CROSS-REFERENCE TO REUATED APPUICATIONS

This application claims the benefit of priority of non-provisional US Application Serial No. 16/219,203, filed on December 13, 2018. The disclosure of the prior application is considered part of and is incorporated by reference in its entirety in the disclosure of this application.

FIEUD OF THE INVENTION

The present invention is in the field of oncology and particularly relates to a method of treatment of a cancer patient with an immune checkpoint inhibitor in combination with another therapeutic agent.

BACKGROUND

One of the major obstacles in clinical oncology is that tumors often develop resistance to therapy even when an initial tumor response to treatment is observed. Many studies have focused on the contribution of mutations and genetic aberrations in the tumor cells which promote drug resistance and can explain tumor re-growth. However, studies have demonstrated that the host, in response to cancer therapy, generates pro-tumorigenic and pro metastatic effects which in turn contribute to tumor re-growth, and therefore negate the anti tumor activity of the drug (for reviews see Katz and Shaked, 2015; Shaked, 2016).

Host-mediated responses to anti-cancer treatment modalities may be molecular and/or cellular responses. Upon treatment with chemotherapeutic drugs, host bone marrow derived cells (BMDCs) are mobilized from the bone marrow compartment, colonize the treated tumor and contribute to tumor angiogenesis and cancer re-growth, thereby promoting therapy resistance (Shaked et ah, 2006, 2008). Cancer therapy also induces pro-tumorigenic activation of various immune cells such as macrophages and antigen presenting cells (Beyar-Katz et ah, 2016; De Palma and Lewis, 2013; Kim et al. 2012; Ma et ah, 2013). Overall, these aforementioned studies indicate that host-mediated molecular and cellular responses to different anti-cancer treatments involve the activation or education of immune cells as well as the secretion of various pro-tumorigenic factors. These combined effects contribute to tumor re-growth and resistance to therapy. This relatively new phenomenon has made a paradigm shift in understanding cancer progression and resistance to cancer therapy.

Recently, a new treatment modality, an immunotherapy using immune checkpoint inhibitors (ICIs), is revolutionizing cancer therapy. Such immune-modulating drugs have shown remarkable successes for the treatment of advanced malignancies (including stage IV) such as melanoma, prostate, non-small cell lung cancer, renal cell carcinoma and also some hematological malignancies (Postow et ah, 2015). Although the human immune system is capable of recognizing and mounting a response to cancerous cells, this response is often circumvented by tumor-derived inhibition resulting in immune tolerance. In this regard, tumor-infiltrating lymphocytes (TILs), such as tumor antigen- specific CD8 ⁺ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, have been found to colonize the tumor microenvironment (Gajewski et ah, 2013). Yet, at the tumor site, they completely lack the ability to act against tumor cells (Ostrand-Rosenberg and Sinha, 2009). This is due to direct inhibitory effects of factors secreted by cancer cells, stromal cells or other suppressive immune cells such as myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs) (Makkouk and Weiner, 2015). For instance, IL-10 is frequently upregulated in various types of cancer, and was shown to suppress the immune system (Sato et ah, 2011). Thus, identifying molecules that negatively regulate the immune system against tumor cells, will lead to the development of immunomodulatory drugs that support the activation of immune cells against tumors.

Of specific interest are immune checkpoint proteins, such as CTLA-4, PD-1 and its ligand, PD-L1. These checkpoint proteins are expressed by tumor cells or other immune cells and contribute to the exhaustion of CTLs (Postow et ah, 2015; Topalian et ah, 2015). Specifically, they keep immune responses in check, and inhibit T cell killing effects against tumor cells. As such, checkpoint inhibitors have been developed in order to inhibit the immune suppression effects. Currently, antibodies blocking the immune checkpoints CTLA-4 and PD-1 or its ligand PD-L1 have been developed (Pardoll, 2012). These ICIs are currently in use in the clinic for the treatment of various malignancies with some promising and remarkable successes (Romano and Romero, 2015). However, ICIs have shown therapeutic benefit only for a limited portion of cancer patients (-10-20%). For example, pooled data from clinical studies of ipilimumab, a CTLA-4 blocking antibody, revealed that the duration of clinical response is around 3 years, and can last up to 10 years. However, this dramatic therapeutic effect is only observed in a subset of patients (-20%). Thus, the majority of patients exhibit intrinsic resistance mechanisms to such therapies. Yet, the molecular aspects that define the subpopulation of patients that are responsive to ICIs are not fully clear. It has been suggested that markers such as PD-L1 expression by tumor cells, mutational burden, and lymphocytic infiltrates could predict the cancer patients that will respond to ICI immunotherapy. However, these aforementioned biomarkers do not always correlate with tumor responsiveness to ICI immunotherapy or resistance of patients to ICIs. Therefore, additional possible mechanisms are still unknown.

In the Applicant’s International Patent Applications No. PCT/IL2018/050608 and No. PCT/IL2018/050609, both filed on June 4, 2018 and published as WO2018/225062 and WO 2018/225063, respectively, the entire contents of which are hereby incorporated herein by reference, a method of predicting personalized response to cancer treatment with a cancer therapy was described by identification of a plurality of factors/biomarkers induced by the cancer patient into the circulation in response to said cancer therapy (“host response”) and determining how a change in the levels of each of one or more of the plurality of factors as compared to a reference level, predicts a favorable or a non-favorable response of the cancer patient to the treatment with said cancer therapy.

It would be highly desirable to unveil host-mediated cellular and molecular mechanisms that contribute to tumor resistance to all modalities of cancer therapy including the promising ICI immunotherapy modality. This will permit development of strategies to block such unwanted host effects and will improve therapeutic outcome and delay resistance to cancer therapy.

SUMMARY OF THE INVENTION

The present invention is based on previous studies mentioned hereinbefore in the Background section of the application that show that a cancer patient (the“host”), in response to a cancer therapy, may generate and induce into the host circulation a set of host-driven resistance factors to cancer immunotherapy with an immune checkpoint inhibitor (hereinafter “ICI”), that may limit or counteract the effectiveness of the patient treatment with the ICI. The determination of these factors allows the prediction in a personalized form of the favorable or non-favorable response of the patient to the treatment with the ICI. These factors, herein designated interchangeably“factors” or“biomarkers”, are factors, mainly cytokines, chemokines, growth factors, soluble receptors, enzymes and other molecules produced by the host cells, either at different organs or at the tumor microenvironment, in response to the cancer therapy with the ICI with which the patient is treated.

Thus, in one aspect, the present invention provides a method of treating a cancer patient with an immune checkpoint inhibitor (ICI), the method comprising the steps of:

(i) performing an assay on a blood sample selected from blood plasma, whole blood, blood serum or peripheral blood mononuclear cells obtained from the cancer patient at a time point after a session of treatment with said ICI, to determine the levels of one or more of a plurality of host-driven resistance factors that are driven by the host (“the cancer patient”) in response to treatment with the ICI, said one or more of the plurality of factors promoting in a personalized form responsiveness or non-responsiveness of the cancer patient to the treatment with the ICI;

(ii) obtaining reference levels for each of the one or more of the plurality of the host- driven resistance factors of step (i) by determining the levels of each of said factors in a blood sample of the same type of the blood sample of step (i), obtained from the cancer patient at a time point before said session of treatment with the ICI;

(iii) establishing the fold change for each of the one or more of the plurality of the host-driven resistance factors of step (i) by comparing the level of each host-driven resistance factor of step (i) with the reference level of step (ii) for the same factor;

(iv) determining that the cancer patient has a favorable or a non-favorable response to the treatment with said ICI based on the fold change established in step (iii) for one or more of the plurality of host-driven resistance factors of step (i); and

(iva) if the cancer patient has a non-favorable response to the treatment with said ICI based on the fold change established in step (iii) for one or more of the plurality of the host- driven resistance factors, then selecting a dominant factor among the one or more host-driven resistance factors showing a fold change indicative of said non-favorable response, and treating the patient with a therapeutically effective amount of the ICI along with a therapeutically effective amount of an agent that blocks the activity of the selected dominant host-driven resistance factor (herein“the dominant factor”) or the receptor thereof; or

(ivb) if the cancer patient has a favorable response to the treatment with said ICI based on the fold change established in (iii) for one or more of the plurality of host-driven resistance factors, then continuing the treatment of the cancer patient with the ICI alone.

In a certain embodiment, the invention relates to a method for treatment of a cancer patient non-responsive to treatment with an immune checkpoint inhibitor (ICI), the method comprising administering to the cancer patient a therapeutically effective amount of the ICI in combination with a therapeutically effective amount of an agent that blocks the activity of a dominant factor, or of the receptor thereof, the dominant factor being selected among the plurality of host-driven resistance factors generated in response to treatment of the cancer patient with the ICI, the plurality of host-driven resistance factors having a fold-change predictive of a non-favorable response of the cancer patient to the treatment with the ICI, wherein the fold change is established by comparing: (i) the level of each host-driven resistance factors in a blood sample selected from blood plasma, whole blood, blood serum or peripheral blood mononuclear cells, obtained from the cancer patient after a session of treatment with the ICI, with (ii) a reference level obtained for the same factor from a blood sample of the same type of (i), obtained from the cancer patient before said session of treatment with the ICI. Preferably, the blood samples of steps (i) and (ii) are both blood plasma.

