, greaterthan or equal to 98%, or even greater than or equal to 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, and optionally SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, and/or SEQ ID NO 015;

-inserting, in an optional second gene transfer step, a transgene encoding a CAR or TgTCR protein as detailed in any one of E to G.

U. method for preparing an isolated cell preparation according to item S or item T, further comprising an optional expansion step, wherein the cells are cultured with CXCL9, CXCL10 and/or CXCL11, in particular with CXCL11 of from 10 to 100ng/ml, optionally in the presence of antigen and/or gamma chain cytokines, in particular IL-2, at a concentration of from 1 to 1000 ng/ml.

A1. Modified CD3 expressing CXCR3 transgene ⁺ T cells, in particular cd3+cd8+ memory T cells, wherein the transgene encodes a recombinant protein comprising a human CXCR3 variant selected from the group consisting of:

CXCR3A, CXCR alt+ and/or CXCR3B,

A2. The modified T cell according to A1, wherein the CXCR3 transgene

A3. The modified T cell according to item A1 or A2, wherein the expression level of CXCR3A and/or CXCR3alt is higher than the expression level of CXCR3B, in particular wherein the ratio of expression level of CXCR3A and/or CXCR3alt compared to CXCR3B is > 1.

A4. A modified T cell according to any one of A1 to A3, which further expresses a Chimeric Antigen Receptor (CAR) comprising

a. A signal peptide, wherein the signal peptide,

b. a target-specific recognition domain, in particular wherein the target is a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen or a virus-specific surface antigen,

d. a linker region connecting domain (b) and domain (c).

A5. The modified T cell according to any one of A1 to A3, further expressing a transgenic T cell receptor (TgTCR) protein, wherein the TgTCR recognizes a target selected from a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen.

A6. The modified T cell according to any one of A4 or A5, wherein the target-specific recognition domain or TgTCR recognizes a target selected from a transgenic T cell receptor specific for an antigen selected from the group consisting of LMPA, CMV _pp65 GD2, L1CAM, her2, IL13Ra2, EGFRvIII, CD133, mesothelin, CAIX, CEACAM5, TAG-72, CEA, COA-1, PSMA or c-MET.

A7. The modified T cell of any one of A1 to A6, wherein the cell further expresses a CXCR3 ligand transgene comprising a CXCR3 ligand transgene promoter sequence and a recombinant human CXCR3 ligand, and wherein the transgene comprises:

c. A nucleic acid sequence encoding an amino acid sequence having at least (. Gtoreq.) 95% sequence identity to the amino acid sequence encoded by SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, and wherein the encoded protein has the same biological activity as SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015,

A8. An isolated immune cell preparation, in particular a T cell preparation,

wherein the isolated immune cell preparation comprises at least (> 50%, specifically 70%, more specifically > 80%, even more specifically > 90% of immune cells, specifically T cells, expressing a human CXCR3 variant selected from CXCR3A, CXCR Alt+ and/or CXCR3B,

A9. The isolated cell preparation according to A8, wherein the cells are derived from a cancer patient sample, in particular a cancer patient sample selected from peripheral blood, tumor tissue and/or tumor draining lymph node tissue.

A10. The isolated cell preparation according to any one of items A8 to A9, comprising at least (gtoreq) 50%, in particular equal to or greater than 70%, more in particular equal to or greater than 80% of any one of the modified immune cells as detailed in items A1 to A7.

A11. The isolated cell preparation according to any one of A8 to a10, wherein the cell does not express any transgene.

A12. The isolated cell preparation of any one of claims A8 to a11, wherein within an immune cell expressing a CXCR3 variant, 50% or more, specifically 70% or more, more specifically 80% or more is:

NK or NKT cells, in particular CD56 ⁺ NK or NKT cells.

A13. A modified immune cell according to any one of A1 to A7, or an isolated cell preparation according to any one of A8 to a12, for use as a medicament.

A14. A modified immune cell according to any one of A1 to A8, or an isolated cell preparation according to any one of A9 to a16, for use in

a. Treating cancer, in particular solid cancer such as squamous cell carcinoma or adenocarcinoma, more particularly a cancer selected from the group consisting of breast cancer, colorectal cancer, neuroblastoma, sarcoma, bladder cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, renal cancer, gastrointestinal cancer or prostate cancer,

b. treating infectious diseases.

A15. A method of obtaining an isolated cell preparation, the method comprising the steps of:

-providing a sample comprising a plurality of immune cells;

b. A nucleic acid sequence encoding an amino acid sequence having at least (gtoreq) 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, and optionally SEQ ID NO 007, ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015, and wherein the encoded amino acid sequence has the same biological activity as SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014 and/or SEQ ID NO 015,

In particular, wherein the transgene encodes an amino acid sequence having greater than or equal to 96%, greaterthan or equal to 97%, greaterthan or equal to 98%, or even greater than or equal to 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, and optionally SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, and/or SEQ ID NO 015;

-inserting in an optional second gene transfer step a transgene encoding a CAR or TgTCR protein as detailed in any one of A5 to A7.

Chemokine biomarkers for prediction and stratification of cancer treatment

The invention also relates to a method for a priori evaluation of CXCR3 splice variants and their ligands CXCL9, CXCL10 and CXCL11 in a patient with Myometrial Invasive Bladder Cancer (MIBC) to enable stratification of the patient according to the patient's predicted response or clinical outcome to treatment with a chemotherapeutic agent. The invention also relates to the treatment of cancer patients who have been identified as being sensitive to certain treatment regimens.

Summary of diagnostic aspects of the invention

The next aspect of the invention relates to a method of measuring the activity status of T cells in a tissue sample of a patient, wherein the method comprises the steps of: first, a patient tissue sample is provided, and then in a measurement step, the biomarker expression level of at least one of the biomarkers CXCL11 and CXCR3A or CXCR3alt is determined. Biomarker expression levels for CXCR3B, CXCL4, CXCL9, and CXCL10 can also optionally be determined. In some embodiments, the tissue sample is assigned to reflect the presence of the CXCR3 cytokine system based on biomarker expression levels, classification of T cell activity status in the patient tissue sample.

Another aspect of the invention relates to a method of specifically measuring the status of T cell activity in a tumor sample of a patient, wherein the method comprises the steps of: providing a cancer tissue sample; the biomarker expression level of the above-mentioned biomarkers is then determined in a measurement step. In an optional classification step, the cancer tissue sample is assigned a value reflecting the number of T cells present and the activation state in the cancer tissue sample based on the biomarker expression level.

The present invention provides in a further aspect determining the expression level of at least one of the biomarkers CXCL11 and CXCR3A or CXCR3alt in a tumor sample taken from a patient who has been previously diagnosed with cancer in order to predict the outcome of an anti-tumor treatment or to classify the patient as a treatment responder or a non-responder.

These methods applied in cancer may optionally include further measurements of CXCR3B splice variants and/or additional chemokine biomarkers CXCL4, CXCL9, and CXCL 10. One particular embodiment relates to classifying cancer patients according to the invention into treatment responders and non-responders. This may be based on inputting detailed biomarkers into an algorithm that provides a probability that the patient will respond favorably to anti-tumor therapy, comparing patient biomarker expression levels to a representative reference sample, or comparing biomarker expression levels in a sample to a list of biomarker expression thresholds generated from previously analyzed patient biomarker expression data groups.

In one embodiment, the expression level of the biomarker CXCL11 is determined at the protein expression level. In certain embodiments of the methods according to the invention, the value is expressed as an unconverted expression level, or an expression level value of CXCL11 is subjected to a normalization process, in particular normalizing the mass of the sample analyzed, or an area sinusoidal hyperbola (Area sinus hyperbolicus (arsin)) normalization, to provide a value of arsin as expressed per gram of tissue. Patients or samples with a CXCL11 expression level above a 13.98 threshold, in particular above 22.4pg, per 10mg sample tissue can be classified as treatment responders according to the invention.

Another specific aspect of the invention is a method of measuring mRNA expression of CXCR3 isoforms CXCR3A, CXCR alt and/or CXCR3B, in particular using a nucleic acid probe that utilizes sequences that distinguish between biomarker CXCR3 variants. The method may optionally comprise normalization of CXCR3 variant expression levels relative to expression of housekeeping genes, in particular housekeeping genes IP08 and CDKN 1B.

In some aspects of the invention, CXCR3 splice variant expression above a threshold level is used to pair a patient cancer sample with a predicted clinical outcome. When CXCR3A is greater than HKG by 2 ^(-12.3) Multiple, in particular 2 more than HKG ^(-11.97) Multiple and/or CXCR3alt 2 more than HKG ^(-13.8) Multiple, in particular 2 more than HKG ^(-11.27) Multiple and/or CXCR3B 2 more than HKG ^(-11.9) Multiple, in particular 2 more than HKG ^(-8.43) At times, patients with tumor samples classified as positive or highly expressed splice variants may respond to anti-tumor therapy. The methods of the invention may be particularly useful in predicting the clinical outcome of anti-tumor therapy for cancer patients diagnosed with renal, prostate, breast, lung, ovarian, gastric, rectal, melanoma, esophageal, or, specifically, bladder cancer, more specifically, myometrial invasive bladder cancer. A particularly useful embodiment of the invention is the measurement of CXCL11 and CXCR3 splice variant biomarkers to predict the outcome of a patient on neoadjuvant chemotherapy treatment, in particular an anti-tumor drug, bcg or immune checkpoint modulator.

Another aspect of the invention is a method of stratifying cancer patients according to their expression levels of detailed biomarkers, according to the priority that they should receive anti-tumor surgical intervention.

Another aspect of the invention provides a method of predicting the clinical outcome of immune cell transfer therapy by matching the expression level of a CXCR3 biomarker in a cell transfer sample to the expression level of a CXCR3 ligand in a target tissue sample.

Another aspect of the invention is a method of comparing the expression levels of CXCL11 and at least one of CXCR3A and/or CXCR3alt, and optionally CXCR3B, CXCL4, CXCL9 and/or CXCL10, in a tumor sample of a patient before and after treatment for the purpose of monitoring the progression of the tumor over time.

Another aspect of the invention is a pharmaceutical composition comprising an anti-tumor platinum drug, such as cisplatin, for treating cancer patients that have been classified as drug responders according to any of the methods described above.

A final aspect of the invention is a system for assigning a value reflecting the T cell activity status of a tissue to a tissue sample of a patient, the system comprising determining the expression level of at least one of the biomarkers CXCL11, CXCR3A, CXCR alt, CXCR3B, CXCL4, CXCL9 and/or CXCL10 in the tissue sample.

Detailed description of diagnostic aspects of the invention

One aspect of the invention relates to a method of measuring the status of T cell activity in a tissue sample of a patient, which method is useful in a clinical setting, wherein information about the number and phenotype of T cells that respond to CXCR3 binding chemokines can inform the patient of a prognosis or clinical treatment choice. This first aspect of the invention may be of importance in patients diagnosed with or suspected of having a condition characterized by chronic inflammation. Biomarker expression may allow a physician to measure whether sufficient pre-existing T cells are present in the target tissue in order to predict whether a drug or cell-based therapy that enhances T cell responses will be effective, e.g., against viral infection or cancer. Instead, the method can be used to examine tissue samples for the presence of detrimental autoimmune T cell recruitment and activation in order to inform decisions about immunosuppressive drugs.

The first step of the method of measuring the state of T cell activity is to provide a patient tissue sample. The sample may be peripheral blood or white blood cells, or in any tissue sample where it is desirable from a clinical point of view to estimate the number or activation state of T cells within the tissue, such as a biopsy, or tissue derived from a tumor, graft moiety, or tissue targeted by infection or autoimmune inflammation.

The second step of the method is to measure the expression level of one or both of the chemokines CXCL11 and CXCR3 variants CXCR3A or CXCR3alt present in the tumor sample. The measuring step may optionally comprise determining the expression level of the further biomarkers CXCL4, CXCL9, CXCL10 and/or splice variant CXCR 3B. In an optional classification step, the biomarker expression level is used to assign a value reflecting the status of T cell activity to the sample or patient, in other words, the number of T cells present in the patient tissue sample and/or the likelihood of inflammation.

The data in the examples show the efficacy of CXCR3 and CXCR3 ligand biomarker families in identifying tissues with an effective T cell population that can be activated by subsequent chemotherapy. Furthermore, it was shown that expression of CXCR3 receptors on certain cd8+ T cell subsets confers migration, proliferation and cytokine secretion potential to Cytomegalovirus (CMV) specific T cells as well as tumor specific T cells in vitro, confirming the broad applicability of this approach to cancer, chronic viral infection and potentially harmful autoimmune or graft specific T cell responses.

The next aspect of the invention relates to a method for specifically measuring T cell activation in a tumor sample of a patient, wherein the method comprises the steps of: providing a cancer tissue sample; the biomarker expression level of the biomarker as detailed in the first aspect of the invention is then determined in a measurement step. The sample may be a peripheral leukocyte sample, a tumor sample from a biopsy, or tissue removed during a surgical intervention, or a cancer patient sample such as peri-tumor tissue, or a tumor draining lymph node. In an optional classification step, the cancer tissue sample is assigned a value reflecting the T cell activity status of T cells present in the cancer tissue sample based on the biomarker expression level. By helping to predict prognosis of patients who have undergone or not undergone cancer treatment, the patient classification provided by the methods described herein may form part of a diagnostic regimen that identifies solid tumors. In other words, the methods of the present invention may facilitate stratification of patients into groups having different recommended treatment regimens or different patient outcomes, and may be useful to a clinician, for example, when assigning patients to groups that will receive medication, surgery, or palliative treatment.

