Tissue nanomechanical signature predicts immunotherapy response

Abstract

In one aspect, the invention relates to a method to predict outcome of immunotherapy. In another aspect, the invention relates to a method of treatment of cancer, the method comprising obtaining a tissue stiffness nanomechanical signature from a tumour sample obtained from the patient, predicting the patient's responsiveness to immunotherapy, and administering neoadjuvant therapy based on the result of the prediction.

Description

[0001] Tissue Nanomechanical Signature Predicts Immunotherapy Response

[0002] Description

[0003] Field

[0004] The present invention relates to a al method to predict the outcome of immunotherapy cancer treatment by detecting and analysing a nanomechanical response profile in an isolated cancer tissue sample.

[0005] Background

[0006] Complex structural remodelling of cells and extracellular matrix during the cancer initiation and progression are accompanied by substantial biomechanical alterations, which can be measured by Atomic Force Microscopy (AFM). Increasing (pre)- and clinical evidence demonstrate the importance of cancer biomechanics in mediating therapy response. Incorporating AFM measurements of clinical biopsies within standard of care is thus a promising avenue to leverage tissue mechanics as prognostic and predictive biomarker for solid cancers.

[0007] Cancer immunotherapy represents a revolutionary approach in the treatment of malignancies by directing the patient's immune system to recognize and eliminate cancer cells. Various strategies within cancer immunotherapy have been developed. Monoclonal antibodies, such as checkpoint inhibitors like anti-PD-1 or anti-CTLA-4 antibodies, work by releasing the brakes on the immune system, enabling T cells to target and destroy cancer cells. Adoptive cell therapy involves infusing patients with genetically engineered T cells or natural killer cells that can recognize and attack specific tumor antigens. Additionally, cancer vaccines aim to stimulate the patient's immune system to recognize and destroy cancer cells bearing the targeted antigens.

[0008] Combining cancer immunotherapy with radiotherapy, known as radioimmunotherapy, holds promise in enhancing treatment outcomes. Radiotherapy delivers ionizing radiation to the tumor, inducing DNA damage and promoting cancer cell death. This process can release tumor antigens and stimulate the immune system, acting as an in-situ vaccine. When paired with immunotherapy, this synergy can amplify the body's immune response against the tumor. Checkpoint inhibitors, for instance, can enhance the immune system's recognition of the released antigens, thereby intensifying the immune-mediated destruction of cancer cells. Additionally, radiation can modulate the tumor microenvironment to make it more immune-responsive, making it easier for immune cells to infiltrate and eradicate the tumor. This combination therapy approach has shown great promise in clinical trials, increasing response rates and improving overall survival in various cancer types.

[0009] Not all patients respond to radioimmunotherapy, however, and as both immunotherapy and radiation treatment are expensive, burdensome for the patient, and challenging to combine effectively from a logistical point of view, the need exists to predict which patients are most likely to benefit from immunotherapy or radioimmunotherapy. If likely non-responders could be identified before or early on during treatment, alternative treatment modalities could be offered to these patients, while resources could be saved for the patient population most likely to benefit from immunotherapy or radioimmunotherapy.

[0010] Cancer biomechanical properties have recently emerged as a potential marker for cancer aggressiveness, progression, and therapy response. Naruse et al., coined the term mechanomedicine in 2013 to describe the medical applications of investigated mechanobiological changes mainly in reproductive and regenerative medicine. However, the terminology is also widely applicable in oncology. Various pre-clinical evidence demonstrates that cellular and extracellular matrix (ECM) stiffness plays an important role in cancer therapy response. Generally, cancer cells are much softer than the cells of the tissue of the origin, and the scale of cell softness reflects the level of malignant transformation. Contrary, the ECM becomes stiffer during malignant transformation and is generally characterized with high mechanical heterogeneity. Soft cancer cells are prone to migration and metastasis, whilst the specific mechanical arrangement of heterogenous ECM contributes to cancer cell directional migration.