In another aspect, the present invention relates to an immune checkpoint inhibitor (ICI), for use in the treatment of cancer in a patient non-responsive to said treatment, comprising administering a therapeutically effective amount of the ICI in combination with a therapeutically effective amount of an agent that blocks the activity of a dominant factor, or of the receptor thereof, the dominant factor being selected among the plurality of host-driven resistance factors generated in response to treatment of the cancer patient with the ICI, the plurality of host-driven resistance factors having a fold-change predictive of a non-favorable response of the cancer epatient to the treatment with the ICI, wherein the fold change is established by comparing: (i) the level of each host-driven resistance factor in a blood sample selected from blood plasma, whole blood, blood serum or peripheral blood mononuclear cells, obtained from the cancer patient after a session of treatment with the ICI, with (ii) a reference level obtained for the same factor from a blood sample of the same type of (i), obtained from the cancer patient before said session of treatment with the ICI. Preferably, the blood samples of steps (i) and (ii) are both blood plasma.

BRIEF DESCRIPTION OF THE FIGURES

Figs. 1A-1C show the effect of blocking MMP9 on primary tumor growth and survival in a mouse model of breast cancer. Fig. 1A shows that mice treated with a combination of anti-PD-Ll and the MMP-2/MMP-9 inhibitor SB-3CT exhibit reduced tumor size compared to mice treated with vehicle, anti-PD-Ll or SB-3CT alone. Fig. IB shows tumor volume at day 27, the last experimental day. Fig. 1C shows better survival of mice treated with the combined therapy compared to the other treatment groups.

Figs. 2A-2C show the effect of blocking host-derived amphiregulin on primary tumor growth and survival in a mouse model of colon cancer. Figs. 2A-2B show tumor volume of mice treated with anti-PD-Ll, anti- amphiregulin antibody (anti-AREG) or anti-PD-Ll and anti-AREG combined therapy. Fig. 2C is a Kaplan-Meier curve demonstrating better survival of mice treated with the combined therapy compared to the other treatment groups.

Fig. 3 shows IL-6 expression following treatment with anti-PD-Ll or anti-CTLA-4.

Figs. 4A-4C show the effect of blocking host-derived IL-6 induced following treatment with anti-PD-Ll on primary tumor growth and mice survival. Fig. 4A shows inhibition in tumor growth in mice treated with a combination of anti-PD-Ll and anti-IL-6 compared to control mice or mice treated with either anti-PD-Ll or anti-IL-6 alone. Fig. 4B corresponds to Fig. 4A, but shows tumor growth in individual animals, and Fig. 4C shows that combined treatment with both anti-PD-Ll and anti-IL-6 also led to better survival relative to the other treatment groups.

Figs. 5A-5B show the effect of blocking host-derived IL-6 induced following treatment with anti-CTLA-4 on primary tumor growth and survival. Treatment of BALB/c mice with anti-CTLA-4 resulted in reduced tumor growth (Fig. 5A) and better survival relative to control mice (Fig. 5B), and these effects were further improved by combined treatment with both anti-CTLA-4 and anti-IL-6 antibodies (Figs. 5A-5B).

DETAILED DESCRIPTION

Before describing the methods of the invention, it should be understood that this invention is not limited to the particular methodology and protocols as described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and, if not defined otherwise, it is not intended to limit the scope of the present invention which will be recited in the appended claims.

It must also be noted that as used herein and in the appended claims, the singular forms “a”,“an”, and“the” include plural reference unless the context clearly dictates otherwise.

As used herein, the term“a cancer therapy” may be used interchangeably with the term “a“a cancer-modality therapy”, and include plural reference, namely, one single modality therapy or a combination of two or more modality therapies. As used herein, the terms“induced”,“driven” and“generated” are used interchangeably to denote the factors induced into the circulation by the cancer patient in response to the cancer therapy (“host-response”).

In cancer therapy, a cycle of treatment means that the drug is administered to the patient at one point in time (for example, injections over a day or two) and then there is some time (e.g., 1, 2 or 3 weeks) with no treatment. As used herein,“a session of treatment” refers to the “one point in time” when the ICI is administered to the patient at the beginning of a cycle of treatment. The treatment and rest time make up one“treatment cycle”. When the patient gets to the end of the cycle, it starts again with the next cycle. A series of cycles of treatment is called“a course”.

As used herein, the term“an immune checkpoint inhibitor (ICI)” refers to a single ICI, a combination of ICIs and a combination of an ICI with another cancer therapy. The ICI may be a monoclonal antibody, humanized antibody, a fully human antibody a fusion protein or a combination thereof.

In one aspect, the invention relates to a method of treating a cancer patient with an immune checkpoint inhibitor (ICI), the method comprising the steps of:

(i) performing an assay on a blood sample selected from blood plasma, whole blood, blood serum or peripheral blood mononuclear cells obtained from the cancer patient at a time point after a session of treatment with said ICI, to determine the levels of one or more of a plurality of host-driven resistance factors that are driven by the host (“the cancer patient”) in response to treatment with the ICI, said one or more of the plurality of factors promoting in a personalized form responsiveness or non-responsiveness of the cancer patient to the treatment with the ICI;

(ii) obtaining reference levels for each of the one or more of the plurality of the host- driven resistance factors of step (i) by determining the levels of each of said factors in a blood sample of the same type of the blood sample of step (i), obtained from the cancer patient at a time point before said session of treatment with the ICI;

(iii) establishing the fold change for each of the one or more of the plurality of the host-driven resistance factors of step (i) by comparing the level of each host-driven resistance factor of step (i) with the reference level of step (ii) for the same factor;

(iv) determining that the cancer patient has a favorable or a non-favorable response to the treatment with said ICI based on the fold change established in step (iii) for one or more of the plurality of host-driven resistance factors of step (i); and (iva) if the cancer patient has a non-favorable response to the treatment with said ICI based on the fold change established in step (iii) for one or more of the plurality of the host- driven resistance factors, then selecting a dominant factor among the one or more host-driven resistance factors showing a fold change indicative of said non-favorable response, and treating the patient with a therapeutically effective amount of the ICI along with a therapeutically effective amount of an agent that blocks the activity of the selected dominant host-driven resistance factor (herein“the dominant factor”), or of the receptor thereof; or

(ivb) if the cancer patient has a favorable response to the treatment with said ICI based on the fold change established in (iii) for one or more of the plurality of host-driven resistance factors, then continuing the treatment of the cancer patient with the ICI alone.

A“favorable response” of the cancer patient indicates“responsiveness” of the cancer patient to the treatment with the ICI, namely, the treatment of the responsive cancer patient with the ICI will lead to the desired clinical outcome such as tumor regression, tumor shrinkage or tumor necrosis; an anti-tumor response by the immune system; preventing or delaying tumor recurrence, tumor growth or tumor metastasis. In this ease, it is possible to continue the treatment of the responsive cancer patient with the ICI alone, as defined in step (ivb) above.

A“non-favorable response” of the cancer patient indicates“non-responsiveness” of the cancer patient to the treatment with the ICI due to induction of host-driven resistance factors that may be pro-tumorigenic, e.g., pro-angiogenic, pro-inflammatory/chemotactic or proliferative growth factors, or pro-metastatic factors, and thus the treatment of the non- responsive cancer patient with the ICI will not lead to the desired clinical outcome but to non- desired outcomes such as tumor expansion, recurrence and metastases. In order to achieve the desired clinical outcome, it is necessary to blockade the dominant factor as defined above in step (iva) and treating the non-responsive cancer patient with a combination of the ICI and a therapeutic agent that blocks the activity of the selected dominant factor.

Thus, in one embodiment, the invention relates to a method for treatment of a cancer patient non-responsive to treatment with an immune checkpoint inhibitor (ICI), the method comprising administering to the cancer patient a therapeutically effective amount of the ICI along with a therapeutically effective amount of an agent that blocks the activity of a dominant factor, or of the receptor thereof, the dominant factor being selected among the plurality of host-driven resistance factors generated in response to treatment of the cancer patient with the ICI, the plurality of host-driven resistance factors having a fold-change predictive of a non-favorable response of the cancer patient to the treatment with the ICI, wherein the fold change is established by comparing: (i) the level of each host-driven resistance factors in a blood sample selected from blood plasma, whole blood, blood serum or peripheral blood mononuclear cells, obtained from the cancer patient after a session of treatment with the ICI, with (ii) a reference level obtained for the same factor from a blood sample of the same type of (i), obtained from the cancer patient before said session of treatment with the ICI.

In a preferred embodiment, the blood samples of steps (i) and (ii) are both blood plasma.

In certain embodiments of the invention, the session of treatment with the ICI is one of multiple sessions of treatment. In certain embodiments, the one of multiple sessions of treatment with the ICI is the first session of treatment with said ICI, the blood sample of step (i) is obtained from the cancer patient at about 20, 24, 30, 36, 40, 48, 50, 60, 72 hours or more, including up to one week or more, up to two weeks or more or up to three weeks or more, after said first session of treatment, and the reference blood sample of step (ii) is obtained from the cancer patient at a time point including at about 72 hours or less, including at about 60, 50, 48, 40, 36, 30, 24 or 20 hours or less or just before said first session of treatment with the ICI.