In the data presented in the examples examining biomarker expression in BC patients, information about CXCL11 expression levels in tumor samples was combined with information about expression of either of CXCR3 splice variants CXCR3A or CXCR3alt, classifying the patient with 100% accuracy as to whether the patient would respond favorably to anti-tumor neoadjuvant chemotherapy treatment. Using a linear regression model, the area under the curve of the Receiver Operating Characteristics (ROC) curve generated from the expression levels of CXCL11 and CXCR3A, CXCL11 and CXCR3alt, or CXCL11 and CXCR3A and CXCR3alt was 1, indicating complete accuracy of NAC response prediction. Also included are fits in which CXCR3B expression levels increase these predictive models. Cox proportional hazard analysis of the second group showed that the correlation between time to live and CXCL11 and CXCL4, CXCL9, CXCL10 mRNA was confirmed, rather than a large number of CXCR3 mRNA levels being correlated with BC treatment results.

In another aspect the invention provides a method of predicting whether a patient bearing a solid tumor will respond to a cancer treatment by measuring the expression of a selected biomarker in a tumor sample as detailed above: CXCL11 and at least one of CXCR3A or CXCR3alt, optionally with the addition of CXCR3B, CXCL4, CXCL9 and/or CXCL10. The methods provided herein can provide information that can assist a clinician in selecting the most appropriate personalized treatment course for a cancer patient. In a classification step of a method of predicting clinical outcome of a cancer patient, the expression level of a CXCR3 chemokine family biomarker determined in a sample is used to classify the cancer patient as likely to be an anti-cancer therapy responder or a therapy non-responder. In other words, the biomarker analysis method is capable of predicting the outcome of a clinical treatment of cancer.

Another aspect of the invention provides a method of measuring the T cell activity status of a tumor, or predicting the outcome of a patient in cancer treatment, by determining the expression level of at least one biomarker selected from the list comprising CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt and/or CXCR3B in a cancer tissue sample. In one embodiment, the expression level of at least two, in particular at least three, provided biomarkers is determined. Because all biomarkers in the CXCR3 system are associated with BC patient NAC treatment outcome, as the number of measured biomarkers increases, increased accuracy can be achieved by statistical models to predict patient outcome.

From analysis of mRNA (CXCR 3) and protein (CXCL 11) expression in the first MBIC patient cohort, the following combinations were identified as having the potential to predict neoadjuvant chemotherapy outcome with both 100% sensitivity and 100% specificity:

CXCL11 together with CXCR3alt, or

CXCL11 together with CXCR3A, or

CXCL11 together with any two CXCR3 variants, or

CXCL11 along with all three CXCR3 variants.

Combining the expression of all four molecules in a predictive linear regression model gives a best fit to the classical cohort data in the examples as reflected by the low AIC and Brier scores of the combination of such predictive biomarkers. In the second group of patients, CXCL11 and CXCL9 and CXCL10 mRNA expression also correlated with the amount of T cell marker CD3 found in the tumor. In this group, all four CXCR3 ligands CXCL4, CXCL9, CXCL10 and CXCL11 are associated with the chemotherapy outcome of bladder cancer patients, providing the following additional biomarker combinations with the potential to predict neoadjuvant chemotherapy outcome:

CXCL4 together with CXCR3alt or CXCR3A, or

CXCL4 together with any two CXCR3 variants,

CXCL4 together with all three CXCR3 variants,

CXCL9 together with CXCR3alt or CXCR3A,

CXCL9 together with any two CXCR3 variants,

CXCL9 along with all three CXCR3 variants,

CXCL10 together with CXCR3alt or CXCR3A,

CXCL10 along with any two CXCR3 variants,

CXCL10 along with all three CXCR3 variants,

CXCL9 and CXCL10 together with CXCR3alt or CXCR3A,

CXCL9 and CXCL10 together with any two CXCR3 variants,

CXCL9 and CXCL10 together with all three CXCR3 variants,

CXCL10 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL10 and CXCL11 together with any two CXCR3 variants,

CXCL10 and CXCL11 together with all three CXCR3 variants,

CXCL9 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL9 and CXCL11 together with any two CXCR3 variants,

CXCL9 and CXCL11 together with all three CXCR3 variants,

CXCL4 and CXCL9 together with CXCR3alt or CXCR3A,

CXCL4 and CXCL9 together with any two CXCR3 variants,

CXCL4 and CXCL9 together with all three CXCR3 variants,

CXCL4 and CXCL10 together with CXCR3alt or CXCR3A,

CXCL4 and CXCL10 together with any two CXCR3 variants,

CXCL4 and CXCL10 together with all three CXCR3 variants,

CXCL4 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL4 and CXCL11 together with any two CXCR3 variants,

CXCL4 and CXCL11 together with all three CXCR3 variants,

CXCL9, CXCL10 and CXCL4 together with CXCR3alt or CXCR3A,

CXCL9, CXCL10 and CXCL4 together with any two CXCR3 variants,

CXCL9, CXCL10 and CXCL4 together with all three CXCR3 variants,

CXCL9, CXCL10 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL9, CXCL10 and CXCL11 together with any two CXCR3 variants,

CXCL9, CXCL10 and CXCL11 together with all three CXCR3 variants,

CXCL9, CXCL4 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL9, CXCL4 and CXCL11 together with any two CXCR3 variants,

CXCL9, CXCL4 and CXCL11 together with all three CXCR3 variants,

CXCL4, CXCL10 and CXCL11 together with CXCR3alt or CXCR3A,

CXCL4, CXCL10 and CXCL11 together with any two CXCR3 variants,

CXCL4, CXCL10 and CXCL11 together with all three CXCR3 variants,

CXCL4, CXCL9, CXCL10 and CXCL11 together with CXCR3alt or CXCR3A,

-CXCL4, CXCL9, CXCL10 and CXCL11 together with two CXCR3 variants, or

CXCL4, CXCL9, CXCL10 and CXCL11 together with all three CXCR3 variants.

In one embodiment according to these aspects of the invention, the expression levels of CXCR3alt and CXCL11 alone are sufficient to determine the outcome of a cancer patient.

In certain embodiments of the method of predicting the outcome of an anti-tumor treatment of a patient bearing a solid tumor, the classifying step includes inputting biomarker expression levels into a model fitting statistical method to generate a value reflecting the probability that the patient will be an anti-tumor treatment responder. Statistical machine learning techniques, particularly supervised machine learning techniques that may be particularly useful for the method, include, but are not limited to, random forest methods or neural networks. Using logistic regression based on previously analyzed results of cancer patients is a particularly useful method of capturing the relationship between expression levels and clinical responses to provide an algorithm that generates probabilities of drug responses based on the input of biomarker data from tumor samples. Classification or regression algorithms used in this aspect of the invention may be applied to improve the predictive ability of biomarkers at the population level. It will be appreciated that these methods may take into account variables other than chemokine biomarker expression levels, such as variables selected from, but not limited to, age, sex, co-morbidities or clinical parameters.

CXCL11 and CXCR3 expression levels in the swedish cohorts shown in the examples were incorporated into predictive logistic regression models to test the benefit of using one, two or more biomarker expression level values to predict BC patient outcome. The performance of each model in predicting MVAC therapy outcome was assessed by the area under the ROC curve, and both AIK and Brier model fitting scores increased with the inclusion of more biomarkers. This analysis identifies the equation by which CXCL11 along with CXCR3A, specifically CXCR3alt, pre-treatment tumor expression levels most accurately separate myometrial invasive bladder cancer patients into MVEC neoadjuvant responders and non-responders.

One possible embodiment of the method of predicting the outcome of an anti-tumor treatment of a patient carrying a solid tumor is to classify the cancer patient as an anti-tumor treatment responder if the level of one or more biomarkers is above a certain threshold. Conversely, if the biomarker expression level is below a particular threshold, cancer patients may be classified as anti-tumor treatment non-responders. Useful thresholds and confidence intervals for these intercept points are provided in embodiments. This information can help the clinician to stratify patients into those who should receive and are new to assist in treatment and those who will benefit from surgery or other treatment options.

One particularly useful embodiment of a method of predicting the outcome of an anti-tumor treatment of a patient with a solid tumor includes measuring the level of CXCL11 protein in a sample. Expression of the markers may be determined at the protein level via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA, or multiplex analysis. Marker expression can also be assessed by measuring expression at the mRNA level by means of quantitative real-time PCR (qPCR), microarray or sequencing assays.

Methods using antibodies or antibody fragments that specifically bind to CXCL9, CXCL10 or CXCL11, such as ELISA or biplex, are particularly useful for determining the expression level of CXCR3 ligand protein in a tumor. Optionally, CXCR3 isoform expression levels are also measured at the protein level using molecular probes that distinguish CXCR3 splice variants. It will be appreciated that accurate measurement of proteins according to certain embodiments of the invention is most effective in samples that are preserved in a manner that the proteins are not degraded. Preferably the sample or a portion of the sample should be frozen in liquid nitrogen immediately after excision from the patient and preferably stored at-80 degrees celsius prior to treatment in the presence of a protein inhibitor.

In an alternative embodiment of the method according to the invention, the cancer patient is classified as a treatment responder or a non-responder by comparing the expression level of the listed biomarkers determined in the tumor sample with the expression level in a reference sample previously analyzed, wherein the matched clinical outcome is known. For example, if biomarker expression is equal to or greater than the expression level in a positive reference sample comprising tumor tissue from a NAC responder, the patient may be classified as likely to respond to NAC. Conversely, if biomarker expression in a patient sample is equal to or less than a negative reference sample derived from tumor tissue of a NAC non-responder, the patient may be classified as unlikely to respond to NAC. For the inversely related markers CXCR3B and CXCL4, this relationship is reversed.

In certain embodiments of the methods provided herein, the expression level of a biomarker, e.g., CXCL11, measured at the protein level is subjected to a statistical normalization process, e.g., expressed as a concentration of protein per milligram of sample. This may be particularly advantageous when analyzing a plurality of samples in large quantities, in order to normalize the variance between samples to achieve a normal distribution and to make biomarker expression levels more suitable for further statistical operations. Biomarker expression values determined by methods such as ELISA or mass spectrometry can be transformed to stabilize the distribution to compensate for the repeated sampling procedure. Normalization may include inputting the expression level into a transformation or scaling function selected from, but not limited to, a double-exponential, logarithmic or logarithmic transformation function, or specifically an arsinih normalization function.

In the data provided in the examples, if the expression level of CXCL11 in a tumor sample taken prior to treatment is higher than 13.98, specifically higher than 22.4pg/10mg sample tissue, then bladder cancer patients can be accurately classified as neoadjuvant therapy responders. The useful threshold is generated by the current model group and may change further as the predictive model is improved by adding more patient data in future use or study.

In a particularly advantageous embodiment of the invention, the expression level of CXCL11, CXCR3A, CXCR alt and/or CXCR3B is measured at the mRNA level. These measurements can be performed with nucleic acid probes, in particular with quantitative PCR methods such as real-time PCR, sequencing reactions or nucleic acid arrays. It will be appreciated that accurate measurement of CXCR3 splice variant expression according to certain embodiments of the invention is most effective in samples stored in a manner that does not degrade mRNA. For example, the sample or a portion of the sample as in the examples should be frozen in liquid nitrogen and stored at-80 degrees celsius. The treatment of the sample should be performed on ice, optionally in the presence of an RNase inhibitor.

One method that is particularly useful for measuring CXCR3 splice variant expression levels is a nucleic acid amplification method using the polymerase chain reaction of RNA extracted from a patient tumor sample. Specific nucleic acid probes, such as the primers of sequences SEQ ID NOs 016 to 021 presented in the examples, can be designed using primers targeting different splicing regions, using standard Taqman ABI assay conditions to distinguish between the three CXCR3 variants.

In the data provided in the examples, the combined information of qPCR measurements derived from CXCR3 splice variants CXCR3A, CXCR alt and CXCR3B both improved the prediction of BC clinical outcome compared to CXCL11 expression levels alone. Expression levels of CXCR3 splice variants can optionally be determined using other techniques designed to quantify nucleic acids, including but not limited to sequencing, microarrays, or gene chips, such as cDNA arrays.

In several embodiments of the invention, it is particularly advantageous to compare or normalize the expression of the biomarker to the expression of one or several housekeeping genes. Two such genes that are particularly useful are the genes IP08 and CDKN1B, but one skilled in the art will recognize that other stably expressed genes may be replaced by genes selected from, but not limited to GAPDH, ACTB, B2M, PPIA, HPRTI, PGKI, TBP or TFRC.

In the data presented in the examples, statistical analysis of a series of housekeeping genes identified IP08 and CDKN1B as the most stably expressed genes in bladder cancer tumor samples. Average expression of IP08 and CDKN1B was used to normalize biomarker expression values in samples of different sizes, mRNA quality, or amplification levels.

In an alternative embodiment of the cancer patient classification step provided by the present invention, if

2 of CXCR3A expression level exceeding the expression level of HKG ^(-12.3) Fold, in particular 2 exceeding the expression level of HKG ^(-11.97) Multiple, and/or

CXCR3alt expression level exceeds HKG expression level 2 ^(-13.8) Fold, in particular 2 exceeding the expression level of HKG ^(-11.27) Multiple, and/or

2 of CXCR3B expression level exceeding HKG expression level ^(-11.9) Fold, in particular 2 exceeding the expression level of HKG ^(-8.43) The number of times of the number of times,

the patient is assigned as an anti-tumor therapy responder.