[0011] The inventors’ team at the University of Basel (Switzerland) optimized the indentation type AFM (IT-AFM) method for the measurement of tissue mechanical properties at nanoscale level in fresh clinical biopsies and developed first Automated and Reliable Tissue Diagnostics (ARTIDIS) investigational device for nanomechanical profiling of clinical biopsy samples at patient bedside. ARTIDIS technology has the distinctive capability to probe the topographical and mechanical properties of fresh human tissue samples in physiological environments at the nanometer level and generate spatial nanomechanical signature within <3h time frame. Most importantly ARTIDIS tissue analysis approach is completely non-destructive and tumor biopsies can enter standard diagnostic process after ARTIDIS measurement.

[0012] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to immunotherapy treatment outcomes in cancer patients. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0013] Summary of the Invention

[0014] In one aspect, the invention relates to a method to predict outcome of cancer immunotherapy for a patient, comprising the steps of: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature. Another aspect of the invention relates to a checkpoint inhibitor agent for use in treatment of a solid tumour, wherein the checkpoint inhibitor agent is administered to a patient having been assigned a likelihood of responsiveness to cancer immunotherapy by a method according to the first aspect of the invention.

[0015] Yet another aspect of the invention relates to a method of treatment of a patient having been diagnosed with a solid tumour, said method comprising the steps: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature as specified herein; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature; applying immunotherapy only if the patient is assigned a high likelihood of being responsive to cancer immunotherapy; applying additional radiotherapy if the patient is assigned a high likelihood of being a resistant to cancer immunotherapy.

[0016] Terms and definitions

[0017] General

[0018] 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.

[0019] 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.”

[0020] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, 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.”

[0021] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0022] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] 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, organic synthesis). 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.

[0024] (Cancer) Immunotherapy

[0025] 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.

[0026] In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitor antibody is meant to encompass a cancer immunotherapy 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 inhibitor antibody is an antibody to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), VISTA (Uniprot Q9H7M9), TIGIT (UniprotQ495A1), TIM-3 (HAVCR2, Uniprot Q8TDQ0), CD158 (killer cell immunoglobulin-like receptor family), TGF-beta (P01137).

[0027] In certain embodiments, the cancer immunotherapy 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.

[0028] 1801342-60-8).

[0029] In the context of the present specification, the term checkpoint agonist agent or checkpoint agonist antibody is meant to encompass a cancer immunotherapy 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, recombinant immune stimulatory proteins, 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 (Uniprot P14784) and CD137 (4-1 BB; Uniprot Q07011), ICOS (Uniprot Q9Y6W8), 0X40 (GP34, Uniprot P43489), or CD40 (Uniprot P25942) .

[0030] In certain embodiments, the cancer immunotherapy is meant to encompass immune cell transfer cancer treatments wherein a patient’s immune cells are activated or expanded in vitro, and / or genetically modified, for example with the addition of a chimeric antigen receptor, before being infused back into the patient to inhibit neoplastic disease. Non-limiting examples of immune cell transfer therapy include chimeric antigen receptor T lymphocytes, and autologous activated T cells or dendritic cells.

[0031] 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 the signal cascade leading to T cell inhibition after T cell activation as part of what is known in the art the immune checkpoint mechanism. Non-limiting examples of a checkpoint inhibitory agent or checkpoint inhibitory antibody include antibodies to CTLA-4 (Uniprot P16410) such as exemplified by ipilimumab (Yervoy; CAS No. 477202-00-9), or antibodies to PD- 1 (Uniprot Q15116) or to PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), such as exemplified by the clinically available antibody drugs 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), and Avelumab (Merck KGaA; CAS No. 1537032-82-8).

