A method for predicting responsiveness of cancer patients to immunological treatments

Abstract

The present invention relates to a method for predicting response of a subject having cancer to an immunological treatment, the method including preparing a tumoroid from tumor cells obtained from the subject; measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9; adding to the tumoroid an immune checkpoint inhibitor (ICI); and measuring an activated level of the at least one chemokine in the tumoroid following addition of the ICI, wherein when the activated level of the at least one chemokine is increased compared to the baseline level, the subject is predicted to be responsive to treatment with the ICI; and when the activated level of the at least one chemokine is not increased, the subject is predicted to be non- responsive to treatment with the ICI and benefit from treatment with the chemokine. The present invention further provides methods of treatment based on the results of the prediction method.

Description

[0001] A METHOD FOR PREDICTING RESPONSIVENESS OF CANCER PATIENTS TO IMMUNOLOGICAL TREATMENTS

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to methods for personalized medicine, more specifically methods for predicting response to immunological therapeutics and especially to immune checkpoint inhibitors and to methods for selecting patients for immunological treatments.

[0004] BACKGROUND OF THE INVENTION

[0005] Thus far the most important breakthrough in cancer therapy has been the development of immune checkpoint inhibitors (ICI). Immune checkpoint therapy has been successfully extended to almost 20 cancer diseases, but only about 10% or less of the patients are positive responders for anti-PDl mAb therapy, which is the leading drug for most of these cancers. The most impressive success is in melanoma, with about 17% positive responders to anti-PDl, about 27% positive responders to anti-CTLA4, and about 55% to their combined therapy.

[0006] CXCR3 is a chemokine receptor with 3 ligands: CXCL9, CXCL10, and CXCL11. CXCL9 and CXCL10 bind a similar site on CXCR3, that differs from the CXCL11 binding site. CXCR3 is primarily expressed on CD4+ and CD8+ T cells, and to some extent on other cells, among them, dendritic cells, macrophages, NK cells, and epithelial cells. Within the CD4+ subset, CXCR3 is mostly abundant in effector T cells, but notably, it is also expressed by a small portion of FOXp3+ regulatory T cells (Tregs) and thus is also associated with their migration, including to tumor sites.

[0007] In numerous cancers, including melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, gastric cancer, and colorectal Cancer, high levels of CXCL9 / CXCL10 indicate good prognosis, and low levels indicate poor prognosis. Moreover, melanoma patients who express low levels of CXCL9 or CXCL10 (either blood or tumor site) are poor responders to ICI, and those with high levels of CXCL9 / CXCL10 are good responders. Likewise, NSCLC patients with high plasma levels of CXCL9 and CXCL10 displayed better responses to anti-PDl or anti-PD-Ll.

[0008] However, there is still a need both to develop a reliable method for predicting which patients should be treated by a specific ICI, as well as methods for improving the immune response of the non-responders.

[0009] SUMMARY OF INVENTION

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0011] In some embodiments, there is provided a method for predicting a response of a subject having a cancer to an immunological treatment, the method including the steps of:

[0012] (a) providing a tumoroid prepared from tumor cells obtained from the subject;

[0013] (b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;

[0014] (c) adding to the tumoroid an immune checkpoint inhibitor (ICI); and

[0015] (d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is predicted to be responsive to treatment with the ICI; and when the activated level of the at least one chemokine is not increased by the predetermined threshold compared to the baseline level, the subject is predicted to be non-responsive to treatment with the ICI and responsive to treatment with the chemokine.

[0016] In some embodiments, the immunological treatment includes an agent selected from an ICI, CXCL10 or a variant or analog thereof, CXCL9 or a variant or analog thereof, and combinations thereof.

[0017] In some embodiments, step (a) includes the steps of: obtaining tumor cells of the subject; and growing the tumor cells in a 3D culture, thereby preparing the tumoroid.

[0018] In some embodiments, preparing the tumoroid further includes adding effector cells obtained from the subject, and incubating until the effector cells infiltrate the tumoroid.

[0019] In some embodiments, adding the effector cells further includes stimulating the effector cells prior to adding them to the tumor cells. In some embodiments, the effector cells are added at a ratio of about 1:1-50:1 effector cells to tumor cells.

[0020] In some embodiments, the ICI is selected from anti-PDl, anti-PDLl, anti-PDL2, anti- CTLA4, anti-CD80 / 86, anti-LAG3, anti-Galectin3, anti-PGLl, anti-TIGIT, anti-CD112, anti- CD155, anti-CD96, anti-BTLA, anti-HVEM, anti-B7-H3, anti- VISTA, anti-Siglec-15, anti-TIM3, and combinations thereof.

[0021] In some embodiments, the ICI is added at a concentration of about 5-100 pg / ml.

[0022] In some embodiments, the predetermined threshold is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the cancer is selected from melanoma, colorectal cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin’s lymphoma, liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

[0023] In some embodiments, the subject is non-responsive to treatment with the ICI.

[0024] In some embodiments, the method further includes treating the subject with an immunological treatment, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

[0025] In some embodiments, the variant or analog of the at least one chemokine is an immunoglobulin Fc region-conjugated chemokine.

[0026] In some embodiments, there is provided a method for treating a subject having a cancer by an immunological treatment, the method including the steps of:

[0027] (a) providing a tumoroid prepared from tumor cells obtained from the subject;

[0028] (b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;

[0029] (c) adding to the tumoroid an immune checkpoint inhibitor (ICI); and

[0030] (d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

[0031] In some embodiments, when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the ICI and the at least one chemokine, or variant or analog thereof.

[0032] In some embodiments, when the activated level of the at least one chemokine is not increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the at least one chemokine, or variant or analog thereof, and the ICI.

[0033] In some embodiments, the at least one chemokine comprises both CXCL10 and CXCL9.

[0034] In some embodiments, there is provided an in vitro tumoroid system for predicting a response of a subject having a cancer to an immunological treatment, the tumoroid system including tumor cells from the subject, an immune checkpoint inhibitor (ICI), and optionally effector cells obtained from the subject.

[0035] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0038] Fig. 1 shows a schematic view of the CXCR3-CXCL9 / 10 and the PD1 - PD-L1 / 2, CTLA4 - CD80 / 86 interplay in regulating the immune response. Left: CXCL10 and CXCL9 induce IFN- y production by T cells that then enhances CXCL10 level on these T cells, resulting in an autocrinepositive loop; the production of CXCL10 by cancer cells that express the IFN-y receptor then further potentiates CD4+ and CD8+ effector cells. Right: the CXCL9 / CXCL10 interaction with CXCR3 on T cells upregulates PD1 which then interacts with PD-L1 and together with CTLA4- CD80 / 86 interaction block effector T cell function.

[0039] Figs. 2A-2B show that immune checkpoint inhibition is CXCR3 dependent. CXCR3 KO (Fig. 2A) or WT (Fig. 2B) mice were orthotopic ally injected with 0.4xl06ret melanoma cells. When tumor size reached about 50mm3they were separated into 3 identical groups of 6 mice each. Each group was treated 3 times, in 3-4 day intervals with lOOpg / mouse aPDl (squares), aCTLA4 (triangles), or with isotype matched control IgG (circles). Tumor volumes were recorded by an observer blind to the experimental protocol. Results present data of one of three independent experiments with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***P< 0.001 were considered significant.

[0040] Figs. 3A-3M show that CXCL10 and CXCL9 drive the polarization of CD4+ T cells into IFN\hlghcytotoxic T cells and Thl7 CD4+ T cells while increasing their Ki-67 and IL-2 level. Spleenocytes were obtained from naive CXCL10 knockout (KO) mice (6 per group) and sorted into CD4+ or CD8+ T cells using a MACS Easy-Sep magnetic separation kit. CD4+ and CD8+ T cells were then cultured at a density of 106cells per well, in the presence of anti-CD3 monoclonal antibody (mAb) at 5pg / ml, anti-CD28 mAb at 3pg / ml, and IL-2 at lOng / ml. Following 4 days of culture, the cells were supplemented with either 100 ng of CXCL9-Fc or CXCLIO-Fc) IgGl for 48 hours and subsequently analyzed via flow cytometry to assess the expression of CXCR3, PD1, IFN-y, Ki-67, IL-2, IL-17, Granzyme B, and Perforin. Figs. 3A-3B. CD4+ and CD8+ T cells activated with anti-CD3 from wild-type (WT, left bars) and CXCR3 knockout (KO, right bars) mice were assessed for the production of CXCL9 (Fig. 3A) and CXCL10 (Fig. 3B). Figs. 3C-3M. CXCLIO-Fc (triangles) and CXCL9-Fc (squares) drove the polarization of CD4+ T cells towards IFN\hlghThl-like cells, Thl7 effector cells, and Granzyme high cytotoxic T cells, while also enhancing expression of Ki-67 and IL-2 (control is shown in the left-most circles). Results present data of one of two independent experimnts with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***p< 0.001 were considered significant.