In certain embodiments, the one of multiple sessions of treatment of the cancer patient with the ICI is the first session of treatment, when the treatment with the ICI is started. In this case, the reference/baseline sample of step (ii), preferably blood plasma, is obtained from the cancer patient at a time point before starting the treatment including at about 72 hours or less, including at about 60, 50, 48, 40, 36, 30, 24 or 20 hours or just before said first session of treatment with the ICI. The comparison is then made between the concentration levels of the factors determined in the blood sample, preferably blood plasma, obtained from the cancer patient at about 20, 24, 30, 36, 40, 48, 50, 60, 72 hours or more, including up to one week or more, up tp two weeks or more, or up to three weeks or more, after said first session of treatment with the ICI.

In certain other embodiments of the invention, the session of treatment of the cancer patient with the ICI is one of multiple sessions of treatment that is not the first session of treatment with the ICI. In this case, the blood sample is obtained from the cancer patient at any time point between two consecutive sessions of treatment with the ICI and serves simultaneously as the blood sample of step (i) and the reference blood sample according to step (ii) for the next session assay according to step (i). The time between two consecutive sessions of treatment depends on the treatment protocol approved for the specific ICI and may be, for example, of 2 or 3 weeks, depending on the ICI, and the blood sample may be obtained at day 1, 2, 3, 7, 14, or 21 days between two consecutive sessions.

In accordance with the invention, the levels of the plurality of factors generated by the host/cancer patient in response to the treatment with the immune checkpoint inhibitor are determined in the blood sample, preferably blood plasma, obtained from the patient after treatment with ICI. The value (factor concentration in pg/mL) obtained for each factor is then compared with a reference level, which is the baseline level of concentration of the same factor determined in a blood sample, preferably blood plasma, obtained previously from the same cancer patient (hereinafter“reference/baseline sample”). The change in the level of one or more of the factors identified in the blood sample obtained from the cancer patient after the treatment with the ICI compared to the reference/baseline level, is defined by the fold change for each factor. The fold change for each factor is determined by calculating the ratio of treatment: reference/baseline values for the factor.

The fold-change of each of the one or more of the plurality of the host-driven resistance factors is considered significant and predictive of a non-favorable response of the cancer patient to the treatment with the ICI if its value is about 1.5 or higher denoting an up- regulation of the factor, or the fold-change is considered significant and predictive of a favorable response of the cancer patient to the treatment with said ICI if its value is about 0.5 or lower denoting down-regulation of the factor.

In accordance with the invention, the prediction of a favorable or a non-favorable response of the cancer patient to the treatment with an ICI is based on significant fold changes of one or more, optionally two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, or twenty-five or more, of the host-driven resistance factors.

The factors/biomarkers induced into the circulation of the cancer patient in response to treatment with the ICI include molecular factors such as cytokines, chemokines, growth factors, enzymes and soluble receptors. The factors may be pro-tumorigenic or pro-metastatic factors. The pro-tumorigenic factors may be pro-angiogenic, pro-inflammatory/chemotactic or proliferative growth factors. In certain embodiments, the session of treatment is the first session of a plurality of sessions of treatment of the cancer patient, when the treatment is started. In this case, the comparison is between the factors determined in the blood sample, preferably blood plasma, obtained from the cancer patient after first starting treatment with the ICI, and the same factors found in the reference/baseline blood sample, preferably blood plasma, obtained from the cancer patient before starting treatment with the ICI. The results may assist the medical oncologists treating the patient to decide if or how to continue the treatment of the cancer patient.

In certain embodiments, the method of the invention is performed for monitoring treatment response in a cancer patient being treated with an ICI. In this case, the session of treatment is one of the sessions of several sessions of treatment, but not the first one. The results will assist the medical oncologist in their decisions if or how to continue the treatment.

In certain embodiments, the fold change determined for pro-tumorigenic factors is predictive of the patient’s favorable response to the cancer therapy and the decision may be to continue the treatment with the same ICI as scheduled.

In certain embodiments, the fold change determined for pro-tumorigenic factors is predictive of the patient’s non-favorable response to the ICI. In this case, depending on the specific biological activity of the pro-tumorigenic factors, the decision may be to continue the treatment with the same ICI but with the addition of a drug that blocks the biological activity of the tumorigenic factors, for example, by adding to the treatment an anti-inflammatory drug if the factors are pro-inflammatory or by adding to the treatment an anti-angiogenic drug if the factors are pro-angiogenic.

Immune checkpoints are regulators of immune activation. They play a key role in maintaining immune homeostasis and preventing autoimmunity. In cancer, immune checkpoint mechanisms are often activated to suppress the nascent anti-tumor immune response. Immune checkpoint molecules are considered as good targets for cancer immunotherapy. Immune checkpoint inhibitors (ICI) that cause blockade of the immune checkpoint molecules are considered good candidates for the development of drugs for cancer immunotherapy with the potential for use in multiple types of cancers and are already in use or are under development.

Examples of immune checkpoints that are candidates as targets for development of immune checkpoint inhibitor (ICI) drugs include PD-1 (Programmed Death- 1) that has two ligands, PD-L1 and PD-L2; CTLA-4 (Cytotoxic T-Lymphocyte- Associated protein 4); A2AR (Adenosine A2A receptor), also known as ADORA2A; BT-H3, also called CD276; BT-H4, also called VTCN1; BT-H5; BTLA (B and T Lymphocyte Attenuator), also called CD272; IDO (Indoleamine 2,3-dioxygenase); KIR (Killer-cell Immunoglobulin-like Receptor); LAG- 3 (Lymphocyte Activation Gene-3); TDO (Tryptophan 2,3-dioxygenase); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3); VISTA (V-domain Ig suppressor of T cell activation).

In certain embodiments of the invention, the ICI is a monoclonal antibody (mAh) against PD-1 or PD-L1 that neutralizes/blocks the PD-1 pathway. In certain embodiments, the anti-PD-1 mAh is Pembrolizumab (Keytruda; formerly called lambrolizumab), approved or tested for treatment of advanced or unresectable melanoma, metastatic non- small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and recurrent squamous cell carcinoma of the head and neck (SCCH). In certain embodiments, the anti-PD-1 mAb is Nivolumab (Opdivo), approved or tested for NSCLC, RCC, melanoma and colorectal cancer (CRC). In certain embodiments, the anti-PD-1 mAb is Pidilizumab (CT0011), approved or tested for non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and acute myeloid leukemia. In certain embodiments, the anti-PD-1 mAb is REGN2810, AMP-224, MEDI0680, or PDR001.

In certain other embodiments of the invention, the immune checkpoint inhibitor is a mAb against PD-L1. In certain embodiments, the anti-PD-Ll mAb is Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi), approved for multiple cancers. Atezolizumab is being tested in combination with one or two other cancer agents such as bevacizumab, gemcitabine, cisplatin, docetaxel, paclitaxel, vinflunine entinostat, daratumumab, MPDL3280A, carboplatin, Nab-paclitaxel, Radium-223 dichloride, obinutuzumab , for multiple cancers.

In certain other embodiments of the invention, the ICI is a mAb antibody against CTLA- 4. In certain embodiments, the and-CTLA-4 is Ipilimumab (Yervoy), approved or tested for advanced/metastatic melanoma and castrate-resistant prostate cancer. In certain other embodiments, the anti-CTLA-4 mAb is Tremelimiimab (formerly ticilimumab).

In certain embodiments, the ICI is an inhibitor including: (i) anti-B7-H3, such as MGA271; (ii) anti-IDO, such as epacadostat; (iii) anti-KIR, such as Lirilumab; (iv) anti-LAG- 3, such as Relatlimab (BMS-986016), LAG 525, REGN3767; (v) anti-TIM-3, such as TSR022 or MBG453; and (vi) anti- VISTA, such as JNJ 61610588. In certain embodiments, a combination of two ICIs is used according to the invention. In certain embodiments, the combination comprises an anti-PD-1 and an anti-CTLA-4, e.g., Nivolumab-Ipilimumab and REGN2810-Ipilimumab. In certain embodiments, the combination comprises an anti-PD-Ll and an anti-CTLA-4, e.g., Durvalumab- Tremelimumab. In certain embodiments, the combination comprises an anti-PD-1 and an anti- PD-Ll, e.g., Nivolumab-Atezolimumab. In certain embodiments, the combination comprises an anti-LAG-3 and an anti-PD-1, e.g., Relatlimab-Nivolumab or REGN3767-REGN2810. In certain embodiments, the combination comprises an anti-PD-1 and an IDO inhibitor, e.g.. Pernbrolizumab and Epacadostat and Nivolumab - Epacadostat.

Costimulatory molecules such as CD137 (4-1BB), CDI34 (QX40), glucocorticoid- induced TNFR (GITR; CD357), and CD40 are expressed by activated T cells, activated natural killer (NK) cells natural killer T (NKT) cells, Tregs, and other Immune cells. The inhibition of die immune checkpoint PD-i and stimulation of costimulatory molecules by agonist antibodies are complementary strategies to enhance immune responses and therefore provide a strong rationale for use in combination. Thus, in certain embodiments, the invention encompasses a combination of an ICI with an agonistic mAh against T-cell co-stimulatory molecules including an anti-ICOS mAh, e.g., MEDI-570 or BMS-986226; an anti-OX40 mAh e.g., MOXR0916, KHK4083, MEDI0562 or MEDI6469; an anti-CD40 mAh; and an anti- CD137 (4-IBB) mAh, e.g., Urelumab or Utomilumab.

