Patent Application
20240066061
The present invention relates to transgenic expression of the chemokine receptor CXCR3 and its ligands in human T cells, thereby enhancing their efficacy and survival to provide an improved cell transfer medicament in patients suffering from cancer, chronic viral infection or autoimmunity.
This application claims the benefit of priority of European patent applications EP21151233.0 and EP21151232.2, both filed 12 Jan. 2021; and EP21151438.5 and EP21151447.6, both filed 13 Jan. 2021, all of which are incorporated herein by reference.
Bladder cancer (BC) ranks among the 10 most frequent malignancies in Europe and the U.S.A. Tumour eradicating T cell responses can be induced via Bacillus Calmette-Guérin (BCG)-therapy in limited, non-muscle-invasive bladder cancer (NMIBC), or sometimes via PD-1/PD-L1-blockade in progressed, muscle-invasive bladder cancer (MIBC). In the current standard-of-care treatment, MIBC-patients receive neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC), which improves overall survival (OS) compared to RC alone (Vale C. Lancet (2003) 361: 1927-1934). Between 25-40% of MIBC patients respond to NAC, as defined by downstaging of the pathology grade of the tumour. Non-responder MIBC-patients do not exhibit tumour reduction, and remain in a muscle-invasive disease state until the RC is initiated. It is therefore vital to effectively identify cancer patients who are most likely to benefit from neoadjuvant drug treatment. Novel stratification systems for predicting the response to NAC are needed to improve the treatment protocols for the clinical benefit of the non-responder MIBC-patients. The benefits of neoadjuvant treatment have also been proven for locally advanced breast cancer (Eltahir A. et al. Am. J. Surg. 1998: 175(2):127-32), gastric cancer (Cunningham D. et al. N. Engl. J. Med. 2006: 355(1): 11-20) and oesophageal cancer (van Hagen P. N. Engl. J. Med. 2012: 366(22):2074-84).
A robust CD8+ T cell response against malignant cells can be induced by chemotherapy, and other types of cancer treatment (Galluzzi, et al. Cancer Cell (2015), 28: 690-714). The chemokine receptor CXCR3 (UniProt P49682), which can be engaged by the IFN-gamma-inducible ligands CXCL9 (MIG, UniProt Q07325), CXCL10 (IP-10, UniProt P02778), and CXCL11 (I-TAC, UniProt 014625), is heterogeneously expressed within the CD8+ T cell compartment. CXCR3 mediates the homing and positioning of CD8+ T cells in the secondary lymphoid compartment, but also to peripheral inflammation and malignant tissues. The activity of the intra-tumoural CXCR3-chemokine system is required for directing CD8+ T cell responses, and the efficacy of anti-PD-1 inhibition in mice. The CXCR3-ligand axis can be activated via chemotherapeutic agents in pre-clinical models supporting anti-tumour efficacy.
In contrast to a single transcript of CXCR3 in the mouse genome, humans express three CXCR3 isoforms, the main isoform CXCR3A, and two splice-variants CXCR3B and CXCR3alt (Ehlert J. et al. J. Immunol. (2004) 172:6234-6240). The CXCR3-isoforms exhibit different affinities for the CXCR3-ligands (CXCL9,10,11) and selectively activate distinct signalling pathways upon binding of different ligands (Metzemaekers M. Front. Immunol. (2018) 8:1970).
The immune compartment in tumours of chemotherapy-treated patients has predominantly been studied post-treatment, and the identified immune markers are rarely tested for their functional anti-tumour potency. In the examples presented here, the inventors stratify the intra-tumoural activity of the CXCR3-chemokine system in a cohort of BC patients, in relation to their response to NAC. The inventors show that the chemokine CXCL11 and its specific receptors CXCR3alt and CXCR3A are powerful predictors of the response of MIBC patients to NAC, and dissect the functional relevance of CXCR3 ligand signals through each receptor in determining T cell potency in response to cancer or viral antigens.
A first aspect of the invention is a T cell, expressing a CXCR3 variant selected from CXCR3A, CXCR3B, and/or CXCR3alt from a transgene, particularly when the CXCR3 variant, or one of the variants is CXCR3A or CXCR3alt. In particular embodiments, the modified T cell is a CD3+ CD8+ T cell. In certain embodiments, the CXCR3 variant transgene comprises the reverse complement of the premRNA, or coding mRNA transcript for CXCR3A, CXCR3B or CXCR3alt, or a sequence encoding an amino acid sequence 95% similar that encoded by the above sequences, retaining the biological function of the CXCR3 variant protein. In some embodiments, the expression of CXCR3A, and/or CXCR3alt is higher than that of CXCR3B, particularly with an expression ratio over 1.
In optional embodiments, the modified T cell additionally expresses a recombinant chimeric antigen receptor (CAR), or transgenic T cell receptor (TgTCR), recognising a cancer, pathogen-derived, or tissue specific antigen. In further embodiments, the modified T cell also expresses one or more CXCR3 ligand transgenes, comprising the reverse complement of the premRNA, or coding transcript of CXCL9, CXCL10, and/or CXCL11.
A second aspect of the invention is an isolated preparation of immune cells, particularly of T cells, of which at least (≥) 50%, particularly 70%, more particularly ≥8.0% are positive for CXCR3A, CXCR3B and/or CXCR3alt expression. In some embodiments the isolated preparation of cells is derived from a cancer patient sample, or comprises cells expressing a CXCR3 variant according to the first aspect of the invention, or conversely, no transgenes.
In particular embodiments, the modified T cell, or the isolated populated of immune cells expressing CXCR3 variants has a chemotactic index over 1 when stimulated with CXCR3 ligands, or proliferates, has enhanced lytic potential, or makes more effector cytokines, compared to unmodified, or unfractionated control immune cells.
A third aspect of the modified T cell, or the isolated preparation of cells according to the above aspects of the invention, provides their use as a medicament, particularly to improve T cell immunity, suppress infection or inflammation, or more particularly to treat solid forms of cancer.
A fourth aspect provided by the invention is a method to isolate CXCR3+ cells from human leukocyte samples. A related aspect is a method to obtain a preparation of CXCR3+ cells according to the invention, which comprises providing a plurality of human immune cells, and inserting a transgene, or transgenes encoding a sequence selected from SEQ ID NO 001 to SEQ ID 006, and optionally, a further sequence selected from SEQ ID NO 007 to SEQ ID NO 015. A final embodiment of the invention is a method to expand or activate an isolated preparation of cells obtained according to the methods provided above, by culturing cells with CXCL9, CXCL10 and/or CXCL11.
For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting 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 dictate 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, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes 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: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.
The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes—and products thereof—of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products.
The term gene expression may also be applied to the transcription and processing of an RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product. The inventors show both methods of CXCL11, CXCL9 and CXCL10 measurement were useful for prediction of OS in chemotherapy-receiving MBIC in data provided in the examples. For the expression of CXCR3 ligand CXCL11 in a sample, 22.4 pg per 10 mg of 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 the level of mRNA expression, but may be measured at the level of surface protein expression with ligands which distinguish between the variants.
The term splice variant or isoform refers here to three polypeptides derived from different splicing arrangements of the CXCR3 gene. CXCR3A refers to the canonical receptor (Loetscher et al. 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. One alternative splicing of the CXCR3 genes is the CXCR3B mRNA (NCBI reference DNA SEQ ID NO 002), producing a polypeptide characterised by an additional N-terminal 51 amino acids (Lasagni et al. J. Exp. Med. 2003 197:1537-1549). The alternative, or CXCR3alt, splice variant mRNA product is truncated (NCBI reference DNA SEQ ID NO 003, Ehlert et al. 2004), producing a polypeptide lacking the 6th and 7th transmembrane helices, and the third intracellular loop, leading to a short cytoplasmic C-terminus. The relative abundance of these splice variants may be measured by means of variant-specific primers or molecular probes such as those used in the examples.
In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively. Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).
As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (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 hereinbelow.
A human T cell according to the invention encompasses both αβ and γδ cell receptors (TCR) expressing cells (expressing CD3, and particularly CD4, or CD8), and natural killer (NK) cells and NK T cells (expressing CD56). These cells can also be characterised by the absence of cell surface markers which characterise myeloid cells, B cells, innate lymphoid cells, endothelial, stromal, or epithelial cells, neurons, erythrocytes, or fibroblasts.
The term neoadjuvant treatment in the context of the present specification relates to pharmaceutical formulations comprising one or more antineoplastic drugs. In the case of bladder cancer, the antineoplastic drugs are most commonly chemotherapy with platinum drug-based treatments, but can also include Bacillus Calmette-Guerin, or cancer immunomodulatory therapy. The neoadjuvant treatment regime may additionally include radiation treatment of the tumour.
The standard of care regime of neoadjuvant chemotherapy is a formulation of the drugs methotrexate, vinblastine, doxorubicin or epirubucin, and cisplatin, but may comprise similar drugs, for example, paclitaxel, carboplatin, adriamycin, gemcitabine, filgramastim, pemetrexed, vinorelbine, oxaliplatin, vinflunine, or doxetaxel.
In the context of the present specification, the term cancer immunotherapy, biological or immunomodulatory therapy is meant to encompass types of cancer treatment that help the immune system to fight cancer. Non-limiting examples of cancer immunotherapy include immune checkpoint inhibitors and agonists, T cell transfer therapy, cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells.
In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitory antibody is meant to encompass an agent, particularly an antibody (or antibody-like molecule) capable of disrupting an inhibitory signalling cascade that limits immune cell activation, known in the art as an immune checkpoint mechanism. In certain embodiments, the checkpoint inhibitory agent or checkpoint inhibitory antibody is an antibody to CTLA-4, PD-1, PD-L1, B7H3, VISTA, TIGIT, TIM-3, CD158, or TGF-beta.
In certain embodiments, the immune checkpoint inhibitor agent is selected from the clinically available antibody drugs ipilimumab (Bristol-Myers Squibb; CAS No. 477202-00-9), nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), avelumab (Merck KGaA; CAS No. 1537032-82-8), durvalumab (Astra Zenaca, CAS No. 1428935-60-7), and cemiplimab (Sanofi Aventis; CAS No. 1801342-60-8).