For the CXCR3 variant biomarker thresholds provided herein, the relationship between the biomarker and HKG is the same when the thresholds are used independently or separately. In other words, using the CXCR3A embodiment described above, if the expression level of CXCR3A is greater than 2 ^(-11.97) This is about 0.00025 times lower than the expression level of HKG, and a threshold for positive results of neoadjuvant therapy can be assigned to the sample. Determining the expression level of more than one provided biomarker will increase the accuracy of the patient outcome prediction provided in the classifying step. If the markers are combined in a multivariate predictive model, in some cases, an inverse correlation between biomarker levels and results can be used to classify the sample. In aspects of the invention that utilize multivariate classification techniques (e.g., random forests), multiple "thresholds" are applied sequentially and can play a role in positive and negative correlations with HKG.

In certain embodiments of the invention, the expression level of the biomarker is compared to a baseline or reference sample in addition to or instead of comparison to HKG. An example of a negative control or negative reference is a sample of healthy tissue from an affected organ, wherein T cell infiltration is low or absent. Examples of positive control or positive reference samples may be examples of previously analyzed samples, wherein high levels or T cell infiltration are present in the tumor or tissue sample. Those skilled in the art will appreciate that in addition to analytical controls as in the examples above, patient samples can be compared to a series of predetermined calibration samples or standards to provide appropriate technical controls and biological controls.

Those skilled in the art will appreciate that the fold change in CXCR3 variant expression values provided above is an example of a differential cycle threshold compared to HKG, i.e., the number of qPCR cycles required to generate a fluorescent signal from the particular nucleic acid probe used, above a user-defined threshold. Thus, these values reflect the proven PCR conditions and cycle thresholds used to generate the main signs of the predictive model, and the exact values are expected to vary in practice.

In certain embodiments of the methods according to the present invention, the cancer tissue sample in which the biomarker levels are determined comprises, or consists essentially of, tumor cells derived from a solid tumor, but may also comprise heterologous cells derived from the immune system or tissue of origin. The method of predicting a clinical outcome of a patient is particularly useful for analyzing tumor cells derived from squamous cell carcinoma such as melanoma or adenocarcinoma, in particular tumor cells derived from tissues selected from, but not limited to, tumors that are treated with a combination of neoadjuvant therapy and surgery selected from breast, lung, kidney, prostate, ovarian, colorectal, gastric, esophageal or bladder cancer. This method is considered particularly effective when the tumor cells themselves are not characterized by significant expression of CXCR 3. In this way, the signal from the CXCR3 measurement may necessarily reflect the phenotype of T cells present in the tumor. In a particular embodiment of the methods provided herein, the cancer patient is a bladder cancer patient, particularly a patient who has been diagnosed with urothelium or squamous cell-derived myometrial invasive bladder cancer.

In the data presented in the examples, the equation capturing the relationship between the expression level of the biomarker CXCL11 and the expression level of one or more CXCR3 splice variants completely accurately classifies myometrial invasive bladder cancer patients as responders or non-responders to subsequent treatment with MVEC neoadjuvant chemotherapy. The data presented in the examples demonstrate that the same immune cells express CXCR3 and CXCL11 in single cell sequencing assays comparing biopsies of bladder cancer and melanoma patients. Furthermore, studies have shown that tumor-infiltrated stem cell memory T cells promote survival in kidney, prostate, bladder, lung and melanoma, and the data provided herein show that these tumor-infiltrated stem cell memory T cells are responsive to CXCR3 ligands (Jansen c.s. Et al Nature (2019) 576:465;Brummelman J. Et al journal of experimental medicine (2018) 215 (10): 2520; siddiqui I. (Immunity) 50:195). Thus, the methods of predicting clinical outcome of cancer provided herein may be widely applicable to squamous cell carcinoma such as melanoma, or adenocarcinoma such as bladder cancer, in which method chemokine biomarkers are used to estimate the efficacy of T cell immunity.

In a specific embodiment, the anti-tumor therapy in question is a neoadjuvant anti-tumor drug, in particular selected from but not limited to cisplatin, methotrexate, vinblastine, doxorubicin, carboplatin, doxorubicin, gemcitabine, paclitaxel, fesegetane, pemetrexed, vinorelbine, oxaliplatin, vinflunine or docetaxel. In other words, the method is particularly useful for predicting the outcome of classical regimens of MVEC or MVAC neoadjuvant chemotherapy containing the drugs methotrexate, vinblastine, doxorubicin or epirubicin and cisplatin, but may be applicable to similar drugs.

In certain embodiments of the methods of predicting clinical outcome of a patient with a solid tumor provided herein, the patient sample is tumor tissue or lymph node tissue, specifically a tumor tissue sample taken during a biopsy performed for tumor pathogenicity typing, or a sample taken from a lymph node in the vicinity of a draining tumor.

In another embodiment of the invention, the classification step of the method can be used to stratify patients with bladder cancer into groups with high or low priority for cystectomy. The methods provided can be used to assign myotic bladder cancer patients classified as non-responders to anti-tumor chemotherapy treatment as detailed above to groups with high priority for receiving radical cystectomy surgery without the need for prior neoadjuvant treatment. Such classification may avoid disease progression or metastasis during ineffective treatment regimens. Additional cancer types include, but are not limited to, breast, rectal, esophageal and gastric cancers, wherein this aspect of the invention may usefully be employed to help clinically stratify patients into groups that may benefit from neoadjuvant therapy or surgical intervention as first line therapy.

The data in the examples show that bladder cancer patients classified as neoadjuvant treatment non-responders are less likely to experience tumor regression during chemotherapy treatment. Clinical outcome of bladder cancer may be improved if these patients instead receive timely surgical intervention.

Another aspect of the invention is a method of matching CXCR3 expression status of an immune cell transfer therapy product to CXCL4, CXCL9, CXCL10 and/or CXCL11 expression status of a patient tissue targeted for immune cell transfer. In other words, a method that matches the positive expression of CXCR3 variants in cells used for metastasis to the expression of their ligands in target tissue in order to ensure treatment may be successful. According to this aspect, the method can predict the outcome of an immune cell metastasis cancer treatment, in particular a T cell metastasis treatment of a patient carrying a solid tumor. First, a patient or product provides a sample of cell metastases, or autoimmune cells, and a sample of target tissue, such as a sample of a patient's tumor. In a first cell transfer measurement step, the expression level of the biomarkers CXCR3A, CXCR alt and/or CXCR3B in the cell transfer sample is determined, followed by a target tissue sample measurement step, the expression level of the biomarkers CXCL4, CXCL9, CXCL10 and/or CXCL11 is determined according to the measurement protocol as detailed in the previous aspect of the invention. Finally, in the classification step, if the biomarker expression levels in both samples are positive or high, the patient is assigned a value reflecting the likelihood or probability that the cell transfer therapy will have a positive clinical outcome. This value reflects the probability of cells migrating to the target tissue, as well as their local probability of proliferation and cytokine production after metastasis. The method may utilize an algorithm, a reference sample, or a threshold value to classify patient samples in biomarker status, as provided in the other methods of the invention provided above. This approach may be particularly desirable for personalized medicine strategies, where the chemokine sensitivity of T cell transfer products, e.g., T cells with recombinant tumor-specific TCRs, or tumor-specific T cell populations expanded from donor or patient's own tumor samples, is assessed for whether the T cell transfer products will respond effectively to chemokine ligands expressed by the patient's tumor prior to administration.

A further aspect of the invention is the use of a provided CXCR3 chemokine system biomarker in a method of monitoring tumor progression over time or monitoring the presence of tumor immunoinfiltration in a patient's tumor over time. The method involves the step of providing two or more consecutive (sequential) tumor samples at different sampling times. In the measuring step, the expression level of CXCL11 and one or both of CXCR3A and CXCR3alt are determined in consecutive tumor samples. Optionally, the expression level of CXCR3B, CXCL4, CXCL9 and/or CXCL10 can be measured. In the final classification step, if the expression level of CXCL11, CXCR3A, CXCR alt, CXCL9 or CXCL10 is increased and/or the expression level of CXCL4 or CXCR3B is decreased in a later sample compared to an earlier sample, the patient is classified as a treatment responder or a patient with increased tumor immunity. This embodiment of the invention is particularly useful when comparing a pre-treatment tumor sample to a post-treatment tumor sample, or providing information to a clinician as to whether a patient is responding to a particular treatment regimen over time.

The data presented in the examples demonstrate that increased expression levels of the biomarkers CXCL11, CXCR3A, CXCR alt, CXCL9 and CXCL10 correlate with improved survival following NAC treatment, while CXCR3B and CXCL4 expression is lower in these patients.

Another aspect of the invention provides a novel adjuvant anti-tumour pharmaceutical formulation for use in the treatment of a patient with a solid tumour, which patient has been classified as a drug responder according to the method detailed previously. Particularly useful antitumor drug formulations according to the present invention are formulations comprising or consisting of antitumor drugs, in particular antitumor drugs selected from but not limited to cisplatin, methotrexate, vinblastine, doxorubicin, paclitaxel, carboplatin, doxorubicin, gemcitabine, febuxostat, pemetrexed, vinorelbine, oxaliplatin, vinflunine or docetaxel.

In a related embodiment, the invention provides an additional T cell stimulating pharmaceutical formulation for use in the treatment of a patient classified as an anti-tumor therapy responder according to the above method. These include, but are not limited to, BCG, the related safety-enhanced recombinant strain VPM1002 (Kaufmann's front of immunology 2020, 11:316; grode et al, vaccine 2013, 31 (9): 1340), and cancer immunotherapy treatments, particularly checkpoint inhibitory antibodies, more particularly checkpoint inhibitor antibodies selected from the group consisting of ipilimumab, na Wu Liyou mab, palbociclizumab, pihuizumab, atilizumab, avermectin, devaluzumab or Sizepine Li Shan.

The data provided in the examples show that these biomarkers can be particularly helpful in predicting whether a patient responds well to anti-tumor immunomodulatory drugs that increase T cell killing of tumor cells, including checkpoint blockade or BCG treatment, because biomarker levels are directly related to the number, activation, or functional phenotype of T cells in a tumor. Studies in mouse models of melanoma and ovarian cancer have shown that the outcome of immune checkpoint blockade correlates with certain components of CXCR3 chemokine signaling in tumors (Chow n.t. et al, immunity (2019), 50 (6): 1498). The data in the MBIC provided show unexpected predictive efficacy of CXCL11 and CXCR3A or CXCR3alt in predicting T cell activation in neoadjuvant chemotherapy, indicating that this approach would be successful in similar treatment options relying on T cell immunity.

Another aspect of the invention provides a system for assigning a value reflecting the T cell activity status of a tissue to a tissue sample of a patient, the system comprising determining the expression level of the genes CXCL11, CXCR3A and/or CXCR3alt, and optionally CXCL4, CXCL9, CXCL10 and CXCR3B, in the tissue sample, and using the biomarker expression level to classify the T cell activity status of the patient sample.

The invention is further illustrated by the following:

a method of measuring the status of T cell activity in a tissue sample of a patient, wherein the method comprises the steps of:

a. providing a patient tissue sample;

b. in the measuring step, a biomarker is determined

CXCL11 and at least one of CXCR3A or CXCR3alt,

and optionally the biomarker expression level of CXCR3B, CXCL4, CXCL9 and/or CXCL10,

c. in an optional classification step, the tissue sample is assigned a value reflecting the number of activated T cells present in the patient tissue sample based on the biomarker expression level.

A method of measuring the state of T cell activity in cancerous tissue, wherein the method comprises the steps of:

a. providing a cancer tissue sample;

b. in the measuring step, a biomarker is determined

CXCL11 and at least one of CXCR3A or CXCR3alt,

and optionally the biomarker expression level of CXCR3B, CXCL4, CXCL9 and/or CXCL 10;

c. in an optional classification step, a value reflecting the number of activated T cells present in the cancer tissue sample is assigned to the cancer tissue sample based on the biomarker expression level.

A method of predicting the outcome of an anti-tumor treatment of a patient bearing a solid tumor, wherein the method comprises the steps of:

a. Providing a cancer tissue sample;

b. in the measuring step, a biomarker is determined

CXCL11 and at least one of CXCR3A or CXCR3alt,

c. in the classifying step, the patient is assigned a predicted therapeutic outcome based on the biomarker expression level.

A method of measuring the status of T cell activity in a tumor or predicting the outcome of anti-tumor drug treatment in a patient bearing a solid tumor, wherein the method comprises the steps of:

a. providing a cancer tissue sample;

b. in the measuring step, the biomarker is determined: biomarker expression levels of at least one of CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt and/or CXCR3B, specifically determining expression levels of CXCL11 and/or CXCR3 alt;

c. in an optional classification step, a value reflecting the number of activated T cells present in the cancer tissue sample is assigned to the cancer tissue sample or a predicted therapeutic outcome is assigned to the patient based on the biomarker expression level.

The method according to item Y, wherein in the step of assaying, the expression level of at least two biomarkers is determined, in particular wherein the expression level of more than three biomarkers selected from CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt and/or CXCR3B is determined.

