Sequential innate and adaptive immune modulation for cancer treatment

Abstract

The present invention relates to the use of TLR9 agonists and checkpoint modulators in biomarker-driven treatment protocols designed to deliver optimal immunomodulatory signals to cancer patients following induction chemotherapy with chemotherapeutic agents.

Description

[Technical field]

[0001] The present invention relates to TLR9 agonist and checkpoint modulator agents for use in the treatment of cancer patients.

[0002] This application claims the benefit of priority to European Patent Application No. 21179848.3, filed June 16, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0003] Toll-like receptors (TLRs) belong to a group of pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides or pathogen-derived nucleic acids, that carry distinct epigenetic modifications or are mistargeted to host cellular compartments; the family of patterns recognized by TLRs is the damage-associated nuclear pattern (DAMP). DAMPs originate from host cells, which die due to pathological processes such as inflammation or cancer. TLRs play a key role in the immediate immune response by enabling immune cells to fight pathogens or pathological cells via the innate immune system. Adaptive immunity then activates antigen-specific effector and memory T cells of the adaptive immune population. Thus, TLR agonists, as well as checkpoint inhibitors, are attractive candidates for the development of therapeutic immunomodulators to treat cancer.

[0004] TLR9 is primarily expressed on plasmacytoid dendritic cells (pDCs) and B cells. It recognizes unmethylated CG motifs that are not present in human / vertebrate nuclear DNA and broadly activates both innate and adaptive immunity. Synthetic DNA molecules have been used in immunotherapeutic approaches, which contain unmethylated CG motifs that act as TLR9 agonists by mimicking pathogen DNA and triggering a broad range of immunological activities.

[0005] Members of the dSLIM® family (dSLIM: double stem loop immunomodulator), a family of TLR9 agonists, consist of dumbbell-shaped covalently closed DNA molecules without any chemical or other artificial modifications of DNA (Kapp K. et al., 2019 Oncoimmunology DOI:10.1080 / 2162402X.2019.1659096; Schmidt M. et al., 2015 Nucleic Acid Therapeut.25(3):140). Protection from nucleolytic degradation is achieved by the covalently closed structure avoiding accessible 3' or 5' ends. Lefitolimod (disclosed in U.S. Pat. No. 6,849,725 B2, incorporated herein by reference in its entirety; otherwise referred to as MGN1703) belongs to this group of TLR9 agonists that exhibit specific immune-modulating sequences and structures. Promising immunogenicity data from exploratory and preliminary mouse studies in combination with chemotherapy in metastatic metastatic colorectal carcinoma (mCRC) and extensive stage small cell lung cancer (esSCLC) suggest that TLR9-induced immune activation could be used to reactivate immune surveillance that recognizes tumor-associated, tumor-specific, and tumor neoantigens on tumor cells in cancer patients; however, it is still unclear which subsets of cancer patients or which clinical treatment settings are associated with extended overall survival (OS) in response to TLR9 agonist treatment. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] United States Patent No. (US) No. 6849725 B2 [Non-patent literature]

[0007] [Non-Patent Document 1] Kapp K. et al., 2019 Oncoimmunology DOI:10.1080 / 2162402X.2019.1659096 [Non-Patent Document 2] Schmidt M. et al., 2015 Nucleic Acid Therapeut.25(3):140 Summary of the Invention [Problem to be solved by the invention]

[0008] Based on the above state of the art, it is an object of the present invention to provide administration schedules and combinations thereof comprising a TLR9 agonist and a checkpoint modulating compound, particularly in a sequential application, capable of modulating the innate or adaptive immune response, for treating cancer patients. [Means for solving the problem]

[0009] This object is achieved by the subject matter of the independent claims herein as well as by further advantageous embodiments described in the dependent claims herein, the examples, the figures and the general description. [Brief description of the drawings]

[0010] [Figure 1A]Insets of all Kaplan-Meier plots show hazard ratios with corresponding confidence intervals (CI) and log rank p-values. Figure 1 shows A: A comparative overlay of IMPULSE progression-free survival (PFS) and overall survival (OS) data from studies used in the comparative analysis and additional representative studies. IMPULSE mPFS1 and mOS1 were calculated from the start of induction chemotherapy. 21d CT: 21-day chemotherapy cycle (CT), ECOG: Eastern Cooperative Oncology Group performance status [Figure 1B] B: Provide a rationale for new treatments. [Diagram 2] Figure 2 shows a comparison of overall survival (OS1) in IMPULSE patients treated with a TLR9 agonist after 4 or 5 cycles of PE chemotherapy. The benefit in mOS was 2.3 months (m), and the plateau region typically observed with immunotherapy became visible. #Chemo=5 is patients treated with 5 cycles of PE chemotherapy or #Chemo=4 is patients treated with 4 cycles of PE induction chemotherapy. [Figure 3A] Figure 3 is a Kaplan-Meier plot comparing OS1 in IMPULSE lefitolimod patients with control (continued maintenance therapy) by B cell activation status (percentage of CD45+CD19+ B cells positive for CD86). Cell activation was measured by flow cytometry in baseline peripheral blood mononuclear cell samples after 24 hours of incubation with MGN1703. A, B: Samples were divided into those with percent activated cells below or above the median of 16.9% or above the median of 16.9%. [Figure 3B] A, B: Samples are divided into those with a percentage of activated cells below the median of 16.9% or above the median of 16.9%. [Figure 3C] C, D show the threshold value of 15.42% applied to the two cohorts of patients in the control group, but not in the lefitolimod group. [Figure 3D] C, D show the threshold value of 15.42% applied to the two cohorts of patients in the control group, but not in the lefitolimod group. [Figure 3E] E and F show the OS between the lefitolimod group and the control group based on a threshold of 15.42% activated B cells. [Figure 3F] E and F show the OS between the lefitolimod group and the control group based on a threshold of 15.42% activated B cells. [Figure 4A-1] Figure 4 shows Kaplan-Meier plots comparing OS1 for IMPULSE patients treated with lefitolimod-switched maintenance therapy with OS1 for the control group (continuation of maintenance therapy), divided according to activated immune cell biomarker status below (left) or above (right) the median thresholds of: A. T cell activation status, percentage of CD69+ positive CD45+CD3+ T cells; [Figure 4A-2] A. T cell activation status, percentage of CD69+ positive CD45+CD3+ T cells [Figure 4B-1] B. Activation status of natural killer T (NKT) cells, percentage of CD69 positive CD45+CD3+CD56+ T cells [Figure 4B-2] B. Activation status of natural killer T (NKT) cells, percentage of CD69 positive CD45+CD3+CD56+ T cells [Figure 4C-1] C. Activation status of plasmacytoid dendritic cells (PDC), percentage of BCDA2 positive lineage (CD3 / CD14 / CD16 / CD19 / CD20 / CD56)-CD45+CD123+HLA-DR+PDC [Figure 4C-2]C. Activation status of plasmacytoid dendritic cells (PDC), percentage of BCDA2 positive lineage (CD3 / CD14 / CD16 / CD19 / CD20 / CD56)-CD45+CD123+HLA-DR+PDC [Figure 4D-1] D. Activation status of myeloid dendritic cells (mDCs), percentage of CD86 positive CD45+ lineage CD11c+HLA-DR+ myeloid dendritic cells [Figure 4D-2] D. Activation status of myeloid dendritic cells (mDCs), percentage of CD86 positive CD45+ lineage CD11c+HLA-DR+ myeloid dendritic cells [Figure 4E-1] E. Activation status of dendritic cells (DCs), percentage of CD40+CD45+ lineage HLA-DR+ myeloid dendritic cells [Figure 4E-2] E. Activation status of dendritic cells (DCs), percentage of CD40+CD45+ lineage HLA-DR+ myeloid dendritic cells [Figure 5-1] Figure 5 shows the determination of optimal progression-free survival (PFS) biomarker thresholds. Left panel, ROC curve; right panel, Youden index. AUC, area under curve. A higher AUC indicates improved outcome in the receiver operating curve (ROC), whereas in the Youden index, a higher intersection point of the two curves indicates optimal biomarker performance. [Figure 5-2] Figure 5 shows the determination of optimal progression-free survival (PFS) biomarker thresholds. Left panel, ROC curve; right panel, Youden index. AUC, area under curve. A higher AUC indicates improved outcome in the receiver operating curve (ROC), whereas in the Youden index, a higher intersection point of the two curves indicates optimal biomarker performance. [Figure 6A] Figure 6 shows a Kaplan-Meier plot comparing IMPULSE patients whose PFS1 exceeded the threshold of 188 days with patients whose PFS1 was less than the threshold of 188 days. A: Switching maintenance therapy with TLR9 agonist [Figure 6B]B: PE chemotherapy as continued maintenance therapy (depending on PFS >1188 days or less). [Figure 7A-1] A: Kaplan-Meier plot comparing IMPULSE patients undergoing switched maintenance therapy with a TLR9 agonist or continued maintenance therapy with control according to whether the percentage of CD45+CD3+CD4+CD25+CD127- regulatory T (Treg) among gated CD45+CD3+CD4+ T cells was above or below the median. [Figure 7A-2] A: Kaplan-Meier plot comparing IMPULSE patients undergoing switched maintenance therapy with a TLR9 agonist or continued maintenance therapy with control according to whether the percentage of CD45+CD3+CD4+CD25+CD127- regulatory T (Treg) among gated CD45+CD3+CD4+ T cells was above or below the median. [Figure 7B-1] B: Kaplan-Meier plot comparing IMPULSE patients who underwent switched maintenance with a TLR9 agonist divided according to whether the median percentage of CD86+ activated B cells after induction or reinduction chemotherapy was 16.9% or less or 21% or less, with IMPULSE patients who underwent continued maintenance with standard of care as control. Patients with activated B cells below the median (16.9%) after induction or reinduction chemotherapy were treated with sequential TLR9 agonist switched maintenance after the fifth cycle of PE chemotherapy, and patients with activated B cells below the median (16.9%, or 21%) after induction or reinduction chemotherapy had a PFS1 of >6.2 months in continued maintenance and were treated with 4, 5, or 6 cycles of PE chemotherapy. [Figure 7B-2]B: Kaplan-Meier plot comparing IMPULSE patients who underwent switched maintenance with a TLR9 agonist divided according to whether the median percentage of CD86+ activated B cells after induction or reinduction chemotherapy was 16.9% or less or 21% or less, with IMPULSE patients who underwent continued maintenance with standard of care as control. Patients with activated B cells below the median (16.9%) after induction or reinduction chemotherapy were treated with sequential TLR9 agonist switched maintenance after the fifth cycle of PE chemotherapy, and patients with activated B cells below the median (16.9%, or 21%) after induction or reinduction chemotherapy had a PFS1 of >6.2 months in continued maintenance and were treated with 4, 5, or 6 cycles of PE chemotherapy. [Figure 8A] Figure 8 shows the second biomarker component analyzed in patients from the IMPALA metastatic colorectal cancer trial treated with lefitolimod or control standard of care. A: Relative distribution of activated B cell biomarker values ​​and percent of CD86 positive CD45+CD19+ B cells in the indicated treatment groups. Kaplan-Meier plots comparing overall survival (OS) of IMPALA patients receiving continued maintenance therapy with a TLR9 agonist or control, divided according to B. progression free survival >225 days or >250 days on maintenance treatment, C. progression free survival >225 days or >250 days on reinduction treatment. [Figure 8B-1] Figure 8 shows the second biomarker component analyzed in patients from the IMPALA metastatic colorectal cancer trial treated with lefitolimod or control standard of care. A: Relative distribution of activated B cell biomarker values ​​and percent of CD86 positive CD45+CD19+ B cells in the indicated treatment groups. Kaplan-Meier plots comparing overall survival (OS) of IMPALA patients receiving continued maintenance therapy with a TLR9 agonist or control, divided according to B. progression free survival >225 days or >250 days on maintenance treatment, C. progression free survival >225 days or >250 days on reinduction treatment. [Figure 8B-2]Figure 8 shows the second biomarker component analyzed in patients from the IMPALA metastatic colorectal cancer trial treated with lefitolimod or control standard of care. A: Relative distribution of activated B cell biomarker values ​​and percent of CD86 positive CD45+CD19+ B cells in the indicated treatment groups. Kaplan-Meier plots comparing overall survival (OS) of IMPALA patients receiving continued maintenance therapy with a TLR9 agonist or control, divided according to B. progression free survival >225 days or >250 days on maintenance treatment, C. progression free survival >225 days or >250 days on reinduction treatment. [Figure 8C-1] Figure 8 shows the second biomarker component analyzed in patients from the IMPALA metastatic colorectal cancer trial treated with lefitolimod or control standard of care. A: Relative distribution of activated B cell biomarker values ​​and percent of CD86 positive CD45+CD19+ B cells in the indicated treatment groups. Kaplan-Meier plots comparing overall survival (OS) of IMPALA patients receiving continued maintenance therapy with a TLR9 agonist or control, divided according to B. progression free survival >225 days or >250 days on maintenance treatment, C. progression free survival >225 days or >250 days on reinduction treatment. [Figure 8C-2] Figure 8 shows the second biomarker component analyzed in patients from the IMPALA metastatic colorectal cancer trial treated with lefitolimod or control standard of care. A: Relative distribution of activated B cell biomarker values ​​and percent of CD86 positive CD45+CD19+ B cells in the indicated treatment groups. Kaplan-Meier plots comparing overall survival (OS) of IMPALA patients receiving continued maintenance therapy with a TLR9 agonist or control, divided according to B. progression free survival >225 days or >250 days on maintenance treatment, C. progression free survival >225 days or >250 days on reinduction treatment. [Figure 9A]Figure 9 shows A: A biomarker-guided, sequential, two-step, branched model of alternating innate and adaptive immune modulation following cancer treatment based on induction chemotherapy (e.g. platinum-etoposide induction chemotherapy for esSCLC, IMPULSE) or reinduction chemotherapy (5-FU / FA or CAPE with OX or IRI, alone or in combination with anti-VEGF or anti-EGFR antibodies for mCRC, IMPALA). The model predicts the most effective effect to apply depending on innate or adaptive immune stimulation based on the median value of biomarkers reflecting the patient's adaptive immune activation level. [Figure 9B] B. Simplified model overview. [Figure 10] FIG. 10 shows representative examples of immunomodulatory treatment regimens with varying duration of platinum-based induction chemotherapy treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Summary of the Invention The present invention provides TLR9 agonist or checkpoint modulator (CPM) drugs for use in subgroups of cancer patients. Experimental studies herein demonstrate the presence of immune correlates in blood samples that indicate whether patients may respond to innate or adaptive immune stimulation after a first induction chemotherapy treatment. Furthermore, the present invention provides specific timing and treatment conditions for administration of said formulations that the inventors have found to be associated with improved clinical outcome.