In certain embodiments of the invention, the ICI is administered in combination with one or more conventional cancer therapy including chemotherapy, targeted cancer therapy and radiotherapy. Combinations of ICI and radiation therapy have been studied in multiple clinical trials.

In certain embodiments, the ICI is used in combination chemotherapy that may be with a single or a combination of chemotherapy drugs, or metronomic chemotherapy. The combinations Pembrolizumab+carboplatin+paclitaxel, Pembrolizumab+gemcitabine+ docetaxel, Nivolumab+gemcitabine+cisplatin, Ipilimumab+carboplatin+paclitaxel, and other combinations were tested or are being tested in clinical trials.

In certain embodiments, the ICI therapy is used in combination with targeted cancer therapy, sometimes called "moleeularly targeted therapy . In certain embodiments, the targeted therapy drugs are small molecules such as bortezomib (Velcade), sunitinib (Sutenf). in certain embodiments, the targeted therapy drugs are monoclonal antibodies such as bevacizumab (Avastin), panitumumab (Vectibix), daratumumab (Darzalex). In certain embodiments, an anti-PD-1 is used in combination with sunitinib (Sutent) or pazopanib (Votrient) that was tested for treatment of RCC, or a combination of anti-CTLA-4 ipilimumab with BRAF inhibitor dabrafenib (Tafinlar).

In certain embodiments, the ICI therapy is used in combination with anti-angiogenic therapy, for example, with a mAb that targets VEGF. Thus, the combination may be of

Ipi limumab and bev acizumab .

In certain embodiments, the ICI therapy is used in combination with other immunotherapies such as cancer vaccines, immunomodulators, immuno stimulatory cytokines, e.g., GM-CSF, IFN-a , TGF-b. IL-10, lL-2, IL-12, 1L-I5, IL-18, and IL-21, or oncolytic viruses. In certain embodiments, anti-CTLA-4 ipilimumab or anti-PD-1 pembrolizumab is used in combination with oncolytic vims ta!irnogene laberparepvec (T-VEC).

In accordance with the invention, the cancer therapy is related to all types of cancer, primary or metastatic, in all stages of the disease, including, but without being limited to, a primary or a metastatic cancer including bladder, bone, breast, brain, cervical, colon, colorectal, esophageal, gastric cancer, gastrointestinal, glioblastoma, head and neck, head and neck squamous cell cancer, hepatocellular cancer, kidney, liver, lung including small cell lung cancer and non-small cell lung cancer (NSCLC), melanoma, nasopharyngeal, ovarian, pancreas, penile, prostate, skin, testicular, thymus, thyroid, urogenital, or uterine cancer, leukemia, lymphoma, multiple myeloma and sarcoma.

In certain embodiments, the cancer is leukemia, a cancer of the body's blood-forming tissues, including the bone marrow and the lymphatic system. In certain embodiments, the leukemia is selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or chronic myeloid leukemia (CML). In certain embodiments, the cancer is multiple myeloma.

In certain embodiments, the cancer is non-small cell lung cancer (NSCLC). In certain embodiments, the cancer is advanced (stage III or IV) or metastatic NSCLC.

In certain embodiments, the cancer is metastatic melanoma, renal-cell carcinoma (RCC), classic Hodgkin ^' s lymphoma (HL), bladder carcinoma, Merkel cell carcinoma head and neck cancer, or solid tumors with mismatch repair-deficiency

The host-driven factors/biomarkers identified by the method of the invention after administration of an immune checkpoint inhibitor to a cancer patient are specific to: (i) the cancer patient; and (ii) the immune checkpoint inhibitor. This is the“host response” that provides specific information about the cancer patient and allows the prediction in a personalized form to help diagnose, plan treatment, find out how well treatment is working, or make a prognosis

If the treatment is with one single ICI, the factors generated by the host/patient are specific to this particular ICI. If the treatment is carried out with a combination of two ICIs, the factors generated by the host/patient are specific to this combination of ICIs. If treatment is with the ICI in combination with another cancer therapy, the factors generated by the host/patient are specific to this combination.

In certain embodiments, the biomarkers are molecular factors such as cytokines, chemokines, growth factors, enzymes or soluble receptors. Some of these factors induce cells that affect the tumor and contribute to tumor angiogenesis and cancer re-growth, thereby promoting resistance to the therapy used. Examples of such cells include bone-marrow derived cells (BMDCs) that are mobilized from the bone-marrow compartment by cytokines and growth factors such as G-CSF and SDF-Ia, and subsequently colonize the treated tumors and promote cancer therapy resistance, particularly, but not exclusively, chemotherapy resistance. Other cells are immune cells such as macrophages and antigen-presenting cells, or stromal cells within the tumor microenvironment which play a pivotal role in tumor progression.

The host-mediated cellular and molecular mechanisms that contribute to tumor resistance to a cancer therapy are based on the biological functions of the factors and/or cells generated in the host by the particular cancer therapy. Each factor may exhibit one or more biological functions or activities.

In certain embodiments, the factors are tumorigenic and contribute to tumor growth. In certain embodiments, the tumorigenic factors are pro-angiogenic. In other embodiments, the tumorigenic factors are pro-inflammatory/chemotactic. In yet other embodiments, the tumorigenic factors are proliferative growth factors.

In certain embodiments, the pro-angiogenic factors include, without being limited to, ANG (angiogenin); angiopoietin-1; angiopoietin-2; bNGF (basic nerve growth factor); cathepsin S; Galectin-7; GCP-2 (granulocyte chemotactic protein, CXCF6); G-CSF (granulocyte-colony stimulating factor); GM-CSF (granulocyte-macrophage colony stimulating factor, also known as colony- stimulating factor 2, CSF2); PAI-1 (plasminogen activator Inhibitor- 1); PDGF (platelet-derived growth factor) selected from PDGF-AA, PDGF-BB, PDGF-AB; P1GF (or PFGF, placental growth factor); P1GF-2; SCF (stem-cell factor); SDF-1(CXCF12, stromal cell-derived factor-1); Tie2 (or TIE-2, an endothelial receptor tyrosine kinase); VEGF (vascular endothelial growth factor) selected from VEGF-A, VEGF-C and VEGF-D; VEGF-R1; VEGF-R2; VEGF-R3.

In certain embodiments, the pro-inflammatory and/or chemotactic factors include, without being limited to, 6Ckine (CCL21, Exodus-2); angiopoietin- 1 ; angiopoietin-2; BLC (CXCL13, B lymphocyte chemoattractant or B cell-attracting chemokine 1 (BCA-1); BRAK (CXCL14); CD186 (CXCR6); ENA-78 (CXCL5, Epithelial cell derived neutrophil activating peptide 78,); Eotaxin-1 (CCL11); Eotaxin-2 (CCL24); Eotaxin-3 (CCL26); EpCAM (Epithelial cell adhesion molecule); GDF-15 (growth differentiation factor 15, also known as macrophage inhibitory cytokine-1, MIC-1); GM-CSF; GRO (growth-regulated oncogene); HCC-4 (CCL16, human CC chemokine 4); 1-309 (CCL1); IFN-g; IL-la; IL-Ib; IL-1R4 (ST2); IL-2; IL-2R ; IL-3; IL-3Ra; IL-5; IL-6; IL-6R; IL-7; IL-8; IL-8 RB (CXCR2, interleukin 8 receptor, beta); IL-11; IL-12; IL-12p40; IL-12p70; IL-13; IL-13 Rl; IL-13R2; IL-15; IL-15Ra; IL-16; IL-17; IL-17C; IL-17E; IL-17F; IL-17R; IL-18; IL-18BPa; IL-18 Ra; IL-20; IL-23; IL-27; IL-28; IL-31; IL-33; IP-10 (CXCL10, interferon gamma-inducible protein 10); I-TAC (CXCL11, Interferon-inducible T-cell alpha chemoattractant); LIF (Leukemia inhibitory factor); LIX (CXCL5, lypopolysaccharide-induced CXC chemokine); LRP6 (low-density lipoprotein (LDL) receptor-related protein-6); MadCAM-1 (mucosal addressin cell adhesion molecule 1); MCP-1(CCL2, monocyte chemotactic protein 1); MCP-2 (CCL8); MCP-3 (CCL7); MCP-4 (CCL13); M-CSF (macrophage colony- stimulating factor, also known as colony stimulating factor 1 (CSF1); MIF (macrophage migration inhibitory factor); MIG (XCL9, Monokine induced by gamma interferon) ; MIP-1 gamma (CCL9, macrophage inflammatory protein-1 gamma); MIP-la (CCL3); MIR-1b; MIR-1d (CCL15); MIP-3a (CCL20); MIR-3b (CCL19); MPIF-1 (CCL23, Myeloid progenitor inhibitory factor 1); PARC (CCL18, pulmonary and activation-regulated chemokine); PF4 (CXCL4, platelet factor 4); RANTES (CCL5, regulated on activation, normal T cell expressed and secreted); Resistin; SCF; SCYB16 (CXCL16, small inducible cytokine B16); TACI (transmembrane activator and CAML interactor); TARC (CCL17, CC thymus and activation related chemokine); TSLP (Thymic stromal lymphopoietins; TNF-a (tumor necrosis factor-a); TNF Rl; TRAIL-R4 (TNF-Related Apoptosis-Inducing Ligand Receptor 4); TREM-1 (Triggering Receptor Expressed On Myeloid Cells 1).