Aa. the method according to any one of the V to Z items, wherein in the measuring step the expression levels of the biomarkers CXCL11 and CXCR3alt are determined.

BB. the method according to any one of the X to AA items, wherein the classifying step comprises inputting biomarker expression levels into an algorithm to provide a probability that the patient will be an anti-tumour therapy responder.

A method of predicting the outcome of an anti-tumor treatment of a patient bearing a solid tumor according to any one of the X to AA items, wherein the classifying step comprises:

if the biomarker expression level is above or below a threshold, classifying the cancer patient as an anti-tumor therapy responder, or

If the biomarker expression level is above or below a threshold, the cancer patient is classified as an anti-tumor treatment non-responder.

DD. the method according to any one of V to CC, wherein the expression level of CXCL11 is determined at the protein level, and wherein the expression level of CXCR3A, CXCR3alt and/or CXCR3B is determined at the mRNA level.

EE method according to DD item, wherein the expression level of CXCR3B is determined using a collection of nucleic acid primers comprising or consisting essentially of the sequences SEQ ID NO 016, SEQ ID NO 017 and SEQ ID NO 018, and/or

The expression level of CXCR3alt is determined using a set of nucleic acid primers comprising or consisting essentially of sequences SEQ ID NO 019, SEQ ID NO 020 and SEQ ID NO 021.

FF. a method of predicting the outcome of an anti-tumour treatment of a patient bearing a solid tumour according to any one of the X to EE items, wherein the classifying step comprises:

-classifying the cancer patient as an anti-tumor therapy responder if the expression level of the biomarker is higher than the expression level in the negative reference sample and/or equal to or higher than the expression level in the positive reference sample, or

-classifying the cancer patient as an anti-tumor therapy non-responder if the expression level of the biomarker is lower than the expression level in the positive reference sample and/or equal to or lower than the expression level in the negative reference sample.

The method according to any one of V to FF, wherein the expression level of CXCL11 is normalized, in particular wherein the expression level of CXCL11 is normalized to the mass of the sample.

HH. the method of predicting the outcome of an anti-tumour treatment of a patient carrying a solid tumour according to any one of the X to GG claims, wherein a cancer patient is classified as an anti-tumour treatment responder if the expression level of CXCL11 is greater than 13.98pg/10mg sample tissue, in particular greater than 22.4pg/10mg sample tissue.

Method of measuring the status of T cell activity or predicting the outcome of an anti-tumor treatment in a patient carrying a solid tumor according to any one of the V to HH claims, wherein the biomarker expression level is normalized to the expression level of at least one Housekeeping (HKG) gene, in particular HKG IP08 and/or CDKN 1B.

Jj. method according to item II, wherein in the classification step, if

-fold change in expression level of CXCR3A exceeds>) HKG expression level 2 ^(-12.3) Specifically > HKG expression level 2 ^(-11.97) Multiple, and/or

-fold change in expression level of CXCR3alt exceeds>HKG expression level 2 ^(-13.8) In particular 2 exceeding the expression level of HKG ^(-11.27) Multiple, and/or

-fold change in expression level of CXCR3B is exceeded>HKG expression level 2 ^(-11.9) In particular 2 exceeding the expression level of HKG ^(-8.43) The number of times of the number of times,

the cancer patient is assigned as an anti-tumor therapy responder.

KK. the method according to any one of W to JJ, wherein the cancer tissue sample comprises, or essentially consists of, tumor cells derived from squamous cell carcinoma or adenocarcinoma, more specifically from renal cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, rectal cancer, melanoma, esophageal cancer or bladder cancer.

LL. the method according to any one of W to KK, wherein the cancer patient is a bladder cancer patient, in particular a patient who has been diagnosed with myometrial invasive bladder cancer.

MM. the method according to any one of X to LL, wherein the anti-tumor treatment is selected from:

antitumor drug, in particular an antitumor drug selected from cisplatin, methotrexate, vinblastine, doxorubicin, carboplatin, doxorubicin, gemcitabine, paclitaxel, febuxostat, pemetrexed, vinorelbine, oxaliplatin, vinflunine or docetaxel,

cancer immunotherapy, in particular checkpoint inhibitory antibodies, more in particular checkpoint inhibitor antibodies selected from the group consisting of ipilimumab, nal Wu Liyou mab, palbociclizumab, pilidazumab, atilizumab, avilamunomab, devaluzumab or cimaprzumab Li Shan-resistant,

BCG, or VPM1002.

NN. the method according to any one of W to MM, wherein the sample is tumor tissue, peripheral blood, white blood cells or lymph node tissue, in particular wherein the sample is a tumor biopsy or draining lymph node tissue from the vicinity of a tumor.

OO. the method of predicting clinical outcome of a cancer patient according to any one of the X to NN claims, wherein the cancer patient has been classified as an anti-tumor therapy non-responder and wherein clinical benefit resulting from a therapy comprising malignant tissue ablation is assigned to the cancer patient as compared to anti-tumor drug therapy.

PP. a method of predicting the clinical outcome of a patient with cancer according to OO, wherein the cancer is myometrial invasive bladder cancer, and wherein the malignant tissue resection is radical cystectomy.

QQ. a method of predicting the outcome of a tissue-targeted immune cell metastasis therapy, in particular a T cell metastasis therapy for cancer, in a patient bearing a solid tumor, wherein the method comprises the steps of:

a. providing a cell transfer sample, in particular a recombinant or amplified TIL;

b. providing a target tissue sample from a patient, in particular a tumor sample from a patient carrying a solid tumor;

c. determining the expression level of at least one of the biomarkers CXCR3A, CXCR alt and/or CXCR3B in a cell transfer measurement step;

d. determining the expression level of at least one of the biomarkers CXCL4, CXCL9, CXCL10 and/or CXCL11 in a target tissue sample measurement step;

e. in the classification step, assigning tissue-targeted immune cell transfer therapy to the patient will have a likelihood of positive clinical outcome based on biomarker expression levels.

RR. a method of monitoring the response of a patient carrying a solid tumour to anti-tumour therapy, wherein the method comprises the steps of:

a. Providing a pre-treatment cancerous tissue sample obtained from a cancer patient prior to initiation of anti-tumor treatment, an

b. Providing a post-treatment cancer tissue sample obtained after initiation of an anti-tumor treatment;

c. in the measuring step, a biomarker is determined

CXCL11 and at least one of CXCR3A or CXCR3alt,

d. in the classifying step, the patient is classified as an anti-tumor therapy responder if the biomarker expression level in the post-therapy sample is higher than the biomarker expression level in the pre-therapy sample.

SS. a pharmaceutical compound for use in the treatment of a patient with a solid tumor, the pharmaceutical compound being selected from the group consisting of:

The vaccine of BCG,

wherein the pharmaceutical compound is administered to a patient who has been classified as an anti-tumor therapy responder by a method according to any one of V to NN.

TT. a system for measuring the activity status of T cells in a tissue sample, wherein the method comprises the steps of:

a. providing a tissue sample;

b. in the measuring step, the biomarker is determined:

biomarker expression levels of at least one of CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt, and/or CXCR 3B;

determining in particular the expression level of CXCL11 and/or CXCR3alt,

c. in the classifying step, the tissue sample is assigned a value reflecting the status of T cell activity present in the tissue sample based on the biomarker expression level.

A method of measuring the status of T cell activity in a tissue sample of a patient, in particular a cancer tissue sample, or predicting the outcome of an anti-tumor treatment of a patient carrying a solid tumor, wherein the method comprises the steps of:

a. providing a patient tissue sample, in particular a cancer tissue sample;

b. in the measuring step, a biomarker is determined

CXCL11

At least one of CXCR3A or CXCR3alt,

c. In an optional classification step, based on biomarker expression levels,

-assigning to the tissue sample a value reflecting the number of activated T cells present in the tissue sample of the patient or in the cancer tissue sample, or

-assigning a predicted treatment outcome to the patient.

VV A method of measuring the T cell activity status in a tumor or predicting the therapeutic outcome of an anti-tumor drug in a patient with a solid tumor, wherein the method comprises the steps of

The steps are as follows:

a. providing a cancer tissue sample;

b. in the measuring step, the biomarker is determined:

biomarker expression levels of at least one of CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt and/or CXCR3B,

in particular determining the expression level of CXCL11 and/or CXCR3 alt;

WW. the method according to the VV item, wherein in the measuring step the expression level of at least two biomarkers is determined, in particular wherein the expression level of more than three biomarkers selected from CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR alt and/or CXCR3B is determined.

XX. the method according to any one of UU to WW, wherein in the measuring step the expression levels of the biomarkers CXCL11 and CXCR3alt are determined.

YY. a method of predicting the outcome of an anti-tumour treatment of a patient bearing a solid tumour according to any one of UU to XX, wherein the classifying step comprises:

-classifying the cancer patient as an anti-tumor therapy responder if the biomarker expression level is above or below a threshold, or

-classifying the cancer patient as an anti-tumor therapy non-responder if the biomarker expression level is above or below a threshold.

ZZ. the method according to any one of UU to YY, wherein the expression level of CXCL11 is determined at the protein level, and wherein the expression level of CXCR3A, CXCR alt and/or CXCR3B is determined at the mRNA level.

Aaa a method of predicting the outcome of an anti-tumor treatment of a patient carrying a solid tumor according to any one of the UU to ZZ items, wherein a cancer patient is classified as an anti-tumor treatment responder if the expression level of CXCL11 is greater than 13.98pg/10mg sample tissue, specifically greater than 22.4pg/10mg sample tissue.

BBB. the method according to any one of UU to AAA, wherein the expression level of CXCR3A, CXCR alt and/or CXCR3B is normalized to the expression level of at least one housekeeping gene (HKG), in particular HKG IP08 and/or CDKN1B, and wherein in the classifying step, if

Fold change in expression level of CXCR3A over (>) (HKG) expression level2 of (2) ^(-12.3) In particular > 2 of HKG expression level ^(-11.97) Multiple, and/or

Fold change in expression level of CXCR3alt over 2 of HKG expression level ^(-13.8) In particular exceeding the level of HKG expression 2 ^(-11.27) Multiple, and/or

Fold change in expression level of CXCR3B exceeding 2 of HKG expression level ^(-11.9) In particular exceeding the level of HKG expression 2 ^(-8.43) More than two times, the cancer patients are allocated as anti-tumor treatment responders.

Ccc. the method according to any one of UU to BBB, wherein the cancer tissue sample comprises, or essentially consists of, tumor cells derived from squamous cell carcinoma or adenocarcinoma, more specifically from renal cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, rectal cancer, melanoma, esophageal cancer or bladder cancer.

Ddd method according to any one of the UU to CCC, wherein the cancer patient is a bladder cancer patient, in particular a patient who has been diagnosed with myometrial invasive bladder cancer.

The method according to any one of UU to DDD, wherein the anti-tumor treatment is selected from:

BCG, or VPM1002.

The method according to any one of UU to EEE, wherein the sample is tumor tissue, peripheral blood, white blood cells or lymph node tissue, in particular wherein the sample is tumor biopsy or draining lymph node tissue from the vicinity of a tumor.

Ggg. the method according to any one of UU to FFF, wherein the cancer patient has been classified as an anti-tumor therapy non-responder, and wherein

The higher likelihood of clinical benefit resulting from treatment involving resection of malignant tissue is assigned to cancer patients than to anti-tumor drug treatment,

In particular, wherein the cancer is myometrial invasive bladder cancer, and wherein the excision of malignant tissue is radical cystectomy.

Hhh a method of monitoring the response of a patient carrying a solid tumor to anti-tumor therapy, wherein the method comprises the steps of:

c. in the measuring step, a biomarker is determined

CXCL11

And

at least one of CXCR3A or CXCR3alt,

A pharmaceutical compound for use in treating a patient with a solid tumor, the pharmaceutical compound selected from the group consisting of:

the vaccine of BCG,

wherein the pharmaceutical compound is administered to a patient who has been classified as an anti-tumor therapy responder by a method according to any one of UU to FFF items.

Further embodiments and advantages will be apparent from the following examples and drawings which illustrate the invention further. These examples are intended to illustrate the invention and not to limit its scope.

Drawings

FIG. 1 shows CD8 on lymph node samples (c-e, LN) derived from healthy donor peripheral blood mononuclear cells (a-b, PBMC n=11), or from Myometrial Invasive Bladder Cancer (MIBC) patients undergoing radical cystectomy ⁺ Flow cytometry analysis of ex vivo chemokine receptor expression on T cell subsets. a, representative human PBMC samples: initial cell (T) _{Initial initiation} ：CCR7 ⁺ CD45RA ⁺ ) Memory stem cells (T) _SCM ：CCR7 ⁺ CD45RA ⁺ ；CD95 ⁺ ) Central memory cell (T) _CM ：CCR7 ⁺ CD45RA ^- ) Effector memory cells (T) _EM ：CCR7 ^- CD45RA ^- ) And terminally differentiated effector T cells (T _EMRA ：CCR7 ^- CD45RA ⁺ ) Is a flow cytometry of (a). b, quantification of Mean Fluorescence Intensity (MFI) of ex vivo expression of specified chemokine receptors in healthy human PBMCs (n=9), median and range for each population in a, respectively. c, CD8 in PBMC and LN derived lymphocytes from PBMC and LN samples from MIBC patients ⁺ Representative FACS plots and quantification of T cell subpopulations (n=5, ln is median of 1 to 3 lymph nodes per patient). And (5) double-tail pairing t-test. ^* p is less than or equal to 0.05.d, CD8 in PBMC and lymphocytes of one LN from one BC patient ⁺ Representative histograms of CXCR3 expression on T cell subsets. e, by LumQuantification of CXCL9/10/11 concentration in serum and bulk LN homogenates measured by inex, n=3 MIBC patients, mean and SEM; friedmann test with Dunn post test ^* P≤0.05；0.01； ^*** P≤0.001。

FIG. 2 a LN CD8 stimulated with matched tumor cells from patients with Myometrial Invasive Bladder Cancer (MIBC) undergoing radical cystectomy ⁺ Flow cytometry quantification of activation frequency of T cells. LN cells were stimulated with autologous bladder tumor lysate for 12 hours and activation was measured by increased CD137 expression. The door is set as shown in fig. 1 c. N=7ln, mean and SEM from 3 MIBC patients are shown. Paired t-test ^* P≤0.05； ^*** P is less than or equal to 0.01.b, LN and PBMC CD8 in response to stimulation as above ⁺ Heat map of background normalized CD137 expression of autologous bladder tumor lysate of T cells. Color indication ΔCD8 ⁺ CD137 ⁺ Above background activation.