[0041] Figs. 4A-4K show that CXCL10 and CXCL9 drive the polarization of CD8+ T cells into IFN\hlghcytotoxic T cells and Thl7 like CD8+ T cells while increasing their Ki-67 and IL-2 expression. Splenocytes were obtained from naive CXCL10 knockout (KO) mice (6 per group) and sorted into CD8+ T cells using a MACS Easy-Sep magnetic separation kit CD8+ T cells were then cultured at a density of 106cells per well, in the presence of anti-CD3 monoclonal antibody (mAb) at 5pg / ml, anti-CD28 mAb at 3pg / ml, and IL-2 at lOng / ml. Following 4 days of culture, the cells were supplemented with either 100 ng of CXCL9-Fc (squares) or CXCLIO-Fc (triangles) for 48 hours and subsequently analyzed via flow cytometry to assess the expression of CXCR3, PD1, IFN-y, Ki-67, IL-2, IL-17, Granzyme B, and Perforin. Results present data of one of two independent experimnts with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***p< 0.001 were considered significant.

[0042] Figs. 5A-5D show that a blockade of PD1 and CTLA4 induces the CXCR3-CXCL10-IFNy cycle in a spheroids and activated CD8+ T cells setup. Fig. 5A. A scheme depicting the coculture protocol between MC38 colon cancer cell line spheroids and activated CD8+ T cells isolated from wild-type C57B1 / 6 mice. MC38 spheroids were cocultured with or without activated CD8+ T cells, supplemented with 10 pg / ml of aPDl or aCTLA4, and analyzed 24 hours post-treatment. Fig. 5B. Flow cytometry analysis conducted on the interior compartment of the spheroid. Fig. 5C. ELISA analysis of CXCL10 and IFN-y levels following the addition of aPDl or aCTLA4. In 5B and 5C, left bars: control; middle bars: anti-PDl; right bars: anti-CTLA. Fig. 5D. Monitoring of spheroid structure using a light microscope. Results present data of one of three independent experiments with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***P< 0.001 were considered significant. Figs. 6A-6P show that CXCLIO-Fc and CXCL9-Fc induce IFNyhlghand IL17hlgheffector / cytotoxic CD8+ T cells that directly limit tumor growth. C57BL / 6 mice were subcutaneously injected with 2xl05ret tumor cells overexpressing mCherry on the right flank. When tumor size reached about 50mm3, they were separated into 3 identical groups of 9 mice each. Each group was injected 3 times, with 200pg CXCLIO-Fc (triangles) or CXCL9-Fc (squares), or with an isotype matched control IgG (circles), on days 8, 10, and 13. Fig. 6A. The kinetics of tumor growth of all groups. Fig. 6B. The effect of CXCL9-Fc or CXCLIO-Fc therapy on the relative number of CD8+, CD4+, and NK cells at the tumor site (as a percentage of total CD45+ population). Figs. 6C-O. CD8+ T Cell FACS analysis. Fig. 6P. Three days after subcutaneous engraftment of the ret melanoma cell line in either C57BL / 6 wild-type (WT) or CXCR3 knockout (KO) donor mice, CD8+ T cells were isolated from the spleen and intravenously transferred (0.5 x 106cells per mouse) into recipient mice lacking CXCR3. All mice received CXCLIO-Fc treatment (40 pg / mouse) twice a week and were monitored for primary tumor development. The kinetics of tumor development in each group and scattered analysis of tumor volume on the final day of the experiment, on day 14. Circles - KO; squares - WT. Results represent data of one of three independent experiments with similar observations and are shown as mean ± standard deviation. Significance is determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***P< 0.001 were considered significant.

[0043] Figs. 7A-7O show that CXCLIO-Fc and CXCL9-Fc induce IFNyhlghand IL17hlgheffctor / cytotoxic CD4+ T cells that directly limit tumor growth. Figs. 7A-7M. CD4+ T Cell analysis of the ex vivo expriment described above with respect to Fig. 6 (in which CD8+ T cells were analyzed). Fig. 7N shows the effect of CXCLIO-Fc therapy on the selection of IL-17hlghCD4+ and CD8+ T cells. Fig. 70. CXCL9-Fc and CXCLIO-Fc limit tumor growth by directly affecting CXCR3+ CD4+ T cells. Three days after subcutaneous engraftment of the ret melanoma line overexpressing OVAII, either in OTII donor mice or in CXCR3 knockout (KO) recipient mice, CD4+ T cells were isolated from the spleen of OTII donor mice and intravenously transferred (0.5 x 106cells per mouse) to three groups of CXCR3 KO mice (7 females / group). Recipient mice were then administrated with CXCL9-Fc (squares) or CXCLIO-Fc (triangles) twice a week, 50pgr / mouse, or with isotype matched control IgG (left, circles). The kinetics of tumor development in each group and scattered analysis of tumor volume on on days 12 & 14. Results present data of one of three independent experimnts with similar observations and are shown as mean ± standard deviation. Significance is determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***P< 0.001 were considered significant.

[0044] Figs. 8A-8E show that CXCL9-Fc and CXCLIO-Fc limit tumor progression in CT26 colon cancer model. BALB / C mice were injected subcutaneously with 0.5xl06CT26 cells at the right flank followed by treatment initiation with 50pg / mouse of either CXCLIO-Fc or CXCL9-Fc three days post-tumor engraftment, along with a control group. Circles: control; squares: CXCLIO-Fc; triangles: CXCL9-Fc. Fig. 8A. Tumor Progression kinetics. Fig. 8B. Scattered analysis on day 20. Fig. 8C. Survival curve with Log Rank test analysis (Log Rank - test=0.0567). Fig. 8D. Upper panel: Histology images of CXCLIO-Fc, or CXCL9-Fc expression compared to control-Fc group using a-cMyc Ab. Lower panels: bars show that CXCL9-Fc and CXCLIO-Fc reach organs that are enriched with CXCR3+ T cells (left bars: control; middle bars: mCXCLIO-Fc; right bars: mCXCL9-Fc). Fig. 8E. Tumor Progression Rate in BALB / C Mice Treated with CXCLIO-Fc, or aPDl. BALB / C mice were injected subcutaneously with 0.5xl06CT26 cells at the right flank. When tumor size reached about 50mm3 they were separated into 3 identical groups of 6 mice each and treated with 200pg / mouse of either CXCLIO-Fc, lOOpg / mouse of aPDl, or with control Fc. Tumor Progression kinetics (left) and scattered analysis on day 20 (right). Circles: control Fc; squares: CXCLIO-Fc; triangles: anti-PDl. Results of panel A and panel B are presented as mean ± standard deviation. Each represents two independent experiments with similar data. In each experiment scoring was done by an observer blind to the experimental protocol. Significance is determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test *P< 0.05, **P< 0.01, ***P< 0.001 were considered significant.

[0045] Figs. 9A-9B. ELISA analysis of mCXCLIO expression in CT26 tumoroids which were cocultured with different ratios of activated CD8+ T cells, supplemented with 100 pg of a control IgG or anti PD1, and analyzed 72 hours post-treatment. Fig. 9A. experimental scheme. Fig. 9B. ELISA results. Effector / target (T cells / tumoroid cells) ratios: 10:1 (left panel), 20:1 (middle panel), or 30:1 (right panel). Left bars: control IgG; right bars: anti-PDl.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0048] CXCL9 and CXCL10 are chemokines secreted by immune cells, affecting effector and cytotoxic CD4+ and CD8+ T cells by binding to the chemokine receptor CXCR3. Through this receptor, they play a role in enhancing the immune response, including chemotaxis, antitumor activity, and inhibition of angiogenesis. As CXCL10 is IFN-y inducible, high IFNy secreted from activated T cells further induces CXCL10 production, not only by T cells but also by the cancer cells. As shown in Fig. 1, which summarizes the mechanism as it is understood based on the experiments disclosed herein, there is a feedback loop in which in high levels of CXCL9 and CXCL10 induce expression of checkpoint molecules such as PD1, which appear to regulate this loop by blocking production of these chemokines. Accordingly, blocking of PD1 further activates this loop.

[0049] For several cancers including melanoma and non-small cell lung cancer (NSCLC), low levels of CXCL9 or CXCL10 were found to be associated with a poor response to immune checkpoint inhibitor (ICI) such as anti-PDl and anti-CTLA4, while high levels were associated with a good response. As low levels of these chemokines are not expected to induce PD1 and CTLA4 expression, this may explain why ICIs directed against these checkpoint molecules are not effective in these patients. As proposed in previous patent applications by the inventors (WO 2021 / 260685 and WO 2021 / 260686), such patients may be treated by externally providing CXCL9 and / or CXCL10, or by a combination of ICIs with the externally provided chemokines.

[0050] In order to be able to assess which patients would not benefit from treatment with ICIs but could benefit from treatment with CXCL9 and / or CXCL10, the inventors have developed a method for predicting the effect of ICIs on tumor cells from a subject by simulating the tumor environment, thereby providing a way to select patients who are expected to respond to the tested ICI. These methods further predict the effect of supplementing the patients with CXCL9 and / or CXCL10.