In certain embodiments, the proliferative factors include, without being limited to, Activin A; Amphiregulin; Axl (AXL, a receptor tyrosine kinase); BDNF (Brain-derived neurotrophic factor); BMP4 (bone morphogenetic protein 4); cathepsin S; EGF (epidermal growth factor); FGF-1 (fibroblast growth factor 1); FGF-2 (also known as bFGF, basic FGF); FGF-7; FGF-21; Follistatin (FST); Galectin-7; Gas6 (growth arrest- specific gene 6); GDF- 15; HB-EGF (heparin-binding EGF); HGF; IGFBP-1 (Insulin-like growth factor binding protein-1); IGFBP-3; LAP (Latency-associated peptide); NGF R (nerve growth factor receptor); NrCAM (neuronal cell adhesion molecule) ; NT-3 (neurotrophin-3); NT-4; PAI-1; TGF-a (transforming growth factor-a); TGF-b; and TGF-P3; TRAIL- R4 (TNF-Related Apoptosis-Inducing Ligand Receptor 4).

In certain embodiments, the pro-metastatic factors include, without being limited to, AD AMTS 1 (A disintegrin and metalloproteinase with thrombospondin motifs 1); cathepsin S; FGF-2; Follistatin (FST); Galectin-7; GCP-2; GDF-15; IGFBP-6; LIF; MMP-9 (Matrix metallopeptidase 9, also known as 92kDa gelatinase or gelatinase B (GELB); pro-MMP9; RANTES (CCL5); SDF-1 (stromal cell-derived factor- 1, also known as CXCL12) and its receptor CXCR4.

The factors may also be anti-tumorigenic factors, e.g., anti- angiogenic, anti inflammatory and/or anti-proliferative growth factors.

In certain embodiments, the circulating factors indicating a host response to ICI include, but are not limited to, ADAMTS 1, amphiregulin; Axl; CCL5/RANTES; CCL17/TARC; EGF; Eotaxin-2; FGF-21; Gas6; G-CSF; GM-CSF; HGF; IFN-gamma; IL-lRalpha; IL-2; IL-6; IL- 7; IL-10; IL-12p40; IL-13; IL-33; I-TAC; MadCAM-1; MCP-5; TACI; M-CSF; MMP-9; PDGF-BB; pro-MMP9; SCF.

In accordance with the present invention, many of the factors that were upregulated in response to anti-PD-1 treatment are key players in pro-tumorigenic and pro-metastatic processes such as angiogenesis, inflammation, chemotaxis and proliferation. Upregulated pro- angiogenic factors include: G-CSF; GM-CSF; and PDGF-BB. Up-regulated pro-inflammatory and/or chemotactic factors include: CCL17/TARC; CCL5/RANTES; G-CSF; GM-CSF; IFN- gamma; IL-lRalpha; IL-2; IL-6; IL-7; IL-10; IL-12p40; IL-13; IL-33; and M-CSF. Upregulated proliferative growth factors include: FGF-21; Gas6; and HGF. Upregulated pro metastatic factors include: MMP-9.

In accordance with the present invention, many of the factors that were upregulated in response to anti-PD-Ll treatment are key players in pro-tumorigenic and pro-metastatic processes such as inflammation, chemotaxis and proliferation. Upregulated pro-angiogenic factors include: G-CSF; and SCF. Upregulated pro-inflammatory and/or chemotactic factors include: Eotaxin-2; G-CSF; IL-lra; IL-6; IL-7; IL-33; I-TAC; MadCAM-1; MCP-5; SCF; and TACI. Upregulated proliferative growth factors include: amphiregulin; Axl; EGF; and HGF. Upregulated pro-metastatic factors include: AD AMTS 1 and pro-MMP9.

As used herein, the term“dominant factor” denotes a potent factor that may be upstream of a signaling pathway that affects a biological process that is vital for the living cell and living organism. These biological processes include proliferation, inflammation, metastasis, and others, and are made of several signaling pathways ultimately leading to activation or inhibition of the biological process. A“signaling pathway” is a row of events in which proteins in the same pathway transfer signal to each other. After the first protein in a pathway receives a signal, it activates another protein which activates another protein and so forth, ultimately leading to activation of one or more cell functions.

A“dominant factor” may also be a key factor that highly interacts with, and highly affects, many other factors/proteins. According to the invention, the dominant factors are selected based on an algorithm which identifies the protein-protein interactions of factors based on the literature. When a factor has more interactions, it serves as a hub and therefore it is a dominant factor. The term“protein-protein interactions” refers to physical interactions or cross-talk between two or more proteins, resulting in activation or inhibition of signal transduction or protein activity. The term“protein hubs” refers to highly connected proteins that play central and essential role in biological processes and thus may confer the host with resistance, limit or counteract the effectiveness of the treatment of the cancer patient with the cancer therapy modality.

The terms“block”,“neutralize” or“inhibit” are herein used interchangeably and refer to the capability of an agent of preventing the selected dominant factor from exerting its function/biological activity.

Examples of dominant factors include, without limitation, Amphiregulin, EGF, EGFR, FGF, IFN-g, IL-Ib, IL-2, IL-6, MMP-9, PDGF, TNF-a and VEGF-A.

To illustrate their qualifications as dominant factors, the properties of some of these factors is provided herein. Interleukin- 1b (IL-Ib, IL-lb) is a cytokine member of the IL-1 family, produced by different immune cells including macrophages. It is a potent mediator of the inflammatory response and also known to be involved in several biological processes such as cell proliferation and apoptosis, as well as cell differentiation. IL-lb was mostly investigated as a protein that initiates the pro-inflammatory cascade. It physically interacts with enzymes such as CASP1, IL1RA, IL1R1, CMA1, IL1RB, ILIA, IL1R2; genetically interacts with MAPK8IP2, ZNF675 and UBEN2N; and is co-expressed with A2M, CXCL8, IL18, CAASpl, IL1R1 and others. Thus, IL-lb serves as a hub for interactions with a large number of proteins that affect several biological pathways including cell proliferation, apoptosis and differentiation as well as inflammation and angiogenesis.

Another dominant factor is Interleukin-6 (IL-6), which is a cytokine that acts mainly as a pro-inflammatory factor but also sometimes as an anti-inflammatory factor produced by muscle cells and as a result downregulate a number of pro-inflammatory proteins such as IL- 1, IL-10 and TNF-a. IL-6 is involved in a number of biological processes including bone formation, disruption of blood brain barrier, macrophage activation and innate immune system contribution, stimulates the synthesis of neutrophils and B cells, and is also involved in neurological activities such as disorders, stress and depression. IL6 interacts and affects a large number of proteins: it physically interacts with HRH1, OSM, IL6ST, IL6R and ZBTB16, and was found to be co-expressed with a large number of proteins such as PTPRE, CSF3, CCL2, CXCL8, CXCL3, ICAM1 SELE, NFKBIZ among others. IL6 is involved in a number of pathways mediated by proteins such as LRPPRC, OSM, PTPRE, PIAS1 and IL6R. As such, IL6 serves as a dominant factor for a number of biological processes involved in immune cell activity, cell genesis, and cell-cell interactions.

A further dominant factor, vascular endothelial growth factor A (VEGF-A), is a growth factor that stimulates the formation of new blood vessels. It is involved in both angiogenesis (endothelial cell proliferation) as well as vasculogenesis (bone marrow-derived endothelial cell precursors and their differentiation). VEGF is important for embryonic cell development and neuronal development in the fetus, and is involved in leukocyte proliferation and differentiation, inflammation and several diseases such as age-related macular degeneration and the majority of cancers. VEGF-A physically interacts with a large number of proteins such as NRP1, NRP2, KDR, FLT1, PGF, THBS1, SPARC, GCP1 and VEGFC; it is co expressed with SEMA3F, SHB, THBS1, FLT1 and VEGFC; it is involved with proteins of various pathways including PGF, CD2AP, IQGAP1, NEDD4; and it affects a number of biological processes such as angiogenesis, tumorigenesis, cell viability, proliferation and differentiation. As such, VEGF-A is considered a dominant factor, and vital factor for various biological processes both in normal physiological conditions as well as in disease states.

In certain embodiments, the selected dominant factor shows a fold change of >1.5 indicative of a non-favorable response of the cancer patient to the treatment with the ICI, and the treatment of the patient with said ICI may proceed in combination with an agent that blocks said dominant factor or the receptor thereof. In certain embodiments, the dominant factor is selected from factors including amphiregulin, EGF, EGFR, FGF, IFN-g, IL-Ib, IL-2, IL-6, MMP9, PDGF, TNF-a and VEGF-A.

In certain embodiments, the dominant factor is MMP9, the ICI is an anti-PD-1 or anti- PD-L1 monoclonal antibody, and the cancer patient is treated with the ICI in combination with a MMP-9 inhibitor including SB-3CT.

In certain embodiments, the dominant factor is amphiregulin, the ICI is an anti-PD-1 or anti-PD-Ll monoclonal antibody, and the cancer patient is treated with the ICI in combination with an anti- amphiregulin antibody.