In the context of the present specification, the term checkpoint agonist agent or checkpoint agonist antibody is meant to encompass an agent, particularly but not limited to, an antibody (or antibody-like molecule) capable of enhancing an immune cell activation signalling cascade. The term checkpoint agonist agent further 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 to CD122, CD137, ICOS, OX40, or CD40.
In certain embodiments, the immune checkpoint agonist agent is selected from the clinically available drugs aldesleukin (Novartis, Cas. No 110942-02-4), interferon alfa-2b (Merck, CAS No. 215647-85-1), imiquimod (Apotex, CAS No. 99011-02-6), PF-8600 (Pfizer), Poly ICLC (Oncovir, CAS No. 59789-29-6), Cabiralizumab (Apexigen, 1613144-80-1) or utomilumab, (CAS No. 1417318-27-4).
The term cycle threshold or CT in the context of the present specification relates to a quantitative nucleic acid measurement, for example a measurement made with a quantitative polymerase chain reactions (qPCR). This method involves repeated cycles of nucleic acid amplification using nucleic acid probes which hybridise the target biomarker, to generate a product emitting a fluorescent signal, which can be measured to determine the amount of starting genetic material. The cycle threshold may be an average value, or the average value of a number of replicate samples. Other quantitative measurements may substitute the cycle threshold, such as a crossing point, or an adjusted inflexion point.
As used herein, the terms 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 terms response or drug responder used here, particularly in regards to the antineoplastic treatment of BC, refers to tumour down-grading as measured by a pathology analysis. The term further encompasses positive clinical outcomes and the presence of immune infiltrate in the tumour, in conditions where a patient's response to treatment can also be linked both to overall survival, and the number of T cells present in the tumour.
The T cell activity state of a sample, is a measure of the potency of T cells in a tissue sample, reflecting their local numbers, activation phenotype, and chemokine milieu. The data presented in the examples demonstrates that the expression levels of the CXCR3 isoforms CXCR3A and CXCR3alt and their ligands, CXCL9, CXCL19 and CXCL11, are directly correlated with the T cell markers CD3, and CD8, and better OS. The presence of CXCR3B and CXCL4 however, correlate with poor OS. Phenotypic analysis shows that CD8+ CXCR3+ stem cell memory cells in healthy and patient lymphocyte samples respond to antigen and CXCR3 ligands cues by migration, proliferation, and production of cytokines. These correlations can be used to quantitatively assess the likelihood of a cancer patient responding favourably to an antineoplastic treatment which relies on T cell activation as one of its mechanisms of action, and is thus associated with overall survival following treatment. The T cell activity state therefore, is a combined measure of the status of T cells present in the sample with respect to the expression of CXCR3+ isoforms and its ligands, in other words the presence of CXCR3A+ and CXCR3alt+ antigen-experienced T cells, and a CXCL9, CXCL10, CXCL11 rich environment, together confer a high potential for local T proliferation and cytokine production. CXCR3 expression is observed on a variety of immune subsets which contribute to tumour infection and immunity, and autoimmunity, including natural killer cells, B cells, and macrophages. The skilled artisan will understand the presence of other immune cells may additionally contribute to the T cell activity state.
A CXCR3+ Cell or Isolated Population of CXCR3+ Cells for Medical Use
A first aspect of the invention is a T cell expressing a CXCR3 variant selected from CXCR3A, CXCR3B, and/or CXCR3alt from an artificially inserted transgene, particularly when the CXCR3 variant includes CXCR3A or CXCR3alt. This can be a cell which does, or does not, natively express CXCR3, particularly a CD4+ or CD8+ T cell, or an NK or NKT cell.
In particular embodiments, depending on the immune modulating effect desired in the end product, the modified immune cell is a CD3+ T cell, selected from, a CD4+ T helper cell or CD8+ T cell (expressing a CD3+, αβ or γδ TCR+, and/or a lineage specific transcription marker such as GATA3, Tbet, or Eomes), or a T regulatory (Treg) cell (expressing CD4, CD25, and the transcription marker forkhead box P3 (foxp3)), more particularly a CD3+ CD8+ T cell. The invention provides a potent T cell product which may deliver diverse immune modulating signals, by conferring enhanced CXCR3-dependent activation and proliferation on either, for example, a suppressive cell (for example a GATA3+ CD4+ T cell, or foxp3+Treg), or an inflammatory cell CD8+ CD45RO+ memory cell or a CD56+ NKT cell.
The data in the examples shows that the CXCR3 variants CXCR3A and CXCR3alt are directly correlated with increased numbers and/or a greater proportion of T cell markers in tumours, which are in turn correlated with better clinical outcomes of BC patients following neoadjuvant treatment providing broad antigenic stimulation of tumour-specific T cells. The differing relationship between each CXCR3 variant, CD3 T cells, and patient outcomes suggests that isoforms have specific downstream effects, which may be harnessed for medical in different settings use by transgenic manipulation of the CXCR3 expression profile. Stimulation of CXCR3high stem cell and central memory cells with CXCR3 ligands in particular is demonstrated to lead to improved proliferation, migration and cytokine secretion of tumour-specific and virus-specific CD8+ T cells in vitro. The inventors propose that the transgenic expression of CXCR3 variants, particularly CXCR3A and CXCR3alt which were associated strongly with the presence and potency of T cell responses, will confer improved proliferation, migration and cytokine secretion on a recipient immune cell, particularly a CD3+ T cell.
In certain embodiments, the modified cell expresses a CXCR3 variant transgene comprising: CXCR3alt, CXCR3B, or particularly CXCR3A only.
In certain embodiments, the modified cell expresses a transgene, or two transgenes comprising: CXCR3A and CXCR3B, CXCR3alt and CXCR3B, or particularly, CXCR3A and CXCR3alt together.
In certain embodiments, the modified cell expresses a transgene, or several transgenes encoding the CXCR3alt, CXCR3A and CXCR3B proteins.
In order to confer the desired functional phenotype, it is understood that these CXCR3 variants should be expressed as a protein present at the cell surface. The CXCR3 variant gene sequences provided here are provisional based on the present knowledge of these newly described isoforms, and specific amino acids, or nucleic acids, may change in the future as data from more human subjects reveals new alleles. The transgenes generally encompass different isoforms of the CXCR3 protein of UniProt P49682.
The following embodiments of the invention address the composition of the CXCR3 transgene, which may either be transiently, or stably incorporated into the cell. In some embodiments, the CXCR3 variant transgene, or transgenes, comprise the reverse complement of the premRNA transcript, containing both introns and exons of the CXCR3 variants CXCR3A, CXCR3B, or CXCR3alt, particularly a sequence selected from SEQ ID NO 001, SEQ ID NO 002 and/or SEQ ID NO 003. In alternative embodiments, the CXCR3 transgene, or transgenes comprise the reverse complement of the coding mRNA transcript, that has only the exons of the CXCR3 variant genes, particularly a sequence selected from SEQ ID NO 004, SEQ ID NO 005, and/or SEQ ID NO 006. In another embodiment, the CXCR3 transgene comprises a sequence≥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, where 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 ≥96%, ≥97, ≥98 or even ≥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 means by comparing a cell, or a population of cells bearing the transgene or transgenes with an unmodified, control cell or population. The first functional assay for CXCR3 provided, is the ability to migrate towards a stimuli of 100 ng/ml CXCL9, CXCL10, or CXCL11 as in
The data in the examples shows the functionality of CXCR3 expression by human T cells from healthy volunteers or cancer patients mediates migration with a chemotactic index over 2 towards CXCL9, CXCL10, or CXCL11 stimulation (
The genetic engineering method by which the transgene or transgenes according to the invention is inserted into the cell is not particularly limited, and may be selected from, but not limited to, the following list of technical approaches:
In certain embodiments, the modified immune cell according to the invention expresses more CXCR3A and/or CXCR3alt, compared to CXCR3B, particularly wherein the ratio of CXCR3A, CXCR3alt, or combined CXCR3A and CXCR3alt expression compared to CXCR3B expression is greater than 1. CXCR3 variants may be measured at the protein level or the nucleic acid level, as shown in
In further embodiments of the modified T cell expressing the CXCR3 variant transgene or transgenes according to the first aspect of the invention, 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:
In another embodiment, the additional transgene expressed by the modified T cell is a transgenic T cell receptor (TgTCR), wherein the TgTCR recognises an immunotherapy target selected from a tumour-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 the TgTCR from the above embodiments, may recognise, in other words, display specific binding towards, an epitope from an molecule selected from, but not limited to, LMP1 (Epstein-Barr virus), CMV (cytomegalovirus), GD2, L1CAM (neuroblastoma), Her2 (colon, sarcoma, glioblastoma, bladder), IL13Ra2, EGFRvIII (glioblastoma), CD133 (HCC, pancreatic and colorectal), mesothelin (pancreatic), CAIX (renal), CEACAM5 (gastrointestinal), TAG-72, CEA (colon), COA-1 (colorectal), PSMA (prostate), or c-MET (breast). These antigens are viral, or tissue antigens, or antigens upregulated in tumour cells, conferring tissue, or disease specific activation signals, which in concert with increased CXCR3-mediated migration towards inflammation characterised by CXCR3 ligands, target T cell activity to prescribed physiological sites (Li et al. Signal Trans. And Targeted Ther. 2019, 4:35).
In another embodiment of modified T cell according to the invention, the cell expresses a CXCR3 ligand transgene encoding a recombinant protein comprising a human CXCR3 ligand, in order to confer an autocrine activation signal to the transgenic CXCR3 receptor. In one embodiment, the CXCR3 ligand transgene comprises both a CXCR3 ligand transgene promotor sequence, and one of the following CXCR3 ligand sequence variants:
In certain embodiments, the CXCR3 ligand transgene promoter is a constitutive promotor, such as the CMV immediate early promoter. In other embodiments, the promoter is conditionally expressed, for example, activated by T cell receptor ligation to allow inducible expression of the CXCR3 ligand transgene downstream of native or transgenic antigen receptor ligation, in order to limit non-specific activation and harmful inflammatory side-effects. This might be achieved by inclusion of antigen response elements such as ARRE-1 or ARRE-2, or a CD28 response region. It is understood that for the CXCR3 ligand to confer a functional advantage on the modified T cell, the ligand must be secreted in order to signal through CXCR3.