[0012] One aspect of the present invention provides a TLR9 agonist for use in a patient who has first undergone induction chemotherapy treatment for cancer and has been found to exhibit markers of immune cell subset activation below a median threshold indicative of an immunologically non-responsive tumor or tumor microenvironment in a first predictive biomarker assay. The TLR9 agonist is then initially administered in the form of one, two or three repeated cycles of a "TLR9 agonist induction immunomodulatory cycle" (wherein the chemotherapy agent is administered in week 1 and the TLR9 agonist is administered in weeks 2 and 3). The TLR9 agonist is then administered as part of a "TLR9 agonist maintenance immunomodulatory regimen" (wherein the TLR9 agonist is administered twice a week, once every week or every two weeks for n weeks of said TLR9 agonist maintenance immunomodulatory regimen, where n is an integer between 5 and 35 weeks, particularly between 9 and 18 weeks, more particularly where n is 12 weeks).

[0013] In current immuno-oncology treatment protocols, each immunotherapy agent is "combined" with chemotherapy or targeted therapy, i.e., applied together or on the same day. For example, in the first line therapy of esSCLC in the trials analyzed in this study, PE chemotherapy is administered together with the PD-L1 inhibitor antibodies Atezolizumab (Tecentriq®, Roche) or Durvalumab (Imfinzi®, AstraZenenca). The results of combination regimes have so far been modest, limited to a small proportion of patients, and have not been able to produce durable responses. In contrast, the present invention provides a dosing regime of checkpoint modulators that differs from previous studies in terms of the target population identified by specific biomarkers and the dichotomized, two-phase sequential nature of the treatment compared to the combination approach.

[0014] Another aspect of the present invention provides a CPM drug for use in patients undergoing induction chemotherapy for cancer and exhibiting markers of immune cell subset activation above a median threshold indicative of immune exhaustion in a first predictive biomarker assay. These patients exhibit an immune "hot" yet exhausted tumor microenvironment, where post-hoc analysis does not associate with substantial benefit from TLR9 agonists as described above, but rather the CPM drug produces significant clinical benefit. Accordingly, the CPM drug is first administered in the form of 1-5 sets of "CPM drug induction immunomodulatory cycles" (chemotherapeutic agent and CPM drug administered together once every 3 weeks). Thereafter, the CPM drug is administered as part of a "CPM drug maintenance immunomodulatory regimen" (wherein the CPM drug is administered twice a week, once every week, or every 2 weeks of said CPM drug maintenance immunomodulatory regimen for n weeks, where n is an integer between 6 and 36 weeks, particularly an integer between 9 and 18 weeks, more particularly n is 12 weeks).

[0015] One aspect of the present invention relates to the allocation of cancer patients to alternating immune-modulating treatment with either a TLR9 agonist or a CPM drug after induction chemotherapy. The timing of said alternating treatment is guided by the outcome of a first and a second predictive biomarker assay measuring an indicator of immune exhaustion. Immune cells activated above a threshold indicate either a "hot" TME (tumor microenvironment) or a previously "hot" but now "exhausted" TME; the immune response cannot be further activated by a TLR9 agonist, regardless of its exhausted state. Immune cells activated below a threshold indicate either a "cold" TME (previously a "hot" TME but now an "exhausted" TME); where the immune response can be activated or reactivated by a TLR9 agonist in a process known as "immune surveillance reactivation" (ISR).

[0016] In another embodiment, the present invention relates to a pharmaceutical composition comprising at least one compound of the present invention or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable carrier, diluent or excipient.

[0017] The present invention provides treatments for use in both patients identified as having an immunologically "cold" or "hot" tumor microenvironment, with targeted provision of immune biomarker assessment following an initial round of standard chemotherapy.

[0018] Terms and Definitions For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0019] As used herein, the terms "comprising," "having," "containing," "including," and other similar forms and their grammatical equivalents are intended to be equivalent in meaning and to be open-ended in that the item or items following any one of these words are not intended to be an exhaustive list of such item or items or are not limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or can include not only components A, B, and C, but also one or more other components. Thus, "comprising" and similar forms and their grammatical equivalents are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0020] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding either or both of those included limits are also included in the disclosure.

[0021] As used herein, reference to a value or parameter "about" includes (and describes) the variation directed to the value or parameter itself. For example, a statement referring to "about X" also includes the statement "X."

[0022] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular biology, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical methods.

[0024] Predictive Biomarker Assays The predictive biomarkers according to the present invention assay the cell surface expression of immune cell subset phenotypic and activation markers in peripheral blood mononuclear cells (PBMCs) isolated from blood samples to determine the immune status of cancer patients. Expression of activation or phenotypic markers can be assayed via techniques such as flow cytometry or multiplex analysis.

[0025] Suitable methods for measuring activated immune cell populations in liquid biopsies are known in the art and include methods for measuring cell phenotype and activation marker expression at the single cell level on slides or in liquid samples to determine the proportion of activated cells within a particular cell subset. Such methodologies include mass spectrometry assays performed by either histology or fluidics-based assays, multiplexed immunohistochemistry, or flow cytometry protocols. Flow cytometry is a method that is particularly used to measure cell subsets and their activation markers according to the present invention.

[0026] As used herein, the term "positive" when used in the context of expression of a marker refers to expression of an antigen as assayed by a labeled molecular probe, particularly a fluorescently labeled antibody, where the signal (e.g., fluorescence) of the label on a structure (e.g., a cell) referred to as "positive" herein is at least 30% higher (≧30%) in median intensity, particularly 50% or more or 80% or more, compared to staining with an isotype-matched labeled antibody that does not specifically bind to the same target. Expression of such markers is indicated by the superscript "plus" ( + ) (e.g. CD4 + ), or by a plus sign after the name of the marker ("CD4+"). When the term "expression" is used herein in the context of "gene expression" or "marker or biomolecule expression" and no further modification of "expression" is mentioned, this includes the meaning of "positive expression" as defined above.

[0027] As used herein, the term "negative" when used in the context of marker expression refers to expression of an antigen assayed by a labeled molecular probe, particularly a fluorescently labeled antibody, where the median labeled signal intensity is less than 30% higher, particularly less than 15% higher, than the median intensity of an isotype-matched antibody that does not specifically bind to the same target. Expression of such markers is indicated by a superscript minus ( - ) (e.g. CD86 - ), or by placing a minus sign after the marker name ("CD86-").

[0028] The term "molecular probe" in the context of this specification refers to a specific ligand, in particular an antibody, an antibody fragment, an antibody-like molecule or an aptamer, more in particular an antibody or an antibody fragment, which binds to a target molecule, such as a specific surface protein, with a binding affinity of (≦) 10 -7 mol / l or less, especially (≦)10 -8 It refers to a molecule capable of binding with a dissociation constant of less than mol / l. Molecular probes include detectable markers such as fluorescent dyes, metal particles, beads, dyes, or enzymes.

[0029] The term "set of molecular probes" refers to a panel of molecular probes specific for the cellular phenotype or activation markers that are measured in a predictive biomarker assay.

[0030] The term "fluorescent label" or "fluorescent dye" as used herein refers to a small molecule that can fluoresce in the visible or near infrared spectrum. Examples of fluorescent labels or labels that exhibit visible colors include, but are not limited to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanin (APC), peridinin chlorophyll (PerCP), phycoerythrin (PE), Alexa Flour (Life Technologies, Carlsbad, CA, USA), dylight fluor (Thermo Fisher Scientific, Waltham, MA, USA), ATTO Dye (ATTO-TEC GmbH, Siegen, Germany), BODIPY Dye (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-based dyes), etc.

[0031] chemotherapy treatment The term "induction chemotherapy" in the context of this specification relates to a period of several weeks or months, particularly 6 to 12 weeks, during which standard of care chemotherapy drugs, i.e. anti-cancer chemotherapy drugs, are administered to cancer patients in order to induce tumor cell death. This releases tumor antigens that initiate an innate and adaptive immune response against the cancer cells, which can later be exploited by administration of an immunomodulatory agent, i.e. TLR9 agonist molecule or check modulator agent (CPM), provided by the present invention. Such "induction chemotherapy" usually comprises or consists of repeated "cycles" of treatment, each cycle consisting of administration of an anti-tumor chemical preparation or targeted monoclonal antibody treatment once in a period of 2 to 4 weeks, usually once every 3 weeks. The anti-cancer agent used in "induction chemotherapy" is not particularly limited according to the present invention, and should be selected and prescribed according to the type and malignancy of the tumor as confirmed by clinical evaluation of the standard of care.

[0032] Examples of induction chemotherapy include, but are not limited to, for esSCLC patients such as those in the IMPULSE trial, 2-6 cycles of platinum-based combination induction chemotherapy, each cycle being administered once every 3 weeks with cisplatin or carboplatin in combination with etoposide. For breast or cervical cancer, induction chemotherapy may consist of cyclic administration of platinum complexes such as cisplatin or carboplatin, or epidermal growth factor or vascular growth factor inhibitor antibodies.

[0033] The term "induction chemotherapy" as used herein encompasses the so-called "reinduction" chemotherapy studied in the IMPALA study according to the mCRC treatment guidelines, where the chemotherapy drug treatment protocol is provided to patients for a second time after a period of remission in case of tumor recurrence and / or in case of diagnosis of metastasis. For metastatic colorectal cancer patients such as those participating in the IMPALA clinical trial, induction (or reinduction) chemotherapy can be cycles of treatment with antineoplastic agents such as, but not limited to, antibodies targeting the growth hormone receptor, platinum-based chemotherapy, platinum complexes such as oxaliplatin, or other antimitotic chemotherapy agents.

[0034] As used herein, the term "platinum-based chemotherapy treatment" refers to a combination drug that includes the following in combination with a second chemotherapeutic agent: - a platinum-containing drug, also known as a platinum-containing complex, selected from, for example, carboplatin, satraplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, picoplatin, and / or triplatin.

[0035] The platinum agent and the second chemotherapeutic agent may be combined in a single tablet or injection, or may be administered simultaneously as two separate formulations.

[0036] Examples of additional anti-tumor chemotherapeutic agents that may be utilized in an "induction chemotherapy" cycle or as a second chemotherapeutic agent that may be used in conjunction with the platinum-containing drugs defined above to provide a platinum-based chemotherapy treatment are known in the art and include, but are not limited to, the following drug classes: - alkylating antineoplastic agents, in particular selected from altretamine, bendamustine, busulfan, carmustine, cyclophosphamide, chlorambucil, dacarbayine, etoposide, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, tiptepa, and trabectedin; - anti-metabolic type antineoplastic agents, in particular selected from azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, hydroxycarbamide, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate and phototrexate; - mitotic inhibitors, in particular selected from cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine and vinorelbine; - anti-metabolic type antineoplastic agents, in particular selected from azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, hydroxycarbamide, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate and phototrexate; anthracycline antineoplastic agents, in particular anthracycline antineoplastic agents selected from daunorubicin, doxorubicin, epirubicin and idarubicin; - selective SERM antineoplastic agents, in particular SERM agents selected from raloxifene, toremifene and tamoxifen; - SERD antineoplastic agents, in particular selected from fulvestrant, brilandestrant and elacestrant; - aromatase inhibitors, in particular aromatase inhibitor antineoplastic agents selected from exemestane, letrozole, vorozole, formestane, fadrozole and anastrozole; and / or - a PI3K pathway inhibitor, in particular a PI3K pathway inhibitor selected from rapamycin, dactolisib, BGT226, SF1126, PKI-587, and NVPBE235; - HER2 targeted antineoplastic agents, in particular selected from trastuzumab, pertuzumab, SYD985, RC48, A166, HER2ALT-P7, T-DM1, ARX788, KN026, BVAC-B, MT-5111, AVX901, TAS0728, MP0274, MM-302, FS102, and H2NVAC; - antineoplastic agents of the class of estrogen growth factor receptor (EGFR) bioactive inhibitors, in particular antineoplastic agents of the class of EGFR bioactive inhibitors selected from gefetinib, erlotinib, lapatinib, cetuximib, neratinib, osimeratib, panitumamib, vandetanib, necitumumab, and dacomitinib; - an angiogenic antineoplastic agent, in particular an angiogenic antineoplastic agent selected from bevacizumab, itraconazole, thalidomide, and lenalidomide, or an antineoplastic agent that inhibits the biological activity of vascular endothelial growth factor (VEGF).

[0037] In the context of this specification, the term "cycle set," "treatment cycle," or "cycle" refers to a treatment in which multiple agents, or combinations of agents, are delivered in a particular order over a particular period of time, and this order is optionally repeated in the same set or cycle or treatment. As used herein, the term "regimen" refers to a single treatment or therapeutic agent, delivered at regular intervals over a particular weekly period of time.

[0038] immunotherapy In the context of this specification, the terms "cancer immunomodulation," "cancer immunotherapy," "biological therapy," or "immunomodulatory therapy" are meant to encompass a type of cancer treatment that helps the immune system fight cancer. Non-limiting examples of cancer immunotherapy include immune checkpoint inhibitors and agonists, T cell transfer therapy, cytokines and their recombinant derivatives, adjuvants, and small molecule or cellular vaccination.

[0039] In the context of the present specification, the term "checkpoint modulator" or "checkpoint inhibitor antibody" is meant to encompass cancer immunotherapeutic agents, particularly antibodies (or antibody-like molecules), capable of disrupting the inhibitory signaling cascade that limits immune cell activation, known in the art as the immune checkpoint mechanism. In the context of the present specification, the term "checkpoint modulator" is meant to encompass agents, particularly antibodies (or antibody-like molecules), capable of disrupting the signaling cascade that leads to T cell inhibition following T cell activation, as part of the immune checkpoint mechanism known in the art. In certain embodiments, a "checkpoint inhibitor" or "checkpoint inhibitor antibody" is an antibody against CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), VISTA (Uniprot Q9H7M9), TIGIT (UniprotQ495A1), TIM-3 (HAVCR2, Uniprot Q8TDQ0), CD158 (killer cell immunoglobulin-like receptor family), TGF-β (P01137).

[0040] Non-limiting examples of "checkpoint modulator agents" or "checkpoint inhibitor antibodies" include ipilimumab (Yervoy; CAS number 477202-00-9), or antibodies against PD-1 (Uniprot Q15116), or antibodies against PD-L1 (Uniprot Q9NZQ7), and antibodies against B7H3 (CD276; Uniprot Q5ZPR3). Clinically available antibody drugs include nivolumab (Bristol-Myers Squibb; CAS number 946414-94-4), durvalumab (Astra Zeneca; CAS number 1428935-60-7), pembrolizumab (Merck Inc.; CAS number 1374853-91-4), pidilizumab (CAS number 1036730-42-3), and atezolizumab (Roche Examples include cemiplimab (Sanofi Aventis; CAS number 1801342-60-8), and avelumab (Merck KGaA; CAS number 1537032-82-8).