[0032] In the context of the present specification, the term adoptive cell therapy (ACT), also used as adoptive therapy or adoptive T-cell therapy represents a recent immunotherapeutic approach within the realm of cancer treatment, which uses the potent anti-tumour capabilities of autologous (a patient's own) or allogeneic (another patient’s) immune cells by engineering or activating the cells for enhanced tumour recognition and destruction. The process of adoptive cell therapy begins with the extraction of specific immune cells, typically T cells, from a patient's bloodstream; alternatively, the cells may be derived from a cell bank. These cells are then modified ex vivo through genetic engineering techniques or activated ex vivo through various stimulation methods, to enhance their tumour-specific recognition and cytotoxicity. The genetic modification may involve the introduction of chimeric antigen receptors (CARs), which are synthetic receptors designed to target tumour-associated antigens, thus endowing the T cells with precise tumour recognition capabilities. Alternatively, T cell receptor (TCR) genes can be transduced to equip the T cells with the ability to recognize tumour-specific antigens. The engineered or activated T cells are then expanded to generate a substantial population, often referred to as a cell product. Once the cell product is prepared, it is infused into the patient. Adoptive therapy encompasses modalities such as Chimeric Antigen Receptor (CAR) T-Cell Therapy, wherein T cells are genetically engineered to express CARs specific to tumour antigens; Tumor-Infiltrating Lymphocyte (TIL) therapy; T-Cell Receptor (TCR) Therapy, which involves genetically modifying T cells to express TCRs specific for tumour antigens; Natural Killer (NK) Cell Therapy.

[0033] In the context of the present specification, the term "radiotherapy^' can particularly denote a cancer treatment approach that utilizes high doses of ionizing radiation to eliminate cancer cells or reduce tumor size. This treatment modality operates by inflicting damage to the DNA within cancer cells, thereby impeding their capacity to proliferate and divide. Although radiotherapy is primarily employed as a localized intervention targeting specific anatomical regions, it may also exert systemic effects when integrated with other therapeutic strategies, including chemotherapy or immunotherapy.

[0034] Further non-limiting examples of radiotherapy encompass external beam radiation therapy (EBRT) and brachytherapy. EBRT typically employs X-rays or gamma rays that are directed toward the cancer from outside the body. In contrast, brachytherapy involves the placement of radioactive materials, such as gamma-emitting isotopes, directly into or adjacent to the tumor. Additionally, proton therapy, a variant of external beam radiation, utilizes protons to achieve high precision in targeting tumors while preserving the surrounding healthy tissue. Radiotherapy may also be used in conjunction with other therapeutic modalities, including immune checkpoint inhibitors, adoptive cell therapy, or small molecule inhibitors, to enhance the overall efficacy of treatment.

[0035] In some embodiments, low-dose radiotherapy may be utilized, which involves administering radiation at doses lower than those conventionally employed for cancer treatment. It has been demonstrated that low-dose radiotherapy possesses immunomodulatory properties, potentially enhancing the immune system's response to cancer when used in conjunction with immunotherapies. This strategy may be especially advantageous for fostering an immune- permissive tumor microenvironment, thereby improving responses to immune checkpoint inhibitors or adoptive cell therapies. Furthermore, low-dose radiotherapy can be effectively applied in palliative care settings to alleviate symptoms in patients with advanced-stage cancer while minimizing associated toxicity.

[0036] The term cancer as used in the context of the present specification relates to malignant neoplastic disease; the terms “cancer” and “malignant neoplastic disease” are used synonymously herein. They specifically include carcinoma (epithelial derived cancer), sarcoma (connective tissue derived cancer), lymphoma and leukemia, germ-cell derived tumours and blastomas. Particular alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the compounds and compositions of the invention in treatment of solid tumours. Other alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the combinations of the invention in treatment of liquid cancers such as myelogenous or granulocytic leukemia, particularly AML, lymphatic, lymphocytic, or lymphoblastic leukemia and lymphoma, polycythemia vera or erythremia.