SEQ NO ID 007 to SEQ NO ID 015 provide splicing variants of the CXCL9, CXCL10 and CXCL11 genes currently known in the art. The application of commercially available CXCL9, CXCL10 and CXCL11 at a concentration of 10 ng/ml to CXCR3 expressing CD8+ T cells from healthy donors or patients, is shown to enhance T cell proliferation and cytokine secretion in the examples, suggesting autocrine production could enhance the functionality of a CXCR3+ modified T cell. The biological function of a CXCL9, CXCL10 and CXCL11 transgene, could be assessed by whether it provokes the same increase in cytokine production compared to the provided CXCR3 ligand transgene sequences, using an assay, or CFSE dilution of antigen stimulated CXCR3+ cells as in
A second aspect of the invention provides an isolated preparation of immune cells, particularly a preparation of T cells, wherein the isolated preparation of immune cells comprises at least (≥) 50%, particularly 70%, more particularly ≥80%, even more particularly ≥90% immune cells, particularly T cells, which express a human CXCR3 variant selected from CXCR3A, CXCR3alt+, and/or CXCR3B, particularly when the human CXCR3 variant, or one of the human CXCR3 variants is CXCR3A and/or CXCR3alt. In some embodiments, the population of T cells expressing CXCR3 variants is a directly ex vivo sample, in others, the isolated population has undergone an enrichment, expansion, or transgene insertion procedure to increase the CXCR3+ population from a starting sample of immune cell precursors.
In certain embodiments, a majority of the preparation of immune cells express only CXCR3alt, only CXCR3B, or only CXCR3A.
In certain embodiments, a majority of the preparation of immune cells express CXCR3A and CXCR3B, or CXCR3alt and CXCR3B. In particular embodiments the majority of immune cells in the preparation of immune cells express CXCR3A and CXCR3alt together.
In certain embodiments, a majority of the preparation of immune cells express all three CXCR3alt, CXCR3A and CXCR3B proteins.
This aspect of the invention encompasses both native CXCR3 variant expression by the preparation of immune cells, or CXCR3 variant expression from a transgene. The CXCR3 expression may be confirmed by realtime qPCR, for example using the probes provided herein, wherein a signal >0.1 fold, particularly >0.2 fold the expression of the house-keeping genes is considered positive. In order to confer the desired functional phenotype, it is understood that these CXCR3 variants should be expressed as a protein present at the cell surface. The cells may be isolated from peripheral blood, or peripheral blood mononuclear cells, a tissue sample, and/or a tumour sample, and may comprise additional transgenic proteins. In certain embodiments of the invention with relevance for cancer immunotherapy, the isolated preparation of immune cells according to the above aspect of the invention, has been isolated from, or expanded from, a plurality of immune cells isolated from a cancer patient sample, such as peripheral blood, or tumour infiltrating lymphocytes (TIL), and/or derived from lymph nodes draining tumour tissue (Poch et al. Oncoimmunol. 2018, 7(9):e1476816); Dudley et al. J. Immunother. 2003, 26: 332; Sakellariou et al. 2018, 36:95).
The data presented in the examples demonstrates the ability of CXCR3+ T cells derived from blood to respond to CXCR3 ligand migration signals (
In another embodiment, the invention provides an isolated population of immune cells comprising 50%, particularly ≥70%, more particularly ≥80% CXCR3+ genetically modified immune cells according to any embodiment of the first aspect of the invention. In other words, primary cells which express CXCR3A, CXCR3B, and/or CXCR3alt from a transgene, and optionally an artificial antigen receptor, and/or a CXCR3-binding chemokine.
In certain embodiments, the isolated preparation of CXCR3+ cells according to this aspect of the invention is ≥50%, particularly ≥70%, more particularly ≥80% enriched in one of the following functionally distinct T cell subsets:
The data presented in the examples demonstrates that CXCR3 variants are highly expressed on CD8+CCR7+CD45RA+CD95+ stem-like memory T cells, and CD8+CCR7+CD45RA− central memory T cells in healthy blood and BC patient LN. These native CXCR3+ cells may be purified to provide an isolated preparation of CXCR3+ cells according to the invention. In embodiments where CD4 help, cytotoxic NKT cell activity, or suppressive Treg cytokines are desirable in the isolated preparation of cells, the alternative embodiments b. through c. may be used. Th1 and NKT cells are useful for fighting cancer, bacterial or viral infections, whereas Tregs may suppress inflammation caused by, for example autoimmune or graft-specific immune activation. The expression of subset-identifying surface molecules can be determined for example, by flow cytometry in comparison to an isotype control, or a cell that is known to lack the marker in question, or by realtime qPCR.
In another embodiment, the modified T cells, or the isolated preparation of CXCR3+ cells according to the first or second aspect of the invention respectively, are defined by their performance in at least one assay which confirms the functionality of the transgenic CXCR3 variant expression. The concentration of CXCL9, CXCL10, or CXCL11 used in any of the following assays may be in the range of 1 to 1000 ng/ml of CXCL11, particularly between 10 and 100 ng/ml. A titration of the concentrations 1, 10, 100 and 1000 ng/ml is of particular use, with an unstimulated control to identify a kinetic, or functional response to stimulation.
According to this embodiment, the cell, or the population of cells may have a chemotactic index>2 upon in vitro stimulation with CXCL9, CXCL10, and/or CXCL11, using an assay such as that provided on p.23, I.30 of the examples.
Alternatively, the cell or population of cells may have a higher frequency of proliferating cells upon stimulation with antigen and CXCL11, compared to non-stimulated cells, in other words the ratio of the measurement of proliferation of the isolated CXCR3+ population, compared to a pre-isolation, or un-isolated sample, must be >1. This may be measured using an in vitro cell division assay such as flow cytometry measurement of the percent of cells which dilute a labelling dye in an assay such as that provided in the methods on p.24 I.23 (alternative dyes, see Parish Immunol. Cell Biol. 1999, 77(6):499), or, for example, by thymidine incorporation. Alternatively, or in addition to proliferation, an assay to measure enrichment of a CXCR3+ antigen specific population may be used, such as the assay measuring CD137 upregulation after antigen culture provided on p.24, I.6. For the current invention, the fold enrichment of the CXCR3+ antigen specific population in response to antigen and CXCL11 stimulation must be >1, particularly over 1.2 times greater than an unfractionated control sample.
In further options provided by this embodiment, in comparison to an unmodified T cell lacking a CXCR3 transgene or transgenes, or an un-fractionated, or pre-isolated preparation of cells, the T cell, or the isolated CXCR3+ population of cells is characterised by having enhanced lytic potential. Lytic potential may be measured by % killing of a target cell population, such as tumour, or virus infected cells marked with a dye or radiolabel, or intracellular measurement of the lytic enzymes granzyme B and/or perforin in a flow cytometric assay (Amini et al. Front. Immunol. 2019, 10:1148; Wagner et al. Nat. Med. 2019, 25(2):242). The ratio of % target cells killed of the CXCR3+ population of cells, must be >1, particularly >1.5 times that of an unfractionated, or pre-isolation sample.
Lastly, the CXCR3+ cell, or population of cells may produce more effector cytokines, particularly effector cytokines selected from IFNγ, TNFα, IL-10, and IL-2, following stimulation with CXCL11, and optionally, antigen-pulsed antigen presenting cells, a TCR crosslinking agent and soluble antigen, or a calcium flux inducing agent. An exemplary protocol of 12 hours stimulation with antigen expressing cells and CXCL11, in the presence of Brefeldin A, followed by intracellular flow cytometry of IFNγ and TNFα, is provided on p.24, I.20. For the purposes of the invention, the % of cytokine+ cells in the expanded, or enriched CXCR3+ population of cells should by >1 times, particularly >1.1 times greater than that of an unenriched, or pre-enrichment control sample.
The data presented in the examples demonstrates that in cancer patients, inflamed lymph nodes rich in CXCR3 ligands are more highly infiltrated with CXCR3+ T cells, and tumours with more CXCR3 biomarker expression, particularly CXCR3A and CXCR3alt, show evidence of containing more T cells. Furthermore, in vitro assays demonstrate that CXCR3+ stem cell memory or central memory cells have a chemotactic index over 2 when stimulated to CXCL9, CXCL10, or CXCL11, and proliferate and produce more cytokines than other subsets in response to viral or tumour antigens when stimulated with CXCL11. These assays form the basis of the functional characterisation presented in this embodiment of the invention.
A third aspect of the invention provides a modified CXCR3+ T cell, or an isolated CXCR3+ preparation of cells, for use as a medicament. In some embodiments, this medicament is used to enhance an aspect of T cell immunity, encompassing both pro- and anti-inflammatory T cell functions provided by the different transgene-bearing sub-populations provided above.
In certain particular embodiments, the modified CXCR3+ T cell, or the isolated CXCR3+ preparation of cells is used to enhance CD8+ T cell immunity, particularly to improve immunity against a solid tumour selected from a squamous cell cancer or adenocarcinoma, more particularly a cancer selected from breast, colorectal, neuroblastoma, sarcoma, bladder, glioblastoma, hepatocellular, pancreatic, renal, gastrointestinal, or prostate cancer. The data in the examples suggests these cells are of particular use to treat a solid cancer, rather than a systemic form cancer derived from a lymphoid cell, as the CXCR3 transgene or enriched CXCR3 expression confers the ability to home to an inflamed tumour tissue expressing CXCL11, CXCL9, or CXCL10.