FIG. 3 shows a, CD8 ⁺ Chemotaxis of T cells to CXCR3 ligand CXCL9/10/11 in vitro, flow cytometry was used and the Chemotactic Index (CI) was calculated relative to the absolute number of migrating cells in the lower chamber without chemokines. CI > 1 (dashed line) indicates chemotaxis of the ligand. The median value is shown. b, quantification of paired frequencies of cd8+cxcr3+ T cell expression in the upper (non-migrating) and lower (migrating) chambers of CXCL9 migration assays.

Figure 4 shows the expansion of CMV-specific cd8+ T cells from purified primary T cells +/-CXCL9/10/11 (n=6 healthy donors). Representative flow cytometry for CMV-induced activation at day 14 (upper panel) and day 21 (lower panel). b, CMV-specific CD8 according to b ⁺ CCR7 ⁺ CD45RA ⁺ Derived T _SCM Multiple enrichment of amplified frequency of +/-CXCL 9/10/11. c, quantification of the frequency of CMV-induced IFN- γ+ cells in CD8+CCR7+CD45RA+ derived T cells amplified in +/-CXCL11 for 14 days. Wilcoxon rank sum test (Wilcoxon rank-sum) ^* P is less than or equal to 0.05, d is the CMV _IE-1/pp65 Representative flow cytometry of CXCR3 expression on T cells derived from cd8+cmv-specific ccr7+cd45ra+ on day 21 overlapping peptide pool pulsed LCL. e, representative streamCD8+T stimulated by cytometry (left) or CXCL11 _SCM Quantification of cell division frequency in cells. Mean and SEM are shown. f, different CD8 in PBMC for mRNA CXCR3 variants (CXCR 3A, CXCR3B and CXCR3 alt) ⁺ Expression in T cell subsets, and model of specific interaction of CXCR3 variants (CXCR 3A/B/alt) with CXCR3 ligands (CXCL 9/10/11). The FAC classification is according to fig. 1 a. CXCR3 isoform expression measured by qPCR was normalized to housekeeping gene (HKG) HPRT (CXCR 3 isoform/HKG). Mean and SEM are shown, healthy donor n=5; one-way analysis of variance ^* P≤0.05； ^* p≤0.01； ^* p≤0.001。

Figure 5 shows the clinical stratification of BC patients and pre-treatment analysis of intratumoral T cell levels. a, workflow for studies on Bladder Cancer (BC) patients and clinical stratification of platinum-based neoadjuvant chemotherapy (NAC) prior to Radical Cystectomy (RC) in myotic bladder cancer (MIBC) patients. The clinical workflow integrates pre-treatment tumor sampling via TURBT (transurethral bladder tumor resection) to assign patients to MIBC versus non-MIBC (NMIBC). Clinically eligible MIBC patients were treated with NAC. After NAC, the pathological anatomical fall in tumor histology of MIBC patients by radical cystectomy samples was identified as responders (resp.) and non-responders with stable or progressive disease (non-resp.). b, estimation of the overall survival (%) of kaplan-mel (NMIBC, MIBC receiving NAC, MIBC without NAC). The lower graph indicates a patient at risk. c, CD3 mRNA expression was measured by qPCR in BC patients from untreated tumor samples. Average values of IPO8 and CDKN1B (HKG) were used for normalization: multiple of Δct CD3 per Δct HKG. The black line in the violin plot shows the median value. Mann-Whitney (Mann-Whitney) test.p.ltoreq.0.01. The kaplan-mel-overall survival estimate (%) shows the division of MIBC patients receiving NAC into CD 3-high (n=10) and CD3-

Low (n=10), giving individual Cox proportional hazards model fitting ^* P is less than or equal to 0.05; the likelihood ratio test of respondents (resp., n=9) and non-respondents (non-resp., n=11). The retrospective observation time of the KM curve was 6 years. In all figures NMIBC patients (n=17), NAC-free patients (n=9) and subjects are shownPatients receiving NAC (n=20) are classified as responders (n=9) and non-responders (n=11) patients.

Figure 6 shows intratumoral cytokine environmental differences between BC patient groups. Non-metered multidimensional scaling (NMDS) of 46 BC patient samples was shown according to their differences in the expression levels of 75 chemotactic cytokines measured by multiplex ELISA. The ellipses enclose individual group masses and the arrows indicate the average contribution of each cytokine cluster to the arrangement.

Fig. 7 shows that CXCL11 is a biomarker for NAC-responsive MIBC patients associated with intratumoral T cell levels. Intratumoral cytokines in primary biopsies of BC patient groups were measured using ELISA-based multiplex assays (table 1, n=46). a, receiver Operating Characteristics (ROC) curves show that biomarker predictions for positive NAC results (=response) are listed as sensitivity, and predictions for negative NAC results (=non-response) are listed as specificity. The ability of each marker to evaluate clinical outcome, including prediction of NAC response in MIBC and prediction of total survival (OS), was examined. Sensitivity and specificity are shown as frequencies, setting 100% to be correctly predicted. Shading indicates that CXCL11 as a biomarker exceeds the good efficacy of all other biomarkers tested. b, intratumoral CXCL11 protein levels of BC-subgroup (NMIBC, MIBC without NAC, MIBC with NAC, including responders and non-responders) measured by multiplex ELISA. The black line shows the median. Post hoc testing of Kruskal-Wallis and Dunn.

^*** P is less than or equal to 0.01.c, linear correlation analysis between intratumoral CXCL11 protein and CD3 mRNA of MIBC patients receiving NAC. Respondents are shown as fully shaded dots, and non-respondents are shown as open dots. Spearman (Spearman) r=0.65 and p=0.0021. d, the Caplan-Mel curve shows a division into CXCL11 ^{High height} (n=11) and CXCL11 ^{Low and low} (n=9) level group of MIBC patients. Improved survival results of MIBC patients receiving NAC with higher intratumoral CXCL11 protein levels as determined by Cox regression ^* P is less than or equal to 0.05; and (5) likelihood ratio test). Showing NMIBC

Patients (n=17), NAC-free patients (n=9) and NAC-receiving patients (n=20), the NAC-receiving patients being divided into respondent (n=9) and non-respondent (n=11) patients.

FIG. 8 shows CXCR3 expression and tissue infiltration CD8 in healthy bladder and MIBC ⁺ T cells are associated. t-SNE plots were generated from a combined single cell RNA sequencing sample dataset from 2 MIBC samples (4080 cells) and 3 healthy bladder samples (13440 cells). Through the source (a),

Normalized logarithmic expression levels of CD8 and CXCR3 (b) individual cells were stained, CD8 and CXCR3 co-localized in immune cell specific clusters (c).

FIG. 9 shows CXCR3 expression and CD8 ⁺ Tumor infiltrating T cells are associated and CXCL9/10/11 expression is associated with macrophages in melanoma. Single cell RNA sequencing data (3187 total cells) of melanoma samples obtained from 19 different patients. T-SNE plots show a, cell type classification, b, log normalized single cell expression levels indicate that CD8 and CXCR3 expression is limited to T cells, and c, CXCL9, CXCL10, and CXCL11 are abundantly expressed in the cd14+ clusters of monocytes/macrophages.

Fig. 10 shows mRNA levels of CXCR3 isoforms measured by qPCR from primary biopsies of swedish BC patient cohorts. a, analysis of linear correlation between intratumoral CXCR3 variants and CD3 mRNA in MIBC patients receiving NAC. B, ROC curve demonstrates CXCR3A, CXCR3B

And CXCR3alt ability to predict positive results (= response) for NAC listed as sensitive and negative results (= non-response) for NAC listed as specific. Sensitivity and specificity are shown as frequencies, setting 100% to be correctly predicted. The shaded area indicates the good efficacy of CXCR3alt (highest AUC).

c, intratumoral mRNA levels of CXCR3alt from a subset of patients were analyzed by qPCR. MIBC that receive NAC are subdivided according to pathological responses to NAC in responders and non-responders. Average values of IPO8 and CDKN1B (HKG) were used for normalization: multiple of Δct CD3 per Δct HKG. Post hoc testing of Kruskal-Wallis and Dunn. ^** p≤0.01； ^*** P is less than or equal to 0.001. The black line in the violin plot shows the median value. d, from the patientThe ratio of CXCR3alt-CD3 mRNA levels in a subset of tumors. According to c, mRNA expression of CXCR3alt was analyzed by qPCR. e, effect of intratumoral CXCR3alt mRNA levels on OS of MIBC patients receiving NAC, as best divided into CXCR3 alt-high (n=12) and CXCL3alt-

Low (n=8) individual Cox proportional hazards model fitting indicates ^* P is less than or equal to +/-0.05; and (5) likelihood ratio test). NMIBC patients (n=17), NAC-free patients (n=9) and NAC-receiving patients (n=20) are shown, respectively, and NAC-receiving patients are classified as responder (n=9) and non-responder (n=11) patients.

Figure 11 illustrates on the figure a cluster of 14 biomarkers of intratumoral cytokines measured in tumor protein lysates by ELISA-based multiplex detection and CXCR3 isoforms measured by qPCR, as well as a partial correlation network analysis of NAC response to highlight independent predictive variables in MIBC patients. The lower graph shows the response to NAC in MIBC with dual layering prediction of CXCL11 and CXCR3 alt. The synergy of the intratumoral receptor CXCR3alt and its ligand CXCL11 response to NAC was assessed by logistic regression analysis for prediction (auc=1, loocv accuracy=0.9). The shading indicates the predicted probability of answer/non-answer.

FIG. 12 shows intratumoral CXCL11 ^hi mRNA predicts improved overall survival of MIBC patients from TCGA cohorts. (a) The kaplan-mel estimate shows the Overall Survival (OS) of MIBC patients receiving chemotherapy (chemo)) and MIBC patients not treated with chemotherapy (non-chemotherapy). (b) Intratumoral mRNA for CXCR3, CXCL9/10/11 and CD3 in MIBC patients receiving chemotherapy (upper panel) and untreated MIBC patients (lower panel)

Linear correlation analysis between levels. (c) Heat map of spearman rank correlation coefficient (including CD 8). (d) The kaplan-mel curve shows OS for MIBC patients receiving chemotherapy, stratified according to mRNA expression levels. The data was split into CXCL9 high (n=21) and CXCL9 low (n=47), CXCL10 high (n=28) and CXCL10 low (n=40), CXCL11 high (n=13) and CXCL11 low (n=55), and CXCR3 high (n=48) and CXCR3 low (n=20), and CXCL4 low (n=56) and CXCL4 high (n=12) using the best split point. Table 1 shows protein analytes measured by multiplex detection ELISA using Luminex.

Table 2 shows the performance of individual biomarker thresholds for predicting clinical outcome of neoadjuvant therapy treatment of NMIBC patients. A decrease in AIC or Brier scores indicates a better model fit.

Table 3 shows the performance of a logistic regression model for predicting clinical outcome of neoadjuvant therapy treatment for NMIBC patients.

Table 4 shows the risk ratio and coefficients for MIBC patients from TCGA cohorts (n=68). The data were split into CXCL9 high (n=21) and CXCL9 low (n=47), CXCL10 high (n=28) and CXCL10 low (n=40), CXCL11 high (n=13) and CXCL11 low (n=55), and CXCR3 high (n=48) and CXCR3 low (n=20) patients, and CXCL4 low (n=56) and CXCL4 high (n=12) using the best split point.

Examples

Method

Patient(s)

During 2010 to 2017, informed consent received 46 patients with BC from different hospital signs in north hygiene areas of sweden. Sample and blood sample in sweden in merco @Sweden) is archived in a university hospital urological (NUS) biological library. The patient was at least 18 years old and the study of patient materials was approved by the regional ethics committee (EPN-mercy, original accession number 2013/463-31M, latest revision 2018/545-32). In addition, all patients have given oral and written consent to donate samples and fluids to the biological libraries and participate in continuous and ethically approved transformation studies. Normalized mRNA expression data for a second group of primary tumor samples were obtained from a cancer genomic map (TCGA, https:// portal. Gdc. Cancer. Gov). Clinical data (BLCA dataset) was used to identify 68 MIBC patients who received chemotherapy (chemotherapy) and 292 MIBC patients who did not receive any chemotherapy (no chemotherapy) within 150 days after sample acquisition.