[0041] In the context of this specification, the term "TLR9 agonist" refers to stimulators of the toll-like receptor 9 that detects pathogenic DNA, in particular members of the dSLIM® family (dSLIM (double Stem Loop Immunomodulator)), a new family of TLR9 agonists consisting of a dumbbell-shaped covalently closed DNA molecule lacking any chemical or other artificial modification of the DNA (Kapp K. et al., 2019 Oncoimmunology DOI: 10.1080 / 2162402X.2019.1659096; Schmidt M. et al., 2015 Nucleic Acid Therapeut. 25(3): 140). Protection against nucleic acid degradation is achieved by the covalently closed structure that avoids the accessibility of the 3' end. Lefitolimod (MGN1703) belongs to this group of TLR9 agonists that represents a specific immunomodulatory sequence and structure.

[0042] In the context of this specification, the term "stem" refers to a DNA duplex formed by base pairing either within the same DNA molecule (partially self-complementary) or within different DNA molecules (partially or fully complementary).

[0043] In the context of this specification, the term "loop" refers to an unpaired single-stranded region either within a stem structure or at the end of a stem structure.

[0044] In the context of this specification, the term "dumbbell-shaped" refers to a polynucleotide that comprises a double-stranded stem (base pairing within the same polynucleotide) and two single-stranded loops at either end of the double-stranded stem.

[0045] As used herein, the term "pharmaceutical composition" refers to a compound of the present invention or a pharma- ceutically acceptable salt thereof, together with at least one pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or injectable administration.

[0046] As used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those of skill in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0047] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., cancer) refers, in one embodiment, to ameliorating the disease or disorder (e.g., delaying, preventing, or alleviating the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible to the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing the treatment and / or prevention of a disease are generally known in the art, unless otherwise described herein below.

[0048] In the context of the present specification, the term "cancer" referred to as a disease to be treated or a disease the recurrence of which is to be prevented, particularly relates to malignant neoplastic diseases, and more particularly includes carcinomas (cancers of epithelial origin), sarcomas (cancers of connective tissue origin), lymphomas and leukemias, germ cell-derived tumors and germinomas. In a particular embodiment, cancer may refer to a neoplastic disease selected from the group consisting of breast cancer, cervical cancer, endometrial cancer, colorectal cancer, metastatic colorectal cancer, and lung cancer. In a more particular embodiment, the TLR9 agonist and / or checkpoint modulator provided herein is used to treat small cell lung cancer, particularly extensive stage small cell lung cancer (esSCLC), or metastatic colorectal cancer.

[0049] Detailed Description of the Invention Sequential / alternate administration schedules of TLR9 agonists A first aspect of the invention relates to a TLR9 agonist for use in the treatment of a patient diagnosed with cancer.

[0050] The TLR9 agonist is administered to patients who have undergone induction chemotherapy consisting of the first set of chemotherapy treatment cycles. Induction chemotherapy refers to standard of care antineoplastic treatment appropriate to the patient's tumor type for a period of several weeks to several months, particularly 6-12 weeks, usually administered in cycles of oral or infusion of drugs every 2-4 weeks. This induction chemotherapy encompasses both primary cancer diagnosis treatment and reinduction chemotherapy, which is administered to patients who have previously undergone cancer treatment and have recently been diagnosed with no recurrence or progression of cancer after a period of disease, or no tumor growth above a baseline measurement or threshold.

[0051] Following induction chemotherapy ("following" in the context of the present invention means preferably starting the week after, or at most 2-3 weeks after, induction chemotherapy), a TLR9 agonist is administered as part of a "TLR9 agonist induction immunomodulatory cycle".

[0052] According to this aspect of the invention, a "TLR9 agonist-induced immunomodulatory cycle" comprises or consists of administering a chemotherapeutic agent or drug, or a combination of drugs, together with a dose of a TLR9 agonist in the first week of said TLR9 agonist-induced immunomodulatory cycle. This is followed by administration of a TLR9 agonist once or twice weekly in the second and third weeks of said TLR9 agonist-induced immunomodulatory cycle. In some embodiments, the TLR9 agonist-induced immunomodulatory cycle utilizes the same therapeutic agent administered in the induction chemotherapy phase. The chemotherapeutic agent can be selected from those used in the induction chemotherapy described above. Certain embodiments relate to the use of platinum-containing complexes or platinum-based chemotherapy treatments in combination with a TLR9 agonist.

[0053] After 1, 2, 3, or 4 of the TLR9 agonist induction immunomodulatory cycles, a TLR9 agonist is administered sequentially as part of a "TLR9 agonist maintenance immunomodulatory regimen."

[0054] According to this aspect of the invention, during the "TLR9 agonist maintenance immunomodulatory regimen", the TLR9 agonist is administered twice a week, once each week, or every two weeks during n weeks of said TLR9 agonist maintenance immunomodulatory regimen, where n is an integer between 5 and 35 (including treatment schedules suitable for mCRC, mBC, or SCLC and other cancers). In a particular embodiment, n is an integer between 9 and 18 weeks (standard treatment timing for SCLC). In a more particular embodiment, n is 12 weeks (demonstrated to include a responsive patient population in the IMPULSE trial evaluated in the Examples). In contrast to the "TLR9 agonist induction immunomodulatory cycle", no additional chemotherapeutic agents are administered to the patient during the "TLR9 agonist maintenance immunomodulatory regimen".

[0055] A particular embodiment of this aspect of the invention relates to a TLR9 agonist for use in patients who have been assigned a "non-matched-in-first" status in a first predictive biomarker assay. The first predictive biomarker assay determines the immune status, i.e., the percentage of activated cells within an immune cell subset selected from B cells, T cells, natural killer T (NKT) cells, dendritic cells (DCs), myeloid dendritic cells (DCs), and / or plasmacytoid dendritic cells (pDCs), or the percentage of regulatory T (Treg) cells within a CD4+ T cell subset, in a blood sample obtained from the patient to measure immune exhaustion. In a particular embodiment, the predictive biomarker assay according to the invention utilizes flow cytometry to identify cell subsets and measure the proportions that display an activated phenotype.

[0056] The percentage of activated immune cells or Treg cells determined in the first predictive biomarker is compared to a threshold of a median value previously determined for blood samples obtained from a cohort of cancer patients diagnosed with the same cancer as the patient and undergoing said first set of treatment cycles prior to biomarker evaluation. If the percentage of activated immune cells is equal to or greater than this median threshold, the patient is assigned as "matched" and if the percentage of activated immune cells or Treg cells is below the threshold, the patient is assigned as "non-matched."

[0057] Particular embodiments of the first predictive biomarker relate to determining the percentage of regulatory T (Treg) cells among CD4+ T cells. According to these embodiments, if the percentage of activated cells in the immune subset is less than the median percentage of activated cells or less than the median percentage of Treg cells among CD4+ T cells, the patient is assigned a "1st round mismatch" status.

[0058] Particular embodiments of the first predictive biomarker according to the first aspect of the invention relate to the use of a flow cytometry assay to determine the percentage of B cells (e.g. defined as CD19+CD45+ cells) expressing an activation marker such as CD80 or CD86, particularly CD86 (Uniprot P42081). In some embodiments, if the percentage of activated B cells falls below a threshold of about 16.9%, the patient is assigned a "first mismatch" status.

[0059] In a further particular embodiment of the TLR9 or CPM for use, or method according to the invention, if the percentage of activated B cells in the blood sample obtained from the patient is at least 15.42%, the patient is assigned a status of "matched-in-first" and if the percentage of activated B cells in the blood sample is less than 15.42%, the patient is assigned a status of "mismatched-in-first".

[0060] In a more particular embodiment of the TLR9, CPM or method for use according to the invention, a patient is assigned a "first-time match" status if the percentage of CD86+CD19+CD45+ B cells in a blood sample obtained from the patient is at least 15.42% of the total CD19+CD45+ B cells, and a patient is assigned a "first-time mismatch" status if the percentage of CD86+CD19+CD45+ B cells in the blood sample is less than 15.42% of the total CD19+CD45+ B cells. High expression of immune activation markers as assessed by the first or second predictive biomarker assays suggests that the patient has received sufficient innate immune stimulant TLR9 agonist and is therefore more likely to benefit from alternative treatments, such as chemotherapeutic agents, or in particular checkpoint modulators.

[0061] In some embodiments, the treatment of patients who have received induction chemotherapy and have been assigned a status of "matched first time" in the first predictive biomarker assay defined by the above-mentioned related aspects of the invention is not particularly limited by the present invention. Patients may be assigned a status of "matched first time" if the percentage of activated cells in the immune subset is equal to or above the threshold, in particular equal to or above the median percentage of activated cells, or if the percentage of Treg cells in CD4+ T cells is equal to or above the threshold. In a particular embodiment, patients are assigned to "matched first time" with a median percentage of B cells equal to or above 16.9%.

[0062] In certain embodiments, if the first predictive biomarker is matched, e.g., the percentage of activated B cells observed in the test sample is equal to or above a predefined threshold of immune activation indicative of immune exhaustion, the TLR9 agonist is discontinued. Certain embodiments relate to returning to standard of care cancer treatment if the biomarker is matched, e.g., continuing maintenance with platinum-based chemotherapy or switching to second-line chemotherapy such as topotecan as demonstrated in the IMPULSE trial.

[0063] In certain embodiments, TLR9 is administered to a cancer patient who has undergone a TLR9 agonist maintenance immunomodulatory regimen as defined above for the previous three weeks and who has also undergone a second predictive biomarker assay, which in certain embodiments determines the percentage of regulatory T cells among CD4+ T cells in a blood sample obtained from the patient.

[0064] In some embodiments, a patient is assigned "non-matched-in-second" status if the percentage of activated Treg cells in the sample is less than the median Tregs obtained from a cohort of patients receiving the same treatment for the same condition. In some embodiments, a patient is assigned "non-matched-in-second" status if the percentage of activated Treg cells in the sample is less than 20%.

[0065] After being assigned a "second mismatch" status, the patient is administered a TLR9 agonist twice weekly, once weekly, or every two weeks for n weeks, where n is an integer between 6 and 24. In a particular embodiment, n is an integer between 6 and 15. In a more particular embodiment, n is 9.

[0066] In certain embodiments, a TLR9 agonist is administered following the induction chemotherapy described above if the second predictive biomarker is: A patient is assigned a "second mismatch" status if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%; or A patient is assigned a "matched-in-second" status if the percentage of Treg cells among CD4+ T cells in the sample is 20% or higher.

[0067] In another embodiment, If the percentage of Treg cells among CD4+ T cells in the sample is less than about 15%, the patient is assigned a "second mismatch" status, or If the percentage of Treg cells among CD4+ T cells in the sample is approximately 15% or higher, the patient is assigned a “second match” status.

[0068] In another embodiment of the second predictive biomarker assay, patients are assigned a biomarker status based on the percentage of activated B cells measured in the ex vivo sample, e.g., patients are assigned a status of "second-time matched" if the percentage of CD19+ cells co-expressing CD80 or CD86 is below a threshold of about 15%, and "second-time mismatched" if the percentage of activated B cells is below 15%.

[0069] After the status of "non-matched 2nd" is assigned, the patient continues to receive the TLR9 agonist twice weekly, once weekly, or every 2 weeks for n weeks, where n is an integer between 6 and 24. In particular embodiments, n is in the range of 6 to 15. In more particular embodiments, n is 9. After the status of "matched 2nd" is assigned, the patient continues to receive the TLR9 agonist twice weekly, once weekly, or every 2 weeks for n weeks, where n is an integer between 3 and 15.

[0070] In some embodiments, a patient is assigned "second-matched" status if the patient reaches the median progression-free survival of a cohort of patients receiving induction or reinduction chemotherapy in the context of clinical standard of care.

[0071] In the context of this specification, the term "tumor progression" or "disease progression" refers to tumor growth, recurrence, or metastasis compared to the tumor size, grade, and severity index assessment obtained at baseline measurement before induction chemotherapy. Disease progression according to the present invention can be assessed using standardized clinical assessment protocols known in the art, such as RECIST 1.1 criteria (Lawrence SH et al., 2016 Eur. J. Cancer 62:138) or irRC criteria (Hoos A. et al., 2007 30(1)1). Disease progression can be assigned or diagnosed, for example, when tumor volume increases by 25% as determined by bidirectional measurement at a time point after baseline measurement and / or when new cancer cell lesions are identified.

[0072] In contrast, patients who are not characterized as having "disease progression" are characterized as having "progression-free survival (PFS)". This term refers to the time period, e.g., days from the start of cancer treatment, without "disease progression" according to the thresholds defined by the irRC and / or RECIST 1.1 criteria. In other words, the time period, counted from the first administration of induction chemotherapy that initiates the patient's treatment protocol according to the present invention, during which the patient's tumor does not grow or metastasize beyond baseline levels or baseline tumor size (e.g., more than 25% increase in tumor volume). "Median progression-free survival" refers to the median number of days until a diagnosis of disease progression is observed / diagnosed in a cohort of patients characterized as having been diagnosed with cancer originating from the same tissue of origin as the cancer patient treated according to the present invention. The size of the cohort must be large enough to perform calculations of medians and confidence intervals that can distinguish between patient outcomes, as demonstrated in the examples (Figure 5).

[0073] In some embodiments, esSCLC patients receiving TLR9 agonist maintenance modulation are assigned "second-matched" status if characterized by the absence of a diagnosis of disease progression for more than 188 days after initiation of induction chemotherapy.

[0074] In some embodiments, colorectal cancer patients who receive TLR9 agonist maintenance modulation are not diagnosed with disease progression between 225 and 250 days after the start of the first dose of reinduction chemotherapy and are assigned a "second-time consistent" status.

[0075] In some embodiments, patients receiving maintenance modulation of a TLR9 agonist are assigned a "second-time match" status if the patient is characterized as free of disease progression at the median progression-free survival time for that particular cancer, e.g., at least 188 days after initiation of initial induction chemotherapy in the case of SCLC, or if the second predictive biomarker indicates that the percentage of Treg cells is above a threshold of 15%, or even above a threshold of 20%, or if the percentage of activated B cells is above 15%.

[0076] Particular embodiments of the TLR9 agonist for use according to the first aspect of the invention defined above relate to the use of the TLR9 agonist such that said second predictive biomarker assay (defined on pages 12 and 13) is performed every 6 to 15 weeks to determine the appropriate timing for switching to a CPM agent. In particular embodiments, the second predictive biomarker assay is performed every 9 weeks. In particular embodiments, the second predictive biomarker assay is performed every 9 weeks until the patient goes into remission or progresses. According to these embodiments: After a "second non-match" status is assigned, the TLR9 agonist continues to be administered twice weekly, once weekly, or every two weeks for n weeks thereafter, where n is an integer ranging from 6 to 24. In particular embodiments, n is in the range of 6 to 15. In a more particular embodiment, n is 9. or After being assigned a “second-match” status, patients will be assigned to switch treatment to a checkpoint modulator (CPM) agent, administered either every 2 weeks or every 3 weeks for the next 3 to 9 weeks.

[0077] In some embodiments, patients who receive a TLR9 agonist following induction chemotherapy are assigned to treatment with a checkpoint modulator (CPM) agent administered every 2 weeks or every 3 weeks for the following 3-9 weeks upon reaching median PFS without a diagnosis of disease progression and without reference to a second predictive biomarker.