In an alternative embodiment of the CXCR3+ T cell, or the isolated CXCR3+ preparation of cells for use as a medicament, the cell, or cells are used to treat an infectious disease. As CXCR3 is shown to enhance inflammatory capability of CD8+ T cells, this treatment may be of particular use in treating disease caused by intracellular pathogens such as a chronic viral infection, where infected cells are vulnerable to killing by cytotoxic CD8 T cells. Relevant viral diseases include, but are not limited to EBV, CMV, human immunodeficiency virus, coronavirus, or hepatitis. Data presented in the examples shows that in vitro stimulation of CXCR3 variant expressing cells by CXCL11 is able to activate CMV and EBV specific CD8 T cells in a human sample (
A fourth aspect of the invention provides a method of obtaining an isolated CXCR3+ preparation of cells, comprising a first step of providing a human sample comprising immune cells, particularly a peripheral blood sample, lymph nodes, or a tumour tissue sample. The data in the examples demonstrates that tumour-draining lymph nodes are enriched for CXCR3+, tumour specific cells. The tumour tissue of neoadjuvant-responsive BC patients was also shown to be enriched in the CXCR3 variant biomarkers.
Likewise, any target tissue, or the lymph nodes draining a target tissue, such as an organ inflicted by harmful immune infiltration, or viral infection, may serve as an appropriate starting sample in which either the desired antigen-specificity of CXCR3 expression profile is present.
The next step is to select, or enrich from the sample the immune cells, particularly T cells, which express the CXCR3 variants CXCR3alt+, CXCR3B and/or CXCR3A+, and/or to remove the cells which do not express the CXCR3 variants CXCR3A, CXCR3B and/or CXCR3alt. This may be achieved, for example, by magnetic sorting with CXCR3alt+ specific antibody bearing a magnetic bead and subsequent retention in a magnetic field, or by flow cytometric sorting of fluorescently labelled cells. The expression of total CXCR3, or individual variant combinations are encompassed by this method.
In some embodiments, the method to obtain an isolated CXCR3+ preparation of cells includes an additional gene transfer step, where a transgene is inserted into the 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, and 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 transgene nucleic acid sequence may encode an amino acid sequence that has ≥95% sequence identity, particularly wherein the transgene encodes an amino acid sequence that has ≥96%, ≥97, ≥98 or even ≥99% sequence identity to the amino acid sequence encoded by a 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, 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 essentially the same biological activity as one of the sequences provided above, particularly in assays such as T cell migration, cytokine production, or proliferation as outlined above and in the examples.
It may also be desirable to add a transgene for a TgTCR or CAR at this step as specified in the first aspect of the invention to provide a modified T cell.
A final embodiment of the method for preparing an isolated preparation of CXCR3 variant expressing cells, provides an expansion step, wherein the cells are cultured with CXCL9, CXCL10, and/or CXCL11 at a concentration between 1 to 1000, or particularly with 10-100 ng/ml of CXCL11. This may optionally be in the presence of antigen, and/or a gamma-chain cytokine, particularly IL-2.
The data in the examples, particularly the CXCR3 ligand titration migration assay shows that 10-100 ng/ml of CXCL9, CXCL10, and/or CXCL11 can enhance the proliferation, migration, and cytokine production of T cell subsets which express CXCR3. The functional importance of the in vitro assays is confirmed in the biomarker analysis capturing the direct correlation between the presence of CXCR3 chemokine family molecules in BC tumours, and positive outcomes of neoadjuvant cancer treatment, and overall survival.
The invention further relates to the following items;
The present invention further relates to a method of a priori assessment of CXCR3 splice variants and its ligands CXCL9, CXCL10, and CXCL11 in muscle-invasive bladder cancer (MIBC) patients, to enable patients to be stratified for their predicted response to a chemotherapy drug treatment, or clinical outcome. The invention further relates to the treatment of cancer patients having been identified as being susceptible to certain treatment regimes.
A next aspect of the invention relates to a method to measure T cell activity state in a patient tissue sample, wherein the method comprises the following steps; Firstly, providing a patient tissue sample, then in a measurement step, determining a biomarker expression level of the biomarkers CXCL11, and at least one of CXCR3A or CXCR3alt. The biomarker expression level of CXCR3B, CXCL4, CXCL9 and CXCL10 may also optionally be determined. In some embodiments, the tissue sample is assigned a classification which reflects the presence of the CXCR3 cytokine system an T cell activity state in the patient tissue sample, based on the biomarker expression levels.
Another aspect of the invention relates to a method to measure T cell activity state specifically in a patient tumour sample, wherein the method comprises the steps of providing a cancer tissue sample; then in a measurement step, determining a biomarker expression level of the biomarkers as specified above. In an optional classification step, the cancer tissue sample is assigned a value which reflects the number and activation status of the T cells present in the cancer tissue sample, based on the biomarker expression levels.
A next aspect provided by the invention is determining the expression level of the biomarkers CXCL11, and at least one of CXCR3A or CXCR3alt in a tumour sample taken from a patient who has been previously diagnosed with cancer, in order to predict the outcome of an antineoplastic treatment, or to classify the patient as a treatment responder or non-responder.
These methods applied in cancer may optionally include further measurements of the CXCR3B splice variant, and/or the additional chemokine biomarkers CXCL4, CXCL9, and CXCL10. A particular embodiment regards the classification of cancer patients according to the invention into treatment responders and non-responders. This may be based on the input of the specified biomarkers into an algorithm which provides a probability that the patient will respond favourably to an antineoplastic treatment, comparing the patient biomarker expression levels to representative reference samples, or comparing the biomarker expression levels in sample to a list of biomarker expression thresholds generated from previously analysed cohorts of patient biomarker expression data.
In one embodiment the expression level of the biomarker CXCL11 is determined at the level of protein expression. In certain embodiments of the method according to the invention, this value is expressed as a non-transformed expression level, or the expression level value of CXCL11 undergoes a normalisation process, particularly to the mass of sample analysed, or Area sinus hyperbolicus (arsinh) normalisation to provide a value as the arsinh of the expression per gram of tissue. Patients, or samples with an expression level of CXCL11 above a threshold of 13.98, particularly more than 22.4 pg per 10 mg sample tissue may be classified as treatment responders according to the invention.
Another particular aspect of the invention is a method to measure the expression of mRNA of the CXCR3 isoforms CXCR3A, CXCR3alt and/or CXCR3B, particularly using a methodology making use of nucleic acid probes that can differentiate between the sequences of the biomarker CXCR3 variants. This method may optionally include the normalisation of the CXCR3 variant expression levels with respect to the expression of house-keeping genes, particularly the house keeping genes IP08 and CDKN1B.
In some aspects of the invention, CXCR3 splice variant expression above a threshold level is used to pair patient cancer samples with a predicted clinical outcome. A patient with a tumour sample classified as positive, or high for a splice variant, is likely to respond to an antineoplastic treatment when CXCR3A is more than 2(−12.3) more than that of the HKG, particularly more than 2(−11.97) times that of the HKG, and/or CXCR3alt is more than 2(−13.8) more than that of the HKG, particularly more than 2(−11.27) times that of the HKG, and/or CXCR3B is more than 2(−11.9) more than that of the HKG, particularly more than 2(−8.43) times that of the HKG. The method of the invention may be particularly useful in predicting the clinical outcome of an antineoplastic treatment in cancer patients diagnosed with kidney, prostate, breast, lung, ovarian, gastric, rectal, melanoma, oesophageal, or particularly bladder cancer, more particularly muscle 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 to a neoadjuvant chemotherapy treatment, particularly an antineoplastic drug, Bacillus Calmette-Guerin, or an immune checkpoint modulator.
Another aspect of the invention is a method to stratify cancer patients according to the priority in which they should receive an antineoplastic surgical intervention, according to the expression levels of the specified biomarkers.
An additional aspect of the invention provides a method to predict the clinical outcome of an immune cell transfer treatment, by matching the expression level of CXCR3 biomarkers in a cell transfer sample, with the expression level CXCR3 ligands in a target tissue sample.
A further aspect of the invention is a method to compare the expression levels of CXCL11 and at least one of CXCR3A and/or CXCR3alt, and optionally CXCR3B, CXCL4, CXCL9, and/or CXCL10, in both a pre-treatment and post-treatment patient tumour sample, for the purpose of monitoring tumour progression over time.
A further aspect of the invention is a pharmaceutical composition which includes antineoplastic platinum drug, such as cisplatin, for use in the treatment of a cancer patient who has been classified as a drug responder according to any of the methods specified above.
A final aspect of the invention is a system for assigning a value to a patient tissue sample that reflects the T cell activity state of the tissue, comprising determining the expression level in the tissue sample of at least one of the biomarkers CXCL11, CXCR3A, CXCR3alt, CXCR3B, CXCL4, CXCL9, and/or CXCL10.
One aspect of this invention relates to a method to measure the T cell activity state in a patient tissue sample, which may be of use in a clinical setting where information about the number and phenotype of T cells responding to CXCR3 binding-chemokines may inform the prognosis, or clinical treatment options of a patient. This first aspect of the invention may have significance in patients diagnosed with, or who are suspected of having, a condition characterised by chronic inflammation. Biomarker expression may allow a physician to measure whether sufficient pre-existing T cells are present in a target tissue, in order to predict whether a drug or cell-based treatment that enhances T cell responses will be effective, for example to combat viral infection or cancer. Conversely, the methodology may be used to examine a tissue sample for the presence of harmful, autoimmune T cell recruitment and activation, in order to inform decisions regarding immune-suppressing medication.
The first step of the method to measure T cell activity state, is providing a patient tissue sample. This may be peripheral blood or white blood cells, or in any tissue sample in which it is desirable from a clinical perspective to estimate the number or activation status of T cells within the tissue, such as a biopsy, or tissue derived from a tumour, graft, transplant, or a tissue targeted by an infection or autoimmune inflammation.
The second step of the method is the measurement of the expression levels of the chemokine CXCL11 and either, or both of, the CXCR3 variants CXCR3A or CXCR3alt that are present in the tumour sample. The measurement step may optionally include the determination of the expression level of the additional biomarkers, CXCL4, CXCL9, CXCL10, and/or the splice variant CXCR3B. In an optional classification step, the biomarker expression levels are used to assign the sample, or patient, a value which reflects the T cell activity state, in other words, the number and/or inflammatory potential of the T cells present in the patient tissue sample.