The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Introduction

As discussed hereinbefore, recent clinical studies report that patients may sometimes develop resistance to ICIs, or may not respond to ICI therapy (Sharma et ah, 2017). We describe herein that the cancer patient, i.e., the host, generates pro-tumorigenic factors in response to ICI therapy, which in turn contribute to tumor re-growth, progression and resistance to therapy. In order to identify the factors that contribute to this mechanism, we perform our in vivo experiments in both non-tumor- and tumor-bearing immunocompetent mice. This approach allows us to distinguish between the therapeutic anti-tumor activity of ICIs and the effect of these drugs on host cells. We focus on ICIs that are extensively used in the clinic, including anti-PDl, anti-PD-Ll and anti-CTL-4 monoclonal antibodies, and use murine tumor models that are known to be responsive or resistant to specific ICIs. For example, CT26 colon and EMT-6 breast carcinoma cell lines respond to anti-CTLA-4 and anti-PD-Ll, respectively (Duraiswamy et al., 2013; Swart et a., 2013), whereas MC38 colon and 4T1 breast carcinoma cell lines are resistant or only modestly responsive to some ICIs (including anti-PD-1) (De Henau et al., 2016; Kodumudi et al., 2016), as also tested in our laboratory (not shown).

Materials and Methods

Materials

The following antibodies were purchased from BioXCell, West Lebanon, NH, USA: InVivoMAb anti-mouse-CTLA-4 (cat. #BE0131); InVivoMAb anti-mouse-IL-6 (cat. #BE0046); InVivoMAb anti-mouse- PD-1 (cat. #BE0146); InVivoPlus anti-mouse-PD-Ll (cat. #BEO101); and InVivoMAb Isotype control IgG2b antibody (cat. #BE0090). SB-3CT (IUPAC name: 2-(((4-phenoxyphenyl) sulfonyl)methyl)thiirane) was purchased from MedKoo Biosciences Inc (cat. #406563). Anti-amphiregulin (cat. #AF989) was purchased from R&D systems. A 10 mM stock solution of SB-3CT was prepared in 100% DMSO (Sigma). For in-vivo experiment, the stock solution was diluted to a final concentration of 1.25 mg/ml in 10% DMSO in normal saline.

(i) Cancer cell culture: Murine EMT6 breast carcinoma and CT26 murine colon carcinoma cells were purchased from the American Type Culture Collection (ATCC, USA). The cells were used in culture for no more than 4 months after being thawed from authentic stocks and were regularly tested and found to be mycoplasma-free (EZ-PCR mycoplasma test kit, Biological Industries, Israel). EMT6 Cells were cultured in Dulbecco’s modified eagle medium (DMEM) and CT26 cells were cultured in RPMI 1640 medium, both supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% sodium-pyruvate and 1% penicillin- streptomycin (Biological Industries, Israel). Cells were cultured at 37°C in 5% CO2.

(ii) Animal treatment protocols and tumor models: Naive 8-10 week old female BALB/c, SCID or NOD-SCID mice (Harlan, Israel) were used in this study. Experiments were performed in accordance with the Animal Ethic Committee at the Technion (Haifa, Israel). The mice were intraperitoneally injected with anti-PD-1 or irrelevant IgG rat-anti- mouse antibodies (BioXCell, West Lebanon, NH, USA).

In other experiments, naive 8- 10- week old female and male BALB/c or C57bl/6 mice (Harlan, Israel) were intraperitoneally injected with anti-PD-Ll or irrelevant IgG rat-anti- mouse antibodies (BioXCell, West Lebanon, NH, USA). In all cases, antibodies were administered at a dose of 200pg/20gr mouse, every other day over the period of 1 week (3 injections in total).

To investigate whether inhibiting host-derived IL-6 improves the efficacy of anti- CTLA-4 treatment, EMT6 murine breast carcinoma cells (5x10 ^s ) were orthotopically implanted into the mammary fat pad of 8- 10-week old BALB/c mice. Tumor size was assessed regularly with Vernier calipers using the formula width xlengthx0.5. In some experiments, mice were injected through the tail vein with EMT6 cells (25x10 ) to form experimental lung metastasis. Mouse survival was monitored daily. When tumors reached a size of 140mm , mice (n=5) were intraperitoneally (IP) injected with 200pg anti-CTLA-4, 200pg anti-IL-6 or a combination of the two antibodies once every three days (a total of 7 injections). Control mice (n=8) were left untreated. Tumors growth was monitored regularly and when tumor size reached 1500 mm , mice were sacrificed

To investigate whether inhibiting host-derived MMP-9 improves the efficacy of anti- PD-Ll therapy, 0.5xl0 ⁶ EMT6 GFP murine breast carcinoma cells (a highly selective metastatic clone, called EMT6-F2, previously described for other cell line in Munoz et ah, 2006) were orthotopically implanted in the mammary fat pad of 8-weeks old female BALB/c mice (n=7). When tumors reached a size of 100 mm , mice were IP injected with 200pg anti- PD-Ll, 50pg SB-3CT (a MMP-2/MMP-9 selective inhibitor) (Kruger A., 2005, Bonfil RD., 2006); or a combination of anti-PD-Ll and SB-3CT. Control mice or mice treated with anti- PD-Ll monotherapy were injected with vehicle control (25%DMSO/65%PEG- 200/10%PBS). Tumor growth and mouse survival were monitored and at endpoint (when tumors reached a size of -1500 mm , or maximum at day 27), mice were sacrificed.

To determine whether inhibiting host-derived amphiregulin improves the efficacy of anti-PD-Ll therapy, 2xl0 ⁶ CT26 cells were subcutaneously implanted in the right flank of 8-

10 weeks old BALB/c mice. When tumors reached a size of 50 mm , mice were IP injected with 200pg anti-PD-Ll, 5pg anti-AREG (as previously demonstrated in Fujiu K., 2017) or a combination of anti-PD-Ll and anti-AREG, every 3 days. Control mice were left untreated. Tumor growth and mouse survival were monitored.

(iii) IL-6 quantification by ELISA: Seven weeks old naive female BALB/c mice (n = 4) were intraperitoneally injected every three days for a total of 3 injections with 200pg anti-PD- Ll or anti-CTLA-4. Control mice (n = 4) were left untreated. One day after the last injection, mice were sacrificed, and blood was collected into EDTA-coated tubes by cardiac puncture. Plasma was isolated by centrifugation of the whole blood at 1300 g for 10 min. at room temperature. Supernatants (representing the plasma layer) were collected and the level of IL-6 was determined by ELISA (IL-6 mouse ELISA Kit, 100712 Abeam) according to the manufacturer’s instruction.

(iv) Plasma samples and conditioned medium preparation: Blood from control IgG-, anti-PD-1-, or anti-PD-Ll -treated mice was collected into EDTA-coated tubes by cardiac puncture. Subsequently, plasma was isolated by centrifugation of whole blood at lOOOg, 4°C, for 20 min. Plasma was stored in aliquots at -80°C until further use. Bone marrow derived cells were flushed from the femurs of IgG or anti-PD-1 treated mice. Bone marrow cells (lxlO ⁶ cells/ml) were cultured in serum-free DMEM for 24 hours to generate conditioned medium (CM).

(v) Antibody arrays: Three protein profiling experiments were performed. In the first experiment, plasma samples extracted from IgG- or anti-PD-1 treated female BALB/c mice were pooled per treatment group (n = 5 per group). Samples were applied to a membrane- based Proteome Profiler Mouse XL Cytokine Array (R&D Systems; Cat no: ARY028) according to the manufacturer’s instruction to screen a total of 111 factors. In the second experiment, plasma samples extracted from IgG- or anti-PD-Ll treated female or male BALB/c or C57bl/6 mice were pooled per group (n = 7 per group). Samples were applied to a glass slide-based Quantibody Mouse Cytokine Array (RayBiotech, Cat no: QAM-CAA-4000) according to the manufacturer’s instruction to screen a total of 200 factors. In the third experiment, plasma samples extracted from IgG- or anti-PD-1 treated female BALB/c or SCID mice were pooled per group (n = 7 per group). Samples were applied to a glass slide- based Quantibody Mouse Cytokine Array (RayBiotech, Cat no: QAM-CAA-4000) according to the manufacturer’s instruction to screen a total of 200 factors. For the membrane-based array, pixel densities on developed X-ray films were analyzed using transmission mode densitometer and image analysis software. For the glass slide-based arrays, the fluorescent readout was detected by a laser fluorescent scanner. In all cases, data was normalized and the fold changes for each factor on the arrays were determined by calculating the ratio of treated: control values.

(vi) Statistical analysis: Data are expressed as mean ± standard deviation (SD). The statistical significance of differences was assessed by one-way ANOVA, followed by Tukey ad hoc statistical test using GraphPad Prism 5 software (La Jolla, CA). Student t-test was used in some experiments when comparing only two groups. Differences between all groups were compared with each other. For the IL-6 quantification by ELISA, the statistical significance of differences was assessed by two tailed unpaired T-test. For tumor growth experiments, statistical significance is assessed by multiple T-test. For survival analysis, differences are assessed by Log-rank Mantle-Cox. Differences were considered significant at p values below 0.05.