[0078] In some embodiments, SCLC patients who have been administered a TLR9 agonist for 188 days after induction chemotherapy and have no evidence of disease progression are switched to treatment with a CPM agent. In another embodiment, mCRC patients receiving a TLR9 agonist immunomodulatory maintenance regimen are switched to receive a CPM maintenance immunotherapy regimen if they reach a PFS threshold of 225 days without a diagnosis of disease progression.

[0079] TLR9 agonists for use according to the present invention TLR9 agonists are mainly oligodeoxynucleotides or their structural derivatives, and the oligodeoxynucleotide sequence contains one or several unmethylated CpG dinucleotides. TLR9 agonists are classified into three classes, A, B, and C, according to the stimulatory effect correlated with their chemical structure. - Class A CpG ODNs (CpG oligodeoxynucleotides) are strong stimulators of IFN-α production but are only weak stimulators of TLR9-dependent NF-κB signaling. Many representatives of this class are characterized by a palindromic motif with a central CpG and a 3'-terminal poly-G stretch with phosphorothioate internucleoside bonds. - Class B CpG ODNs activate B cells very effectively but only weakly stimulate the secretion of IFN-α. The main examples of this class are characterized by all internucleoside linkages being formed by phosphorothioates (thiophosphates). - Class C CpG ODNs activate IFN-α production as well as B cell stimulation. Structurally, C-type CpG ODNs often combine properties of both classes, possessing both a full-length PTO backbone and a CpG-containing palindromic motif in the middle of the sequence.

[0080] Many different TLR9 agonist small molecule structures and deoxyribonucleotide oligomers have been clinically developed as immunomodulatory agents, including, but not limited to, the TLR9 agonist molecules disclosed in US2010144846A1 and its family, US2007142315A1 and its family, US2004143112A1 and its family, US2007066553A1 and its family, US2006241076A1 and its family, US10894963B2 and its family.

[0081] PF-35112676 (Pfizer) is a class B TLR9 agonist, also called CPG7909 (Coley). Class B TLR9 agonists are not approved for oncological indications, as they are associated with adverse side effects and have not been demonstrated to increase patient survival. This highlights the need for alternative approaches to target immune cold tumors to initiate new (de novo) T cell responses, which the present invention answers. Class B CpG-ODNs are good vaccine adjuvants, but have proven poor for cancer therapy, whereas class A TLR9 agonists, such as lefitolimod, or potentially class C molecules, are more suitable for therapeutic efficacy for this purpose. In a particular embodiment, the TLR9 agonist for use according to the present invention comprises at least one unmethylated CG dinucleotide, which is part of the term "CpG motif" referred to below, and which has a CpG motif at the 5' side and a CpG motif at the 5' side and a CpG motif at the 5' side. 1 N 2 and N on the 3' side 3 N 4 consists of adjacent unmethylated CGs, where N 1 N 2 is AA, TT, GG, GT, or AT, and N3 N 4 is CT, TT, TC, TG, or CG, where C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine, and T is deoxythymidine.

[0082] In a particular embodiment, the TLR-9 agonist for use according to the invention comprises at least one unmethylated CG dinucleotide and a stretch of at least three, in particular four, consecutive deoxyguanosines, where the deoxyribose moieties of the oligodeoxyribonucleotide are linked by phosphodiester bonds. In a particular embodiment, the stretch of at least three, in particular four, consecutive deoxyguanosines is located at the 5' end of the oligodeoxyribonucleotide.

[0083] In certain embodiments, a stretch of at least three consecutive deoxyguanosines is located between two CG dinucleotides, more particularly between two CpG motifs.

[0084] In a particular embodiment, the TLR9 agonist for use according to the invention comprises a double-stranded stem, two single-stranded loops, forming a dumbbell shape, and comprises one or several CpG motifs. In a particular embodiment thereof, the CpG motif is comprised in the stem portion of the dumbbell. In another particular embodiment, the CpG motif is comprised in one or both loop portions of the dumbbell. In yet another particular embodiment, the CpG motif is comprised in both the stem and loop portions of the dumbbell.

[0085] In certain embodiments of a TLR9 agonist for use according to the invention, or a CPM for use according to the invention, the TLR9 agonist is characterized as a dumbbell-shaped, covalently closed polynucleotide comprising a double-stranded stem and at least two single-stranded loops on either end of the double-stranded region.

[0086] In a particular embodiment of the TLR9 agonist according to the invention, at least one, more particularly at least three CG dinucleotides are located in each of the single-stranded loops of the dumbbell-shaped polynucleotide structure.

[0087] In certain embodiments of the TLR9 agonist for use according to the invention, the TLR9 agonist is an oligonucleotide characterized by a covalently closed structure lacking an accessible 3' end, comprising at least three unmethylated CG dinucleotides located in each of the single-stranded loops of the dumbbell-shaped polynucleotide structure.

[0088] In certain embodiments of the TLR9 agonist defined in the preceding paragraph, the unmethylated CG dinucleotides used to define the TLR9 agonist motif consist of the following (all nucleotides written in the conventional 5' to 3' orientation): the two nucleotides N 1 N 2 , followed immediately by an unmethylated CG, followed immediately by two consecutive nucleotides N on the 3' side 3 N 4 where N 1 N 2 is AA, TT, GG, GT, AT, or GA, and N 3 N 4 is CT, TT, TC, TG, CG, AT, or CT.

[0089] In a particular embodiment of the TLR9 agonist for use according to the present invention, it belongs to class A with respect to established classifications.

[0090] In certain embodiments of the TLR9 agonist for use according to the invention, the TLR9 agonist is not a class B TLR9 agonist molecule. In certain embodiments of the TLR9 agonist for use according to the invention, the TLR9 agonist is a class A TLR9 agonist molecule. In other certain embodiments of the TLR9 agonist for use according to the invention, the TLR9 agonist is a class C TLR9 agonist molecule.

[0091] In certain embodiments of the TLR9 agonist for use according to the invention, the TLR9 agonist is an oligodeoxynucleotide containing only phosphodiester bonds in the DNA backbone.

[0092] In a particular embodiment of the TLR9 agonist for use according to the invention, the TLR9 agonist is an oligodeoxynucleotide that does not contain chemically modified nucleotides, in other words it consists of a chain of the four DNA-forming nucleotides deoxyguanosine, deoxyadenine, deoxythymine and deoxycytidine linked by phosphodiester bonds.

[0093] In a further particular embodiment of the TLR9 agonist for use according to the invention, the secondary structure of the TLR9 agonist is characterized by a double-stranded DNA stem, both ends of which are covalently closed by single-stranded DNA loops, said structure lacking a 3' end capable of degradation by a 3' exonuclease.

[0094] In a further particular embodiment of the TLR9 agonist for use according to the present invention, the secondary structure of TLR9 is characterized as dumbbell-shaped.

[0095] In a further particular embodiment of the TLR9 agonist for use according to the invention, at least one unmethylated CG dinucleotide is located within the single-stranded loop.

[0096] In a particular embodiment, the TLR9 agonist for use according to the invention is a single cyclic deoxyribonucleotide oligomer having a length of 20 to 150, in particular 30 to 60, deoxyribonucleotides.

[0097] In certain embodiments, the TLR9 agonist for use according to the present invention is Lefitolimod, also known as MGN1703 (CAS:1548439-51-5) (described in US6849725B2, which is incorporated herein by reference in its entirety). The following items relating to TLR9 agonists are encompassed by the invention disclosed herein:

[0098] Item AA: A TLR9 agonist for use according to any one of the aspects, embodiments or claims herein, wherein the TLR9 agonist is an oligodeoxynucleotide sequence comprising one or more unmethylated CpG dinucleotides, in particular the TLR9 agonist is not characterized as a class B TLR9 agonist molecule.

[0099] BB: A TLR9 agonist for the use according to paragraph AA, wherein said TLR9 agonist is a class A TLR agonist with regard to established classifications.

[0100] CC: A TLR9 agonist for the use according to paragraphs AA or BB, wherein said TLR9 agonist consists of a chain of four DNA-forming nucleotides linked by phosphodiester bonds.

[0101] DD: A TLR9 agonist for use according to any one of paragraphs AA to CC, wherein said TLR9 agonist does not contain a phosphorothioate moiety.

[0102] EE: The TLR9 agonist for use according to any one of paragraphs AA to DD, wherein said TLR9 agonist is an oligodeoxynucleotide comprising one unmethylated CG dinucleotide.

[0103] FF: A TLR9 agonist for use according to any one of paragraphs AA to EE, wherein said TLR9 agonist comprises at least three unmethylated CG dinucleotides.

[0104] GG: A TLR9 agonist or CPM for use according to any one of paragraphs AA to FF, wherein said TLR9 agonist is in a covalently closed structure.

[0105] HH: The TLR9 agonist for use according to item GG, wherein the TLR9 agonist is characterized as a dumbbell-shaped covalently closed polynucleotide, and the dumbbell-shaped covalently closed polynucleotide comprises a double-stranded stem, and a first single-stranded loop, and a second single-stranded loop, the first and second single-stranded loops being located at either end of the double-stranded stem.

[0106] II: A TLR9 agonist for use according to paragraph HH, wherein at least one unmethylated CG dinucleotide is located in each of the single-stranded loops of the dumbbell-shaped polynucleotide structure.

[0107] JJ: A TLR9 agonist for use according to paragraph HH or II, wherein at least three unmethylated CG dinucleotides are located in each of the single-stranded loops of the dumbbell-shaped polynucleotide structure.

[0108] KK: The TLR9 agonist has an N 1 N 2 (CpG dinucleotide) and its 3' end N 3 N 4 an unmethylated CG dinucleotide immediately adjacent to the sequence, 1 N 2 is selected from the list consisting of AA, TT, GG, GT, AT, or GA, and N 3 N 4 The TLR9 agonist for use according to any one of items AA to JJ, wherein is selected from the list consisting of CT, TT, TC, TG, CG, AT, or CT.

[0109] LL: The TLR9 agonist for use according to any one of items AA to KK, wherein said TLR9 agonist is a single cyclic deoxynucleotide oligomer having a length of 20 to 150 deoxyribonucleotides, in particular a length of 30 to 60 deoxyribonucleotides.

[0110] These definitions of TLR9 agonists of CpG dinucleotides are intended to apply to any aspect or embodiment, clause or claim herein directed to a use or dosing schedule of a checkpoint modulator agent (CPM) as contemplated herein in which a TLR9 agonist is present.

[0111] Sequential / alternate administration schedules for checkpoint modulators (CPMs) A further aspect of the invention relates to a CPM agent for use in the treatment of cancer, said CPM agent being administered to a patient who has initially undergone induction chemotherapy comprising a first set of treatment cycles.

[0112] Furthermore, the patient who received the induction chemotherapy has been assigned a status of "matched first time" in a first predictive biomarker assay measuring immune cell activation in a patient sample, as described on pages 10 and 11. A patient is assigned a status of "matched first time" if the percentage of activated cells in an immune subset is equal to or exceeds a threshold provided for activated immune cells, in particular the median percent of activated cells, or if the percentage of Treg cells in CD4+ T cells is equal to or exceeds a threshold. In certain embodiments, a CPM is provided for use in a patient who has been assigned a status of "matched first time" if the percentage of activated B cells determined in the patient sample exceeds a threshold obtained by determining the median activated B cells of a cohort of patient samples. In certain embodiments, a patient is assigned a status of "matched first time" if the activated B cells in the sample are equal to or exceed a threshold of about 16.9% of activated B cells.

[0113] Additionally, following induction chemotherapy, the CPM agents are administered as part of a "CPM induction immunomodulatory cycle," particularly a three week CPM induction immunomodulatory cycle, which comprises or consists of administration of a chemotherapy agent and a CPM agent once during the first week of the cycle, followed by one or two weeks of no treatment.

[0114] After one cycle or repetition of 2, 3, 4, or 5 cycles of the CPM induction immunomodulatory cycle, the CPM agent is administered as part of a "CPM maintenance immunomodulatory regimen," which comprises or consists of administering the CPM agent once every 2 or 3 weeks for n weeks, where n is an integer between 6 and 36. In a particular embodiment, n is an integer between 9 and 18. In a more particular embodiment, n is the administration of a "CPM maintenance immunomodulatory regimen" for 12 weeks.

[0115] In certain embodiments of CPM maintenance immunomodulation for the treatment of lung cancer, patients assigned a "1st match" status in the first predictive biomarker assay are administered 9-18 weeks of the CPM by infusion. In certain embodiments, a 12 week CPM maintenance immunomodulation regimen is administered to lung cancer patients assigned a "1st match" status in the first predictive assay.

[0116] In certain embodiments relating to CPM maintenance immunomodulation administered for the treatment of reinduction chemotherapy of metastatic cancer, particularly mCRC, in a CPM maintenance immunomodulatory regimen, the CPM agent is administered by infusion for 9-18 weeks, hi certain embodiments, the CPM maintenance immunomodulatory regimen is administered for 24 weeks.

[0117] A particular embodiment of a CPM for use according to this aspect of the invention relates to a patient who has undergone a previous 3 week CPM maintenance immunomodulatory regimen, where a second predictive biomarker assay value has been determined in a patient sample, and where the second predictive biomarker determines that the percentage of Treg cells in CD4+ T cells in a blood sample obtained from the patient is above a median threshold value. In a particular embodiment, if the percentage of Treg cells in CD4+ T cells is equal to or above a threshold value of 15-20%, the patient is assigned a "2nd match" status and is then administered a CPM maintenance immunomodulatory regimen in which a CPM drug is administered every 2 or 3 weeks for n weeks, where n is an integer between 6 and 36. In a particular embodiment, n is an integer between 9 and 12. In a more particular embodiment, n is administration of a CPM maintenance immunomodulatory regimen for 12 weeks.

[0118] Other embodiments relate to a CPM for use in patients who have undergone a CPM maintenance immunomodulatory regimen as defined in one of the five preceding paragraphs and who have also undergone a second predictive biomarker assay. According to these embodiments: If the percentage of Treg cells among CD4+ T cells in the sample is below a threshold of 20%, the patient is assigned a "second mismatch" status, or · If the percentage of activated Treg cells in the sample is equal to or above the threshold of 20%, the patient is assigned a “second match” status.

[0119] In another embodiment, the threshold for the second predictive biomarker is 15% Tregs among CD4+ T cells.

[0120] After being assigned a "second mismatch" status, the treatment of the patient on the CPM maintenance immunomodulatory regimen is switched to administration of a TLR9 agonist twice weekly, once weekly, or every two weeks for n weeks, where n is an integer between 6 and 24. In a particular embodiment, n is an integer between 6 and 15. In a more particular embodiment, n is 9 weeks.

[0121] In some embodiments, patients who have undergone a CPM agent maintenance immunomodulatory regimen are subsequently switched to administration of a TLR9 agonist as described above upon reaching a median PFS survival threshold characterizing the particular cancer disease without reference to or application of a second predictive biomarker assessment, in certain embodiments, this switching occurs after a PFS of 188 days for esSCLC patients or after a PFS of 225 days for mCRC patients.