The data in the examples shows the efficacy of the CXCR3 and CXCR3 ligand biomarker family in identifying tissue with a potent T cell population that can be activated by subsequent chemotherapy. Furthermore, the expression of CXCR3 receptors on certain CD8+ T cell subsets is shown to confer migration, proliferation, and cytokine secretion potential to cytomegalovirus (CMV) specific T cells, as well as tumour-specific T cells in vitro, confirming the broad applicability of this method to both cancer, chronic viral infection, and potentially harmful autoimmune or graft-specific T cell responses.
A next aspect of the invention relates to a method to measure T cell activation specifically in a patient tumour sample, wherein the method comprises the steps of providing a cancer tissue sample; then in a measurement step, determining a biomarker expression level of the biomarkers as specified in the first aspect of the invention. This may be a peripheral white blood cell sample, a tumour sample from a biopsy, or tissue removed in a surgical intervention, or a cancer patient sample such as peri-tumoural tissue, or a tumour-draining lymph node. In an optional classification step, the cancer tissue sample is assigned a value which reflects the T cell activity state of the T cells present in the cancer tissue sample, based on the biomarker expression levels. The patient classification provided by the methods described herein may form part of the diagnostic protocols identifying a solid tumour, by helping to predict the prognosis of a patient with, or without cancer treatment. In other words, the method of the invention may be useful to stratify patients into groups with different recommended treatment protocols, or distinct patient outcomes, and may be of use to clinicians when, for example, assigning patients to groups which will receive either drug treatment, surgery, or palliative care.
In the data presented in the examples examining biomarker expression in BC patients, combining information about the level of CXCL11 expression in the tumour sample, with information about the expression of either of the CXCR3 splice variants CXCR3A, or CXCR3alt, classified patients with 100% accuracy as to whether they will respond favourably to antineoplastic neoadjuvant chemotherapy treatment. Using a linear regression model, the area under the curve of a Receiver operator characteristic (ROC) curve generated from the expression levels of either CXCL11 and CXCR3A, CXCL11 and CXCR3alt, or CXCL11 and CXCR3A and CXCR3alt is 1, showing complete accuracy in the prediction of NAC response. Further including CXCR3B expression level increased the fit of these predictive models. Cox-proportional hazard analysis of a second cohort showed confirmed an association between survival time and CXCL11, and showed CXCL4, CXCL9, CXCL10 mRNA, but not bulk CXCR3 mRNA level was correlated with BC treatment outcome.
Another aspect of the invention provides a method to predict whether a patient bearing a solid tumour will respond to a cancer treatment, by measuring the expression of selected biomarkers in a tumour sample as specified above: CXCL11 and at least one of CXCR3A or CXCR3alt, with the optional addition of CXCR3B, CXCL4, CXCL9 and/or CXCL10. The method provided herein may provide information that can help a clinician to choose the most appropriate personalised treatment course for the cancer patient. In the classification step of the method to predict the clinical outcome of a cancer patient, the expression levels of the CXCR3 chemokine family biomarkers determined in the sample are used to classify the cancer patient as either likely to be an anticancer treatment responder, or a treatment non-responder. In other words, the biomarker analysis method can make a prediction regarding the outcome of a clinical treatment for cancer.
A further aspect of the invention provides a method to measure the T cell activity state of a tumour, or a method to predict patient outcome in cancer treatment, by determining the expression level in a sample of cancer tissue of at least one of the biomarkers selected from a list comprising CXCL4, CXCL9, CXCL10, CXCL11, CXCR3A, CXCR3alt and/or CXCR3B. In one embodiment, the expression level of at least two, particularly at least three of the provided biomarkers are determined. As all the biomarkers in the CXCR3 system are associated with BC patient NAC treatment outcome, as the number of biomarkers measured increases, increasing accuracy can be achieved by in the statistical model to predict patient outcome.
From an analysis of mRNA (CXCR3) and protein (CXCL11) expression in a 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:
Combining the expression of all four molecules in a predictive linear regression model gave the best fit to the Swedish cohort data in the examples, as reflected by the low AIC and Brier scores for this combination of predictive biomarkers. In the second cohort of patients, CXCL11, but also CXCL9 and CXCL10 mRNA expression also correlated with the amount of the T cell marker CD3 found in the tumour. In this cohort all four CXCR3 ligands CXCL4, CXCL9, CXCL10 and CXCL11 were associated the outcome of chemotherapy in bladder cancer patients, providing the following additional biomarker combinations which the potential to predict neoadjuvant chemotherapy outcome:
In one embodiment according to these aspects of the invention, the expression level of CXCR3alt and CXCL11 alone is sufficient to determine cancer patient outcome.
In certain embodiments of the method to predict the outcome of an antineoplastic treatment in a patient bearing a solid tumour, the classification step comprises inputting the biomarker expression levels into a model-fitting statistical methodology in order to generate a value reflecting the probability that a patient will be an antineoplastic treatment responder. Statistical machine learning techniques, particularly supervised machine learning techniques which may be particularly useful for this method include, but are not limited to, random forest methodology, or neural networks. The use of logistic regression based on the results of previously analysed cancer patients is a particularly useful method to capture the relationship between expression levels and clinical response to provide an algorithm which generates a probability of drug response upon the input of biomarker data from the tumour sample. The classification or regression algorithms used in this aspect of the invention may be applied in order to improve the predictive power of the biomarkers at population level. It is understood that these methods may take variables other than the chemokine biomarker expression levels into account, such as variables selected from, but not limited to, age, gender, comorbidities, or clinical parameters.
The CXCL11 and CXCR3 expression levels in the Swedish cohort shown in the examples were incorporated into a predictive logistic regression model to test the benefit of using one, two, or multiple biomarker expression level values to predict BC patient outcome. The performance of each model in terms of predicting outcome to MVAC therapy was assessed by the area under the ROC curves, and both the AIK and Brier model fitting scores increased as more biomarkers were included. This analysis identified equations with which the pre-treatment tumour expression levels of CXCL11, together with CXCR3A, and particularly CXCR3alt, most accurately segregate muscle-invasive bladder cancer patients into MVEC neoadjuvant treatment responders and non-responders.
One possible embodiment of the method of predicting the outcome of an antineoplastic treatment in a patient bearing a solid tumour, is the classification of the cancer patient as an antineoplastic treatment responder if the level of one, or several biomarkers is above a certain threshold. Conversely, the cancer patient can be classified as an antineoplastic treatment non-responder if the level of biomarker expression is below a particular threshold. Useful thresholds and confidence intervals for these cut-offs are provided in the examples. This information could help a clinician to stratify patients into those who should receive prompt neoadjuvant treatment, and those who will benefit from surgery or other treatment options.
A particularly useful embodiment of the method to predict the outcome of an antineoplastic treatment in a patient with a solid tumour comprises measuring the level of CXCL11 protein in the sample. The expression of a marker may be assayed at the protein level via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA or multiplex analyses. Marker expression may also be evaluated by measuring the expression at the level of mRNA by means of quantitative realtime PCR (qPCR), microarray, or sequencing assays.
Methods that use an antibody, or antibody fragment, which binds specifically to CXCL9, CXCL10, or CXCL11, such as ELISA, or bioplex, are particularly useful for determining the expression level of CXCR3 ligand protein in a tumour. Optionally, CXCR3 isoform expression levels can also be measured at the protein level, using molecular probes that distinguish between the CXCR3 splice variants. It is understood that the accurate measurement of protein according to certain embodiments of the invention is most effective in samples that have been preserved in such a way that protein is not degraded. It is preferable that the sample, or a portion of the sample, should be immediately frozen in liquid nitrogen upon resection from the patient, and stored at −80 degrees Celsius before processing, preferably in the presence of protein inhibitors.
In an alternative embodiment of the method according to the invention, the cancer patient is classified as a treatment responder, or non-responder, by comparing the expression levels of the listed biomarkers determined in the tumour sample with the expression levels in previously-analysed reference samples, where the matched clinical outcome is already known. For example, if the biomarker expression is equal to, or more than the expression level in a positive reference sample comprising tumour tissue from a NAC responder, the patient may be classified as likely to respond to NAC. Conversely, if the biomarker expression in a patient sample is equal to, or less than a negative reference sample of tumour tissue derived from a NAC non-responder, the patient may be classified as unlikely to respond to NAC. For the inversely correlated markers CXCR3B and CXCL4, this relationship is reversed.
In certain embodiments of the method provided by the invention, the expression level of biomarkers measured at the protein level, for example, CXCL11, undergoes a statistical normalisation process, for example, is expressed as a concentration per milligram of sample protein. This can be particularly advantageous when multiple samples are analysed in bulk, in order to standardise variance between samples to achieve normal distribution, and make biomarker expression levels more amenable to further statistical manipulations. The biomarker expression value determined by a method such as ELISA, or mass spectrometry, can be transformed to stabilise the distribution to compensate for repeated sampling procedures. The normalisation may include inputting the expression level into a transformation or scaling function, selected from, but not limited to, biexponential, logicle, or log transformation functions, or particularly an arsinh normalisation function.
In the data provided in the examples, bladder cancer patients can be accurately classified as neoadjuvant treatment responders, if the expression level of CXCL11 in the tumour sample taken prior to treatment is above 13.98, particularly more than 22.4 pg per 10 mg sample tissue. This useful threshold value was generated from the current model cohort, and may be subject to further changes as the predictive model is refined by the addition of more patient data in future use or studies.
In particularly advantageous embodiments of the invention, the expression levels of CXCL11, CXCR3A, CXCR3alt and/or CXCR3B are measured at the mRNA level. These measurements can be made with nucleic acid probes, particularly with a quantitative PCR methodology such as real time PCR, sequencing reactions, or a nucleic acid array. It is understood that accurate measurement of CXCR3 splice variant expression according to certain embodiments of the invention is most effective in samples that have been preserved in such a way that mRNA is not degraded. 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. Processing of the samples should be carried out on ice, optionally in the presence of RNase inhibitors.
One methodology that is particularly useful for measuring the CXCR3 splice variant expression level, is a nucleic acid amplification method conducted using polymerase chain reaction of the RNA extracted from the patient tumour sample. Specific nucleic acid probes, such as the primers of sequences SEQ ID NO 016 to 021 presented in the examples, can distinguish between the three CXCR3 variants using primer designs which target differently spliced regions, using standard Taqman ABI assay conditions.