[0051] The method of the invention includes preparing a tumoroid including tumor cells obtained from a biopsy of the subject, treating the tumoroid with the tested ICI, such as anti-PDl, anti- PDL1, anti-PDL2, and / or anti-CTLA4, and measuring the levels of CXCL10 and / or CXCL9 in response to the treatment. If chemokine levels are increased in response to ICI treatment of the tumoroid, then the ICI treatment is predicted to be successful and the subject may be a candidate for ICI treatment. However, if levels of the measured chemokine are not increased in response to the ICI treatment, then the subject is likely to be non-responsive to the ICI treatment. In this case, treatment with CXCL9 and / or CXCL10 may be recommended instead, or in addition to, ICI treatment.

[0052] As opposed to existing ways to measure chemokines, such as measuring in peripheral blood, which are very variable and do not necessarily correlate with the tumor environment, the method of the invention provides a simulation which is most similar to the behavior of the tumor in the subject’s body. Another advantage is that it allows testing the response to treatment before actually treating the patient, thereby preempting unnecessary side effects and costs.

[0053] A proof of concept for the method is presented in Example 3 (and Fig. 5). In this example, a coculture was prepared between an MC38 colon cancer cell line spheroids and activated CD8+ T cells isolated from wild-type C57B1 / 6 mice. Following infiltration of the T cells into the spheroids, the ICIs anti-PDl or anti-CTLA4 were added, and various cytokines were measured. As shown, the addition of the ICIs caused an increase in CXCL10, as well as other cytokines indicating an increased immune response against the tumor. This example shows that the spheroid / tumoroid system simulates the in vivo response of the immune system. Additionally, Example 6 (and Fig. 9) shows a mouse tumoroid system derived from a mouse tumor obtained by engrafting a mouse with CT26 colon cancer cells, also showing that addition of anti PD1 to the tumoroid causes increased production of mCXCLIO by the tumoroid cells. This response was proportional to the amount of effector cells added.

[0054] A method for predicting response to ICI

[0055] In some embodiments, there is provided a method for predicting a response of a subject having a cancer to an immunological treatment, the method including the steps of:

[0056] (a) providing a tumoroid prepared from tumor cells obtained from the subject;

[0057] (b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;

[0058] (c) adding to the tumoroid an immune checkpoint inhibitor (ICI); and

[0059] (d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is predicted to be responsive to treatment with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is predicted to be non- responsive to treatment with the ICI and responsive to treatment with the chemokine.

[0060] The method of the invention is intended to evaluate whether a subject having cancer is a suitable candidate for an immunological treatment.

[0061] The term “immunological treatment”, as used herein, is meant to encompass cancer treatments including ICIs and / or chemokines. More specifically, it is intended to encompass treatments including ICIs which may be tested by the method of the invention, such as anti-PDl, anti-PDLl, anti-PDL2, and CTLA4, as well as treatments including CXCL9 and / or CXCL10, or variants or analogs of these chemokines, as defined herein below.

[0062] In some embodiments, the ICI is any ICI which causes an elevation of the level of CXCL9 and / or CXCL10 as part of a successful treatment.

[0063] In some embodiments, the ICI is a checkpoint antagonist, e.g. of an inhibitory checkpoint molecule such as PD1, PDL1, PDL2, CTLA1, TIM-3, VISTA, B and T lymphocyte attenuator (BTLA), or sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15).

[0064] In some embodiments, the ICI is a checkpoint agonist, e.g. of a co- stimulatory molecule such as inducible T cell co-stimulator (ICOS), CD40, 4- IBB, or OX-40.

[0065] In some embodiments, the ICI is selected from anti-PDl, anti-PDLl, anti-PDL2, anti- CTLA4, anti-LAG3, anti-TIGIT, anti-TIM3, anti-CD80 / 86, anti-Galectin3, anti-PGLl, anti- CD112, anti- CD155, anti-CD96, anti-BTLA, anti-HVEM, anti-B7-H3, anti- VISTA, anti-Siglec- 15, and combinations thereof. In some embodiments, the ICI is selected from anti-PDl, anti-PDLl, anti-PDL2, CTLA4, and combinations thereof. In some embodiments, the ICI is anti-PDl.

[0066] The term “tumoroid”, as used herein, is the same as a “cancer organoid” or a “tumor organoid”, and relates to an organoid grown in a 3D culture from cancer cells obtained from a subject, and which self-organize into multicellular structures within the tumoroid. Tumoroids are generally known to maintain the mutational status, gene levels, and phenotypes observed in the original tumors, and therefore they are acceptable models for tumors.

[0067] Examples 3, providing a proof of concept for the method, uses a similar model - i.e. a spheroid, in which a spherical cluster is grown in suspension from an immortalized cancer cell line. In order to simulate a tumoroid, CD8+ T cells were added to the spheroid. Example 6 shows a tumoroid derived from a tumor in a mouse engrafted with CT26 colon carcinoma cells.

[0068] In some embodiments, providing the tumoroid includes preparing the tumoroid.

[0069] Preparing tumoroids may be conducted according to methods known in the art (and see Sayed et al. Adv Cancer Res 151, 345-383 (2021), and Examples 5 and 6).

[0070] In some embodiments, preparing the tumoroid includes obtaining tumor cells of the subject, such as from a biopsy. In some embodiments, preparing the tumoroid further includes breaking the tumor into individual cells by physical means (e.g. grinding or micing) and / or by enzymatic methods, such as adding collagenase or hyaluronidase. In some embodiments, preparing the tumoroid further includes growing the tumor cells in suspension (i.e., a 3D culture). In some embodiments, the tumoroid grown in suspension is passaged when the tumoroid has a size of about 200-300 pm in diameter, or once every about 7 days. In some embodiments, the tumoroid includes CD8+ T-cells.

[0071] In some embodiments, the tumoroid includes at least one cell type selected from T-cells (CD8+ T-cells and / or CD4+ T-cells), B-cells, NK cells, monocytes, and macrophages (e.g., dendritic cells).

[0072] In some embodiments, the method further includes adding factors and / or effector cells during growth of the tumoroids, i.e., during step (a), before step (b) or before step (c).

[0073] Factors which may be added include growth factors, factors promoting cell survival and differentiation, factors promoting the formation of spheroids, and factors inhibiting apoptosis and cell death. Nonlimiting example for suitable factors include EGF, Rho-kinase inhibitor Y-27632, nicotinamide, and A83-O1.

[0074] Effector cells are cells that have an immune function, and which participate in the immune response. In some embodiments, the effector cells are selected from peripheral blood lymphocytes (PBL)s, T-cells (CD8+ T-cells and / or CD4+ T-cells), B-cells, NK cells, monocytes, and macrophages (e.g., dendritic cells).

[0075] In some embodiments, the effector cells are obtained from the subject. However, the effector cells may also be obtained from a different source, such as from a different subject, from pooled cells, or from a commercial product.

[0076] In some embodiments, the method further includes a step of stimulating the effector cells. In some embodiments, the stimulating is done by incubating the effector cells with a composition including anti-CD3, or a combination of anti-CD3 and IL2. In some embodiments, the step of stimulating is done before adding the effector cells during tumoroid growth. In some embodiments, the step of stimulating is done after adding the effector cells during tumoroid growth.

[0077] In some embodiments, the method further includes a step of incubating the effector cells with the tumoroid until the effector cells infiltrate the tumoroid.

[0078] In some embodiments, the effector cells are added at a ratio of about 1:1-100:1 effector cells to tumor cells. In some embodiments, the effector cells are added at a ratio of at least about 1:1, 10:1, 20:1, or 30:1 effector cells to tumor cells.

[0079] The term “baseline level” relates to level of the chemokines CXCL9 and CXCL10 prior to adding ICI to the tumoroid, reflecting the endogenous level of these chemokines in the tumoroid.

[0080] The term “activated level” relates to level of the chemokines CXCL9 and CXCL10 following addition of ICI to the tumoroid, reflecting the level of these chemokines in response to the ICI.

[0081] It is appreciated that measuring baseline levels and measuring activated levels may be done at different times, e.g., measuring baseline level before adding ICI, and measuring activated levels after adding ICI (e.g., for the same tumoroid). Alternatively, the tumoroid may be divided into at least two tumoroids, or more than one tumoroid may be grown from the cells of the same tumor, and measuring baseline and activated levels may be done in parallel, by testing a tumoroid treated with ICI and another tumoroid of the same tumor not treated with ICI.

[0082] Measuring baseline levels and activated levels may be done by any suitable method known in the art, including by immunofluorescence (e.g. by using an fluorescence-activated cell sorter (FACS)), enzyme-linked immunosorbent assay (ELISA), etc. Usually, antibodies against CXCL9 or CXCL10 would be used in such assays to identify the target.

[0083] In some embodiments, the ICI is added to the tumoroid at a concentration of about 5-100, 10-50, or 10-20 pg / ml. In some embodiments, when the ICI is aPDl or aCTLA4, the ICI is added to the tumoroid at a concentration of about 10 pg / ml.

[0084] After obtaining both the baseline level and the activated level of the at least one chemokine, the levels are compared to determine whether the ICI caused an increase in the chemokine levels above a certain threshold, indicating that treatment of the subject with the ICI is expected to have the same effect.

[0085] In some embodiments, the predetermined threshold is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0086] While an increase above the threshold is predictive of a positive response to the ICI, when the chemokine levels are not increased at least by the predetermined threshold, this indicates that the subject is likely to be unresponsive to treatment with the ICI, and also that the subject may benefit from administration of the chemokine instead.