Example 1. The effect of immune checkpoint inhibitor therapy on circulating host- derived factors - a protein profiling approach in mice

Previous experiments suggest that anti-PD-1 therapy induces an upregulation of factors in the circulation which ultimately promotes tumor cell aggressiveness. Such effects may occur in response to other types of immune checkpoint inhibitor therapies. To identify host- derived circulating factors whose levels change in response to anti-PD-1 and anti-PD-Ll therapies, we performed 3 protein array-based screens using naive (non-tumor bearing) mice. The use of naive mice allows us to identify factors specifically generated by the host in response to therapy, independent of the tumor.

In the first screen, naive 8-10 week old female B ALB/c mice (n = 3) were intraperitoneally injected with anti-PD-1 rat anti-mouse antibody (BioXCell, West Lebanon, NH, USA) at a dose of 200pg/20gr mouse every other day over a period of 1 week (3 injections in total). Control mice (n = 3) were similarly injected with a rat-anti-mouse IgG antibody at the same dose. One week after the first injection, mice were sacrificed, and blood was collected into EDTA-coated tubes by cardiac puncture. Plasma was isolated by centrifugation of whole blood at 1300 g for 10 min. at room temperature. Supernatants (representing the plasma samples) were collected and pooled per group. Aliquots were stored at -80°C until further use. Plasma samples were applied to a membrane-based Proteome Profiler Mouse XL Cytokine Array (R&D Systems; Cat no: ARY028) to screen a total of 111 factors. A full list of cytokines, enzymes and growth factors detected by the array is shown in Table 1. Pixel densities on developed X-ray films were analyzed using transmission mode densitometer and image analysis software. Normalized data was analyzed to identify factors whose circulating levels were changed in response to anti-PD-1 treatment. Specifically, the fold change was determined for each factor by calculating the ratio of treatment: control values. Factors exhibiting a fold change of more than 1.5 or less than 0.5 were defined as being up- or down-regulated, respectively, in response to anti-PD-1 treatment. These factors and their respective fold changes are listed in Table 2. Many of the factors that were upregulated in response to anti-PD-1 treatment are key players in pro-tumorigenic and pro metastatic processes such as angiogenesis, inflammation, chemotaxis and proliferation. Upregulated pro-angiogenic factors include: G-CSF; GM-CSF; and PDGF-BB. Up-regulated pro-inflammatory and/or chemotactic factors include: CCL17/TARC; CCL5/RANTES; G- CSF; GM-CSF; IFN-gamma; IL-lRalpha; IL-2; IL-6; IL-7; IL-10; IL-12p40; IL-13; IL-33; and M-CSF. Upregulated proliferative growth factors include: FGF-21; Gas6; and HGF. Upregulated pro-metastatic factors include: MMP-9.

In the second screen, naive 8-10 week old female B ALB/c, male B ALB/c, female C57B1/6 or male C57B1/6 mice (n=7 mice per group) were intra-peritoneally injected with anti-PD-Ll or control IgG antibodies (BioXCell, West Lebanon, NH, USA) every other day over a period of 1 week (3 injections in total) at a dose of 200pg/20gr mouse per injection. Twenty-four hours after the last administration, mice were sacrificed, blood was drawn and plasma was prepared. Plasma samples from each group were pooled and applied to a glass slide-based Quantibody Mouse Cytokine Array (RayBiotech, Cat no: QAM-CAA-4000) according to the manufacturer’s instruction to screen a total of 200 factors. A full list of cytokines, enzymes and growth factors detected by the array is shown in Table 3. The fold changes were determined for each factor on the protein array by calculating the ratio of treated: control values. Factors exhibiting a fold change of more than 1.5 or less than 0.5 were defined as being up- or down-regulated, respectively, in response to anti-PD-Ll treatment. These factors, and their respective fold changes are listed in Table 4. The data demonstrate that the profiles of up- and down-regulated factors do not completely overlap when comparing between the different mouse strains or when comparing between males and females of the same strain. This suggests that the response to anti-PD-Ll treatment is genotype-dependent. This may reflect differences known to exist also among cancer patients, and therefore provides a rationale for testing the response of the host in patients in a personalized manner. Many of the factors that were upregulated in response to anti-PD-Ll treatment are key players in pro-tumorigenic and pro-metastatic processes such as inflammation, chemotaxis and proliferation. Upregulated pro-angiogenic factors include: G- CSF; and SCF. Upregulated pro-inflammatory and/or chemotactic factors include: Eotaxin-2; G-CSF; IL-lra; IL-6; IL-7; IL-33; I-TAC; MadCAM-1; MCP-5; SCF; and TACI. Upregulated proliferative growth factors include: amphiregulin; Axl; EGF; and HGF. Upregulated pro metastatic factors include: AD AMTS 1 and pro-MMP9.

To gain insight into which host cell types secrete these pro-tumorigenic factors, we performed a similar screen, comparing between BALB/c and SCID mice treated with anti- PD-1 or control IgG antibodies. SCID mice carry the severe combined immune deficiency (SCID) mutation on the BALB/c background, and therefore lack functional adaptive immune cell types (B cells and T cells). Naive 8-10 week old female BALB/c or SCID mice (n=7 mice per group) were intraperitoneally injected with anti-PD-1 or control IgG antibodies (BioXCell, West Lebanon, NH, USA) every other day over a period of 1 week (3 injections in total) at a dose of 200pg/20gr mouse per injection. Twenty-four hours after the last administration, mice were sacrificed, blood was drawn and plasma was prepared. Plasma samples from each group were pooled and applied to a glass slide-based Quantibody Mouse Cytokine Array (RayBiotech, Cat no: QAM-CAA-4000) according to the manufacturer’s instruction to screen a total of 200 factors. A full list of cytokines, enzymes and growth factors detected by the array is shown in Table 3. The fold changes were determined for each factor on the protein array by calculating the ratio of treated: control values. Factors exhibiting a fold change of more than 1.5 or less than 0.5 were defined as being up- or down- regulated, respectively, in response to anti-PD-1 treatment. These factors, and their respective fold changes are listed in Table 5. Several factors were found to be up-regulated in response to anti-PD-1 treatment, some of which were specific to BALB/c and not SCID mice, e.g., ADAMTS1; amphiregulin, I-TAC and SCF. These results suggest that these specific factors are secreted by cells of the adaptive immune system in response to anti-PD-1 treatment. Collectively, these results demonstrate that anti-PD-1 and anti-PD-Ll treatments induce a response in the host that supports tumor progression, counteracting the desired therapeutic effects of immune checkpoint inhibitor therapy.

Example 2. The effect of reducing host-derived MMP-9 levels on ICI therapy in a primary breast tumor model

Matrix metallopeptidase 9 (MMP9) is an enzyme that belongs to the zinc- metalloproteinases family involved in the degradation of the extracellular matrix. MMP-9 is involved in a variety of biological processes, among them are pro-tumorigenic and tissue regeneration biological processes, including epithelial to mesenchymal transition, cell proliferation, angiogenesis, bone formation and wound healing. MMP-9 is one of the key factors involved in invasion and metastasis. Due to its involvement in various key dominant biological pathways in cancer, for which drugs have been developed as anti-cancer agents, MMP-9 serves as a dominant factor induced in response to ICI therapy, and whose inhibition may improve therapeutic outcome.

MMP-9 was found to be induced in B ALB/c mice following treatment with anti-PD-1 or anti-PD-Ll, which demonstrate that: i) MMP-9 is secreted by bone marrow-derived cells of naive B ALB/c mice in response to anti-PD-1 treatment; ii) the plasma level of MMP9 is increased 5.4 fold in response to anti-PD-1 treatment in naive B ALB/c mice (Table 2); and iii) the plasma level of pro-MMP-9 is increased by 2-3 fold in response to anti-PD-Ll treatment in naive BALB/c mice (Table 4). To investigate whether inhibiting host-derived MMP9 improves the efficacy of anti-PD-1 or anti-PD-Ll therapy, the MMP2/MMP9 selective inhibitor SB-3CT is used in combination with anti-PD-1 or anti-PD-Ll antibodies. EMT6 murine breast carcinoma cells (5x10 ^s ) are orthotopically implanted in the mammary fat pad of BALB/c mice, age 8-weeks, (Harlan, Israel). Tumor size is assessed regularly with Vernier calipers using the formula width xlengthx0.5. When tumors reach a size of 100 mm , mice are randomly assigned to the following treatment groups (n=6 mice per group): i) control; ii) anti-PD-1 monotherapy; iii) anti-PD-Ll monotherapy; iv) MMP2/MMP9 selective inhibitor SB-3CT monotherapy; v) anti-PD-1 and SB-3CT combination therapy; and vi) anti- PD-Ll and SB-3CT combination therapy. Anti-PD-1, anti-PD-Ll and IgG control antibodies are administered by intraperitoneal injections at a dose of 200pg/20g mouse every 3 days. SB- 3CT is administered by intraperitoneal injections at a dose of 1 mg/20g mouse every 3 days. Control mice are injected with IgG antibody and vehicle (10% DMSO in normal saline). Mice receiving either anti-PD-1 or anti-PD-Ll monotherapies are also injected with vehicle (10% DMSO in normal saline). Mice receiving SB3-CT monotherapy are also injected with IgG control antibodies. Tumor growth and mouse survival are monitored. At endpoint (when tumors reach a size of -1500 mm or until experiment reached 27 days), mice are sacrificed. The results, presented in Figs. 1A and IB (tumor volume over time and at day 27, the final experimental day, respectively), demonstrate that mice treated with a combination of anti-PD-Ll and the MMP-2/MMP-9 inhibitor SB-3CT, exhibited reduced tumor size compared to mice treated with vehicle, anti-PD-Ll or SB-3CT alone. The advantage of the combined treatment over treatment with the anti-PD-Ll alone was demonstrated by better response rate in individual mice (Fig. 1C, black versus grey, respectively), where it is clearly shown that 3 out of the 8 mice treated with the combined treatment demonstrated reduced tumor size at end point (day 27) compared to the other mice. Moreover, not only the combined treatment inhibited tumor growth, but also improved mice survival (Fig. ID). Namely, mice treated with the combination therapy exhibited increased survival compared to the control mice, mice treated with anti-PD-Ll or mice treated with SB-3CT. These results suggest that inhibition of MMP-2/MMP-9 together with anti-PD-Ll treatment may improve treatment efficacy.