[0037] The term inhibitor in the context of the present specification relates to any pharmaceutically acceptable agent or compound that may be used to interact with and specifically interfere with the biological activity of its designated target (PARP, specifically PARP1 , or UBE2L3 in the case of the present specification). Inhibitors include small molecule drugs that fulfil the criteria summarized as Lipinski’s Rules of five (the drug fulfils at least three of the following rules: number of H-bond donors is < 5; number of H-bond acceptors is < 10; molecular mass is <500Da; octanol / water partition coefficient < 5). Specific examples of such inhibitors are mentioned herein.

[0038] 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 here in below.

[0039] Detailed Description of the Invention

[0040] In one aspect, the invention relates to a method to predict outcome of cancer immunotherapy for a patient, comprising the steps of: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature.

[0041] Different timepoints can be chosen to obtain the cancer biopsy tissue sample, depending on the clinical context in which the analysis is made. In certain embodiments the biopsy is taken in routine diagnostic settings, before the start of any treatment.

[0042] In certain embodiments the biopsy is taken during an on-going treatment, either as a first sample, or to allow following up on the analysis of a first sample having been obtained prior to commencement of treatment. In certain embodiments the biopsy is taken in routine diagnostic setting and follow-up biopsies are taken during treatment.

[0043] In case of biopsy following routine diagnosis (such as ultrasound, MRI, liquid biopsy, etc. that lead to taking a biopsy), there is not pre-selection of the patient, any biopsy is analysed with regard to its nanomechanical profile.

[0044] No control or healthy tissue is required. The components of the biopsy (cancer cells, stromal cells, immune cells, connective tissue, ... ) each carry their own characteristic signature and a modification or exchange of components changes to overall biopsy signature without the need for further control.

[0045] In certain embodiments, the nanomechanical profile is obtained by atomic force microscopy (AFM).

[0046] In certain embodiments, the nanomechanical profile comprises obtaining a plurality of tissue stiffness values by AFM measurement from the sample.

[0047] The whole biopsy is measured with "force maps" covering the complete sample, placing typically 10 to 30 maps, also depending on the biopsy size. At least 5 maps are taken. A single force map can be configured to measure typically 10 x 10 curves over 10 x 10 pm2up to 100 x 100 force curves over 100 x 100 urn2and combinations thereof.

[0048] The minimum amount of force curves is thus 100 force curves for a 10x10 map at 5 maps per biopsy, giving 500 force curves in total up to 12'000 force curves for example for 30 maps a 20x20 (400) force curves.

[0049] There is computation that extracts from raw force-distance curves a set of contact mechanics values, not limited to but, such as sample stiffness, elastic modulus, dissipation, adhesion and also surface topography. These derived contact mechanics values are used to derive thresholds or decision trees for predicting the response to a particular treatment.

[0050] In particular embodiments, the nanomechanical profile comprises 10 x 10 tissue stiffness values. In more particular embodiments, the nanomechanical profile comprises 20 x 20 or 24 x 24 tissue stiffness values. Even more particular embodiments have 50 x 50 or even 100 x 100 stiffness values.

[0051] In particular embodiments, the patient is assigned a high likelihood of being a responder to cancer immunotherapy if no frequency of values of modulus between 0 and 2 kPa (averaged over a range of 200 Pa) in the plurality of stiffness values exceeds a frequency of values of modulus between 3 and 4 kPa (averaged over a range of 200 Pa) by more than a factor of three.

[0052] In other particular embodiments, the assignment of the patient to a high likelihood of being a responder to cancer immunotherapy is based on the absence of “soft peak” between 0 and 2 kPa is present in the distribution of stiffness values across the sample. Such soft peak may be characterized by a median value below 1 kPa and a standard deviation of less than 0.5 kPa, or the presence of a peak which is significantly softer and narrower than a 2nd broad peak at 5kPa). The soft peak is characterized by a median value below 1 kPa and a standard deviation of less than 0.5 kPa OR significantly softer (p < 0.05 for modulus median) AND narrower (p < 0.05 for modulus Standard deviation) than the 2nd broad peak.