In the data provided in the examples, combining information derived from qPCR measurements of the CXCR3 splice variants CXCR3A, CXCR3alt and CXCR3B all improved the prediction of BC clinical outcomes, compared to the CXCL11 expression level alone. The expression levels of the CXCR3 splice variants may optionally be determined using other technologies designed to quantify nucleic acids, including, but limited to, sequencing, microarrays or gene chips, for example a cDNA array.
In several embodiments of the invention it is particularly advantageous to compare, or normalise the expression of the biomarkers to the expression of one, or several, housekeeping genes. Two such genes which are of particular use are the genes IP08 and CDKN1B, but the skilled artisan will recognise that other stably expressed genes may be substituted 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 range of housekeeping genes identified IP08 and CDKN1B as those most stably expressed in bladder cancer tumour samples. The mean expression of IP08 and CDKN1B was used to normalise biomarker expression values across samples of differing size, mRNA quality, or amplification level.
In an alternative embodiment of the cancer patient classification step provided by the invention, the patient is assigned as an antineoplastic treatment responder if the expression level of
For the CXCR3 variant biomarker thresholds provided here, the relationship between the biomarker and the HKG is the same when thresholds are used independently, or separately. In other words, using the example of CXCR3A above, the threshold for a positive outcome for neoadjuvant treatment may be assigned to a sample if the expression level CXCR3A is more than 2(−11.97), which is approximately 0.00025 times less than the expression level of the HKG. Determining the expression level of more than one of the provided biomarkers will increase the accuracy of the patient outcome prediction provided in the classification step. If markers are combined in a multivariate predictive model, in some cases an inverse correlation between biomarker level and outcome may be used to classify samples. In aspects of the invention utilising multivariate classification techniques (e.g. Random Forest), multiple “thresholds” are sequentially applied, and can function in both positive and negative correlation to the HKG.
In certain embodiments of the invention, in addition to, or instead of comparison to HKG, the expression level of the biomarker is compared to a baseline, or reference sample. One example of a negative control, or negative reference is a sample of healthy tissue from the affected organ, in which T cell infiltration is low, or absent. An example of a positive control, or positive reference sample, may be an example of a previously analysed sample in which there is a high level or T cell infiltration in the tumour or tissue sample. The skilled artisan will appreciate that in addition to analytical controls such as the examples presented above, patient samples may be compared to a range of pre-determined calibration samples, or standards, to provide appropriate technical and biological controls.
The skilled artisan will appreciate that the fold change CXCR3 variant expression values provided above are examples of differential cycle thresholds compared to HKG, i.e. the number of qPCR cycles needed to generate a fluorescence signal from the specific nucleic acid probes used, above a user-defined threshold. These values therefore reflect the PCR conditions and cycle threshold used to generate proof of principal evidence for the predictive model, and the exact values are expected to vary in practice.
In certain embodiments of the method according to the invention, the cancer tissue sample in which the biomarker levels are determined comprises, or essentially consists of, neoplastic cells derived from a solid tumour, but may further include heterogenous cells derived from the immune system or the tissue of origin. The method for predicting the clinical outcome of a patient is particularly useful for analysing neoplastic cells derived from squamous cell cancer, such as melanoma, or adenocarcinoma, particularly neoplastic cells from tissues selected from, but not limited to, tumours frequently treated with a combination of neoadjuvant treatment and surgery selected from breast, lung, kidney, prostrate, ovarian, colorectal, gastric, oesophageal or bladder cancer. The methods are thought to be particularly effective when the neoplastic cells themselves are not characterized by significant expression of CXCR3. In this way, the signal from the CXCR3 measurements can be certain to reflect the phenotype of T cells present in the tumour. In a particular embodiment of the method provided by the invention, the cancer patient is a bladder cancer patient, particularly a patient having been diagnosed with muscle-invasive bladder cancer of urothelial, or squamous cell origin.
In the data presented in the examples, an equation which captures the relationship between the expression levels of the biomarker CXCL11, together with the expression level of one or more of the CXCR3 splice variants, classified muscle-invasive bladder cancer patients with perfect accuracy as either responders, or non-responders to a subsequent treatment with MVEC neoadjuvant chemotherapy. The data presented in the examples demonstrates that in a single cell sequencing analysis of comparing bladder cancer and melanoma patient biopsies, the same immune cells express CXCR3 and CXCL11. In addition, studies show tumour-infiltrating, stem cell memory T cells, which the data provided here show are responsive to CXCR3 ligands, promote survival in kidney, prostrate, bladder, lung cancer and melanoma (Jansen C. S. et al. Nature (2019) 576:465; Brummelman J. et al. J. Exp. Med. (2018) 215(10):2520; Siddiqui I. Immunity (2019) 50:195). The methods to predict clinical outcomes in cancer in which chemokine biomarkers are used to estimate the potency of T cell immunity provided here, are therefore likely to be broadly applicable in squamous cell cancers, such as melanoma, or adenocarcinomas, such as bladder cancer.
In particular embodiments, the antineoplastic treatment in question is a neoadjuvant antineoplastic drug, particularly a neoadjuvant antineoplastic drug selected from, but not limited to, cisplatin, methotrexate, vinblastine, doxorubicin, carboplatin, adriamycin, gemcitabine, paclitaxel, filgramastim, pemetrexed, vinorelbine, oxaliplatin, vinflunine, or doxetaxel. In other words, the method is of particular use for predicting the outcome of a classical regime of MVEC or MVAC neoadjuvant chemotherapy containing the drugs methotrexate, vinblastine, doxorubicin or epirubucin, and cisplatin, but is likely to apply to similar drugs.
In certain embodiments of the method to predict the clinical outcome of a patient with a solid tumour provided by the invention, the patient sample is tumour tissue or lymph node tissue, particularly a tumour tissue sample taken during a biopsy to perform pathology typing of the tumour, or a sample taken from a lymph node draining the vicinity of the tumour.
In another embodiment of the invention, the classification step of the method can be of use to stratify bladder cancer patients into groups with high or low priority for cystectomy. The method provided can be used to assign muscle invasive bladder cancer patients classified as antineoplastic chemotherapy treatment non-responders as specified above, into a group with high priority for receiving a radical cystectomy procedure, without prior neoadjuvant treatment. This classification can avoid disease progression or metastases during an ineffective treatment regime. Additional cancer types wherein this aspect of the invention may be usefully employed to aid clinical stratification of patients into groups which are likely to benefit from either neoadjuvant treatment or surgical intervention as a first line of treatment includes, but is not limited to, breast cancer, rectal cancer, oesophageal cancer and gastric cancer.
The data in the examples shows that bladder cancer patients classified as neoadjuvant treatment non-responders are unlikely to experience tumour down-grading upon chemotherapy treatment. Clinical outcomes in bladder cancer can be improved if these patients instead receive prompt surgical intervention.
Another aspect of the invention is a method to match the CXCR3 expression status of an immune cell transfer treatment product, with the CXCL4, CXCL9, CXCL10 and/or CXCL11 expression status of the patient tissue being targeted by the immune cell transfer. In other words, a method to match positive expression of the CXCR3 variants in the cells for transfer, with expression of their ligands in the target tissue, in order to ensure the treatment is likely to succeed. According to this aspect, the method can predict the outcome of an immune cell transfer cancer treatment, particularly a T cell transfer treatment in a patient bearing a solid tumour. Firstly, a patient or product provides a cell transfer sample, or an autologous immune cell sample, in addition to a target tissue sample, such as a patient tumour sample.
In a first cell transfer measurement step, the expression level of the biomarkers CXCR3A, CXCR3alt, and/or CXCR3B in the cell transfer sample is determined, and next, in a target tissue sample measurement step, the expression level of the biomarkers CXCL4, CXCL9, CXCL10, and/or CXCL11 is determined, according to the measurement protocols as specified in previous aspects of the invention. Lastly, in a classification step, the patient assigned a value, reflecting the likelihood, or probability that the cell transfer treatment will have a positive clinical outcome, if the biomarker expression levels are positive, or high, in both samples. This value reflects the potential of the cells to migrate to the target tissue, and their local potential for proliferation and cytokine production after transfer. This method may utilise algorithms, reference samples or thresholds to classify the patient sample for biomarker status, as provided in other methods of the invention provided above. This method may be particularly desirable for a personalised medicine strategy, wherein the chemokine sensitivity of a T cell transfer product, such as, for example, a T cell with a recombinant tumour-specific TCR, or a tumour-specific T cell population expanded from a donor, or patient's own tumour sample, is assessed for whether it will effectively respond to the chemokine ligands expressed by the patient's tumour prior to administration.
A next aspect of the invention, is the use of CXCR3 chemokine system biomarkers provided in a method to monitor tumour progression over time, or to monitor the presence of tumour immune infiltrate in a patient tumour over time. The method involves the step of providing two, or more than two, sequential tumour samples at different sampling times. In a measurement step, the expression level of CXCL11 and either, or both of CXCR3A and CXCR3alt are determined in the sequential tumour samples. Optionally, the expression level of CXCR3B, CXCL4, CXCL9 and/or CXCL10 can be measured. In a final classification step, the patient is classified as a treatment responder, or as a patient with increased tumour immunity, if the expression levels of the CXCL11, CXCR3A, CXCR3alt, CXCL9, or CXCL10 increase, and/or CXCL4 or CXCR3B decrease in later samples, compared to earlier samples. This embodiment of the invention is of particular use when comparing pre-treatment tumour samples, to post-treatment tumour samples, or providing information to a clinician on whether a patient is responding to a particular treatment regime over time.
The data presented in the examples demonstrates that increasing expression levels of the biomarkers CXCL11, CXCR3A, CXCR3alt, CXCL9, and CXCL10 correlates with improved survival following NAC-treatment, whereas CXCR3B and CXCL4 expression is lower in these patients.
Another aspect of the invention provides a neoadjuvant antineoplastic pharmaceutical formulation for use in the treatment of a patient with a solid tumour who has been classified as a drug responder according to the previously specified methods. Antineoplastic pharmaceutical formulations of particular utility according to the invention are those which comprise, or consist of an antineoplastic drug, particularly an antineoplastic drug selected from, but not limited to, cisplatin, methotrexate, vinblastine, doxorubicin, paclitaxel, carboplatin, adriamycin, gemcitabine, filgramastim, pemetrexed, vinorelbine, oxaliplatin, vinflunine, or doxetaxel.