Diagnosis and NAC treatment

Diagnosis of urethral BC is established based on tumor histology of samples received at transurethral bladder tumor resection (TURBT). In TURBT samples, MIBC disease is defined by the histological invasion of tumors into the detrusor muscle; cT2-T4 (29/46 patients). Next, clinical studies were conducted on the eligibility of MIBC patients to receive NAC based on good performance status, including Charleson's age co-morbid index (CACI) 6, age 77 years and no severe kidney injury (GFR 55-60) or any other related co-morbid condition. NAC treatment contains high doses of the drugs methotrexate, vinblastine, doxorubicin or epirubicin and cisplatin according to the following protocol, in most cases:

methotrexate 30mg/m on day 1 ²

Vinblastine 3mg/m on day 2 ² Doxorubicin 30mg/m ² Cisplatin 70mg/m ² (at most 140 mg)

Day 3 subcutaneous PEGylated febuxostat 6mg

Furthermore, lymph node disseminated and organ disseminated were excluded by radiological Computed Tomography (CT); cN0M0 ³⁵² . Eligible MIBC patients (20/29) received 2 to 4 cycles of NAC treatment prior to radical surgery (i.e., radical cystectomy: RC). NAC is used as a combination chemotherapy based on cisplatin, mainly cisplatin, methotrexate, vinblastine, doxorubicin (MVAC). The response to NAC was defined as the pathological anatomical decline of the tumor in the RC sample, based on which MIBC patients receiving NAC were defined as responders (9) or non-responders (11). These two groups have equivalent clinical manifestations, exemplified by similar ranges of CACI index, american society of anesthesiologist classification (ASA) score, and patient age. Furthermore, based on tumor histology, the response to NAC is subdivided into a Complete Response (CR) with p0N0M0, a Partial Response (PR) with pTa/T1/TisN0M0, a Stable Disease (SD) with ≡p2N0M0 and a Progressive Disease (PD) with any pT and N1/2 and/or M1. 5 patients exhibited CR,4 patients exhibited PR,4 patients exhibited SD, and 7 patients exhibited PD.7/29 MIBC patients did not meet NAC (i.e., MIBC patients without NAC; see above standard) and received direct RC (4/7), or no RC (3/7) was applied for palliative reasons. If tumor infiltration in TURBT samples is limited to The subepithelial or epithelial layer, a tumor is defined as non-myogenic invasive bladder cancer (NMIBC). NMIBC patients receive non-systemic treatment, such as local administration of BCG vaccine, and, if necessary, further TURBT treatment.

Patient sample processing

Tumor samples were collected during TURBT and lymph nodes were collected during RC. All samples were immediately frozen in liquid nitrogen and stored at-80 ℃. For treatment, the samples were kept on ice at all times, cut into two parts with a scalpel, and the mass was weighed. Next, the protein extraction buffer (T-PER ^TM The method comprises the steps of carrying out a first treatment on the surface of the Race feeil technologies (Thermo Fisher Scientific)) is applied to one part and RNA/DNA lysis buffer with 2M DTT (RLT; the company qiagen (qiagen)) is applied to another part. Tubes with ceramic beads were used in a tissue homogenizer system (all from Bertin instruments (Bertin Instruments)) to mechanically break up the sample. Concomitant DNA/RNA extraction was performed using AllPrep DNA/RNA minikit according to manufacturer's instructions (qiagen). 13 lymph nodes were kept intact after RC in order to isolate viable lymphocytes. After soaking in cold AIM-V medium (Siemens technologies), the samples were dissected with a scalpel and the cells were gently filtered through a 40. Mu.M cell strainer.

PBMC formulations

Blood samples were collected from healthy volunteers after informed consent was obtained. Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from heparinized whole blood of healthy donors by density gradient centrifugation with a Biocoll isolation solution (Biochrom GmbH, berlin) from Berlin. Isolated PBMCs were resuspended in PBS and maintained at 4 ℃. The study on PBMCs was approved by the institutional review board (institutional review board) university medical college ethics committee Xia Ruidi.

Flow cytometry analysis

For analysis of T cell phenotype, CD3 (BV 650, clone OKT 3), CD4 (PerCP-Cy 5.5, clone SK 3), CD8 (BV 570, clone RPA-T8), CCR7 (AF 647, clone G043H 7), CD45RA (PE/dazle 594, clone HI 100) and CD95 (PE/Cy 7/Brilliant Violet (BV) 421, clone DX 2) were used; BD Biosciences), CXCR3 (PE, clone G025H 7), PBMC and lymph node derived cells were stained at 4 ℃ for 30 min. To exclude DEAD cells, blue DEAD cell staining dye (Sesameiser technologies) was fixed by addition of LIVE/DEAD. Similarly, chemokine receptors (CXCR 1, CXCR3, CXCR4, CCR3, CCR5, CCR6, CCR 7) were stained on the cell surface using a human cell surface marker screening panel (BD biosciences). All antibodies were purchased from BioLegend, unless otherwise indicated. Cells were analyzed on LSR-II FORTESSA flow cytometer (BD biosciences) and FlowJo software version 10 (Tree Star). Lymphocytes are gated based on Forward Scatter (FSC) versus Side Scatter (SSC) patterns, followed by FSC area gating based on FSC height to exclude doublets. In the stimulation experiments, fixation/permeabilization was performed with the eBioscience FoxP 3/transcription factor staining buffer group (sameimer feishier technologies) according to the manufacturer's instructions. After washing, the fixed cells were stained with fluorochrome conjugated monoclonal antibodies of IFN-. Gamma. (eF, clone 4S. B3), TNF-. Alpha. (Alexa Fluor 700, clone MAb 11) and CD137 (PE/Cy 7, clone 4B 4-1) for 30 min at 4 ℃. The background response was assessed using a non-stimulated control and subtracted from antigen-reactive cytokine production.

Chemotaxis assay of CD8+ T cell subsets.

Will initially be 1x10 ⁶ Millions of human PBMCs were inoculated into the upper chamber of 24 transwell plates (Corning) with a 3 μm pore size in 200 μl RPMI,10% fcs,1% penicillin/streptomycin (fig. 4 a). 600. Mu.L of CXCL9, CXCL10 or CXCL11 in the medium is added to the lower chamber and the migration measurement is carried out for 3 hours. Chemotaxis Index (CI) describes the absolute number of migrating cells in the lower chamber, with CXCL9/10/11 normalized to the absolute number of migrating cells in the lower chamber without the chemokine. To calculate the absolute number of migrated subsets of cd8+ T cells, the fraction of each T cell subset was determined by flow cytometry of migrated cells, as characterized by the phenotypic subset (fig. 2 e). The frequency of the migrated cd8+ T cell subpopulations was assessed by flow cytometry.

T _SCM Amplification protocol

Using the gating strategy in fig. 1a, PBMCs were CD3 via FACS on BD FACS Aria II SORP (BD biosciences) ⁺ CCR7 ⁺ CD45RA ⁺ And (5) enriching the T cell population. The sorted T cells were allowed to stand overnight, activated by irradiated (30 Gy) and CD3 depleted (MicroBeads (microblads); methaemal-Twai Biotech (Miltenyi Biotech)), autologous PBMC were previously activated with CMV _pp65/IE1 The overlapping peptide pools were pulsed at a ratio of 10:1 (T cells: feeder cells). CMV (CMV) _pp65/IE1 The peptide pool consisted of 15-mer peptides with 11 amino acid overlaps (JPT peptide technologies company of Berlin, germany (JPT Peptide Technologies, berlin, germany)) and was reconstituted in DMSO. After stimulation, at 37℃and 5% CO ₂ Next, the cells were cultured in a humidified incubator in complete medium comprising recombinant human IL-7 and IL-15, each IL-7 and IL-15 being 10ng ml ^-1 (CellGenix). On day 7, cultured cells were re-stimulated with freshly isolated, peptide pool pulsed and irradiated CD3 depleted autologous PBMCs at a ratio of 10:1 (T cells: feeder cells). On days 14 and 21, the amplified T cells were tested for their antigen specificity by measuring their ability to recognize peptide-loaded target cells by CD137 up-regulation. The target cells were autologous lymphoblast B cell lines (LCL) transformed with B95-8 EBV strain and generated as described previously (Heslop, H. Et al Nature medicine (1996) 2:551-555). The restimulation of cytokine measurements was performed in the presence of 1 μg ml-1 brefeldin a (Sigma-Aldrich) for 12 hours, 11 hours. CCR7 ⁺ CD45RA ⁺ Antigen-specific T within a T cell population _SCM Is assessed via peptide pool stimulation of freshly isolated PBMCs. For measuring proliferation, T is marked with CFSE according to the manufacturer's instructions (Semer Feishul technologies Co., ltd.) _SCM And let T at its initial frequency _SCM Incorporation of T _{Initial initiation} Is a kind of medium. As indicated, the cells were stimulated with CMVIE-1/pp65 overlapping peptide pool pulsed antigen presenting cells. CD8 ⁺ Proliferation of T cells was assessed by the percentage of CFSE diluted cells after 96 hours of incubation in the presence or absence of CXCL11 and CMVIE-1/pp65 overlapping peptide pools.

Intratumoral cytokine measurement

Cytokines were evaluated in the extracted proteins. Application of Luminex technology using multiplex detection assay (Merck), inc200 systems, burle (BioRad)) (table 1). For each sample, the respective optical density values of analyte concentrations were evaluated via a calibration curve and subtracting a blank. The mean concentration and standard deviation of the samples were calculated.

Intratumoral analysis of mRNA CXCR3 variants

1. Mu.g of RNA from TURBT samples from 46 BC patients was used for cDNA synthesis according to the QuantiTect reverse transcription kit handbook (Kaiji). Quantitative real-time PCR (qRT-PCR) analysis was performed using TaqMan PCR with FAM-BHQ1 labeled probes. mRNA CXCR3 variants were measured via a TaqMan qRT-PCR assay. To measure the major variant CXCR3A mRNA (NCBI reference sequence: NM-001504.1), a TaqMan universal PCR master mix was used with the probe Hs 00171041-m 1 (ABI). To measure CXCR3 splice variants, two RT-qPCR plates specific for CXCR3B and CXCR3alt were designed (fig. 1). The probe of CXCR3B (5 '-TCACTATCCCAGAGCCCAG-3') (SEQ ID NO 016) was designed to be specific for the extension site of CXCR3B and primers were set to F:5 '-CCGTACTTCCTCAACTCCATCCGCT-3' (SEQ ID NO 017) and R:5 '-TCCTATAACTGTCCCCGCC-3' (SEQ ID NO 018) based on NCBI reference sequence NM-001142797.2. The probe of CXCR3alt (5 '-CCGGAACTTGACCCCTGTGGGAAG-3') (SEQ ID NO 019) was designed to hybridize to a CXCR3alt specific sequence generated from the ligating base due to post-transcriptional exon skipping (Ehlert, J.2004), and the primer of CXCR3alt was set to forward (F): 5 '-CACGACGAGCGCCTCAA 3' (SEQ ID No. 020) and reverse (R): 5 '-GTTGGGGCAGCCCAGG-3' (SEQ ID No. 021) based on NCBI reference sequence XM_ 005262257.3. For the design, snapgene software 4.3.11 (GSL biotechnology liability company (GSL Biotech LLC)) was used. The expression level of the target gene was measured in duplicate using the ABIPrism 7500 sequence detection system and associated software (all from ABI). For normalization, mRNA of hypoxanthine-guanine phosphoribosyl transferase (HPRT) was used as housekeeping gene (HKG), and the expression level of the target gene was calculated as a multiple of HKG. All expression levels were analyzed in duplicate using the ABIPrism 7500 sequence detection system and associated software (all from ABI). The geometric mean of IPO8 and CDN1B was used to normalize tissue expression of the target gene.