Example 3. The effect of reducing host-derived amphiregulin levels on ICI therapy in a primary breast tumor model

Amphiregulin is one of the ligands of the epidermal growth factor receptor (EGFR). Studies have demonstrated a functional role of amphiregulin in several aspects of tumorigenesis. Amphiregulin was chosen in light of our findings described in Example 1 above, which demonstrate that the plasma levels of amphiregulin are increased 2-3 fold in response to anti-PD-Ll treatment in naive B ALB/c and C57/bl/6 mice (Table 4), and 3.7 fold in response to anti-PD-1 treatment in B ALB/c mice (Table 5).

Amphiregulin is known to be involved in number of biological processes crucial for tumor development such as proliferation, invasion, angiogenesis, metastasis and resistance to apoptosis, proposing that its expression is indicative of non-responsiveness to cancer treatment. Moreover, Amphiregulin is one of the ligands of an important cancer promoting receptor, Epidermal Growth Factor Receptor (EGFR). As such, it is considered a dominant factor with pro-tumorigenic activities, and its inhibition may improve treatment efficacy.

To investigate whether inhibiting host-derived amphiregulin (which is upregulated in B ALB/c mice in response to either anti-PD-1 or anti-PD-Ll treatment) improves the efficacy of anti-PD-Ll therapy, CT26 colon carcinoma cells were subcutaneously implanted into

BALB/c mice. When tumors reached a size of 50 mm , mice were either treated with anti-PD- LI, anti-AREG or anti-PD-Ll and anti-AREG combination therapy while control mice were left untreated. Tumor growth and mouse survival were monitored.

As demonstrated in Fig. 2A, treatment with anti-AREG in combination with anti-PD- Ll, exhibited lower tumor growth compared to control mice and anti-AREG-treated mice. While no major difference between the average tumor volume of mice treated with the combined therapy and the anti-PD-Ll was observed, the combined therapy demonstrated a complete response (disappearance of the tumors) in 25% of the treated mice while none of the mice treated with anti-PD-Ll demonstrated this phenomenon (Fig. 2B; The dashed vertical lines represent the days of treatment administration). The higher efficacy of the combined treatment is directly correlated with mice survival (Fig. 2C). Accordingly, mice treated with the amphiregulin inhibitor in combination with anti-PD-Ll have indicated better survival rates compared to control mice, mice treated with anti-PD-Ll or mice treated with anti-AREG monotherapy.

Example 4. IL-6 expression is induced in response to ICI treatment

IL-6 is another dominant factor that was demonstrated above to be highly induced in response to ICI therapy. As presented, IL-6 was increased by 15.6-fold in response to anti- PD-1 treatment in BALB/c mice (Table 2), 1.7- 1.8-fold in response to anti-PD-Ll treatment in naive BALB/c and C57/bl/6 mice (Table 4) and 1.8-fold in response to anti-PD-1 treatment in SCID mice (Table 5).

The induction of IL-6 expression following ICI treatment was further validated using ELISA. Mice injected with anti-PD-Ll, anti-CTLA-4 or untreated (control mice) were analyzed for IL-6 level in the plasma using ELISA (IL-6 mouse ELISA Kit, ab 100712, Abeam). As shown in Fig. 4, mice treated with anti-PD-Ll and anti-CTLA-4 exhibited a pronounced induction of IL-6 concentration (13.4 and 6.15 pg/ml, respectively) compared to control mice in which IL-6 was undetectable (p values 0.03 and 0.02, respectively). Since this experiment was performed using naive mice, it demonstrates that IL-6 is produced by host cells in response to immune checkpoint inhibitors therapy, independently of tumor presence.

Example 5. Blocking of anti-PD-Ll-induced host-derived IL-6 inhibits primary tumor growth and improves mice survival

IL-6 is known to be involved in number of biological processes crucial for tumor development including proliferation, angiogenesis, inflammation, differentiation and resistance to apoptosis. In addition, IL-6 is a pro-inflammatory cytokine that have been described as a prognostic factor in cancer. Since IL-6 is located at the top of the pro- inflammatory cascade and have been demonstrated to correlate with metastasis it is considered as a dominant factor with pro-tumorigenic and pro-metastatic activities.

For this reason, we tested whether blocking of host-derived IL-6 improves the efficacy of anti-PD-Ll treatment. BALB/c mice were subcutaneously injected with CT26 murine colon carcinoma cells. When tumors reached a size of 50mm , mice were IP injected with anti-PD-Ll, anti-IL-6, or a combination of the two antibodies and control mice were left untreated. Tumors growth was monitored regularly and when reached a size of -2000 mm mice were sacrificed. As demonstrated in Fig. 5A, mice treated with the combination of anti- PD-Ll and anti-IL-6 exhibited reduced tumor growth compared to control, anti-PD-Ll or anti-IL-6 treated mice (p values of 0.003, 0.192 and 0.009, respectively). The advantage of the combined treatment compared to anti-PD-Ll or anti-IL-6 treatments in inhibiting tumor growth was more emphasized when comparing tumor growth in single mice. As demonstrated in Fig. 5B, four out of six mice (57%) treated with anti-PD-Ll in combination with anti-IL-6 showed complete response to the treatment and their tumors disappeared, while in the anti- PD-Ll treated group only one mouse out of the six (16.7%) showed complete response and disappearance of the tumor.

As demonstrated in Fig. 5C, the combined treatment with anti-PD-Ll and anti-IL-6 not only reduced tumor growth but also improved mice survival (undefined median survival) compared to control, anti-PD-Ll or anti-IL-6 treatments (median survival of 31, 39.5 and 31 days, respectively) (p values 0.0026, 0.3552 and 0.0386, respectively), with about 70% survival after 40 days, compared to 50% for the anti-PD-Ll alone group.

Example 6. Blocking of anti-CTLA-4-induced host-derived IL-6 inhibits primary tumor growth and improves mice survival

To study whether blocking of host-derived IL-6 upregulated in response to anti-CTLA-4 treatment (demonstrated in Fig. 4) improves the efficacy of the treatment, BALB/c mice were orthotopically injected with EMT6 murine breast carcinoma cells into the mammary fat pad.

When tumors reached a size of 100mm , mice were IP injected with anti-CTLA-4, anti-IL-6 or a combination of anti-CTLA-4 and anti-IL-6 once every three days (a total of 4 injections). Control mice were left untreated. Tumor growth was monitored regularly and when a size of

1500 mm was reached, mice were sacrificed. Fig. 5A clearly demonstrates enhanced anti-tumor effect of the combined treatment (anti-CTLA-4 and anti-IL-6) compared to the control, anti-CTLA-4 alone or anti-IL-6 treated mice (p values 0.002, 0.137 and <0.001 respectively). Moreover, three out of five mice (60%) treated with the combined treatment of anti-CTLA-4 and anti-IL-6 showed complete response to the treatment and their tumors completely disappeared at about day 35, in contrast to one mouse in the anti-CTLA-4 group, showing that the combined treatment not only inhibited tumor growth but also eradicated the tumor.

Blocking host-derived IL-6 in combination with anti-CTLA-4 also improved mice survival. As shown in Fig. 5B, mice treated with anti-CTLA-4 in combination with anti-IL-6 exhibited enhanced survival rate (undefined median survival) compared to mice treated with anti-CTLA-4, anti-IL-6 or control mice (median survival of 49, 27 and 27 days, respectively), p values 0.076, 0.0008 and 0.0005, respectively.

APPENDIX

Table 1:

List of 111 factors participating in the antibody array screen performed with plasma from mice receiving immune-checkpoint inhibitor anti-PD-1 therapy

Table 2:

Summary of fold changes in the levels of circulating factors in anti-PDl-treated vs control BALB/c mice

Table 3:

List of 200 factors participating in the antibody array screen performed with plasma from mice receiving immune-checkpoint inhibitor (anti-PD-1 or anti-PD-Ll) therapy

Table 4:

Summary of fold changes in the levels of circulating factors in anti-PD-Ll-treated vs control BALB/c and C57bl/6 mice

Table 5:

Summary of fold changes in the levels of circulating factors in anti-PDl-treated vs control BALB/c and SCID mice

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