In a related embodiment, the invention provides additional T cell-stimulating pharmaceutical formulations for use in the treatment of patients classified as antineoplastic treatment responders according the methods described above. These include, but are not limited to, Bacillus Calmette-Guérin (BCG), the related increased safety recombinant strain VPM1002 (Kaufmann Frontiers Immunol. 2020, 11:316; Grode et al., Vaccine 2013, 31(9):1340) and cancer immunotherapy treatments, particularly checkpoint inhibitory antibodies, more particularly a checkpoint inhibitor antibody selected from ipilimumab, nivolumab, pembrolizumab, pidilizumab, atezolizumab, avelumab, durvalumab, or cemiplimab.
The data provided in the examples shows that these biomarkers may be particularly helpful for predicting whether a patient will respond well to an antineoplastic immunomodulatory medication that increases T cell killing of tumour cells, included checkpoint blockade or BCG treatment, as the biomarker levels are directly correlated with the number, and activation, or functional phenotype of T cells in the tumour. Studies in mouse models of melanoma and ovarian cancer have suggested that the outcome of immune checkpoint blockade correlates with certain components of the CXCR3 chemokine signals in tumours (Chow N. T. et al. Immunity (2019), 50(6):1498). The human data in MBIC provided shows the unexpected predictive efficacy of CXCL11 and CXCR3A or CXCR3alt in predicting T cell activation in neoadjuvant chemotherapy, suggesting the methodology would be successful in similar treatment options which rely on T cell immunity.
A further aspect of the invention provides a system for assigning a value to a patient tissue sample that reflects the T cell activity state of the tissue, comprising determining the expression level in the tissue sample of the genes CXCL11, CXCR3A and/or CXCR3alt, and optionally CXCL4, CXCL9, CXCL10 and CXCR3B, and using the biomarker expression levels to classify the T cell activity state of the patient sample.
The invention is further illustrated by the following items:
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Tab. 1 shows the protein analytes measured by multiplex ELISA using Luminex.
Tab. 2 shows the performance of individual biomarker thresholds for predicting the clinical outcome of neoadjuvant therapy treatment in NMIBC patients. Decreasing AIC or Brier scores indicated a better model fit.
Tab. 3 shows the performance of logistic regression models for predicting the clinical outcome of neoadjuvant therapy treatment in NMIBC patients.
Tab. 4 Table shows hazard ratios and coefficients of the MIBC patients from the TCGA cohort (n=68). Data were dichotomized using optimal split points into CXCL9high (n=21) and CXCL9low (n=47), CXCL10high (n=28) and CXCL10low (n=40), CXCL11high (n=13) and CXCL11low (n=55), and CXCR3high (n=48) and CXCR3low (n=20) patients, and CXCL4low (n=56) and CXCL4high (n=12).
46 patients with BC were recruited with informed consent from different hospitals in the northern health region of Sweden during the years 2010-2017. Specimens and blood samples were archived in the biobank of the department of urology at the university hospital in Umeå (NUS), Sweden. Patients were at least 18 years of age, and the study on patient material was approved by the regional ethical board (EPN-Umeå, original registration number: 2013/463-31M, with latest amendment 2018/545-32). Further, all patients had given verbal and written consent to contribute with specimens and fluids to the biobank and to participate in consecutive and ethically approved translational research. A second cohort of normalized mRNA expression data of primary tumour samples was obtained from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov). Clinical data (BLCA dataset) were used to identify 68 MIBC patients that received chemotherapy (chemo) within 150 days after sample procurement and 292 MIBC-patients that did not receive any chemotherapy (no-chemo).
Diagnosis and NAC-Treatment
The diagnosis of urinary BC was established based on tumour histology of the specimen that was received at transurethral resection of the bladder tumour (TURBT). In the TURBT sample, MIBC disease was defined by the histological invasion of the tumour into the detrusor muscle; cT2-T4 (29/46 patients). Next, MIBC-patients were clinically investigated on eligibility to receive NAC containing based on a good performance status including Charleson age comorbidity index (CACI) ≤6, age≤77 years and no major renal impairment (GFR≥55-60) or any other relevant comorbidity. NAC treatment in most cases contained a high dose of the drugs methotrexate, vinblastine, doxorubicin or epirubucin, and cisplatin according to the following regime:
Further, by radiological computer tomography (CT) nodal and organ-dissemination was excluded; cN0M0352. Eligible MIBC-patients (20/29) received 2-4 cycles of NAC-treatment before radical surgery (i.e. cystectomy with radical intention: RC). NAC was applied as Cisplatin-based combination chemotherapy (predominantly: cisplatin, methotrexate, vinblastine, doxorubicin (MVAC). Response to NAC was defined as pathoanatomical downstaging of the tumour in the RC-specimen and based on this, NAC-receiving MIBC patients were defined as responders (9) or non-responders (11). These two groups had equivalent clinical performance status exemplified by similar ranges in the CACI index, the American Society of Anaesthesiologists Classification (ASA)-score and patient age. Further, response to NAC was subdivided based on the tumour histology into complete response (CR) with p0N0M0, partial response (PR) with pTa/T1/TisN0M0, stable disease (SD) with ≥p2N0M0 and 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 were ineligible for NAC (i.e. no-NAC MIBC patients; see criteria above) and underwent direct RC (4/7) or due to palliative reasons, RC was not applied (3/7). If the tumour infiltration in TURBT-specimen was limited to the subepithelial or epithelial layer, the tumour was defined as non-muscle invasive bladder cancer (NMIBC). NMIBC patients underwent non-systemic treatment such as local administration of Bacillus Calmette-Guérin (BCG) vaccine and when indicated, re-TURBT treatment.
Patient Sample Processing
The tumour samples were taken during TURBT and lymph nodes were taken during RC. All specimens were immediately frozen in liquid nitrogen and stored at −80° C. For processing, specimens were kept on ice at all times, cut in two parts with a scalpel and the mass was scaled. Next, protein extraction buffer (T-PER™; Thermo Fisher Scientific) was applied to one part and RNA/DNA lysis buffer (RLT; Quiagen) with 2 M DTT was applied to the other part. Specimens were mechanically disrupted using tubes with ceramic beads in a tissue homogenizer system (all from Bertin Instruments). Concomitant DNA/RNA extraction was performed using the AllPrep DNA/RNA Micro Kit following the manufacturer's instructions (Quiagen). 13 lymph nodes were kept non-disrupted after RC in order to isolate live lymphocytes. After immersion in cold AIM-V medium (Thermo Fisher Scientific), the specimen was cut with a scalpel and cells were gently filtered through a 40 μM cell strainer.
PBMC Preparation
Blood samples were collected from healthy volunteers after obtaining informed consent. Human peripheral blood mononuclear cells (PBMCs) were separated from the heparinized whole blood of healthy donors by lymphoprep density gradient centrifugation with a Biocoll separating solution (Biochrom GmbH, Berlin). Isolated PBMC were re-suspended in PBS and kept at 4° C. The study on PBMC was approved by the Charité University Medical School Ethical Committee (institutional review board).
Flow Cytometric Analysis
For analysis of T cell phenotypes, PBMCs and lymph node-derived cells were stained using fluorescently conjugated monoclonal antibodies for CD3 (BV650, clone OKT3), CD4 (PerCP-Cy5.5, clone SK3), CD8 (BV570, clone RPA-T8), CCR7 (AF647, clone G043H7), CD45RA (PE/Dazzle 594, clone HI100), and CD95 (PE/Cy7/Brilliant Violet (BV) 421, clone DX2; BD Biosciences), CXCR3 (PE, clone G025H7) at 4° C. for 30 min. To exclude dead cells, LIVE/DEAD Fixable Blue Dead Cell Stain dye (Thermo Fisher Scientific) was added. Analogously, chemokine receptors (CXCR1, CXCR3, CXCR4, CCR3, CCR5, CCR6, CCR7) were stained on the cell surface using the human cell surface marker screening panel (BD Biosciences). All antibodies were purchased from BioLegend, unless otherwise indicated. Cells were analysed on an LSR-II FORTESSA flow cytometer (BD Biosciences) and FlowJo software version 10 (Tree Star). Lymphocytes were gated on the basis of the forward scatter (FSC) versus side scatter (SSC) profile and subsequently gated on FSC-Height versus FSC-Area to exclude doublets. In stimulation experiments, fixation/permeabilization was performed with an eBioscience FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions. After washing, fixed cells were stained with the fluorochrome-conjugated monoclonal antibodies for IFN-γ (eF405, clone 4S.B3), for TNF-α (Alexa Fluor 700, clone MAb11) and for CD137 (PE/Cy7, clone 4B4-1) at 4° C. for 30 min. Background response was assessed using non-stimulated controls and subtracted from the antigen-reactive cytokine production.
Chemotaxis Assay for CD8+ T Cell Subpopulations.
1×106 million human PBMC were initially seeded in 200 μL RPMI, 10% FCS, 1% Penicillin/Streptomycin into the upper chamber of 24 transwell plates with 3 μm pore size (Corning) (
TSCM-Expansion Protocol
PBMCs were enriched via FACS for a CD3+CCR7+CD45RA+ T cell population on a BD FACS Aria II SORP (BD Bioscience) using the gating strategy in
Intra-Tumoural Cytokines Measurement
Cytokines were assessed in protein extracted. Luminex technology (Bio-Plex® 200 System, BioRad) was applied using multiplex assays (Merck) (Tab. 1). For each sample, the respective optical density values of the analyte concentration were assessed via a calibration curve and subtraction of the blank. The mean concentrations and standard deviations of the samples were calculated.