[0087] As noted above, a direct correlation between CXCL9 / CXCL10 levels and prognosis for ICI treatment has been found with various cancers.

[0088] In some embodiments, the cancer is selected from melanoma, colorectal cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin’s lymphoma, liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

[0089] In some embodiments, the subject is already known to be non-responsive to the ICI. In this case, the method of the invention predicts the suitability of the subject for treatment by administration of CXCL9 and / or CXCL10.

[0090] A method for predicting response to a chemokine

[0091] In some embodiments, the present invention further provides a method for predicting a response of a subject having a cancer to a chemokine, the method including the steps of: (a) providing a tumoroid prepared from tumor cells obtained from the subject;

[0092] (b) measuring in the tumoroid a baseline level of at least one molecule associated with immune activation related to CXCL9 and / or CXC110, such as IFN-y, Granzyme-B, Peripherin, Ki67, CXCR3, IL-2, and CD69.

[0093] (c) adding to the tumoroid a chemokine selected from CXCL10 and CXCL9; and

[0094] (d) measuring an activated level of the molecule associated with immune activation following the addition of the chemokine, wherein: when the activated level of the at least one molecule associated with immune activation is increased by at least a predetermined threshold compared to the baseline level, the subject is predicted to be responsive to treatment with the chemokine; and when the activated level of the at least one molecule associated with immune activation is not increased by the predetermined threshold compared to the baseline level, the subject is predicted to be non-responsive to treatment with the chemokine.

[0095] Definitions, terms, and embodiments mentioned above and which may be relevant to the present embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0096] In some embodiments, the method of predicting a response to a chemokine includes a further step after step (d), including treating the subject with the chemokine.

[0097] Methods of treatment including patients selection

[0098] In some embodiments, the method further includes treating the subject with an immunological treatment. The treatment depends on the result of the method, namely: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

[0099] In some embodiments, there is provided a method for treating a subject having a cancer by an immunological treatment, the method including the steps of:

[0100] (a) providing a tumoroid prepared from tumor cells obtained from the subject;

[0101] (b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;

[0102] (c) adding to the tumoroid an ICI; and

[0103] (d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

[0104] Definitions and embodiments mentioned above and which may be relevant to the method of treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0105] In some embodiments, when the subject is treated with the ICI, the subject may further be treated with the chemokine. Accordingly, when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the ICI and the at least one chemokine, or variant or analog thereof.

[0106] In some embodiments, when the subject is treated with the chemokine, the subject may further be treated with the ICI. Accordingly, when the activated level of the at least one chemokine is not increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the at least one chemokine, or variant or analog thereof, and the ICI.

[0107] In some embodiments, the at least one chemokine comprises both CXCL10 and CXCL9. In some embodiments, the at least one chemokine comprises CXCL9. In some embodiments, the at least one chemokine comprises CXCL10.

[0108] The expression “variant or analog thereof’, as used herein, is intended to encompass proteins which are not identical to the natural proteins CXCL9 and CXL10 (the human wild-type proteins, e.g., as defined in GenBank accessions NP_002407 and NP_001556, respectively), but are similar in sequence (such as having at least 80%, or 90% sequence identity) and have the same function, including binding to the CXCR3 receptor and activating immune cells, as further detailed below.

[0109] WO 2021 / 260685 and WO 2021 / 260686 disclose various variants of CXCL9 and CXCL10, specifically modifications and fusion proteins thereof.

[0110] Modified proteins disclosed in WO 2021 / 260685 include, for example insertion of nucleotides, such as insertions at the N-terminal end of the mature human CXCL10 protein sequence (between positions 21 and 22 of the precursor protein including signal peptide), more specifically insertions of glutamine, asparagine, proline, phenylalanine, leucine, isoleucine, valine, tyrosine, histidine, lysine, aspartate, glutamate, arginine or glycine. This document further discloses amino acid substitutions, e.g. R5H and R5K (of the mature protein).

[0111] Modified proteins disclosed in WO 2021 / 260686 include, for example insertion of nucleotides, such as insertions at the N-terminal end of the mature human CXCL9 protein sequence (between positions 22 and 23 of the precursor protein including signal peptide), more specifically insertions of glutamine, asparagine, proline, phenylalanine, leucine, isoleucine, valine, tyrosine, histidine, lysine, aspartate, glutamate, arginine or glycine.

[0112] Additionally these two publications include modified proteins which are fusion proteins of CXCL9 and CXCL10 with immunoglobulin Fc regions. More specifically, various options for Fc configurations are mentioned, such as Fc from IgG, Fc including hinge-CH2-CH3 regions (heavy chain CHI region deleted to prevent antibody-dependent cellular cytotoxicity (ADCC)), and GC- rich linkers linking the chemokines to the Fc region.

[0113] Accordingly, the variants or analogs of the chemokines, as mentioned herein, are intended to encompass the wild-type CXCL9 and CXCL10 proteins, as well as all of the modified and / or fused proteins mentioned in WO 2021 / 260685 and WO 2021 / 260686 or otherwise known. Additionally, the variants and analogs of the mentioned chemokines are further intended to encompass obvious alternatives of the above-mentioned wild-type of modified proteins, including sequences having at least about 90, 95, or 99% sequence identity to the protein sequence of any of the wild-type of modified proteins mentioned above; sequences having at least about 90, 95, or 99% sequence identity to the DNA sequence encoding any of the wild-type of modified proteins mentioned above; and / or sequences which cover a length of at least about 90, 95, or 99% of the length of any of the wild-type of modified proteins mentioned above, as long as they are capable of carrying the same functions, including binding to the CXCR3 receptor and activating immune cells. To further clarity the intended scope of the variants, also encompassed are proteins, variants, or analogs according to the above definitions, which include at least one non-conventional amino acid, amino acid analog, or modified amino acid. Especially noted are sequences which are modified for practical reasons to facilitate production, or large-scale production, or stability, of the proteins, as long as the produced proteins comply with the above definitions. One specific modification intended to be covered by the invention is amino acid insertions, deletions, substitutions, or modifications, which render the protein resistant to cleavage by dipeptidyl peptidase 4 (DPP4, also known as CD26) that acts on the proline at position 2 and cleaves the two N-terminal amino acids, resulting in a nonfunctional protein that may also act as an antagonist to intact the chemokine.

[0114] In some embodiments, the chemokine is a human chemokine.

[0115] In some embodiments, the chemokine is a nonhuman chemokine. In some embodiments, the subject is treated with a chemokine and / or ICI included in a pharmaceutical composition further including a pharmaceutically acceptable carrier.

[0116] In some embodiments, the pharmaceutically acceptable carrier is a buffer, diluent, adjuvant, excipient, or vehicle suitable for administration with the peptide of the invention. In some embodiments, the pharmaceutically acceptable carrier may be suitable for intravenous infusion. In some embodiments, the pharmaceutically acceptable carrier may be suitable as a cryoprotectant. In some exemplary embodiments, the carrier may be DMSO (for example, at about 10%). In some embodiments, the pharmaceutically acceptable carrier may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0117] The term “treating”, as used herein, refers to means of obtaining a desired physiological effect, in this case, ameliorating the symptoms of the cancer, or reducing tumor size.

[0118] The administering may be by any method or route suitable for treating the cancer. The administration regimen may be determined by a physician based on the condition of the subject.

[0119] The administration may be a systemic or a local administration. Non-limiting examples for administration routes include intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, oral, sublingual, enteral, intranasal, buccal, vaginal, rectal, intraocular, intrathecal, topical, transdermal, and intradermal administration.

[0120] In some embodiments, the route of administration is selected from intravenous, subcutaneous, intramuscular intratumoral, intraperitoneal, intranasal, intratumoral, and topical administration.

[0121] A tumoroid system

[0122] Tumoroids are 3-D cultures that resemble organoids and are commonly used for drug testing. They could be generated from biopsy samples and best represent the tumor set up (including tumor microenvironment) in an in vitro system.

[0123] In some embodiments, there is provided an in vitro tumoroid system for predicting a response of a subject having a cancer to an immunological treatment, the tumoroid system comprising tumor cells from the subject, an immune checkpoint inhibitor (ICI), and optionally effector cells obtained from the subject.

[0124] Definitions and embodiments mentioned above and which may be relevant to the method of treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0125] 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 to which the invention pertains.

[0126] The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0127] The term "about" when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0128] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0129] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0130] EXAMPLES

[0131] Materials and Methods

[0132] Mice strains and maintenance:

[0133] In all experiments, 8-10 weeks old female mice were used. C57BL / 6 (wild-type (WT)) and BALB / C (WT) mice were purchased from Harlan (Israel). CXCR3 KO mice, CXCLIOKO mice, OTII mice, and OTI mice were purchased from JAX lab (Bar Harbor, Maine). All mice were maintained under Individually Ventilated Caging Systems (IVC). All experiments were approved by the Technion Committee for Care and Use of Laboratory Animals which operates under the NIH guideline (Technion animal experimentation protocol No: IL-072-05-2021, valid until June

[0134] 2025 ).