[0053] In particular embodiments, the patient is assigned a high likelihood of being a resistant to cancer immunotherapy if in the plurality of stiffness values, a frequency of values of modulus between 0 and 2 kPa (averaged over a range of 200 Pa) exceeds a frequency of values of modulus between 3 and 4 kPa (averaged over a range of 200 Pa) by more than a factor of three. Alternatively, this determination can be made in certain embodiments by determining if a “soft peak” between 0 and 2 kPa is present that is characterized by a median value below 1 kPa and a standard deviation of less than 0.5 kPa, or a peak which is significantly softer and narrower than a 2nd broad peak at 5kPa.

[0054] In certain embodiments, a plurality of adhesion energy values is obtained across the sample.

[0055] In particular embodiments, the patient is assigned a high likelihood of being a responder to cancer immunotherapy if in the plurality of adhesion energy values, more than 80% of adhesion energy values are below 4 x 10'16J. In other embodiments, the patient is assigned a high likelihood of being resistant to cancer immunotherapy, if in the plurality of adhesion energy values, more than 40% of adhesion energy values are above 4 x 10'16J.

[0056] In particular embodiments, a plurality of dissipation contact area values is obtained across the sample.

[0057] In some embodiments, the cancer immunotherapy comprises administration of a checkpoint inhibitor antibody agent. The checkpoint inhibitor antibody agent may be selected from, but is not limited to, a member of the group of an antibody against PD-1 , an antibody against PD-L1 , an antibody against CTLA-4, an antibody against B7H3, an antibody against VISTA, an antibody against TIGIT, an antibody against TIM-3, an antibody against CD158, an antibody against TGF- beta. In particular embodiments the checkpoint inhibitor antibody agent is selected from the group consisting of an antibody against PD-1 , an antibody against PD-L1 , and an antibody against CTLA- 4.

[0058] In some embodiments, the cancer immunotherapy is combined with radiotherapy.

[0059] Another aspect of the invention relates to a checkpoint inhibitor agent for use in treatment of a solid tumour, wherein the checkpoint inhibitor agent is administered to a patient having been assigned a likelihood of responsiveness to cancer immunotherapy by a method according to the first aspect of the invention. In other words, the nanomechanical signature disclosed herein is used to decide whether a patient is likely to benefit from checkpoint inhibitor therapy alone, or whether a more aggressive treatment, particularly by radiotherapy, is warranted.

[0060] In some embodiments, the patient has been assigned a high likelihood of responsiveness. In some embodiments, the patient has been assigned a low likelihood of responsiveness, or a high likelihood of being resistant to immunotherapy, and the administration of a checkpoint inhibitor agent is prior to, concomitant with or subsequent to radiation treatment, particularly by X-ray radiation therapy.

[0061] In particular embodiments, the checkpoint inhibitor antibody agent is selected from the group consisting of an antibody against PD-1 , an antibody against PD-L1 , and an antibody against CT LA-4.

[0062] Medical treatment

[0063] Similarly, within the scope of the present invention is a method or treating cancer in a patient in need thereof, comprising administering to the patient an immunotherapy cancer treatment, optionally in combination with radiotherapy, particularly low-dose X-ray therapy, according to the above description.

[0064] Thus, yet another aspect of the invention relates to a method of treatment of a patient having been diagnosed with a solid tumour, said method comprising the steps: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature as specified in any one of claims 1 to 12; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature; applying immunotherapy only if the patient is assigned a high likelihood of being responsive to cancer immunotherapy; applying additional radiotherapy if the patient is assigned a high likelihood of being a resistant to cancer immunotherapy.

[0065] In particular embodiments, the radiotherapy comprises low-dose X-ray therapy.

[0066] The invention further encompasses the use of nanomechanical profiling identified herein for use in the manufacture of a system enabling the prediction of outcome of immunotherapy therapy in cancer, particularly in breast cancer.