Intra-Tumoural Analysis of mRNA CXCR3-Variants
1 μg RNA from TURBT-specimens of the 46 BC-patients was used for cDNA synthesis according to the QuantiTect Reverse Transcription Kit manual (Qiagen). Quantitative real-time PCR (qRT-PCR) analysis was performed using TagMan PCR, containing FAM-BHQ1—labelled probes. mRNA CXCR3-variants were measured via TagMan qRT-PCR assays. To measure the main variant CXCR3A mRNA (NCBI reference sequence: NM 001504.1), the TagMan Universal PCR Master Mix was used with the probe Hs00171041_m1 (ABI) was used. To measure the CXCR3-splicing variants, two RT-qPCR panels specific for CXCR3B and CXCR3alt were designed (
Statistical Analysis
GraphPad Prism 8 (GraphPad Software) and R17 (version 3.5.2) were used to generate graphs and carry out the statistical analysis of data. To test for a normal Gaussian distribution, the Kolmogorov-Smirnov test was employed. RT-PCR data were log 2- and protein data were arsinh-transformed for display and prior to statistical analyses. Ct values below detection limit, i.e. above 40 (11%), were imputed using nondetects R package. In tumour samples, non-detects were not present, but 15 out of 90 measured protein analytes did not show sufficient expression, i.e. median absolute deviation above 0 across 46 tumour specimens, and were therefore excluded from statistical analyses. In serum samples, missing protein data (1.1%) were imputed using missForest R package. Cox proportional hazards models were fitted using coxph and cutp functions (survival package) to determine optimal split points to display Kaplan-Meyer curves of dichotomized data. Individual patients' restricted mean survival times as shown in
To unveil the functional relevance of the treatment-naïve CXCR3-chemokine system associated with human anti-tumour immunity, primary tumour biopsies, routinely taken prior to the onset of platinum-based NAC, were collected from BC-patients that were categorised as either NMIBC or MIBC. A comprehensive retrospective characterisation of intra-tumoural cytokines and CXCR3-isoform expression in relation to anti-tumour responses induced by NAC was then performed.
CXCR3 is Highly Expressed on Early-Differentiated Peripheral CD8+ T Cells and Enriched in CXCL9/10/11 high Lymph Nodes of MIBC-Patients.
To investigate the heterogeneous chemokine receptor expression on CD8+ T cells, CXCR3 expression was compared with CXCR1, CXCR4, CCR3, CCR5, CCR6, and CCR7 on discreet CD8+ T cell functional subsets from human healthy donors (
Indeed, when LN-derived cells were stimulated for 12 h with autologous bladder tumour lysates, antigen specific activation measured by increased upregulation of CD137 could be observed in memory and effector CD8+ population compared to the naïve compartment, suggesting an enrichment of tumour specific T cells (
High CXCR3-Isoform Expression on Early-Differentiated CD8+ T Cells Associates with Differential Functional Outcome Mediated by the CXCR3-Ligand Family
In-vitro migration assays (
CXCL11 is Associated with Intra-Tumoural T Cell Infiltration Marks NAC-Responsive Patients A cohort of 46 BC-patients was used to dissect the putative roles of the CXCR3-chemokine system in anti-tumour responses induced by chemotherapy (
The formation of functional intra-tumoural T cell structures requires effective chemotactic homing within a favourable inflammatory milieu. However, it remains unknown whether the CXCR3-ligands, CXCL9/10/11, are part of the BC-specific cytokine signature or whether the distinct CXCR3-ligands are associated with the anti-tumour response. A multiplex-based detection of pre-treatment cytokines was performed on lysate from primary BC-biopsies. In NAC-receiving MIBC, the tumours were assigned to high versus low inflamed states characterised by cytokines and chemokines in two distinct clusters (4 & 6) that segregated the NAC-responding MIBC-patients from the remaining BC-subgroups in a multidimensional scaling model (non-responding MIBC, no-NAC MIBC, NMIBC) (
Receiver operating characteristic (ROC)-curves were generated to analyse the diagnostic ability of all significantly different cytokines to predict the response to NAC. CXCL11 was the most sensitive marker for predicting the response to NAC (
CXCL11 and CXCR3alt as Dual Stratification to Predict the Response to NAC in MIBC
To dissect which cells in the healthy bladder and the bladder tumour express CXCR3, CXCR3-expression was measured in human bladder by accessing publicly available single-cell RNA-sequencing data of three healthy bladder homogenates and two cancer cell-enriched MIBC-specimens (see data availability). This data indicated an absence of CXCR3 expression in healthy bladder cells as well as in cancer cells, whereas CXCR3 was expressed in tissue-infiltrating T cells (
The alternative spliced transcript CXCR3alt has been reported to exclusively bind CXCL11 (Ehlert, J. 2004) and elicit downstream signalling upon CXCL11-ligation (Berchiche, Y. A. and Sakmar T. P. (2016) 90: 483-495). To determine whether the CXCR3-isoforms expressed by different CD8+ T cell subsets have a functional significance in BC, a RT-qPCR-panel for the CXCR3A/B/alt-variants was employed to measure variant expression in patient samples. In the BC-cohort, intra-tumoural mRNA expression levels of the CXCR3-isoforms (CXCR3A/B/alt) were tested for correlation with T cell levels. The mRNA-expression levels of CXCR3A/alt, but not CXCR3B, significantly correlated with the T cell levels in NAC-receiving MIBC (
To scrutinize the dependencies between the CXCR3-chemokine system and the inflammatory tumour milieu, pairwise correlation analysis was used to detect intra-tumoural co-regulation between the CXCR3-isoforms, T cell levels and cytokine expression, including the mRNA of the CXCR3-isoforms, the mRNA of CD3 and the cytokine protein levels. Using a robust clustering technique, the three CXCR3-isoforms and CD3 grouped with the three CXCR3-ligands (CXCL9/10/11), and IFN-gamma, CCL3, CCL4, IL-16, CCL19, CXCL12, CXCL13 in one specific cluster (
Expression levels thresholds and the 95% confidence interval (CI) for CXCL11 protein, or the CXCR3 isoforms measured by quantitative PCR which predict a positive outcome to NA treatment were as follows:
Applying CXCR3alt-CXCL11 as a dual marker stratification for NAC-receiving MIBC patients using a logistic regression model, responding and non-responding MIBC patients could be completely separated prior to NAC treatment (
For external validation, the pre-treatment mRNA expression levels were analysed in tumour specimens of an independent MIBC patient cohort provided by the TCGA (The Cancer Genome Atlas: a cohort of 68 chemotherapy-receiving MIBC patients to 292 chemo-naïve MIBC patients). In this cohort, chemotherapy treatment was associated with slightly improved OS (
Statistical models to predict the clinical outcome of the NIMBC patients to neoadjuvant therapy were developed based on the expression levels of the biomarkers, CXCL11 and CXCR3 splice variants in pre-treatment tissue samples. The predictive performance of biomarkers was assessed for either individual marker thresholds (Tab. 2), or predictive logistic regression models using two or more biomarker values (Tab. 3). The performance of each model in terms of predicting outcome to MVAC therapy was assessed by the AUC of ROC curves, and both the AIK and Brier model fitting scores.
The presence of CXCR3 in the cancer tissue samples was measured by real time quantitative PCR, using Taqman probes, providing a CT value. The CT value, or threshold cycle, is the cycle number at which the fluorescent signal of the reaction crosses a user-defined threshold, i.e. exceeds background level. The CT value is inversely related to the starting amount of target DNA. The Δ CT value is the difference in expression (CT) between the target gene and the CT of a control gene, a stable expressed housekeeping gene. Here the control CT is the arithmetic mean of two house-keeping genes, IPO8 and CDKN1B identified by the genorm algorithm. In the context of the present examples, the value for the biomarker is given by:
CXCR3alt=ΔCT CXCR3alt=CT(CXCR3alt)−((CT(IPO8)+CT(CDKN1B))/2)
CXCL11 was measured by a multiplexed cytokine bead array system, giving a concentration in pg per 10 mg of tumour sample. This value was then normalized to stabilize the variance of multiple measured proteins of different intensity measured by the multiplex system using a quasilogarithmic transformation described by:
CXCL11=arsinh(CXCL11 concentration in pg per 10 mg of tumour sample)=ln(x+(x2+1)0.5).
The probability of responding to NAC is calculated by the formula:
p=1/(1+exp(−y)), where
The linear combination can be calculated including estimates for an intercept a and two regression factors β1, β2 for the variables:
y=α+β1(CXCL11)+β2(CXCR3alt)
Estimates of the regression coefficients obtained by maximum likelihood estimation with Firth's bias reduction method for the logistic regression model generated in example 1:
α=6.045
β1=1.303
β2=0.904
Probability of a patient responding favourably to NAC using levels of the two markers where unnormalized CXCL11=2.77 pg per 10 mg of tumour sample, and the CXCR3alt=ΔCT−16.426758 is:
p=1/(1+exp(−(6.045+10.303(CXCL11)+0.904(CXCR3alt))))
p=1/(1+exp(−(6.045+1.303(arsinh(2.77))+0.904((−16.426758)))))
Analogously, a formula for clinical application of a predictive logistic regression model can be developed based on the value of the two biomarkers, CXCL11 and CXCR3A:
y=α+β1(CXCL11)+β2(CXCR3A) with
α=9.558
β1=1.547
β2=1.327
The prediction performance can be improved by including a CXCR3 score which captures the opposing function of the CXCRB splice variant as a second variable in the logistic regression model. The CXCR3 score is a linear combination of the ΔCT values of the three splice variants describing a negative correlation of CXCR3B to CXCR3A and CXCR3alt, respectively:
CXCR3score=CXCR3alt+CXCR3A−CXCR3B
y=α+β1(CXCL11)+β2(CXCR3score) with
α=0.765
β1=1.246
β2=0.353
Using the above regression coefficients, the probability of a patient responding favorably to NAC using levels of four markers where unnormalized CXCL11=348.9 pg per 10 mg of tumour sample, CXCR3alt=ΔCT−8.752, CXCR3A=ΔCT−11.12, and CXCR3B ΔCT=−6.039 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, therefore a 98.3% probability of a favourable response to NAC, and is therefore classified as a NAC responder.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21151232.2 | Jan 2021 | EP | regional |
| 21151233.0 | Jan 2021 | EP | regional |
| 21151438.5 | Jan 2021 | EP | regional |
| 21151447.6 | Jan 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/050571 | 1/12/2022 | WO |