[0135] Cell lines and culture Ret (murine melanoma cell line) were kindly provided by Prof. Neta Erez from Tel Aviv University, with permission of Prof. Viktor Umansky (DKFZ). CT26 (murine colon cancer cell line) and MC38 colon cancer cell line were purchased from ATCC. Cells were routinely tested for mycoplasma contamination and maintained under 37°C and 5% CO2 conditions in RPMI Medium 1640 (Gibco, Rhenium, Israel, Cat. 21875034). Cho-Ki cells were purchased from Perkin Elmer (USA) and maintained under 37°C and 8% CO2 conditions in F-12 (Nutrient Mixture F-12(Ham), Gibco, Rhenium, Israel, Cat. 21765029). All media were supplemented with 10% fetal calf serum (Fetal Bovine Serum Heat Inactivated, Merck Fife Science, Israel, Cat. F9665-500ML), 1% L- glutamine (Gibco, Rhenium, Israel, Cat. 25030024), and 1% Pen-Strep solution (Gibco, Rhenium, Israel, Cat. 15140122).

[0136] Tumor engraftment.

[0137] Ret and CT26 cells were collected after trypsinization, washed with PBS, and resuspended in lOOpl PBS per mouse (4.5xl05 / 100pE in PBS). Cells were injected subcutaneously into the right flanks of 6-8 week-old female C57BL / 6, CXCR3KO, or BALB / c mice. In all experiments, when tumors reached a volume of ~50 mm3, mice were randomized into different experiment groups, monitored daily for evidence of illness, and treated with mouse anti-PDl (clone RMP1- 14, BioXCell Cat. BE0146), mouse anti-CTLA4 (clone 9H10, BioXCell Cat. BE0131) antibodies and mouse CXCEIO-Fc and CXCE9-Fc proteins. Control groups were treated with IgG isotype control (BioXCell Cat. BE0089). Tumor volume was measured manually using an electronic caliper. Tumor volume was calculated using the formula 7t / 6 x a x b x c, where a represents the length, b represents the width and c represents the height of the tumor.

[0138] Western bloting:

[0139] Protein samples were separated on 4%-20% SDS-PAGE mini gel (GeneScript) and transferred to nitrocellulose membrane using a semi-dry blotter (Trans blot Turbo transfer system, Biorad) at 1.3A / 25V per mini gel for 7 min, in transfer buffer (250mM Glycine, 25mM Trizma base, 20% methanol). The membrane was blocked with 1% BSA in PBS for Ih at RT and incubated with 1:5000 anti-HIS tag HRP-conjugated primary antibody, diluted in blocking solution. The membrane was shaken for 2 hours at RT, followed by three washes with TBST (150mM NaCl, 20mM Tris-HCl pH=8, 0.1% Tween 20), incubated for 1 min in EZ-ECE solution (Advansta K- 12045-D20) and developed on VILBER Fusion FX7 Machine.

[0140] Cytokines quantification by indirect ELISA ( sandwich )

[0141] Cytokine concentrations in culture media were assessed using commercial ELISA kits: Murine IP- 10 (CXCL10) Standard ABTS ELISA Development Kit, (900-K153, Peprotech), Human IP- 10 (CXCL10) Standard ABTS ELISA Development Kit (900-K39, Peprotech), ELISA Kit for MOUSE CXCL9 (UCSN SEB928Mu), Human MIG (CXCL9) Standard ABTS ELISA Development Kit (900-K87, Peprotech) and Murine IFN gamma Standard ABTS ELISA Development Kit, (900-K98, Peprotech) according to manufacturer's instructions.

[0142] Spleenocyte isolation and activation

[0143] CD4+and CD8+T cells were isolated from whole spleens by EasySep™ CD4+(StemCell Technologies, Cat. 19852A ) or CD8+(StemCell Technologies, Cat. 19853A ) T cells enrichment magnetic beads according to manufacturer standard protocol. Spleenocytes were activated with 5 pgr / ml of anti-mouse CD3s (Biolegend Cat. 100302), 3 pgr / ml of anti-mouse CD28 (Biolegend Cat. 102116) and 10 ng / ml of Murine IL-2 (Peprotech Cat. 212-12-20). They were separately cultured in vitro in DMEM media.

[0144] TILs isolation

[0145] TILs isolation was performed as follows: 1 gr of tumor tissue was cut into < 5mm pieces, transferred into a GentleMacs tube (Cat. 130-096-334 purple cap) containing 5ml of cold RPMI 1640 medium, supplemented with 20% FCS, 2% P / S and run on the GentleMacs device, program m_impTumor_02*, for three times. 75 pl of freshly prepared collagenase I solution (Sigma, Cat. c0130) and 150 pl Dispase II solution (Roche, Cat. 04942078001) were added to the minced tissue and incubated at 37 °C, on a shaker 1 lOrpm, 40min. The tubes were run again on the GentleMacs device, program m_impTumor_04*, three times.

[0146] The liquid was passed through a 40pm cell strainer (Bar Naor, Cat. BN93040S), washed with cold PBSxl, and completed the volume to 25ml with cold PBSxl in a 50 ml tube. The solution was centrifuged at 1500 rpm, for 5 min, at 4°C and resuspended with 5 ml of cold RBC lysis (41.4 gr NH4CI buffer, 5 gr NaHCOs, 0.189 gr EDTA, 500 ml DDW) vortexed and incubated for 7 min at RT. 45 ml of cold PBSX1 was added to the tube, centrifuged at 1500 rpm, for 5 min, at 4°C, and resuspended with the FACS buffer (500ml PBSX1, 10 ml FCS, 10 ml 50Mm EDTA) / medium for further procedure. Spleenocyte isolation was performed as follows: a spleen was minced in 10 ml of cold PBSX1 using a 40pm cell strainer (Bar Naor, Cat. BN93040S), centrifuged at 1500 rpm, for 5 min, at 4°C and resuspended with 0.5 ml of cold RBC lysis buffer (Rhenium, Cat. 00-4333- 57), for 20 seconds at RT. The final volume was completed to 10 ml with PBSX1 and centrifuged again under the same conditions. The pellet was resuspended in 2 ml of FACS buffer for further analysis.

[0147] Flow cytometry acquisition and analysis.

[0148] Before immunostaining, cells were plated at a density of IxlO6cells / well in an appropriate medium and stimulated for 4-6 hours under 37 °C and 5% CO2 conditions with Cell Activation Cocktail (Biolegend 423303). Tumor and spleen cells were immuno-stained for the following surface and intracellular markers. Cell permeabilization was carried out using the BD Biosciences kit (BD Biosciences 554714) according to the manufacturer’s instructions. All monoclonal antibodies were purchased from BD Biosciences and BioLegend. Flow cytometry data was acquired on BD LSRFortessa using and analyzed with FlowJo V.10 software (FlowJo, Ashland, Oregon, USA).

[0149] Paraffin embedding of Tumor, Spleen, and Liver tissues

[0150] Tumor and spleen tissues were immediately fixated in 4% formaldehyde PH=7.2 overnight, the next day they were placed in tissue cassettes and kept in ethanol 70% overnight. The next day, Tumor tissue was subjected to the processes of dehydration - three exchanges of ethanol 95% (20min each), three exchanges of ethanol 100% (20min each), clearing - two exchanges of chloroform (lOmin each), and embedding - two exchanges of paraffin (Ih each, 60°C), followed by a third exchange of paraffin overnight. Tumor tissue was then molded into paraffin blocks and 5pm sections were made, and were let dry overnight at 37°C until stained.

[0151] Immunohistological staining

[0152] Slides were de -paraffinized at 600C for 1 hour and incubated in K-Clear solution for 2 repeats of 5 min each and in 100% EtOH for 2 repeats of 5 min each. Endogenous peroxidase blocking was performed using freshly prepared 100% Methanol with 1% H2O2, followed by 1-2 min wash in 70% EtOH and 3x rinsing with DDW. Antigen retrieval was performed by microwave boiling the slides in 10 Sodium Citrate buffer, pH 6 for 22 minutes. The slides were slowly cooled down to RT and washed twice with PBSX1. Blocking was performed using 10% normal goat serum, for 1 hour at RT. Following blocking, slides were incubated with primary antibodies, overnight, at 40C. On the next day, the slides were washed 4x5 min in PBS. 2-3 drops of HISTOFINE Simple Stain Max Po (Multi) Universal Immuno peroxidase Polymer anti- rabbit / mouse (Nichirei) were added followed by 1-hour incubation, RT. Washed 4x5 min in PBS. Incubation with HISTOFINE Simple Stain AEC Solution (10 min, RT) (Nichirei). Washed until clear and counter- stained for 30 sec with hematoxylin. Washed and left in DDW for 10 min, at RT. Mounted and sealed with mounting medium (Immuno Mount, Thermo Shandon). Statistical analysis was performed using Fiji software.

[0153] Calcium assay:

[0154] Calcium assay was performed in CHO cells which overexpress human CXCR3A. GPCR stimulation with a CXCR3A ligand induces Ca++ flux. The assay is based on a reporter system that includes calcium binding to the aequorin oxidation of coelenterazine which leads to the emission of light (469nm). Ca++ flux in CHO cells was induced by 100 ngr of CXCL10 (Peprotech) and CXCL9 (Peprotech) chemokines and detected using Calcium Assay Kit (Zotal, AB-abl 12114-10,). The OD measurements were performed on a plate reader (Infinite M200 PRO).