Statistical analysis

Using GraphPad Prism 8 (GraphPad software) and R ¹⁷ (version 3.5.2) to generate a graph and to perform statistical analysis of the data. To verify the normal Gaussian distribution, a Kelmogorov-Scorvo (Kolmogorov-Smirnov) test was used. RT-PCR data was log2-, and protein data was arginh transformed for display prior to statistical analysis. Non-detection (candidates) R packets are used to interpolate Ct values below the detection limit, i.e. above 40 (11%). In tumor samples, no undetected presence was present, but 15 of the 90 measured protein analytes did not show sufficient expression, i.e. the median absolute deviation of 46 tumor samples was greater than 0, and therefore was excluded from statistical analysis. Missing protein data (1.1%) was interpolated using the missfast R package in serum samples. The cox proportional hazards model was fitted using coxph and curp functions (survival packages) to determine the best segmentation points, showing the kaplan-mel curve for the bipartite data. As shown in fig. 6b, the limited mean survival time of individual patients is given by the area under the survival curve from the start point to the time point of each event. Hierarchical clustering of patient cytokine data was performed within patient subgroups (NMIBC, MIBC with NAC, MIBC without NAC) alone using the euclidean distance between the rank transform data and the full linking approach. The overall differences between patient subgroups in the chemokine environment were examined by analysis of variance and visualized by non-metric multidimensional scaling (vegan R package). Clustering of markers (using Ward method) was performed on the contracted partial rank correlation matrix of all 46 BC patients using the consinsus clusteriplus package with 1000-fold resampling scheme to identify co-regulated protein functional modules. Markers identified within the CD3 connected module are selected to obtain a sparse gaussian mixture graphical model in which responses to NAC are included as binary variables. For this purpose, according to 20, based on the potentially Gaussian Copula model The observed mixed-type data (14 continuous variables and responses) of the patient receiving NAC estimated a semi-parametric correlation matrix, which was subsequently selected using a graphical lasso with extended bayesian information criteria and presented in qgraph packages. A logistic regression model with CXCR3alt and CXCL11 as predictors was fitted using the offset reduction method of fith (implemented in the brglm R package). Published single cell RNA sequencing (scRNAseq) data was obtained from the NCBI gene expression integrated database (GEO). The scRNAseq data for two primary bladder cancer samples (MIBC) with GEO series accession numbers GSE130001 and three primary healthy bladder samples (BH) with GEO series accession numbers GSE129845 were downloaded as UMI count matrices generated by the cellrange procedure (10X Genomics). Count data were normalized to remove low quality cells and low abundance genes, followed by clustering of 17520 cells (BH: 337,3527,9576; MIBC:3486, 594) and t-SNE dimension reduction. The melanoma dataset of GEO series accession number GSE72056 (GSE 72056 _melanoma_single_cell_restored_v2.txt) was a log normalized single cell expression profile and cell type signature of 19 tumor samples. To visualize the expression of selected genes in different cell types, each tumor was downsampled to a maximum of 100 cells per cell type to a total of 3187 cells input, and the t-SNE algorithm was performed without further filtering (using rtstone package with default hyper parameters). Bladder oncogene expression data as prepared by cancer genomic profiling (TCGA, https:// portal. Gdc. Cancer. Gov) and pre-processed by a firehose pipeline to RSEM normalized gene expression values (https:// gdac. Broadenstitute. Org) were downloaded using the curatedTCGAData R package.

Example 1: predictive biomarkers of NAC response in BC

To reveal the functional relevance of the pre-treatment CXCR3 chemokine system in relation to human anti-tumor immunity, primary tumor biopsies, routinely taken prior to the start of platinum-based NAC, were collected from BC patients classified as NMIBC or MIBC. A full retrospective characterization of intratumoral cytokine and CXCR3 isoform expression associated with NAC-induced anti-tumor responses was then performed.

CXCR3 peripheral CD8 differentiated at early stage in MIBC patients ⁺ Highly expressed on T cells and expressed on CXCL9/10/11 ^{High height} Enrichment in lymph nodes.

To study CD8 ⁺ Heterogeneous chemokine receptor expression on T cells, CXCR3 expression versus discrete CD8 from human healthy donors ⁺ Comparison of CXCR1, CXCR4, CCR3, CCR5, CCR6 and CCR7 on T cell functional subpopulations (fig. 1 a). High CXCR3/CCR7 expression characterizes early differentiated CD8 ⁺ Stem cell memory (T) _SCM ) T cells and central memory (T _CM ) T cells, but in CD8 ⁺ Primary T cells (T) _N ) Advanced differentiated CD8 ⁺ Effector memory T cells (T) _EM ) And CD8 ⁺ Terminally differentiated effector memory T cells (T _EMRA ) Upper, CXCR3 expression was low (fig. 1 b). CXCR3 promotes CD8 ⁺ T cells home to secondary lymphoid organs and their presets in the lymph nodes. Thus, tumor adjacent lymph nodes are early differentiated tumor-reactive CD8 ⁺ CXCR3 ⁺ An important reservoir for T cells. T cell subpopulation distribution, T cell mediated anti-tumor reactivity and CXCR3 receptor/ligand expression within the tumor-adjacent microenvironment were analyzed in tumor-adjacent lymph nodes of 5 MIBC patients undergoing RC. Higher frequency of T was observed in lymph nodes compared to peripheral blood of MIBC patients _SCM Cells and T _CM Cell (FIG. 1 c), and in lymph node CD8 ⁺ Higher CXCR3 expression was detected on the T cell subpopulation (fig. 1 d). The CXCR3 ligand CXCL9/10/11 is higher in the lymphoid tissue of the patient compared to serum levels (FIG. 1 e), reflecting the chemokine gradient that promotes LN homing of the early differentiated CXCR3+ T cells. Indeed, when LN-derived cells were stimulated with autologous bladder tumor lysate for 12 hours, antigen-specific activation, measured by increased up-regulation of CD137, could be observed in the memory and effector cd8+ populations compared to the initial compartment, indicating an enrichment of tumor-specific T cells (fig. 2).

High expression of CXCR3 isoforms on early differentiated cd8+ T cells is associated with differential functional consequences mediated by the CXCR3 ligand family

In vitro migration assay (fig. 3 a) found that CXCL9, CXCL10 and CXCL11 induced CXCR3 high cd8+ TSCM and TCM chemotaxis(FIG. 3 b). At any CXCL9/10/11 of 100ng/ml, CD8+T is observed _SCM Maximum cell migration of (a). This means that all cd8+ T cell subsets, in particular cxcr3+ stem cell memory cells, are given an enhanced responsiveness to specific CXCR3 ligands. To determine CXCL11 versus CD8 ⁺ Action of T cell subpopulations, examination of CXCR 3-linked early differentiated CD8 important for mediating anti-tumor responses using antigen specific in vitro amplification cultures ⁺ T _SCM Functional consequences of the cells. CXCL11 rather than CXCL9/10 expands antigen-specific CD8 with CXCR3 down-regulation ⁺ T _SCM Enrichment of cells (FIGS. 4 a-d). Furthermore, CXCL11 accelerated in vitro proliferation of cd8+tscm in short term cell division assays (fig. 4 e). CXCL 11-mediated viral-specific CXCR3 due to in vitro application ^{High height} CD8 ⁺ Activation of T cells, tumor expression of CXCL11 can expand the cancer-directed response of early differentiated T cells. Alternatively spliced transcripts CXCR3alt have been reported to bind exclusively to CXCL11 (Ehlert, J.2004) and trigger downstream signaling upon CXCL 11-ligation (Berchiche, Y.A. and Sakmar T.P. (2016) 90:483-495). To determine the difference between CD8 ⁺ Whether CXCR3 isoforms expressed by T cell subsets are functionally significant in BC, variant expression in patient samples was measured using RT-qPCR plates for CXCR3A/B/alt variants. With CD8 ⁺ In contrast to the T cell subpopulation, T is peripheral _SCM Cells and T _CM The highest transcriptome activity was found in cells for all three CXCR3 variants. At T _SCM The highest expression of the alternatively spliced transcript CXCR3alt was detected in the cells. Furthermore, CXCR3alt is the CXCR3 transcript that was most differentially expressed within the T cell subpopulation (fig. 4 f). Thus, high expression of CXCR3alt recognizes a trend toward T that is functionally responsive to CXCL11 ligation in tumor microenvironment _SCM And (3) cells.

CXCL11 is associated with intratumoral T cell infiltration markers of NAC responsive patients

A group of 46 BC patients was used to profile the putative role of the CXCR3 chemokine system in chemotherapy-induced anti-tumor responses (fig. 5 a). Via primary endoscopic biopsy; i.e., TURBT (transurethral cystectomy), assign patients to NMIBC (17/46) or MIBC (29/46). In the follow-up, 20/29 MIBC patients were clinically appropriate, so compliance was met with platinum-based NAC prior to RC. Responders to NAC were identified due to the pathoanatomical degradation phase of tumor histology in RC. Responses to NAC are also surrogate markers for long-term survival. In this group, 9/29 MIBC patients were clinically unsuitable for NAC (NAC-free) due to age/co-morbidity/impaired renal function. In the patient group, good OS for NMIBC, moderate OS for MIBC receiving NAC and poor OS for MIBC patients without NAC were observed (fig. 5 b). To examine whether these prognostic differences are associated with the overall levels of tumor infiltrating T cells, pre-treatment CD3 mRNA expression levels were assessed as surrogate markers for T cell infiltration, reflecting the ability to protective anti-tumor immunity. Considerable levels of intratumoral T cell infiltration were detected in the three BC subset. In MIBC patients receiving NAC, significantly higher intratumoral T cell levels were found in responders compared to non-responders (fig. 5 c).

The formation of functional intratumoral T cell structures requires efficient chemotactic homing in a favorable inflammatory environment. However, it is not clear whether the CXCR3 ligand CXCL9/10/11 is part of the BC-specific cytokine profile or whether a different CXCR3 ligand is associated with an anti-tumor response. Multiple assays of pretreatment cytokines were performed on lysates from primary BC biopsies. In MIBC receiving NAC, tumors were assigned to high and low inflammatory states characterized by cytokines and chemokines in two different clusters (4 and 6) separating MIBC patients responding to NAC from the remaining BC subgroup (non-responding MIBC, non-NAC MIBC, NMIBC) in a multidimensional scaling model (fig. 6). Cluster 4 contains CXCR3 ligands CXCL9/10/11, which include IFN- γ, CCL2/3/4/19, CXCL12/13, and IL-16. Furthermore, NMIBC-associated features (cluster 1: IFN-. Beta., IL-28-. Beta., IFN-. Alpha. -2, IL-13, IL-29, IL-34, IL-19, IL-11, XCL1, IL-3) were isolated from the MIBC environment. In MIBC without NAC, the inflammatory profile is significantly reduced. Correlation analysis was used to isolate individual markers that were significantly associated with T cell infiltration and response to NAC, respectively. Cluster 4, which includes CXCR3 ligands, shows the strongest correlation with T cell levels in all BC sub-groups. In summary, 24 cytokines were significantly associated with T cell infiltration, and 9 cytokines were significantly associated with response to NAC, CXCR 3-receptor ligand CXCL11 exhibited the highest level of significance (p < 0.001) among all markers, indicating CXCL11 could be used as an effective marker to predict response to NAC.

Receiver Operating Characteristics (ROC) curves were generated to analyze the diagnostic ability of all significantly different cytokines to predict responses to NAC. CXCL11 is the most sensitive marker for predicting response to NAC (fig. 7 a). MIBC patients responding to NAC showed significantly higher CXCL11 intratumoral concentrations than non-responding MIBC patients and NMIBC patients (fig. 7 b). Cytokine pre-treated serum levels were analyzed without significant elevation of CXCL11 in patients responding to NAC. Furthermore, with CXCL11 in MIBC receiving NAC ^{Low and low} In contrast to tumors, there was a positive correlation between intratumoral CXCL11 levels and tumor-infiltrating T cell levels (fig. 7 c) and CXCL11 ^{High height} Is improved (fig. 7 d). Taken together, these data indicate that intratumoral CXCL11 is a key component of an immune response that mediates the beneficial effects of NAC, and that CXCL11 is a biomarker that accurately identifies NAC responder patients before they receive NAC treatment.

CXCL11 and CXCR3alt as dual stratification to predict response to NAC in MIBC

To dissect which cells in healthy bladder and bladder tumors expressed CXCR3, CXCR3 expression in human bladder was measured by obtaining publicly available single cell RNA sequencing data (see data availability) for 3 healthy bladder homogenates and 2 cancer cell enriched MIBC samples. This data indicates that CXCR3 expression is absent in healthy bladder cells as well as cancer cells, whereas CXCR3 is expressed in tissue-infiltrating T cells (fig. 8). In the human melanoma cancer dataset, CXCR3 expression was also limited to CD8 in melanoma in previously disclosed single cell RNA sequencing data ⁺ Tumor infiltrating T cells (fig. 9). In BC and melanoma, CXCR3 expression is limited to tumor-infiltrating immune cells that predominantly include T cells, although other cancer types may employ CXCR3 expression for tumor progression and metastasis (billotet c. (2013) 1836:287-295).

Alternative splicing transcripts have been reported to bind CXCR3alt exclusively to CXCL11(Ehlert, J.2004) and induce downstream signaling upon CXCL11 ligation (Berchiche, Y.A. and Sakmar T.P. (2016) 90:483-495). To determine the difference between CD8 ⁺ Whether CXCR3 isoforms expressed by T cell subsets are functionally significant in BC, variant expression in patient samples was measured using RT-qPCR plates for CXCR3A/B/alt variants. In the BC cohort, the correlation of intratumoral mRNA expression levels of CXCR3 isoforms (CXCR 3A/B/alt) with T cell levels was examined. mRNA expression levels of CXCR3A/alt, but not CXCR3B, were significantly correlated with T cell levels in MIBC receiving NAC (fig. 10 a). Like CXCL11 concentration, CXCR3 isoform expression can also predict patient response to NAC. CXCR3A and more strictly CXCR3alt mRNA expression accurately predicts response to NAC (fig. 10 b). Notably, non-responding MIBC patients exhibited significantly lower mRNA expression of CXCR3alt (fig. 10 c) compared to all other BC subgroups including responding MIBC patients, which was also confirmed after additional normalization to T cell levels (fig. 10 d). The clinical significance of CXCR3alt in response to NAC was confirmed by a strong association with OS in MIBC patients (fig. 10 e).