[0122] In another embodiment, patients who have been assigned a "2nd match" status and who have been placed on a CPM maintenance immunomodulatory regimen continue to receive a CPM agent every 2 weeks or every 3 weeks for n weeks, where n is an integer ranging from 6 to 36. In a particular embodiment, n is in the range of 9 to 12. In a more particular embodiment, patients who have been assigned a "2nd match" status are administered a CPM agent for 12 weeks.

[0123] Biomarker-Driven Immunomodulation Alternatives Platinum-based induction chemotherapy generates two populations of patient samples from patients with tumors defined by a first biomarker: one with immunologically cold tumors, i.e., the respective rates of innate and adaptive immune cell activation are below the threshold median biomarker value, and the other with immunologically hot tumors, i.e., the respective rates of innate and adaptive immune cell activation are above the threshold biomarker value (these tumors are immunologically "hot", but due to immune exhaustion mediated by checkpoint inhibition pathways, the underlying immune response may no longer be effective in terms of antitumor activity).

[0124] In the treatment algorithm described by the present invention, immune responses are promoted in tumors lacking biomarkers of immune cell activation (sometimes called immune "cold" tumors). They are converted into immune "hot" tumors during induction immunomodulation by subsequent cycles employing chemotherapy in combination with TLR9 agonists. We conclude from the evidence presented in the examples that once cold tumors are converted and maintained into immune hot tumors, natural counterregulation by regulatory B populations, represented by Treg and CD86+ B cell populations, may be initiated. Thus, when the activation markers of one or more patient samples match the second biomarker, the immunomodulation is switched from TLR9 agonist maintenance therapy to CPM therapy according to a treatment regime aimed at resuming the mechanisms of neoantigen presentation and cross-presentation for effective antitumor adaptive immunity. In repeated application of the second biomarker at intervals of several months, when the values ​​of the patient samples match the second biomarker once again, the maintenance immunomodulation is switched from CPM maintenance therapy to TLR9 agonist therapy.

[0125] Rather than combining innate and adaptive immune stimulation as per the prior art, in some embodiments, the TLR9 agonist and CPM agent are delivered in a sequential manner, i.e., through timed and quantitatively controlled stimulation of alternative innate and adaptive immune pathways.

[0126] In certain embodiments, the second biomarker determines the appropriate timing for switching or repeating or alternating administration of these adaptive immune stimulating compounds (CPM agents) or innate immune stimulating compounds (TLR9 ​​agonists). According to these embodiments, the CPM or TLR9 agonist is administered to the patient having the second predictive biomarker assay measured in a blood sample every 6-15 weeks, in particular every 9 weeks. When the results of the second predictive biomarker assay indicate that the immune system displays signs of exhaustion in the form of activated B cells or Treg cells above a threshold, the CPM is administered to counteract inhibitor checkpoint signals on the adaptive immune cells. Alternatively, the TLR9 agonist is administered to convert an immune "cold" tumor into an active immune environment.

[0127] In another embodiment, administration of the adaptive or innate immune stimulatory agent specified in the paragraph above is alternated when the patient meets the "matched for second time" criteria or when the patient reaches the median progression-free survival for patients receiving standard of care chemotherapy, e.g., in the case of SCLC, a progression-free survival of 188 days from the start of the induction chemotherapy cycle, whichever occurs first.

[0128] In certain embodiments, after application of the first predictive biomarker, the patient has undergone a "CPM maintenance immunomodulatory regimen" within the previous three weeks and then: After "second-matched" status is assigned (or the median PFS threshold is reached), the TLR9 agonist is administered twice weekly, once weekly, or every two weeks for the subsequent n weeks, where n is an integer ranging from 6 to 24. In a particular embodiment, n is in the range of 6 to 15 weeks. In a more particular embodiment, n is 9 weeks of administration of the TLR9 agonist; or After a status of "2nd non-match" is assigned, CPM continues to be administered every 2 weeks or every 3 weeks for the next n weeks, where n is an integer in the range of 6-24 weeks. In a particular embodiment, n is in the range of 6-12 weeks. In a more particular embodiment, n is 9 weeks.

[0129] Conversely, if after application of the first predictive biomarker, the patient received a "TLR9 agonist maintenance immunomodulatory regimen" in the previous 3 weeks, then: After the "second non-match" status is assigned, continue to administer the TLR9 agonist twice weekly, once weekly, or every two weeks for the following n weeks, where n is an integer ranging from 6 to 24. In a particular embodiment, n is in the range of 6 to 15 weeks. In a more particular embodiment, n is 9 weeks; or After "second matching" status is assigned (or the median PFS threshold is reached), CPM is administered every 2 weeks or every 3 weeks for the following n weeks, where n is an integer ranging from 6 to 24 weeks. In a particular embodiment, n is in the range of 6 to 12 weeks. In a more particular embodiment, n is 9 weeks.

[0130] In some embodiments, after repeated application of the second predictive biomarker, the treatment is switched from twice weekly, once or every 2 weeks administration of the TLR9 agonist to every 2, 3 weeks administration of CPM, or vice versa, if a median PFS survival threshold characterizing the cancer disease is reached, as shown in the examples, without reference to evaluation of the second predictive biomarker, e.g., a PFS of 188 days for esSCLC patients or a PFS of 225 days for mCRC patients.

[0131] Predictive biomarkers In a particular embodiment, the TLR9 agonist and / or checkpoint modulator according to any one of the above aspects or embodiments is administered in a biomarker-driven treatment protocol to a patient diagnosed with a type of cancer that is sensitive to chemotherapeutic agents in combination with immunoadjuvant therapy. In particular, said cancer is selected from breast cancer, cervical cancer, endometrial cancer, colorectal cancer, metastatic colorectal cancer, lung cancer. In a more particular embodiment, the TLR9 agonist and / or checkpoint modulator is used to treat small cell lung cancer, in particular extensive stage small cell lung cancer (esSCLC), or metastatic colorectal cancer, as investigated in the clinical trials analyzed in the examples.

[0132] In other embodiments of the TLR9 agonist and / or checkpoint modulator used according to any one of the preceding aspects or embodiments for treating lung cancer, in particular esSCLC, a patient is assigned a "first time mismatch" status if the first predictive biomarker result is below a median threshold value obtained from analysis of a cohort of esSCLC patient samples. In certain embodiments, a patient is assigned a "first time mismatch" status if the patient's biomarker values ​​measured in samples analyzed in the first predictive biomarker assay are characterized as follows: - the proportion of activated B cells is less than about 16.9% of B cells; - the proportion of activated T cells is less than about 0.76% of T cells; - The proportion of Treg cells is less than approximately 7.6% of CD4+ T cells; - the proportion of activated NKT cells is less than 4.1% of NKT cells; - the proportion of activated pDCs is less than 7.6% of pDCs; - mDC percentage is less than 0.48%; and / or - The proportion of activated DCs is less than 54.51% of DCs. In certain embodiments of the invention, a patient is assigned a "first time mismatch" status if the percentage of activated B cells in the patient sample is below a threshold of about 16.9% of B cells.

[0133] In another embodiment, a patient is assigned a "first match" status if the first predictive biomarker result is characterized as follows: - the proportion of activated B cells is 16.9% or more of B cells, and in particular 21% or more of B cells; - the proportion of activated T cells is 0.76% or more of T cells; - the proportion of activated Treg cells is 7.6% or more of Treg cells; and / or - The proportion of activated NK cells is 4.1% or more of NK cells; - the ratio of activated pDCs is 7.6% or more of pDCs; - mDC rate ≥ 0.48%; and / or - The ratio of activated DCs is 54.51% or more of DCs.

[0134] In certain embodiments, a patient is assigned "first-time match" status if the percentage of activated B cells in the patient's PBMC sample is 16.9% or greater of B cells. In certain embodiments, a patient is assigned "first-time match" status if the percentage of activated B cells in the patient's sample is 21% or greater of B cells.

[0135] In certain embodiments, the percentage of B cells expressing the activation marker CD86 is determined in the patient PBMC sample in a first predictive biomarker assay, and the patient is assigned a "first-time match" status if the percentage of CD86+ B cells is equal to or greater than 16.9% of CD45+CD19+ B cells. In certain embodiments, the patient is assigned a "first-time mismatch" status if the biomarker value in the patient sample falls below a threshold proportion of activated CD86+ B cells that is less than about 16.9% of total B cells.

[0136] In certain embodiments, the allocation of patients to treatment is performed by determining the percentage of activated immune cells using the following markers quantified by flow cytometry: B cells expressing the marker CD86, activated T cells, and / or NK cells according to the percentage of CD69+, activated mDCs or pDCs that are CD40+ or ​​BDCA-2+, in particular BDCA-2+, and / or activated DCs expressing CD40.

[0137] In certain embodiments, the percentage of activated cells in B cells, T cells, NKT cells, pDCs, or DCs is determined in the first predictive biomarker assay.

[0138] A particular embodiment is an assay in which the percentage of one or more of: activated B cells; and activated T cells, NKT cells, PDC cells, or DCs is determined in the first predictive biomarker assay.

[0139] In other embodiments, the percentage of activated B cells, T cells, and NKT cells is determined in the first predictive biomarker assay.

[0140] In certain embodiments, the percent of activated B cells is determined in the first predictive biomarker assay.

[0141] In certain embodiments, the percentage of regulatory B (Breg) cells is the assay used in the second predictive biomarker assay.

[0142] In certain embodiments, activated B cells are defined as CD86+ in the first or second predictive biomarker assay.

[0143] In certain embodiments of the invention, the percentage of activated CD86+ B cells and the percentage of Treg cells among CD4+ T cells are the assays used in the second predictive biomarker assay.

[0144] In certain embodiments of the invention, an assay in which the percentage of Treg cells among CD4+ T cells is determined is used in the second predictive biomarker assay.

[0145] The median percentage of activated cell subsets that serves as a threshold according to the present invention can be calculated from data obtained from the analysis of biomarker expression obtained from a cohort of patient samples, as demonstrated in the examples. Said cohort of patients is characterized by having been diagnosed with cancer originating from the same tissue as the patient being treated, and preferably by having undergone induction chemotherapy at the same time point or within 1-2 months of the patient undergoing the first or second predictive biomarker assay. The number of patients in such a cohort is not particularly limited, but should be sufficiently powered to be able to distinguish between patients with different outcomes, as demonstrated in the study of lefitolimod shown in the examples.

[0146] In a particular embodiment, the percentage of activated immune cells or the percentage of Treg cells is determined in a peripheral blood sample in the first or second predictive biomarker assay according to the aspect of the invention defined above. In a particular embodiment, the blood sample is processed, for example by gradient centrifugation, to provide a peripheral blood mononuclear cell (PBMC) sample free of platelets, neutrophils and erythrocytes, which is assessed for the presence of immune cell subsets and activation markers, as demonstrated in the examples. In another embodiment contemplated by the inventors as possible, whole blood, or a blood smear, histological or biopsy sample is analyzed to assess immune activation parameters.

[0147] In certain embodiments of the invention, a PBMC sample or a whole blood sample is used in a predictive biomarker assay following a cell culture step in which the sample is stimulated with an immune agonist, such as a TLR9 agonist, for a period ranging from 4 to 48 hours. In certain embodiments, a patient's PBMC sample stimulated with lefitolimod for about 24 hours is used in the first and / or second predictive biomarker assay.

[0148] Induction chemotherapy In certain embodiments, TLR9 agonists and / or CPMs are used in patients who have received platinum-based chemotherapy, including platinum-containing agents selected from carboplatin, satraplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, picoplatin, and triplatin tetranitrate, in combination with etoposide or topotecan, which are induction chemotherapy agents that include the most commonly utilized agents in the treatment of lung cancer.

[0149] The TLR9 agonist or checkpoint modulator for use according to the invention may be followed by different types of induction chemotherapy according to certain embodiments of the invention. In certain embodiments, the induction chemotherapy comprises a first set of treatment cycles comprising 2, 3, 4 or 5 treatment cycles. In certain embodiments, the patient receives 4 sets or repeated 3-week cycles of said induction chemotherapy, and the chemotherapeutic agent is administered in the first week of said cycle.

[0150] In certain embodiments, the TLR9 agonist and / or checkpoint modulator is administered to the patient after an induction chemotherapy period in which the chemotherapeutic agent administered comprises an antineoplastic platinum complex drug. In certain embodiments, the antineoplastic platinum complex drug is selected from cisplatin, oxaliplatin, or carboplatin and is administered together with an antineoplastic alkaloid drug. In certain embodiments, the antineoplastic platinum complex drug is administered in combination with an antineoplastic alkaloid drug selected from etoposide or topotecan.

[0151] In a particular embodiment, the induction chemotherapy according to the present invention is a form of reinduction chemotherapy, in which chemotherapy is administered or resumed after tumor recurrence occurs after a period of remission or after tumor progression is diagnosed.In the IMPALA Phase III study, lefitolimod switched maintenance therapy had inferior median progression-free survival (mPFS) compared to the control group.However, in patients who received reinduction therapy and then cycled chemotherapy and lefitolimod, mPFS was significantly better in the chemotherapy / lefitolimod group compared to the control.

[0152] In certain embodiments, the antineoplastic chemotherapeutic agent is administered once every 2-4 weeks, which results in one cycle of induction chemotherapy. In certain embodiments, the antineoplastic chemotherapeutic agent is administered once every 3 weeks of the induction chemotherapy cycle.

[0153] Cycles of induction chemotherapy are common in chemotherapy regimes, such as in metastatic breast cancer, using, for example, EGF, VEGF specific monoclonal antibody therapy, etc. In certain embodiments, TLR9 agonists or immune checkpoint inhibitors are used according to the present invention in patients who have received induction chemotherapy that comprises or consists of intravenous infusion of hormone receptor targeted monoclonal antibody therapy.

[0154] In certain embodiments, the TLR9 agonist and / or CPM are administered to SCLC or NSCLC patients (as second line treatment) after induction chemotherapy comprising administration of carboplatin or cisplatin in combination with etoposide or topotecan.

[0155] In certain embodiments, the TLR9 agonist and / or CPM are administered to patients with breast, ovarian, or endometrial cancer following induction chemotherapy that comprises or consists of a treatment cycle with carboplatin.

[0156] In certain embodiments, the TLR9 agonist and / or CPM are administered to cervical cancer patients following induction chemotherapy that comprises or consists of the administration of a cycle of treatment with cisplatin.

[0157] In certain embodiments, the TLR9 agonist or CPM is used in patients after induction chemotherapy for colorectal cancer (CRC) that comprises or consists of administration of oxaliplatin. In certain embodiments, the induction chemotherapy comprises administration of oxaliplatin in a reinduction setting for treating metastatic CRC.

[0158] In certain embodiments, the cancer patient receives a total of six cycles of treatment with antineoplastic agents, for example, four cycles of antineoplastic agents alone and two cycles of treatment using antineoplastic agents in combination with a TLR9 agonist or CPM agent in an induction immunomodulatory treatment cycle of the TLR9 agonist or CPM agent.