[0155] Adoptive transfer protocol:

[0156] Three days after ret melanoma line engraftment in either WT or CXCR3KO donor mice, CD8+ T cells were isolated from the spleen and transferred (0.5X106cells per mouse) into CXCR3KO mice 3 days following engraftment with ret melanoma line. All mice were treated twice a week with mCXCLIO-Ig (200pg / mouse) monitored for primary tumor development. On day 20, mice were sacrificed and tumor weight was measured.

[0157] Expression and Purification of Fusion Proteins

[0158] Fusion proteins were expressed and purified using the Expi293™ Expression System according to the protocol (Thermo Fisher Scientific, Cat. Number A14635, Publication Number MAN00 19402) and purified on the Ni-NTA column

[0159] Spheroids

[0160] MC38 spheroids were generated by seeding 1000 cells per well on Nunclon Sphera (ThermoFisher) round bottom 96 wells plates in complete DMED medium. 4 days later, cocultures were started by adding 10xl03total of activated CD8+ T cells extracted from WT C57BI / 6 mice, per well. 24 hours later, 10 pgr / ml of anti PD1 or anti CTLA4 or PBS were added to each well. For flow cytometry analyses, 12 wells per condition were seeded and divided into 4 groups. Spheroids were isolated from wells, gently resuspended, and trypsinized to obtain a single-cell suspension further analyzed by flow cytometry.

[0161] Statistical analysis

[0162] Statistical analyses were performed using Graph Pad Prism software version 8.0. For comparison, two samples mean t-test was used, and for multiple experiments, the statistical method of choice was the one-way analysis of variance (ANOVA) test using the Tukey multiple comparisons test, log-rank test was used to compare the survival distributions of two samples. P< 0.05 was considered statistically significant.

[0163] Example 1: Immune checkpoint inhibition is CXCR3 dependent.

[0164] The response to anti-PDl and anti-CTLA4 in WT and CXCR3 knockout (KO) mice has been evaluated using a melanoma model based on the Ret transgenic mouse model. A skin malignant melanoma that is characterized by the overexpression of the human Ret transgene in melanincontaining cells. These transgenic mice spontaneously develop skin tumors with metastases in lymph nodes, lungs, liver, brain, and bone marrow. A low passage pre-line has been isolated from these mice, and then subjected to overexpression of mCherry and is currently being used as a highly reliable model for autotropic melanoma, with clear metastatic spread.

[0165] Figs. 2A-2B show that blockade of either PD 1 or CTLA4 had a significantly lower effect on tumor development in CXCR3 KO mice (Fig. 2A), as opposed to WT mice (Fig. 2B, P<0.05 for CTLA4 and P<0.01 for PD1 blockade). From the translational perspective, the findings that CXCL101hlgh / CXCL9hlghpatients display less severe forms of several cancer diseases, and also that melanoma patients that are CXCL101hlghand / or CXCL9hlghare good responders to anti-PDl, may suggest that the major role of the PD 1 and CTLA4 axis is to regulate the CXCR3-CXCL9 / CXCL10 interplay.

[0166] Example 2: CXCL9 and CXCL10 directly induce different subtypes of effector and cytotoxic CD4+ T cells and CD8+ T cells in a self-feeding loop and are likely to be regulated by anti- PDl.

[0167] A major drawback in all in vitro studies thus far has been that CXCR3 ligands, particularly CXCL10, are also produced, to a varying degree, by the cultured T cells undergoing activation. The inventors therefore generated a setup (see Fig. 3 description) in which the response to an added CXCR3 ligand would be recorded in the absence of any endogenous CXCR3 ligands. Under these conditions, upon induced activation by anti-CD3 & anti-CD28 mAbs CD4+ T cells and CD8+ T cells produce CXCL10, but not CXCL9, whereas CD4+ T cells and CD8+ from CXCL10 KO mice do not produce CXCL10 and there is no compensation by CXCL9 (Figs. 3A-3B). C57BL / 6 mice also do not produce CXCL11 due to a mutation in the open reading frame of CXCL11. This setup was used for further analysis of the differential contribution of CXCL9 and CXCL10 to the biological characteristic of CXCR3+ T cells undergoing activation. Figs. 3C-3M show the differential effect of CXCL10 (right bars - triangles) and CXCL9 (middle bars - squares) on cultured CD4+ T cells, and Figs. 4A-4K show the corresponding analysis of CD8+ T cells. The effect of CXCL9-Fc and CXCLIO-Fc on T cell polarization of CD4+ and CD 8+ T cells is similar, with minor differences. For both, it mostly includes upregulation of ki-67 (Fig. 3D, Fig. 4B), IFN- y (Fig. 3E, Fig. 4C), and IL-2 (Fig. 3F, Fig. 4D) indicating a Thl-like CD4+ T cells and IFN-Yhlgl1effector / cytotoxic CD8+ T cells selection. Additionally, a significant increase was seen in Thl7- like CD4+ T cells and ILl-17-producing CD8+ T cells, also known as Tcl7 cells (Fig. 3H, Fig. 4F) that are known to hold significant anti-tumor properties. Collectively it is implied that both CXCL9 and CXCL10 induce proliferation and increased effector function in both CD4+ T and CD8+ T cells, with relevance for cancer therapy. Remarkably, CXCL9 and CXCL10 induce IFN- y expression in both CD4+ T cells and CD8+ T cells (Fig. 3E and Fig. 4C), however, CXCL10 is IFN-y inducible. This suggests a self-feeding loop that amplifies effector / cytotoxic T-cell functions. Further analysis shows that this interaction also upregulates the expression of PD1 (Fig. 3J, Fig. 4H), possibly counterbalancing this loop. Moreover, a comparative analysis of IFNy upregulation and PD1 expression shows their elevated expression in CXCR3+ T cells (Figs. 3K- 3L, Figs. 4I-4J), as well as PD1 expression on IFN-y111811producing cells (Fig. 3M, Fig. 4K).

[0168] Example 3: Crosstalk between cancer and the immune cells extends the self- feeding loop and its association with immune checkpoint blockade.

[0169] To evaluate the possible extension of the CXCLIO-IFN-y axis to cancer cells (since CXCL10 is IFN-y inducible) it was first examined whether IFNy also induces CXCL10 production in various murine and human cancer cell lines. In four out of five different cell lines that were examined, IFN-y significantly increased CXCL10 production and did not affect CXCL9 production (not shown). Remarkedly, human A375 melanoma cells produce a similar base level of CXCL10 and CXCL9 (about 200 pg / ml) but in response to IFN-y it shows about a 6-fold increase in CXCL10 production, but no increase in CXCL9 production (not shown). To detect how anti-PDl or anti- CTLA4 checkpoint blockade would interfere in the above cycle, a 3D spheroid system was used (Friedrich et al. (2009). Nat Protoc 4, 309-324), including spheroids and CD8+ T cells from MC38 cancer-developing donors as described in Fig. 5A. Cultures were or were not supplemented with 10 pg / ml aPDl or aCTLA4 and analyzed 24h later by flow cytometry, or by ELIZA (Figa. 5B- 5C). The results clearly show that blockade of either PD1 or CTLA4 triggers the IFNy-CXCLlO- CXCR3 cycle that includes a significant increase in the relative number of CD8+ T cells, their proliferation rate (Ki67+), CXCR3 expression by CD8+ T cells, IFNy production, and CXCL10 production. Collectively, this implies that immune checkpoint inhibitors induce the IFNy- CXCL10- CXCR3 cycle and its amplification is in a CXCLIO-dependent manner. CXCL9 is not produced by CD8+ T cells or cancer cells and so it has not been investigated as part of the loop but should be taken into consideration at the tumor site where it is largely produced by CD 106+ DC.

[0170] Example 4: Administration of CXCLIO-Fc and CXCL9-Fc limits melanoma while selecting subtypes of effector and cytotoxic CD4+ and CD8+ T cells with an IFN-y111"11signature. that limit cancer

[0171] The inventors have previously developed a fusion protein that includes a murine IgGl Fc linked to CXCL10 (CXCLIO-Fc) for cancer therapy and a CXCLl l-Fc for therapy of autoimmunity (Zohar et al. (2014), J Clin Invest 124, 2009-2022; Barash et al. (2014), Leukemia 28, 2178-2187). Both fusion proteins, as well as CXCL9-Fc lack the CHI domain to limit possible induction of ADCC. Before being administered during cancer development, each purified fusion protein was subjected to Western Blot analysis and Ca++ flux using CHO cells overexpressing human CXCR3A (not shown). Then each fusion protein was administered to mice engrafted with the ret melanoma cell line. Fig. 6A shows that CXCL9-Fc and CXCLIO-Fc significantly limited tumor growth (Fig. 6A, day 13 P<0.05). To test whether the ex-vivo setup showed a similar effect as that observed in the in vitro setup, the effect of therapy on the relative number of CD8+ T cells, CD4+ T cells, and NK cells (out of total CD45+ cells) at the tumor site was evaluated. The relative number of CD8+ T cells increased from about 3% to about 20% and 15% following CXCL9-Fc or CXCL120-Fc administration, and for CD4+ T cells from about 5% to about 18% and 13% accordingly (Fig. 6B). As for NK cells, their relative number in control mice was about 1.8% in control mice and increased about 2-fold in mice treated with CXCLIO-Fc (Fig. 6B).