To carefully study the dependence between the CXCR3 chemokine system and the inflammatory tumor environment, pairwise correlation analysis was used to detect intratumoral co-regulation between CXCR3 isoforms, T cell levels and cytokine expression, which includes mRNA of CXCR3 isoforms, mRNA of CD3 and cytokine protein levels. Using robust clustering techniques, three CXCR3 isoforms and CD3 were grouped with three CXCR3 ligands (CXCL 9/10/11) in one specific cluster, IFN- γ, CCL3, CCL4, IL-16, CCL19, CXCL12, CXCL13 (fig. 11, top). To profile functional dependence and estimate the ability to target anti-tumor responses in this cluster, network analysis was used with clinical responses to NAC as binary variables. CD3 is recognized as a central node within the network, with IFN- γ modules (IFN- γ, CXCL10, CCL3, CCL 4), lymphoid modules (IL-16, CCL19, CXCL12, CXCL13, CXCL 9), a strong co-regulation between CXCR3A and CXCR3alt, and finally CXCR3B as a negative factor. Notably, a direct relationship was found between CXCL11 and response to NAC and independently between CXCR3 variant expression and response to NAC (fig. 11, table 2).

The threshold expression level of CXCL11 protein or CXCR3 isoform and 95% Confidence Interval (CI) measured by quantitative PCR to predict positive outcome of NA treatment are as follows:

CXCL11 exceeds 22.4pg/10mg tissue, CI: [13.98, 35.44]

CXCR3A exceeds HKG 2 ^(-11.97) Multiple times. CI: [2 ^(-12.3) ，2 ^(-11.0) ]

CXCR3alt exceeds HKG 2 ^(-11.27) Multiple times. CI: [2 ^(-13.8) ，2 ^(-10.4) ]

CXCR3B 2 over HKG ^(-8.43) Multiple times. CI: [2 ^(-11.9) ，2 ^(-4.9) ]

Using a logistic regression model, CXCR3alt-CXCL11 was applied as a dual marker stratification for MIBC patients receiving NAC, and responding and non-responding MIBC patients could be completely distinguished prior to NAC treatment (fig. 11 lower panel, table 3). In summary, assessment of CXCL11 protein levels and CXCR3alt mRNA expression in BC biopsies from MIBC patients enabled prediction of response to NAC.

For external validation, the patients with MIBC who received chemotherapy from TCGA (cancer genomic profile: 68) were treated with 292 patients without chemotherapy (chemo-) Is provided) and pre-treatment mRNA expression levels are analyzed in tumor samples of the independent MIBC patient cohort. In this group, chemotherapy treatment was associated with slightly improved OS (fig. 12 a). Before all treatments (treatment-)>) In the samples, there was a significant positive correlation between intratumoral CD3 and CXCL9/10/11mRNA expression levels, confirming that the expression of this chemokine system correlates with the level of T cell activity present in the tumor (FIGS. 12b and 12 c). In the MIBC group receiving chemotherapy, but not the MIBC patient group without chemotherapy, CXCL11 mRNA high tumor is a sign of OS improvement, as is CXCL9/10mRNA expression levels in the large group, confirming that the amount of T cell activity measured by the levels of CXCR3 ligand prior to treatment is comparable to the outcome of neoadjuvant therapy Effective biomarkers of interest (fig. 12d, table 4). This larger cohort additionally recognizes CXCL9 and CXCL10 as potent positive predictors. Although not recognized in the T cell response cluster of chemokine protein analysis, the final member CXCL4 of the CXCR3 cytokine family was also examined in the TCGA cohort and shown to be a negative predictor of the neoadjuvant response of BC patients. TCGA data includes intratumoral mRNA gene expression levels in contrast to protein levels, thus not allowing for discrimination of CXCR3alt isoforms. Bulk CXCR3 high mRNA tumors indicated improved OS compared to CXCR3 low mRNA tumors (fig. 12 d). The overall CXCR3 high mRNA tumor predicted improved OS less well than the discrimination provided by each variant in the Swedish group (table 4). The validation group demonstrated robust predictions of CXCR3 chemokine family in the second group of MIBC receiving NAC with different sample measurement protocols. The modest predictive ability of a large array of CXCR3 measurements suggests that CXCR3 variants are excellent biomarkers.

Example 2: BC patient classification using CXCL11 and CXCR3A or CXCR3alt expression

Based on the expression levels of the biomarkers CXCL11 and CXCR3 splice variants in pre-treatment tissue samples, statistical models were developed to predict clinical outcome of NIMBC patients on neoadjuvant therapy. Using two or more biomarker values (table 3), the predictive performance of the biomarkers is evaluated against individual marker thresholds (table 2) or predictive logistic regression models. The performance of each model in predicting the outcome of MVAC therapy was assessed by AUC of ROC curve, AIK and Brier model fitting scores.

The presence of CXCR3 in cancer tissue samples was measured by real-time quantitative PCR using Taqman probes, providing CT values. The CT value or threshold cycle is the number of cycles that the fluorescent signal of the reaction crosses a user-defined threshold, i.e. exceeds the background level. The CT value is inversely related to the initial amount of the target DNA. The Δct value is the expression difference (CT) between CTs of the target gene and the control gene, the stably expressed housekeeping gene. Here, control CT is the arithmetic mean of the two housekeeping genes IPO8 and CDKN1B identified by the gene algorithm. In the context of the present embodiment, the value of the biomarker is given by:

CXCR3alt＝ΔCT CXCR3alt＝CT(CXCR3alt)-((CT(IPO8)+CT(CDKN1B))/2)

CXCL11 is measured by a multiplex assay cytokine bead array system to give concentrations in pg/10mg tumor samples. This value was then normalized to stabilize the variance of multiple measured proteins of different intensities measured by the multiplex detection system using a quasi-logarithmic transformation as described below:

cxcl11=arsinih (CXCL 11 concentration in pg/10mg tumor sample) =ln (x+ (x2+1) ^0.5 )。

The probability of responding to NAC is calculated by:

p=1/(1+exp (-y)), where

y is a linear combination of two explanatory variables.

A linear combination can be calculated comprising estimates of two regression factors β1, β2 of the intercept a and the variable:

y＝ɑ+β1(CXCL11)+β2(CXCR3alt)

For the logistic regression model generated in example 1, an estimated value of the regression coefficient was obtained by maximum likelihood estimation using the offset reduction method of Firth:

ɑ＝6.045

β1＝1.303

β2＝0.904

levels of both markers were used, with non-normalized cxcl11=2.77 pg/10mg tumor sample, and cxcr3alt=Δct-16.426758, the probability that the patient favorably responded to NAC was:

p＝1/(1+exp(-(6.045+1.303(CXCL11)+0.904(CXCR3alt))))

p＝1/(1+exp(-(6.045+1.303(arsinh(2.77))+0.904((-16.426758)))))

p=0.0015, and thus the probability of being a NAC responder is 0.15%, and is therefore classified as a non-responder.

Similarly, formulas for predicting clinical use of logistic regression models can be developed based on the values of two biomarkers CXCL11 and CXCR 3A:

y=a+β1 (CXCL 11) +β2 (CXCR 3A), wherein

ɑ＝9.558

β1＝1.547

β2＝1.327

Example 3: BC patient classification using CXCL11 and CXCR3A, CXCR3alt and CXCR3B expression

By including in the logistic regression model the CXCR3 score that captures the inverse function of the CXCRB splice variant as a second variable, predictive performance can be improved. CXCR3 scores are linear combinations of Δct values describing three splice variants of CXCR3B negatively correlated with CXCR3A and CXCR3alt, respectively:

cxcr3 score = cxcr3alt+cxcr3a—cxcr3b

y=a+β1 (cxcl11) +β2 (CXCR 3 score), where

ɑ＝0.765

β1＝1.246

β2＝0.353

Using the regression coefficients described above, levels of four markers were used, with non-normalized cxcl11= 348.9pg/10mg tumor sample, cxcr3alt=Δct-8.752, cxcr3a=Δct-11.12, and cxcr3bΔct= -6.039, the probability that the patient favorably responded to NAC is:

p＝1/(1+exp(-(0.765+1.246(CXCL11)+0.353(CXCR3alt+CXCR3A-CXCR3B))))

p＝1/(1+exp(-(0.765+1.246(arsinh(348.9))+0.353((-8.752-11.12+6.039)))))

P=0.983, and thus advantageously has a probability of responding to NAC of 98.3% and is therefore classified as a NAC responder.

TABLE 1

TABLE 2

TABLE 3 Table 3

dCT-cycle threshold difference

Area under AUC-receiver operating characteristics

AIC-erythro information criterion, model fitting score

Brier score-model fitting score

TABLE 4 Table 4

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Claims

1. Modified CD3 for use in the treatment of cancer ⁺ T cells, in particular cd3+cd8+ memory T cells, expressing a CXCR3 transgene, wherein the transgene encodes a recombinant protein comprising a human CXCR3 variant selected from the group consisting of:

CXCR3A, CXCR alt+ and/or CXCR3B,

in particular, wherein the human CXCR3 variant, or one of the human CXCR3 variants, is CXCR3A and/or CXCR3alt.

2. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes only the CXCR3 variant CXCR3alt.

3. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes only the CXCR3 variant CXCR3A.

4. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes only the CXCR3 variants CXCR3alt and CXCR3A.

5. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes only the CXCR3 variants CXCR3alt and CXCR3B.

6. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes only the CXCR3 variants CXCR3A and CXCR3B.

7. The modified T cell for use according to claim 1, wherein the CXCR3 transgene encodes the CXCR3 variants CXCR3alt, CXCR3A and CXCR3B.

8. The modified T cell for use according to any one of claims 1 to 7, wherein the CXCR3 transgene

c. Encodes an amino acid sequence having at least (. Gtoreq.) 95% sequence identity to the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006, and wherein the encoded protein has the same biological activity as the amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005 and/or SEQ ID NO 006,

In particular, wherein said CXCR3 transgene encodes an amino acid sequence having greater than or equal to 96%, greaterthan or equal to 97%, greaterthan or equal to 98%, or even greater than or equal to 99% sequence identity to said amino acid sequence encoded by SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, and/or SEQ ID NO 006.

9. The modified T cell for use according to any one of claims 1 to 8, wherein the expression level of CXCR3A and/or CXCR3alt is higher than the expression level of CXCR3B, in particular wherein the ratio of the expression level of CXCR3A and/or CXCR3alt compared to CXCR3B is greater than 1.

10. The modified T cell for use according to any one of claims 1 to 9, which further expresses a Chimeric Antigen Receptor (CAR), the CAR comprising

a. A signal peptide, wherein the signal peptide,

b. a target-specific recognition domain, in particular, wherein the target is a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen,

d. a linker region connecting domain (b) and domain (c).

11. The modified T cell for use according to any one of claims 1 to 9, further expressing a transgenic T cell receptor (TgTCR) protein, wherein the TgTCR recognizes a target selected from a tumor-associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen.

12. The modified T cell for use according to any one of claims 10 or 11, wherein the target-specific recognition domain or the TgTCR recognizes a target selected from a transgenic T cell receptor specific for an antigen selected from LMPA, CMV _pp65 GD2, L1CAM, her2, IL13Ra2, EGFRvIII, CD133, mesothelin, CAIX, CEACAM5, TAG-72, CEA, COA-1, PSMA or c-MET.

13. The modified T cell for use of any one of claims 1 to 12, wherein the cell further expresses a CXCR3 ligand transgene comprising a CXCR3 ligand transgene promoter sequence and a recombinant human CXCR3 ligand, and wherein the transgene comprises:

In particular, wherein said CXCR3 transgene encodes an amino acid sequence having No. 96%,. Gtoreq.97%,. Gtoreq.98% or even No. 99% sequence identity to said amino acid sequence encoded by SEQ ID No. 007, SEQ ID No. 008, SEQ ID No. 009, SEQ ID No. 010, SEQ ID No. 011, SEQ ID No. 012, SEQ ID No. 013, SEQ ID NO 014 and/or SEQ ID No. 015.

14. The modified T cell for use according to any one of claims 1 to 13, wherein the cancer is a solid cancer, such as squamous cell carcinoma or adenocarcinoma, more particularly a cancer selected from breast cancer, colorectal cancer, neuroblastoma, sarcoma, bladder cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, renal cancer, gastrointestinal cancer or prostate cancer.

15. An isolated immune cell preparation, in particular a T cell preparation,

wherein the isolated immune cell preparation comprises at least (> 50%, specifically 70%, more specifically > 80%, even more specifically > 90% of immune cells, specifically T cells, expressing one or more human CXCR3 variants selected from CXCR3A, CXCR alt+ and/or CXCR3B,

wherein the human CXCR3 variant, or one of the human CXCR3 variants, is CXCR3A and/or CXCR3alt.

16. The isolated cell preparation according to claim 15, wherein the cells are derived from a cancer patient sample, in particular a cancer patient sample selected from peripheral blood, tumor tissue and/or tumor draining lymph node tissue.

17. The isolated cell preparation according to any one of claims 15 or 16, comprising at least (No. 50%), in particular No. 70%, more in particular No. 80% of any one of the modified immune cells according to any one of claims 1 to 14.

18. The isolated cell preparation of any one of claims 15 or 16, wherein the cell does not express any transgene.

19. The isolated cell preparation of any one of claims 15 to 18, wherein within the immune cell expressing CXCR3 variant, 50% or more, specifically 70% or more, more specifically 80% or more is:

NK or NKT cells, in particular CD56 ⁺ NK or NKT cells.

20. The isolated cell preparation according to any one of claims 15 to 19 for use in a.treating cancer, in particular a solid cancer, such as squamous cell carcinoma or adenocarcinoma, more particularly a cancer selected from breast cancer, colorectal cancer, neuroblastoma, sarcoma, bladder cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, renal cancer, gastrointestinal cancer or prostate cancer.