[0067] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. 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.

[0068] Description of the Figures

[0069] Fig. 1 shows understanding resistance to immunotherapy;

[0070] Fig. 2 shows ARTIDIS approach: dissecting the role of nanomechanics in response to immunotherapy;

[0071] Figs. 3a-c show the nanomechanical signature of low dose-mediated response to immune checkpoint inhibitors, immunofluorescence analysis reveals downregulated ECM degradation, reduced T cell, NK and B cell infiltration, increased presence of M2 and ARG1 + cells, Nanostring analysis shows downregulation of ECM degradation in the resistant model;

[0072] Figs. 4a, b show ARTIDIS nanomechanical signature predicts low dose-mediated response to immune checkpoint inhibitors; and

[0073] Figs. 5a-c show the nanomechanical signature of low dose-mediated CAR-T cell infiltration.

[0074] Examples

[0075] Example 1

[0076] Ectopic mice models are often employed in preclinical research to establish or confirm mechanism of action, due to the simplicity of establishment and ease of access. Immune competent models are essential to establish immune-mechanisms of tumor control. By performing early biopsies on ectopic tumor models in immune-competent mice that are treated with combination immunotherapy and radiation and followed until death or tumor resolution, we effectively established a clinical trial in mouse protocol, where the baseline ARTIDIS signature can be used prospectively to predict overall outcome and optimize therapeutic strategies based on predicted response. By performing biopsies at different times during treatment, we established a protocol to retrospectively determine early response signature to treatment, and prospectively determine if that treatment will be sufficient or if escalation is necessary.

[0077] The results in the Figures Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5 below show that detection of the ARTIDIS nanomechanical signature of imminent aggressiveness in combination with the ARTIDIS nanomechanical signature of heterogeneity supports the use of stroma remodulation agents (e.g. low-dose radiation therapy) in combination with immunotherapy (e.g. CAR-T cells and / or immune checkpoint inhibitors). Mechanistically, the use of low-dose radiation therapy acts both as an immune stimulant by promoting the release of antigens, and stroma homogenization agent to remove stromal barriers to T cell tumor penetration, enhancing T cell accumulation within the tumor region as well as T cell activation.

[0078] References US2014007309A1 (METHOD FOR STAGING CANCER PROGRESSION BY AFM; Plodinec et al., to Uni Basel).

[0079] US2017299570A1 (Method for predicting cancer progression by nanomechanical profiling; Loparic et al.; to Uni Basel)

[0080] US2020253590A1 (CORE BIOPSY NEEDLE; Plodinec et al., to Uni Basel). US2015369838A1 (METHOD AND DEVICE FOR CONTROLLING A SCANNING PROBE

[0081] MICROSCOPE; Lim et al. to Uni Basel).

[0082] All scientific publications and patent documents cited in the present specification are incorporated by reference herein or mentioned directly on figures.

Claims

Claims1 . A method to predict outcome of cancer immunotherapy for a patient, comprising the steps of: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature.

2. The method according to claim 1 , wherein the nanomechanical profile is obtained by atomic force microscopy (AFM).

3. The method according to claim 1 or 2, the nanomechanical profile comprises obtaining a plurality of tissue stiffness values by AFM measurement from the sample, particularly 10 x 10, tissue stiffness values, more particularly 20 x 20 or 24 x 24 tissue stiffness values, even more particularly 50 x 50 or even 100 x 100 stiffness values.

4. The method according to any one of the preceding claims, wherein: if in the plurality of stiffness values, no frequency of values of modulus between 0 and 2 kPa (averaged over a range of 200 Pa) exceeds a frequency of values of modulus between 3 and 4 kPa (averaged over a range of 200 Pa) by more than a factor of three (i.e. there is no “soft peak” between 0 and 2 kPa is present that is characterized by a median value below 1 kPa and a standard deviation of less than 0.5 kPa, or a peak which is significantly softer and narrower than a 2nd broad peak at 5kPa), the patient is assigned a high likelihood of being a responder to cancer immunotherapy.