[0172] As for CD8+ T cells, not only their relative number dramatically increase, but also within the CD8+ T cells the relative number of tumor- specific T cells, as determined by TRP-2 pentamers, show about 12-folds and a 7-fold increase in CXCL9-Fc and CXCLIO-Fc treated mice (Fig. 6C). A 3-fold increase was also recorded in the expression of CXCR3 on these cells (Fig. 6D). An about 5-fold increase in Ki67 accompanied by s similar increase in IL-2 production indicates an increase in the proliferation and activation state of these cells (Figs. 6E-6F). Most importantly, both CXCL9-Fc and CXCLIO-Fc polarized T cells with IFN-y high signature (Fig. 6G). Further analysis of IFNy Vs IL-2 (Fig. 6H) indicates a clear association between increased IL-2 production and IFN-y production in these cells. A very similar increase in Perforin and Granzyme-B, as well as the analysis of PerforinhlghGranzyme-BhlghCD8+ T cells (Figs. 6I-6K), shows that 20% of CD8+ T cells induced by CXCL9-Fc or CXCLIO-Fc are cytotoxic CD8+ T cells, compared to less than 3% in control mice. Finally, as a possible counter mechanism, CXCL9-Fc and CXCLIO-Fc led to a significant increase in PD1 expression from about 2% to about 10% in CXCL9-Fc and about 7% in CXCLIO-Fc treated mice (Fig. 6L). The significant increase in PD1 expression, IL- 2, and IFN-y are all associated with increased CXCR3 expression on CD8+ T cells (Figs. 6M-6O). In conclusion, systemic administration of either CXCL9-Fc or CXCLIO-Fc markedly increases the relative number of activated highly potent tumor- specific CD8+ T cells at the tumor site, and to some extent PD1 expression on about 7-10% of these cells.

[0173] To test the importance of the direct effect of CXCLIO-Fc on the ability of CXC3+ CD8+ T cells to limit cancer, an adoptive transfer set-up in which CD8+ T cells were isolated from the spleen of either WT or CXCR3 KO tumor-developing mice and transferred into cancer-developing CXCR3 KO recipient mice was conducted (explained in more detail in Fig. 6P). Both recipient groups were then treated with CXCLIO-Fc. Only the administration of CXCLIO-Fc to mice transferred with CXCR3+ CD8+ T cells led to a significant decrease in tumor Vol and weight (Fig. 6P). The results represent data from one of two independent experiments with very similar data. In conclusion, CXCL10, which directly polarizes and potentiates CXCR3+ CD8+ T cells (see Fig. 4) also directly potentiates these cells to limit melanoma. An in vivo adoptive transfer experiment with CXCL9-Fc instead of CXCLIO-Fc is also conducted, as described above, mutatis mutandis.

[0174] Figs. 7A-7M show the corresponding analysis of CD4+ T cells, as was done for CD8+ T cells (Figs. 6C-6O) in the same experimental setup. The results are very similar to those obtained for CD8+ T cells and include a highly significant increase in CXCR3 expression, with about 6- folds following CXCL9-Fc therapy, and about 10 folds following CXCLIO-Fc therapy (Fig. 7A). A significant upregulation in T cell proliferation (Ki67, Fig. 7B), IL-2 (Fig. 7E), and IFNy production (Thl polarization) (Fig. 7D) and polarization of PerforinhlghGranzyme Bhlghcytotoxic CD4+ T cells (for Granzyme-B from about 2% to about 12% in CXCLIO-Fc treated mice, Fig. 7G). The relative number of FOXp3+ Tregs was relatively low (about 3%) and gradually increased to about 4% following CXCL9-Fc and 5% following CXCLIO-Fc therapy (Fig. 7H, not significant). Similarly to CD8+ T cells here CD4+ T cells upregulate PD1 expression from about 6% to about 18% (Fig. 7C) with a high association with CXCR3 expression (Fig. 7K). Collectively the results are very similar to those obtained for CD8+ T cells and imply increased proliferation and shifting T cell polarization into IFN-yhlghThl -like cells and IFN-yhlghcytotoxic CD4+ T cells. In another experiment done by the same protocol, IL- 17 expression in CD4+ T cells and CD8+ T cells following CXCLIO-Fc therapy was evaluated (Fig. 7N) showing a significant increase in Thl7-like CD4+ T cells.

[0175] An adoptive transfer set-up in which CD4+ T cells were isolated from OTII developing melanoma into CXCR3 KO mice was conducted by a similar protocol to that used for CD8 cells (see Fig. 6P) with the difference that donor CXCR3 KO OTII cell are used. Briefly, CD4+ T cells from OTII mice (CXCR3+) engrafted with a ret melanoma cell line transduced to stably express OVA II (ret-OVAII melanoma cell line) were injected into CXCR3 KO mice also engrafted with ret-OVAII melanoma cell line and then treated with CXCL9-Fc, CXCLIO-Fc or control IgGl (explained in more detail in Fig. 70) and monitored for tumor development. Fig. 70 shows that both CXC19-Fc and CXCLIO-Fc significantly suppressed tumor development. CXCLIO-Fc was superior to CXCL9-Fc (p<0.05). Collectively these results further highlight the role of CXCL9 and CXCLO in potentiating not only anti-tumor CD8+ T cells but also anti-tumor CD4+ T cells, including cytotoxic CD4+ T cells.

[0176] Taken together, the above data imply that CXCL9 and CXCL10 directly polarize different subtypes of effector / cytotoxic T cells that are either CD4+ or CD8+, both with IFNyhlghsignature that can induce an IFNy dependent self-feeding loop to further amplify these activities. The results also indicate that the direct effect on either CD4+ or CD8+ T cells alone is sufficient to restrain cancer diseases.

[0177] Finally, a comparative analysis between the spleen and tumor cells of mice treated with either CXCLIO-Fc or control IgG showed successful therapy led to a significant increase in CD4+ T cells and CD8+ T cells at the tumor site, but not spleen (not shown). Similarly, the relative number of NK cells also exclusively increased at the tumor site. Ki67 significantly increases for CD8+ T cells (Figure S3D) and CD4+ T cells (in both the spleen and the tumor site, indicating that CXCLIO-Fc induces these cells also in the periphery, but the most significant result is at the tumor site where the relative tumor- specific CD8+ T cells are relatively high. Other parameters include intracellular cytokine staining, further confirming the IFNy signature described above. Finally, ki67 staining of NK cells showed a significant increase at the tumor site, but not the spleen (not shown).

[0178] Example 5: Exploring CXCL9-Fc and CXCL-10-Fc immunotherapy in immunocompetent BALB / C mice: Both could be preferentially identified at sites enriched with CXCR3+ cells.

[0179] CXCL9-Fc and CXCLIO-Fc therapy was further tested in the CT26 Clone cancer model in BALB / C mice (as detailed in the methods). These mice fully express functional CXCL9, CXCL10 and CXCL11. CXCL10 is also produced by the cancer cells. In the first set of experiments, a relatively low dose of (50pg / mouse) of CXCL9-Fc or CXCLIO-Fc was administered, starting 3 days after tumor engraftment (See more details in the explanation to Fig. 8). Both significantly inhibited tumor growth (Fig. 8A-8B, day 20 p<0.01) and increased survival (Fig. 8C) with an advantage to CXCL120-Fc (Log Rank - test p=0.0567). To follow the fate of CXCL9-Fc and CXCLIO-Fc 24h post systemic administration, histological sections from the liver, spleen, or tumor site of representative mice injected with either control Fc, CXCLIO-Fc, or CXCL9-Fc were subjected to immunostaining of a-cMyc (included in the plasmid used for generation of recombinant proteins), showing that CXCL9-Fc and CXCLIO-Fc could be identified at the spleen and tumor site (where CXCR3+ T cells are present), but not in the liver (Fig. 8D).

[0180] Finally, in further expanding CXCLIO-Fc therapy, it was administered 3 times starting when tumor size reached vol of 50mm3. Anti-PDl mAb was used as a positive control. Fig. 8E shows that both equally and significantly limited tumor growth.

[0181] Example 6: Generation of a tumoroid platform for testing response to ICI in colon cancer

[0182] In order to test the response of a specific tumor to ICI in vitro, a tumoroid system was used. The tumoroid system is similar in principle to the spheroid system described in Example 3, however, as opposed to the spheroid system, the tumoroid system is based on tumors obtained from a living body (rather than from a cell line) and therefore also include the tumor microenvironment including anti-tumor CD 8+ T cells. Preparation of a colon cancer tumoroid was conducted generally based on the protocol described in Sayed et al. Adv Cancer Res 151, 345-383 (2021).