[0159] Medical Treatments, Dosage Forms and Salts Also included within the scope of the present invention is the treatment or therapy of cancer in a patient in need thereof, comprising administering to the patient a TLR9 agonist or CPM, optionally in combination with an anti-neoplastic chemotherapeutic drug, or a combination of drugs, such as a platinum-based drug combination as described above.

[0160] Similarly, there is provided a dosage form for the prevention or treatment of cancer comprising a TLR9 agonist molecule according to any of the above aspects or embodiments of the invention, and a CPM agent.

[0161] In certain embodiments of the TLR9 agonist for use according to any of the above aspects or embodiments, the TLR9 agonist is administered by subcutaneous injection.

[0162] In certain embodiments, the TLR9 agonist is: 50-80 mg twice a week, especially 60 mg, with an interval of 2 or 3 days; Doses of 30-60 mg, especially 60 mg, once a week a dose of 100-150 mg, especially 120 mg, once a week, or At a dose of 100-150 mg, especially at a dose of 120 mg, once every two weeks Administer.

[0163] In a particular embodiment of the TLR9 agonist for use according to the invention, it is administered once a week at a dose of 60 mg.

[0164] In certain embodiments, the checkpoint modulator for use according to the invention is an antibody specific for PD-1 or PDL-1. In certain embodiments, the CPM is an antibody selected from atezolizumab, durvalumab, pembrolizumab, or nivolumab. In more particular embodiments, the CPM is an anti-PD-L1 antibody selected from atezolizumab or durvalumab.

[0165] Administration protocols for checkpoint modulators such as those described above are well known in clinical use. CPMs are generally administered by intravenous or subcutaneous infusion, and dosages may vary depending on the type and severity of cancer, or on the occurrence of side effects. Certain embodiments relate to the use of CPMs delivered by intravenous infusion every three weeks as part of a combined "CPM induction immunomodulatory cycle" or "single agent CPM maintenance immunomodulatory regimen."

[0166] Those skilled in the art will recognize that the drug compounds specifically mentioned herein may exist as pharma- ceutically acceptable salts of the drug, which contain an ionized drug and an oppositely charged counterion. Non-limiting examples of pharma- ceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, ebonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, dibasic phosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, and valerate salts. Non-limiting examples of pharma-ceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0167] The administration form, particularly for chemotherapeutic drugs, can be enteral, such as nasal, buccal, rectal, transdermal or oral administration. Alternatively, particularly for TLR9 agonists or CPMs, parenteral administration can be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0168] Pharmaceutical Compositions and Administration Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers as described herein.

[0169] In certain embodiments of the present invention, the compounds of the present invention are generally formulated into pharmaceutical dosage forms to provide easily controllable administration of the drug and to provide the patient with a clear, easy to handle product.

[0170] The pharmaceutical composition may be formulated for enteral administration, particularly oral or rectal administration. Furthermore, the pharmaceutical composition of the present invention may be in a solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or in a liquid form (including, but not limited to, solutions, suspensions, or emulsions).

[0171] The pharmaceutical composition can be formulated for parenteral administration, eg, by intravenous (iv) injection, intradermal, subcutaneous or intramuscular administration.

[0172] The dosing regimen of the compounds of the present invention will vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration; the recipient's species, age, sex, health, medical condition, and weight; the nature and extent of the condition; type of concurrent treatment; frequency of treatment; route of administration, the patient's renal and hepatic function, and the desired effect. In certain embodiments, the compounds of the present invention may be administered once daily or the total daily dosage may be administered in divided doses two, three, or four times daily.

[0173] In certain embodiments, the pharmaceutical composition or combination of the present invention may be a unit dose of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the subject's species, weight, age, individual condition, and the disorder or disease being treated or its severity. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient required to prevent, treat, or inhibit the progression of a disorder or disease.

[0174] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization, and / or can contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They can be produced by standard processes, such as conventional mixing, granulation, dissolving, or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al., Theory and Practice of Industrial Pharmacy, 4th ed., 2013 (ISBN 8123922892).

[0175] Methods of manufacture and treatment according to the present invention The present invention further encompasses, as an additional aspect, the use of a TLR9 agonist, or a CPM, or a pharma- ceutically acceptable salt thereof, as identified herein, for use in a method for the manufacture of a medicament for the treatment or prevention of cancer, in treating a subset of cancer patients, as identified by activated immune cell biomarkers, according to a treatment protocol as defined in detail above.

[0176] Similarly, the invention encompasses a method of treating a patient diagnosed with a cancer disease associated with sensitivity to immune modulation, such as a TLR9 agonist or immune checkpoint modulation, e.g., a tumor characterized by multiple mutations, a tumor that has already responded to immune modulation therapy in the past, or a tumor that has been determined to be likely sensitive to immune modulation by certain genetic characteristics, comprising administering to the patient an effective amount of a TLR9 agonist or CPM, as identified herein, or a pharma- ceutically acceptable salt thereof, as defined in detail herein.

[0177] The invention further encompasses the use of molecular probes that evaluate immune cell subset activation markers identified herein, such as antibodies that identify T cells, Tregs, B cells, NKT cells, pDCs, mDCs, or DCs, or their specific activation markers, for use in the manufacture of a kit for detecting a cancer state in a patient that may be treated with TLR9, or CPM, or alternating administration of TLR9 and CPM, via a biomarker-driven treatment protocol identified by the aspects and embodiments of the invention provided herein.

[0178] Where alternatives to a single separable feature, such as a cell surface immune cell activating protein or a TLR9 agonist, a CPM, or a particular type of cancer, are presented herein as "embodiments," it is understood that such alternatives can be freely combined to form separate embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a detectable biomarker immune cell population can be combined with any of the alternative embodiments of a TLR9 agonist or a CPM, and these combinations can be combined with any of the cancer types or diagnostic methods referred to herein.

[0179] The present invention further includes the following:

[0180] A. A TLR9 agonist for use in the treatment of cancer, comprising: - the TLR9 agonist is administered to a patient who has received induction chemotherapy comprising a first set of treatment cycles; - the patient, after receiving induction chemotherapy, is assigned a status of "discordant 1" in a first predictive biomarker assay; the first predictive biomarker assay determines the percentage of activated cells within an immune cell subset selected from B cells, T cells, natural killer T (NKT) cells, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and / or the percentage of regulatory T (Treg) cells among CD4+ T cells in a blood sample obtained from the patient; and if the percentage of activated cells in the immune subset is less than the median percentage of activated cells or less than the median percentage of Treg cells among CD4+ T cells, the patient is assigned a status of "first mismatch"; - administering said TLR9 agonist as part of a TLR9 agonist induction immunomodulatory cycle following said induction chemotherapy; wherein a chemotherapeutic agent is administered during the first week of said TLR9 agonist-induced immunomodulatory cycle; and the TLR9 agonist is administered once or twice weekly during weeks 2 and 3 of the TLR9 agonist-induced immunomodulatory cycle; and - administering said TLR9 agonist as part of a TLR9 agonist maintenance immunomodulatory regimen after 1 to 4 sets of said TLR9 agonist induction immunomodulatory cycles; wherein the TLR9 agonist is administered twice weekly, once each week, or every two weeks for n weeks of the TLR9 agonist maintenance immunomodulatory regimen, where n is an integer between 5 and 35, particularly an integer between 9 and 18, more particularly n is 12; The TLR9 agonist.

[0181] B. - the patient has received said TLR9 agonist maintenance immunomodulatory regimen as defined in item 1 during the previous 3 weeks; - a second predictive biomarker assay determines a percentage of Treg cells among CD4+ T cells in a blood sample obtained from the patient; - if the percentage of activated Treg cells in the sample is less than 20%, the patient is assigned a "second mismatch" status; and A TLR9 agonist for use according to item A, wherein after a status of "second non-match" is assigned, administration of the TLR9 agonist continues twice weekly, once weekly or every two weeks for n weeks, where n is an integer between 6 and 24, particularly an integer between 6 and 15, more particularly n is 9.

[0182] C. - in the second predictive biomarker assay as defined in item B, A patient is assigned a "second mismatch" status if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%, or Patients were assigned a "second match" status if the percentage of Treg cells among CD4+ T cells in the sample was 20% or greater; - after the status of "second non-match" is assigned, continue to administer the TLR9 agonist twice weekly, once weekly, or every two weeks for n weeks, where n is an integer between 6 and 24, particularly an integer between 6 and 15, more particularly n is 9; or - after the "second-matched" status is assigned and / or if the patient does not have a diagnosis of disease progression and exceeds a median progression-free survival threshold from the start of induction chemotherapy, in particular a median progression-free survival threshold of about 188 days, a checkpoint modulator (CPM) is administered once every 2 weeks or once every 3 weeks for n weeks, where n is an integer between 3 and 15; A TLR9 agonist for the use according to item B.

[0183] D. - the second predictive biomarker assay is performed every 6 to 15 weeks, in particular every 9 weeks; A TLR9 agonist for the use according to paragraphs B or C.

[0184] E. A CPM agent for use in the treatment of cancer, comprising: - the CPM agent is administered to a patient who has received induction chemotherapy comprising a first set of treatment cycles; - the patient who received the induction chemotherapy is assigned a "matched first" status in a first predictive biomarker assay that determines the percentage of activated cells in an immune cell subset as defined in section A; If the percentage of activated cells in the immune subset is greater than or equal to the median percentage of activated cells, or the percentage of Treg cells among CD4+ T cells is greater than or equal to the median percentage of Treg cells among CD4+ T cells, the patient is assigned a “first match” status; - after said induction chemotherapy, said CPM agent is administered as part of a CPM induction immunomodulatory cycle; wherein the chemotherapy agent and the CPM agent are administered once during the first week of said CPM induction immunomodulatory cycle; and - after a set of 1 to 5 of the CPM induction immunomodulatory cycles, the CPM agent is administered as part of a CPM maintenance immunomodulatory regimen; the CPM agent is administered once every 2 weeks or every 3 weeks of the CPM maintenance immunomodulatory regimen for n weeks, where n is an integer between 6 and 36, particularly an integer between 9 and 18, more particularly n is 12; The CPM agent.

[0185] F. - the patient has received a CPM maintenance immunomodulatory regimen as defined in section G for the previous 3 weeks; - determining the percentage of Treg cells among CD4+ T cells in a blood sample obtained from said patient by a second predictive biomarker assay; If the percentage of Treg cells among CD4+ T cells is 20% or more, the patient is assigned a "second match" status; and - a CPM agent for use according to item E, wherein after the status of "second match" is assigned, the CPM agent is administered every 2 weeks or every 3 weeks for n weeks, where n is an integer between 6 and 36, in particular an integer between 9 and 12, in particular n is 12.

[0186] G. - in the second predictive biomarker assay determined as defined in item F; A patient is assigned a "second mismatch" status if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%; or - If the percentage of activated Treg cells in the sample is 20% or more, the patient is assigned a "second match" status; - wherein the TLR9 agonist is administered twice weekly, once weekly or every two weeks for n weeks after the status of "second mismatch" is assigned and / or when the patient exceeds the median progression-free survival from the start of induction chemotherapy without a diagnosis of disease progression, in particular a median progression-free survival threshold of about 188 days, where n is an integer between 6 and 24, in particular an integer between 6 and 15, more in particular n is 9; or - After the status of "second match" is assigned, the CPM agent is administered once every 2 weeks or once every 3 weeks for n weeks, where n is an integer between 6 and 36, particularly an integer between 9 and 12, more particularly n is 12; A CPM agent for use as described in item F.

[0187] H. - the second predictive biomarker assay is performed every 6 to 15 weeks, particularly every 9 weeks; - If the patient has received a CPM maintenance immunomodulatory regimen as defined in section E within the previous 3 weeks, then after a status of "second-time discordant" has been assigned and / or the patient has exceeded a median progression-free survival threshold time from the start of induction chemotherapy without a diagnosis of disease progression, in particular a median progression-free survival threshold time of about 188 days, the TLR9 agonist is administered twice weekly, once weekly or every two weeks for the subsequent n weeks, where n is an integer between 6 and 24, in particular an integer between 6 and 15, more in particular n is 9; or after a status of "second match" is assigned, continuing to administer said CPM once every two or three weeks for the following n weeks, where n is an integer between 6 and 24, particularly an integer between 6 and 12, more particularly n is 9; or - If the patient has received a TLR9 maintenance immunomodulatory regimen as defined in item A within the previous 3 weeks; after a status of "second non-match" is assigned, continue to administer the TLR9 agonist twice weekly, once weekly, or every two weeks for the subsequent n weeks, where n is an integer between 6 and 24, particularly an integer between 6 and 15, more particularly n is 9; or after a "second-match" status has been assigned and / or if the patient has exceeded a median progression-free survival threshold time from the start of induction chemotherapy without a diagnosis of disease progression, in particular a median progression-free survival threshold time of about 188 days, said CPM being administered once every 2 weeks or once every 3 weeks for the subsequent n weeks, where n is an integer between 6 and 24, in particular an integer between 6 and 12, more in particular n is 9; A TLR9 agonist for use according to any one of items B to D, or a CPM agent for use according to items F or G.

[0188] I. The TLR9 agonist is an oligodeoxynucleotide that contains an unmethylated CG dinucleotide; especially - said unmethylated CG dinucleotide has at its 5' end an N 1 N 2 is adjacent to the 3' end of 3 N 4 are adjacent, where N 1 N 2 is AA, TT, GG, GT, or AT, and N 3 N 4 is CT, TT, TC, TG, or CG, and / or - the TLR9 agonist comprises a stretch of at least three or four consecutive deoxyguanosines, and / or - the TLR9 agonist is a dumbbell deoxynucleotide; Even more particularly, said TLR9 agonist is lefitolimod (CAS: 1548439-51-5), A TLR9 agonist for use according to any one of items A to D, or H, or a CPM agent for use according to item G or H.

[0189] J. The TLR9 agonist is administered by subcutaneous injection: - 50-80 mg twice a week, especially 60 mg, with an interval of 2 or 3 days; - once a week at a dose of 30-60 mg, especially at a dose of 60 mg; - once a week at a dose of 100-150 mg, especially at a dose of 120 mg, or - 100-150 mg, especially 120 mg, once every 2 weeks; Administered at In particular, the TLR9 agonist is administered at a dose of 60 mg once a week, A TLR9 agonist for use according to any one of items A to D, H or I, or a CPM agent for use according to any one of items G to I.

[0190] K. The checkpoint modulator is an antibody specific for PD-1 or PDL-1, in particular an antibody selected from atezolizumab, durvalumab, pembrolizumab, or nivolumab, more particularly an anti-PD-L1 antibody selected from atezolizumab or durvalumab; A TLR9 agonist for use according to any one of items A to D, or H to J, or a CPM agent for use according to any one of items E to J.