5. The method according to any one of the preceding claims, wherein if in the plurality of stiffness values, a frequency of values of modulus between 0 and 2 kPa (averaged over a range of 200 Pa) exceeds a frequency of values of modulus between 3 and 4 kPa (averaged over a range of 200 Pa) by more than a factor of three (i.e. there is a “soft peak” between 0 and 2 kPa present that is characterized by a median value below 1 kPa and a standard deviation of less than 0.5 kPa, or a peak which is significantly softer and narrower than a 2nd broad peak at 5kPa), the patient is assigned a high likelihood of being a resistant to cancer immunotherapy.

6. The method according to any one of the preceding claims, wherein a plurality of adhesion energy values is obtained across the sample.

7. The method according to claim 7, whereinif in the plurality of adhesion energy values, more than 80% of adhesion energy values are below 4 x 10-16J, the patient is assigned a high likelihood of being a responder to cancer immunotherapy.

8. The method according to claim 7, wherein if in the plurality of adhesion energy values, more than 40% of adhesion energy values are above 4 x 10'16J, the patient is assigned a high likelihood of being resistant to cancer immunotherapy.

9. The method according to any one of the preceding claims, wherein a plurality of dissipation contact area values is obtained across the sample.

10. The method according to claim 9, wherein distribution of dissipation contact area values is shifted to significantly higher values relative to a sample taken earlier during or before treatment, the patient is assigned a high likelihood of being resistant to cancer immunotherapy.

11. The method according to any one of the preceding claims, wherein the cancer immunotherapy comprises administration of a checkpoint inhibitor antibody agent.

12. The method according to any one of the preceding claims, wherein the checkpoint inhibitor antibody agent is selected from an antibody against PD-1 , an antibody against PD-L1 , an antibody against CTLA-4, an antibody against B7H3, an antibody against VISTA, an antibody against TIGIT, an antibody against TIM-3, an antibody against CD158, an antibody against TGF-beta; particularly wherein the checkpoint inhibitor antibody agent is selected from the group consisting of an antibody against PD-1 , an antibody against PD-L1 , and an antibody against CTLA-4.

13. The method according to any one of the preceding claims, wherein the cancer immunotherapy is combined with radiotherapy.

14. A checkpoint inhibitor agent for use in treatment of a solid tumour, wherein the checkpoint inhibitor agent is administered to a patient having been assigned a likelihood of responsiveness to cancer immunotherapy by a method according to any one of claims 1 to 12.

15. The checkpoint inhibitor agent for use according to claim 14, wherein the patient has been assigned a high likelihood of responsiveness.

16. The checkpoint inhibitor agent for use according to claim 14, wherein the patient has been assigned a low likelihood of responsiveness, and the administration of a checkpoint inhibitoragent is prior to, concomitant with or subsequent to radiation treatment, particularly by X- ray radiation therapy.

17. The checkpoint inhibitor agent for use according to claim 14 to 16, wherein the checkpoint inhibitor antibody agent is selected from the group consisting of an antibody against PD-1 , an antibody against PD-L1 , and an antibody against CTLA-4.

18. A method of treatment of a patient having been diagnosed with a solid tumour, said method comprising the steps: subjecting a cancer tissue sample obtained from a patient ex-vivo to nanomechanical profiling to obtain a tissue stiffness nanomechanical signature as specified in any one of claims 1 to 10; assigning to said patient a likelihood of being responsive to cancer immunotherapy based on said nanomechanical signature; applying immunotherapy only if the patient is assigned a high likelihood of being responsive to cancer immunotherapy; applying additional radiotherapy if the patient is assigned a high likelihood of being a resistant to cancer immunotherapy.

19. The method according to claim 18, wherein the radiotherapy comprises low-dose X-ray therapy.

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