[0183] CT26 Colon cancer cells were trypsinized, washed with PBS, and resuspended in 100 pl PBS per mouse (4.5xl05cells / 100 pL in PBS), and BALB / c mice were engrafted with the cells. When tumors reached a volume of ~ 100 mm3, they were excised and cut into < 5mm pieces, transferred into a GentleMacs™ tube (Cat. 130-096-334 purple cap) containing 5ml of cold RPMI 1640 medium, supplemented with 20% FCS, 2% Penicillin / Streptomycin and run on the GentleMacs™ device, program m_impTumor_02*, for three times. 75 pl of freshly prepared collagenase I solution (Sigma, Cat. c0130) and 150 pl Dispase II solution (Roche, Cat. 04942078001) were added to the minced tissue and incubated at 37°C, in a shaker 110 rpm, 40 min. The tubes were run again on the GentleMacs™ device, program m_impTumor_04*, three times.

[0184] The liquid was passed through a 40pm cell strainer (Bar Naor, Cat. BN93040S), washed with cold PBSxl, and completed the volume to 25ml with cold PBSxl in a 50 ml tube. The solution was centrifuged at 1500 rpm for 5 min at 4°C and then resuspended with 5 ml of cold RBC lysis (41.4 gr NH4CI buffer, 5 gr NaHCOs, 0.189 gr EDTA, 500 ml DDW), vortexed and incubated for 7 min at RT. 45 ml of cold PBSX1 was added to the tube, centrifuged at 1500 rpm, for 5 min, at 4°C, and resuspended with RPMI medium (Gibco).

[0185] Cells were counted and seeded in Nunclon Sphera (ThermoFisher) round bottom 96 wells plates in complete RPMI medium, 104cells per well. 4 days later, co-cultures were started by adding 10xl04, 20xl04, or 30xl04total of activated CD8+ T cells extracted from WT BALB / c mice, per well. This results in ratios of 10:1, 20:1, and 30:1, respectively, of T cells (effector) to tumor cells (target). 24 hours later, 100 pgr / ml of anti PD1 or Control IgG were added to each well. Concentrations of mouse CXCL10 (mCXCLIO) in culture media following addition of anti PD1 or control IgG were assessed using commercial ELISA kit: Murine IP-10 (CXCL10) Standard ABTS ELISA Development Kit, (900-K153, Peprotech).

[0186] Fig. 9 presents the experimental scheme (Fig. 9A) and the ELISA results at the three ratios of T cells to tumoroid cells (Fig. 9B). As shown, a dose dependent effect of the ability of anti PD- 1 to increase CXCL10 production we observed, in which at a ratio of 30: 1 (T cells to tumoroid cells) produced the largest effect. Example 7: Generation of a tumoroid system for testing response to ICI in colon cancer from a patient

[0187] Briefly, tumor sample obtained from a patient biopsy are washed with Advanced DMEM medium (Gibco). All procedures are carried out on ice. Tumor samples are minced into small pieces in 2-3 ml media with a surgical blade and the pieces are collected in a new tube and digested in 10ml of BM (Advanced DMEM / F12 +1% P / S +Glutamine +HEPES), with the addition of O.lg / ml collagenase I and 20mg / ml hyaluronidase. Digestion is be carried out for 30 minutes at 37°C in a water bath. Cells are filtered with a 70 pm strainer and 1ml fetal bovine serum (FBS) is added to the digested solute to block the reaction. Cell suspension is plated in non-adherent 96- well U-bottom plates in 500pl / well T cells medium containing (per 500 ml in RPMI 1640): 432ml Glutamax™, 50ml FBS (10%), 50nM 2- mercaptoethanol, 5ml Penicillin / Streptomycin, and 12.5ml HEPES IM (25mM).

[0188] Optionally, peripheral blood lymphocytes (PBL)s, isolated T cells, or isolated CD8+ T cells are added to the tumor cells. The added cells may be stimulated by incubating with anti-CD3, or with CD3 + IL2, or left to be stimulated directly by the tumor.

[0189] For 3D culture, cells are cultured in non-adherent 96-well U-bottom plates in 500pl / well RPMI 1640 medium containing EGF, Noggin, Rho-kinase inhibitor Y-27632, SB202190, A83- 01, Wnt3A, R-spondin, Gastrin, and Nicotinamide.

[0190] When the tumoroid is around 300-400 pm in diameter, about 7 days since establishment, the tumoroid is ready for testing response to ICI (including anti-PDl, anti-PDLl, anti-PDL2, anti- CTLA4, anti-LAG3, anti-TIGIT, and / or anti-TIM3). The tumoroid is first tested to obtain baseline levels of CXCL9 and CXCL10, by ELISA (or optionally by FACS). The tested ICI is added to the tumoroid, followed by incubation of the tumoroid with the ICI. The tumoroid is then tested again for CXCL9 and CXCL10 levels after ICI treatment as above, and the level after treatment is compared to the level before treatment, to assess the response to the ICI.

[0191] Example 8: Generation of a tumoroid platform for testing response to ICI in melanoma from a patient

[0192] A melanoma tumoroid is prepared as detailed above with respect to the colon cancer tumoroid, but the 3D culture media includes 500pl / well of medium (such as RPMI 1640) enriched with Primocin 1:500, Thiazovivin 1:5000, R-Spondin 1.5:1000, EGF, Noggin, A83-01 (TGFpi), SB202190 (p38i), Wnt3A, Rspondin, Gastrin, Nicotinamide, Prostaglandin-E2. Cells are optionally added, as also detailed above, with respect to the colon cancer tumoroid.

[0193] Cells are then tested for CXCL9 and CXCL10 expression before and after addition of ICI, as explained above.

Claims

CLAIMS1. A method for predicting a response of a subject having a cancer to an immunological treatment, the method comprising the steps of:(a) providing a tumoroid prepared from tumor cells obtained from the subject;(b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;(c) adding to the tumoroid an immune checkpoint inhibitor (ICI); and(d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is predicted to be responsive to treatment with the ICI; and when the activated level of the at least one chemokine is not increased by the predetermined threshold compared to the baseline level, the subject is predicted to be non-responsive to treatment with the ICI and responsive to treatment with the chemokine.

2. The method of claim 1 , wherein the immunological treatment comprises an agent selected from an ICI, CXCL10 or a variant or analog thereof, CXCL9 or a variant or analog thereof, and combinations thereof.

3. The method of claim 1 or 2, wherein step (a) comprises the steps of: obtaining tumor cells of the subject; and growing the tumor cells in a 3D culture, thereby preparing the tumoroid.

4. The method of claim 3, wherein preparing the tumoroid further comprises adding effector cells obtained from the subject, and incubating until the effector cells infiltrate the tumoroid.

5. The method of claim 4, wherein adding the effector cells further comprises stimulating the effector cells prior to adding them to the tumor cells.

6. The method of claim 4 or 5, wherein the effector cells are added at a ratio of about 1:1-50:1 effector cells to tumor cells.

7. The method of any one of claims 1-6, wherein the ICI is selected from anti-PDl, anti-PDLl, anti-PDL2, anti-CTLA4, anti-CD80 / 86, anti-LAG3, anti-Galectin3, anti-PGLl, anti-TIGIT,anti-CD112, anti- CD155, anti-CD96, anti-BTLA, anti-HVEM, anti-B7-H3, anti- VISTA, anti- Siglec-15, anti-TIM3, and combinations thereof.

8. The method of any one of claims 1-7, wherein the ICI is added at a concentration of about 5- 100 |ig / ml.

9. The method of any one of claims 1-8, wherein the predetermined threshold is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

10. The method of any one of claims 1-9, wherein the cancer is selected from melanoma, colorectal cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin’s lymphoma, liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

11. The method of any one of claims 1-10, wherein the subject is non-responsive to treatment with the ICI.

12. The method of any one of claims 1-11, further comprising treating the subject with an immunological treatment, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; and when the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

13. The method of claim 12, wherein the variant or analog of the at least one chemokine is an immunoglobulin Fc region-conjugated chemokine.

14. A method for treating a subject having a cancer by an immunological treatment, the method comprising the steps of:(a) preparing a tumoroid from tumor cells obtained from the subject;(b) measuring in the tumoroid a baseline level of at least one chemokine selected from CXCL10 and CXCL9;(c) adding to the tumoroid an immune checkpoint inhibitor (ICI); and(d) measuring an activated level of the at least one chemokine in the tumoroid following the addition of the ICI, wherein: when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with the ICI; andwhen the activated level of the at least one chemokine is not increased by at least the predetermined threshold compared to the baseline level, the subject is treated with the at least one chemokine or variant or analog thereof.

15. The method of any one of claims 12-14, wherein when the activated level of the at least one chemokine is increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the ICI and the at least one chemokine, or variant or analog thereof.

16. The method of any one of claims 12-14, wherein when the activated level of the at least one chemokine is not increased by at least a predetermined threshold compared to the baseline level, the subject is treated with a combination of the at least one chemokine, or variant or analog thereof, and the ICI.

17. The method of any one of claims 12-16, wherein the at least one chemokine comprises both CXCL10 and CXCL9.

18. An in vitro tumoroid system for predicting a response of a subject having a cancer to an immunological treatment, the tumoroid system comprising tumor cells from the subject, an immune checkpoint inhibitor (ICI), and optionally effector cells obtained from the subject.

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