[0191] L. The TLR9 agonist or CPM agent for use according to any one of paragraphs A to K, wherein said cancer is a cancer sensitive to chemotherapeutic agents in combination with immunoadjuvant therapy, in particular a cancer selected from breast cancer, cervical cancer, endometrial cancer, colorectal cancer, metastatic colorectal cancer, or lung cancer, more in particular small cell lung cancer or metastatic colorectal cancer, and even more in particular extensive stage small cell lung cancer (SCLC).

[0192] M. - the first predictive biomarker result is i. the percentage of activated B cells is less than 16.9% of B cells; ii. The percentage of activated T cells is less than 0.76% of T cells; iii. The proportion of Treg cells is less than 7.6% of CD4+ T cells; iv. The percentage of activated NKT cells is less than 4.1% of NK cells; v. the proportion of activated mDCs is less than 0.48% of pDCs; vi. The percentage of activated pDCs is less than 5.0% of pDCs; and / or vii. The proportion of activated DCs is less than 54.51% of DCs; the patient is assigned a status of "first mismatch" when compared to a threshold selected from: - The results of the first predictive biomarker are i. The percentage of activated B cells is 16.9% or more of B cells, and in particular 21% or more of B cells; ii. The percentage of activated T cells is 0.76% or more of T cells; iii. The percentage of activated Treg cells is 7.6% or more of Treg cells; and / or iv. The ratio of activated NKT cells to NK cells is 4.1% or more; v. The ratio of activated mDCs to pDCs is 0.48% or more; vi. The percentage of activated pDCs is 5.0% or more of pDCs; and / or vii. The proportion of activated DCs is 54.51% or more of DCs; A status of "first match" is assigned to the patient when the IL-164A-specific IL-164A-binding protein is compared to a threshold selected from In particular, the percentage of activated B cells is determined in the first predictive biomarker assay. A TLR9 agonist or a CPM agent for use according to any one of items A to L, in particular for use in the treatment of lung cancer, more in particular for use in the treatment of extensive stage SCLC.

[0193] N. The induction chemotherapy - a first set of treatment cycles comprising 2, 3, 4 or 5 cycles, in particular 4 cycles, each cycle consisting of 3 weeks; and - a chemotherapy agent or a combination of chemotherapy agents is administered during the first week of said induction chemotherapy cycle; - in particular, said induction chemotherapy cycle comprises administering a combination of platinum-based chemotherapy agents during the first week of said induction chemotherapy cycle; A TLR9 agonist or CPM agent for use according to any one of items A to M.

[0194] O. The induction chemotherapy comprises: - antineoplastic platinum complex drugs, in particular antineoplastic platinum complex drugs selected from cisplatin, oxaliplatin or carboplatin, and - antineoplastic alkaloid drugs, in particular antineoplastic alkaloid drugs selected from etoposide or topotecan, comprising or consisting of treatment with A TLR9 agonist or CPM agent for use according to any one of items A to N.

[0195] The present invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived, these examples being intended to illustrate the invention without limiting its scope. EXAMPLES

[0196] Examples, Methods IMPULSE trial, NCT02200081 An international, multicenter, open-label, phase II study was conducted in 102 patients with extensive-stage small cell lung cancer after first-line platinum-based therapy. Patients who received four cycles of first-line induction chemotherapy (platinum-based) were randomized to receive either lefitolimod combination maintenance therapy or local standard of care (control). After progression, patients received second-line treatment.

[0197] IMPALA Trial, NCT02077868 An international, multicenter, open-label, phase III study was conducted in 549 patients with metastatic colorectal cancer in eight European countries. Patients who demonstrated an objective response to any first-line induction chemotherapy (5-FU / FA, or CAPE, with OX, or IRI, alone or in combination with anti-VEGF or anti-EGFR antibodies) were randomized to receive either lefitolimod monotherapy or local standard of care (control). After progression, patients started reinduction therapy, while patients in the experimental arm continued to receive additional lefitolimod.

[0198] In vitro biomarker testing in cell cultures Approximately 20 mL of whole blood collected in lithium heparin tubes at scheduled visits was analyzed within 24 hours after collection and transported at room temperature (18°C–24°C). Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll). To determine each patient's individual response to in vitro lefitolimod MGN1703 stimulation, PBMCs were analyzed for cytokine and chemokine production. PBMCs were treated with MGN1703 for 24 hours. Immune cell activation was measured by flow cytometry and membrane-associated proteins were stained by staining intact cells with monoclonal antibodies in phosphate-buffered saline containing 10% (v / v) human serum, 2.5% (v / v) fetal calf serum, and 0.1% (w / v) azide on ice. The following antibodies were used: anti-lineage cocktail1* CD3, CD14, CD16, CD9, CD20, CD56), anti-CD123 (7G3), anti-HLA-DR (L243), anti-CD40 (5C3), anti-CD11c (B-ly6), anti-CD86 (2331FUN-1), anti-CD19 (4G7), anti-CD69 (FN50), all from BD Biosciences; anti-CD14 (61D3), anti-CD169 (7-239), anti-CD3 (OKT-3), anti-CD56 (MEM188), anti-CD80 (2D10), all from eBioscience.

[0199] Example 1: Sequential, two-step, biomarker-guided, divergent alternation of innate and adaptive immune modulation following induction or reinduction chemotherapy The phase II IMPULSE trial investigated the OS benefit of immunotherapeutic switch maintenance with the TLR9 agonist lefitolimod (dSLIM-30L1) in ES-SCLC. Comparisons were made with various regional standards of care (SoC), typically carboplatin and etoposide or cisplatin and etoposide. No additional benefit was observed with lefitolimod switch maintenance, as assessed by median progression-free survival (mPFS) or median overall survival (mOS). However, a specific subgroup of patients showed a strong improvement in median OS and a biological mechanism of action confirmed by immunogenicity analysis of patient samples, namely reactivation of immune surveillance (Thomas M. et al., Oncol. 29(10):2076). A combinatorial post hoc analyse was performed on all available immunological data and relevant clinical parameters from the IMPLUSE trial. Additionally, to determine under what conditions stimulation of adaptive or innate immunity may improve chemotherapy outcomes, we incorporated published data on mPFS and mOS for each treatment and control arm of a PD-L1 inhibitor-antibody chemotherapy immunomodulatory maintenance trial and compared the use of checkpoint inhibitors with the TLR9 agonist used in IMPULSE.

[0200] Lefitolimod immunomodulatory maintenance therapy was compared with IMpower133 (Horn L. et al., 2018 N. Engl. J. Med. 379:2220), (atezolizumab) in ClinicalTrials.gov Identifier: NCT02763579, and CASPIAN (Paz-Ares L. et al., 2019 Lancet 394(10212):1929), (durvalumab) in ClinicalTrials.gov Identifier: NCT03043872. Outcomes from the Keynote-604 (Rudin C. et al., 2020 J. Clin. Oncol. 38:9001), (ClinicalTrials.Gov Identifier: NCT03066778, pembrolizumab), and EA5161 (Leal T. et al., 2020, J. Clin. Oncol. 38:9000), (ClinicalTrials.Gov Identifier: NCT03382561, nivolumab) trials were considered as comparisons.

[0201] All lung cancer trials used (IMPULSE, IMpower133, CASPIAN, Keynote-604, and EA5161) are "maintenance" trials, in which immune induction therapy (a set of chemotherapy cycles) is applied to initiate an antitumor response (as defined by RECIST guidelines), followed by maintenance therapy until tumor progression. IMpower133, CASPIAN, Keynote-604, and EA5161 are "continuation maintenance" trials, whereas IMPULSE is a "switch maintenance" trial. The four continuation maintenance trials provide an immunomodulatory product (IMP), i.e., the PD-L1 checkpoint inhibitors (CPIs) atezolizumab, durvalumab, pembrolizumab, or nivolumab, respectively, during induction chemotherapy and during the maintenance phase. In the switch maintenance trial IMPULSE, the IMP lefitolimod is not used during induction chemotherapy, but is introduced ("switched") during the maintenance phase of the trial (Figure 1). Therefore, to compare the PFS and OS data from IMPULSE with other studies, the mPFS1 and mOS1 from IMPULSE were calculated from the start of induction chemotherapy, and therefore, all subsequent analyses use the respective mOS1 values ​​from IMPULSE.

[0202] Table 1 provides a comparison of mOS between IMPULSE and the control and treatment arms of IMpower133, CASPIAN, Keynote-604, and EA5161. Clearly, the mOS reached by IMPULSE, IMpower133, and CASPIAN are similarly high, while the mOS of Keynote-604 and EA5161 are several months lower. The mOS achieved by the pivotal Phase III studies IMpower133 and CASPIAN led to the approval of atezolizumab (Tecentriq® by Roche) and durvalumab (Imfinzi® by AstraZeneca). Different definitions of clinical success (IMpower133 and CASPIAN) or failure (IMPULSE) relate to the mOS of the control arms of each study. The mOS of the control arm of IMpower133 is 10.3 months (m), and the corresponding mOS of CASPIAN is also 10.3m. The mOS from both studies was close to the previous mOS of 10.7m for these patients (Steffens CC et al., 2019 Lung Cancer 130:216) and was therefore considered comparable to the IMPLUSE data for the purposes of this study and was combined.

[0203] I. Advantages of a sequential immunomodulatory strategy Analysis comparing lefitolimod treatment in induction chemotherapy (IMPULSE, as first-line treatment for esSCLC) with reinduction chemotherapy (as first-line treatment for metastatic colorectal cancer (mCRC)) and immune modulation with a TLR9 agonist (IMPALA, Figure 8 ) showed that the TLR9 agonist was more effective in terms of mOS when applied after platinum-based chemotherapy rather than in combination.

[0204] Example: platinum / etoposide (PE) chemotherapy for 2, 3, or 4 qw3 cycles (the first cycle is PE chemotherapy administered in week 1 of each cycle and no treatment in weeks 2 and 3 of each cycle; the second cycle, etc.) followed sequentially by PE chemotherapy qw3 cycles in which PE is administered in week 1 of each cycle and a TLR9 agonist is administered in weeks 2 and 3 of each cycle.

[0205] Evidence: Four qw3 cycles of PE chemotherapy followed sequentially by a fifth cycle of PE chemotherapy, in which PE chemotherapy was administered in week 1 of the cycle followed by a TLR9 agonist in weeks 2 and 3 of the cycle, improved patients' mOS by 2.3 months (Figure 2).

[0206] II: Predictive biomarker analysis for optimal induction immunomodulation A collection of predictive biomarkers was determined after the first, second and third qw3 cycles of induction or reinduction chemotherapy and before qw3 cycles of sequential addition of TLR9 agonist treatment. To evaluate the utility of biomarker thresholds applicable to cancers of different tissue origins, overall survival was analyzed in patient groups divided according to median thresholds of percentage of activated immune cells in samples from a cohort of cancer patients. Patients with activated immune cells below the median threshold showed little evidence of adaptive immune induction, whereas patients above it were classified as having "above median activated immune cells" or "high activated immune cells". high " which indicate the respective percentage of activated B cells, activated T cells, activated regulatory T cells, or activated NKT cells, dendritic cells, or plasmacytoid dendritic cells exceeds the median determined for a cohort of patients diagnosed with cancer originating from the same tissue of origin and with the same time period since the start of induction chemotherapy.

[0207] Example: Extensive-stage small cell lung cancer (esSCLC) - Median percentage of activated B cells exceeds 16.9% - The percentage of activated T cells exceeds the median value of 0.76% - Median percentage of activated regulatory T cells exceeds 7.6% - Median percentage of activated natural killer T cells exceeds 4.1% - The percentage of activated pDCs is less than 5.1% of pDCs - Percentage of activated mDCs is less than 0.48%, and / or - The percentage of activated DCs is less than 54.51% of DCs.

[0208] Evidence: Patients with activated immune cells above the median of the cohort show increased overall survival. For example, patients with B cells above the median (above 16.9%) who received PE chemotherapy as a continuation maintenance had an mOS of 17.9 months, whereas patients with activated B cells below the median (below 16.9%) had an mOS of only 10.8 months (Figure 3). The threshold of 15.42% applied to the patient groups shown separated the two populations in the control arm but not in the lefitolimod arm, indicating that for patients exhibiting immune exhaustion characterized by an increase in activated B cell subsets, checkpoint inhibitor modulation is likely to provide substantial early benefit during early chemotherapy induction and maintenance immune modulation. Similar results were observed for additional immune subsets (Figure 4).

[0209] An important observation from the present study is that the threshold value of activated cells, especially the median percentage of activated B cells, identifies a strong preventive effect in mOS only in the control group of the study. This contradicts what was disclosed for TLR9 agonists in EP 339245 A1. The analysis provided here isolates the difference between lefitolimod treatment and immunomodulation with TLR9 agonists in induction chemotherapy (as first-line treatment for esSCLC) compared to reinduction chemotherapy (e.g., first-line metastatic colorectal cancer), showing that TLR9 agonists are more effective in terms of mOS when applied subsequently than in combination with platinum-based chemotherapy. Figure 3 shows that survival improves 3- to 10-fold (depending on the threshold value chosen) when a control patient population with activated B cells >15.42% is selected.

[0210] In the proposed therapeutic model design that is sensitive to these groupings, the predictive biomarker “activated immune cells” high Patients who match the criteria, and among them those who show the most statistically significant values ​​of activated B cells, are assigned to the "top branching pathway" that includes the period of checkpoint inhibition. high Patients who do not match the "targeted" path are assigned to a "branched subpathway" that includes TLR9 agonist treatment (Figures 9 and 10).

[0211] III. Predictive biomarker analysis for optimal maintenance immunomodulation SCLC patients who reach a certain threshold of progression-free survival (PFS) are likely to show a significant benefit in overall survival (OS). The method chosen to define the optimal threshold is a combination of the "Youden index" and the "Receiver Operating Characteristic Curve (ROC Curve)". Among the many Youden indexes and ROC curves compared, the pair of curves shown in Figure 5 represents the optimal threshold.

[0212] For example, esSCLC patients who achieve a PFS1 of >188 days (>6.2 months) are most likely to achieve an OS1 of >350 days (>11.5 months). This optimal threshold closely resembles the PFS1 values ​​found in the full analysis set (FAS) for patients on switched maintenance with TLR9 agonists (6.1 months) and for patients on continued maintenance PE chemotherapy (6.7 months).

[0213] Evidence: Using the above derived PFS threshold of >6.2 months in a Kaplan-Meier plot, mOS1 was calculated for the subgroup of patients on switched and continued maintenance therapy with the TLR9 agonist lefitolimod, with values ​​of 17.1 and 16.7 months, respectively (see Figure 6). The corresponding mOS1 values ​​for a PFS threshold of ≤6.2 months reached 10.7 and 10.2 months, which was only consistent with the mOS (10.3 months) observed in the control arms of the IMpower133 and CASPIAN studies.

[0214] The proportion of regulatory T cells (Tregs) increases by 20% or more after the start of "induction immunomodulation" (baseline, 90-104 days after the start of "induction chemotherapy") and at "maintenance immunomodulation" (visit 2, 118-125 days; visit 3, 146-153 days; visit 4, 188-195 days). Visit 4 corresponds to a statistically determined optimal PFS threshold (see Table 2). This provides an absolute measurement of a "second predictive biomarker". This corresponds to a patient having a Treg proportion of 20% or more at 188 days, approximately 6 months or more after the start of "induction chemotherapy" (Figure 7A). Similarly, the median excess of activated B cells, corresponding to the optimal PFS threshold, can also be used to identify patients with a significant benefit in overall survival at subsequent time points (Figure 7B).

[0215] Targeted Therapeutic Algorithms for Cancer Immunomodulation The results of the deep combined post-hoc analyses of the clinical trial data presented under I, II and III established essential elements for the treatment algorithm of first-line induction chemotherapy or reinduction (platinum-based) chemotherapy. These essential elements are: 1) "sequential"; 2) "two-step, biomarker-guided, branching"; and 3) "Alternation between innate and adaptive immune regulation."

[0216] 1) "Sequential" refers to adopting the natural order of activation in the innate and adaptive immune pathways. In all approved immuno-oncology treatment protocols, each immunotherapeutic agent is "combined" with chemotherapy or targeted therapy, i.e., applied together or on the same day. For example, in the first line therapy of esSCLC in the trials analyzed in this study, PE chemotherapy is administered together with the PD-L1 inhibitor antibody atezolizumab (Tecentriq®, Roche) or durvalumab (Imfinzi®, AstraZenenca).

[0217] Th1 immunity is provided by chemotherapy. Furthermore, the above data demonstrate the existence of two patient subgroups after PE “induction chemotherapy”, namely, those with more than median activated immune cells after four cycles of PE “induction chemotherapy” and those with more than median activated immune cells after four cycles of PE “induction chemotherapy”. high " (predominantly activated B cells, but also activated T cells, activated regulatory T cells, and activated NKT cells), and patients with less than the median number of activated immune cells, "activated immune cells low", demonstrating the presence of patients with a CRPC-like response. Of note, this distinction only occurs after several cycles of "induction chemotherapy" have generated different immune responses that may distinguish the two patient populations, so the combination with CPM agents such as TLR-9 agonists or immune checkpoint inhibitors (ICIs) from the start of "induction chemotherapy" is not justified by immune measurements. However, this is the current standard of care (SoC) as the outcomes of the IMpower-133 and CASPIAN clinical trials are very similar (Figure 1 and Table 1).

[0218] Post-hoc analyses of the IMPULSE and IMPALA clinical trials suggest that an immune response must first be generated by "induction chemotherapy" (four cycles of PE "induction chemotherapy" in IMPULSE) before the natural downregulation response can be effectively countered by applying an ICI targeting adaptive immunity (such as PD-L1 blockade). The proposed treatment algorithm therefore begins with 2, 3, 4, or 5 cycles of platinum-based "induction chemotherapy" (shown as "induction or re-induction chemotherapy" in Figures 9 and 10) before predictive biomarker match is determined and the respective sequential treatment ("induction immunomodulation") is initiated.

[0219] The prediction of moving from "maintenance immunomodulation" to "alternation of maintenance immunomodulation" is also sequential. Here, the "sequential alternation" of either treatment with a TLR9 agonist or treatment with an ICI is presented as an alternative to the "combination" of both simultaneous adaptive and natural immune stimulants. The rationale for the "alternation" will be explained in 3).

[0220] 2) "Two-phase, biomarker-guided, branched" means that the evaluation of predictive immune biomarkers allows the selection of a treatment program for patients associated with the best possible clinical outcome. In this "branched pathway" approach, patients are not excluded from treatment, but are assigned to either the up or down pathway, whether or not they match the first biomarker (Figures 9 and 10). "Two-phase" refers to the application of two biomarkers: a first biomarker to select the appropriate induction immunomodulator, and a second biomarker to guide the immunomodulatory maintenance therapy, or possibly the timing of the replacement therapy. Patients are not excluded from treatment, whether or not they match the second biomarker, but are assigned a time window to replace either TLR9 (natural) or ICI (adapted) maintenance immunomodulation.

[0221] First predictive biomarker: - The percentage of activated B cells exceeded the median (Figure 3) - The percentage of activated T cells exceeded the median (Figure 4) - The percentage of activated regulatory T cells (Treg) exceeded the median (Figure 4) - The percentage of activated natural killer T cells exceeded the median (Figure 4) - The percentage of activated dendritic cells exceeded the median (Figure 4) - Percentage of activated myeloid dendritic cells above the median - The percentage of activated plasmacytoid dendritic cells exceeded the median (Figure 4).

[0222] Second predictive biomarker: - Median progression-free survival (PFS) was 188 days, exceeding 6.2 months (Figures 5 and 6) - The percentage of activated regulatory B cells (Bregs) was equal to or greater than the median value more than 6 months after the start of induction chemotherapy (Figure 7). - The percentage of activated regulatory T cells (Treg) is ≥ 20% (preferred standard) more than 6 months after the start of induction chemotherapy (Figure 7).

[0223] 3) "Alternation between innate and adaptive immune regulation" This is another key concept of the treatment algorithm presented here. Many "combination" approaches of immune checkpoint inhibitors (PD-1 blockade, PD-L1 blockade are regulatory approved examples) with immunomodulators (e.g., TLR agonists, cGAS-STING, RIG-I) do not take into account the time course and quality of the immune response to tumor therapy, but the quality and quantity of immune cells, as well as chemokines and cytokines, analyzed by "liquid biopsy" can reflect the immunobiological situation in the tumor micro-environment (TME).

[0224] After induction or reinduction chemotherapy, measurements of activated immune cells in liquid biopsy samples high Patients classified as above (or above the median) are likely to reflect an immunologically "hot" but "immunoedited" tumor microenvironment (TME). Here, we predict that treatment with TLR9 agonists is likely to increase the proportion of mistargeted activated B cells, exhausted T cells, and regulatory cells (Bregs and Tregs). These patients are correlated with patients who match the first predictive biomarker and are assigned to the upper pathway of the branch (Figures 9 and 10). The "induction immunomodulation" for these patients consists of platinum-based chemotherapy and ICI (preferably PD-L1 blocking antibodies). The platinum-based (preferably PE) chemotherapy will not only continue to destroy the patient's tumor cells, but may also eliminate activated B cells and exhausted T cells. The "maintenance immunomodulation" will continue to block immune checkpoints until the "second predictive biomarker" is matched, i.e., progression-free survival (PFS) lasts longer than the respective tumor entity median (mPFS) or parameters of adaptive immune activation fall below the median threshold. The excess median PFS indicates that the ongoing antitumor immune response is being overwhelmed by further immunoediting.

[0225] Patients whose liquid biopsy samples measure consistent with the second predictive biomarker will then switch from adaptive immunity with ICI maintenance to innate immunity with TLR9 agonist maintenance, a "maintenance immune modulation." Activation of antigen-presenting cells (APCs) (i.e., plasmacytoid dendritic cells (pDCs) are activated by TLR9 agonists) transmits activation to myeloid dendritic cells, thereby reactivating tumor (neo) antigen presentation for a new, yet unedited, antitumor immune response.

[0226] After induction or reinduction chemotherapy, measurements of activated immune cells in liquid biopsy samples low Patients classified as above (or below the median) are likely to reflect an immunologically “cold” tumor microenvironment. In such tumors, “induction immunomodulation” and “maintenance immunomodulation” therapy with TLR9 agonists would improve OS by immune surveillance reactivation (ISR). ISR is required to transform the immunologically “cold” tumor microenvironment (TME) into an immunologically “hot” one. This is achieved during “maintenance immunomodulation” with TLR9 agonists. TLR9 activation broadly activates not only innate immunity with antigen-presenting apparatus (pDCs and mDCs) and effector cells (NK cells and NKT cells), but also adaptive immunity via T cells and B cells. Then, if the “second predictive biomarker” is matched, i.e., progression-free survival (PFS) lasts longer than the respective tumor real median (mPFS) or adaptive immune activation parameters measured in liquid biopsies rise above the median threshold. A PFS above the median indicates that the progression of antitumor immune responses is losing efficacy due to downregulation control by regulatory immune cells (Treg and Breg). Patients who match the second predictive biomarker will then switch from innate immunity with TLR9 agonists to adaptive immunity with ICIs for "maintenance immune modulation". Blocking immune checkpoints with ICIs will downregulate the activity of Treg and Breg, thereby allowing the progression of antitumor adaptive immunity.

[0227] overview The combined post-hoc analysis associated with the examples combines the accessible relevant clinical parameters and immunological data from the IMPLUSE study with published mPFS and mOS data from the respective treatment and control arms of the PD-L1 inhibitor-antibody chemotherapy immunomodulatory maintenance study, comparing the use of checkpoint inhibitors with the TLR9 agonist used in IMPULSE. The analysis identifies immune parameters related to the underlying immune status that reflect the suitability of the patient for one of the two treatment protocols, which is only evident after the first round of chemotherapy cycles.

[0228] The immune analysis identifies patients with "first predictive biomarkers," measurements in liquid biopsy samples classified based on whether the percentage of activated immune cells, particularly activated (e.g., CD86+) B cells, after induction or reinduction chemotherapy is above or below the median, thereby distinguishing between immunologically "cold" or "hot" tumor microenvironments in which treatment with CPMs such as cancer therapeutic TLR9 agonists or ICIs, respectively, may provide effective treatment.

[0229] The development of such adaptive immunity and the eventual development of an exhausted phenotype are subsequently tracked by the "second predictive biomarker" according to the invention by identifying inflection points where the response fades, i.e., continued exceedance of the median progression-free survival (mPFS) for each tumor entity, or an increase above the median threshold of adaptive immune activation reflected by the percentage of Tregs among T cells measured in liquid biopsies. A PFS above the median or an exhausted Treg compartment indicates that the ongoing antitumor immune response is losing potency due to downregulation control by regulatory immune cells (Tregs and Bregs). Thus, patients who match the second predictive biomarker will switch from innate immunity with TLR9 agonists to adaptive immunity with ICIs, a "maintenance immune modulation". ICIs will block immune checkpoints.

[0230] Table 1 summarizes the median overall survival (mOS) using mOS1 (see Figure 1) and intention to treat (ITT) analyses in IMPULSE. Treatment and control groups are shown for IMPULSE, IMpower133, CASPIAN, Keynote-604, and EA5161.

[0231] Table 2 shows descriptive statistics and paired comparisons of regulatory T cells (%); two-sample t-tests; comparison of treatment groups; paired t-tests; differences from baseline within treatment groups. The percentage of regulatory T cells (Tregs) increases with duration of TLR9 agonist; PE chemotherapy; platinum / etoposide for induction chemotherapy, then maintained until progression; baseline, at the end of cycle 4 of induction PE chemotherapy; visit 2, 118-125 days; visit 3, 146-153 days; visit 4, 188-195 days are calculated from start of induction PE chemotherapy, IMPULSE.

[0232] [Table 1]

[0233] [Table 2]

Claims

1. 1. A TLR9 agonist for use in the treatment of cancer, comprising: - administering said TLR9 agonist to a patient who has undergone induction chemotherapy consisting of a first set of treatment cycles; - said patient, after receiving induction chemotherapy, has been assigned a status of "non-matched-in-first" in a first predictive biomarker assay, wherein the first predictive biomarker assay determines a percentage of activated cells within an immune cell subset selected from B cells, T cells, natural killer T (NKT) cells, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and / or a percentage of regulatory T (Treg) cells among CD4+ T cells in a blood sample obtained from the patient; and if the percentage of activated cells in the immune subset is less than the median percentage of activated cells or less than the median percentage of Treg cells among CD4+ T cells, the patient is assigned a status of "1st mismatch"; - administering said TLR9 agonist after said induction chemotherapy as part of a TLR9 agonist induction immunomodulatory cycle, wherein a chemotherapeutic agent is administered during week 1 of said TLR9 agonist-induced immunomodulatory cycle; and administering the TLR9 agonist once or twice weekly during weeks 2 and 3 of the TLR9 agonist-induced immunomodulatory cycle; and - after 1 to 4 sets of said TLR9 agonist induction immunomodulatory cycles, said TLR9 agonist is administered as part of a TLR9 agonist maintenance immunomodulatory regimen; wherein said TLR9 agonist is administered twice weekly, once weekly, or every two weeks during n weeks of said TLR9 agonist maintenance immunomodulatory regimen, where n is an integer between 5 and 35; and - determining the percentage of Treg cells among CD4+ T cells in a blood sample obtained from said patient by a second predictive biomarker assay after at least three weeks of said TLR9 agonist maintenance immunomodulatory regimen; - if the percentage of Treg cells among CD4+ T cells in the sample is at least 20% (or higher), the patient is assigned a status of "matched-in-second"; and After the status of "second match" has been assigned here, and / or if the patient exceeds a threshold time of median progression-free survival from the start of the induction chemotherapy without a diagnosis of disease progression; a checkpoint modulator (CPM) agent administered once every two or three weeks for n weeks, where n is an integer between 3 and 15; or - if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%, the patient is assigned a status of "non-matched-in-second"; and wherein after a status of "second non-match" is assigned, the TLR9 agonist continues to be administered twice weekly, once weekly, or every two weeks for n weeks, where n is an integer between 6 and 24; and The TLR9 agonist is lefitolimod, and the CPM agent is an antibody specific for PD-1 or PD-L1. The TLR9 agonist.

2. - said second predictive biomarker assay is performed every 6 to 15 weeks; A TLR9 agonist for use according to claim 1.

3. 1. A CPM (checkpoint modulator) agent for use in the treatment of cancer, comprising: - said CPM agent is administered to a patient who has undergone induction chemotherapy comprising a first set of treatment cycles; - the patient, after receiving induction chemotherapy, is assigned a status of "matched-in-first" in a first predictive biomarker assay determining the percentage of activated cells in an immune cell subset as defined in claim 1, wherein if the percentage of activated cells in said immune subset is greater than or equal to the median percentage of activated cells, or the percentage of Treg cells among CD4+ T cells is greater than or equal to the median percentage of Treg cells among CD4+ T cells, then said patient is assigned a status of "matched first time"; - administering said CPM agent as part of a CPM induction immunomodulatory cycle following said induction chemotherapy, wherein the chemotherapy agent and the CPM agent are administered once during the first week of said CPM induction immunomodulatory cycle; and administering said CPM agent as part of a CPM maintenance immunomodulatory regimen after sets 1 to 5 of said CPM induction immunomodulatory cycles; the CPM agent is administered once every two or three weeks of n weeks of the CPM maintenance immunomodulatory regimen, where n is an integer between 6 and 36; and The CPM agent is an antibody specific for PD-1 or PD-L1; The CPM agent.

4. - said patient has received a CPM maintenance immunomodulatory regimen as defined in claim 3 during the preceding 3 weeks; - determining the percentage of Treg cells among CD4+ T cells in a blood sample obtained from said patient by a second predictive biomarker assay; if the percentage of Treg cells among CD4+ T cells is 20% or greater, the patient is assigned a status of "second match"; and After a "second match" status is assigned, the CPM agent is administered every two weeks or every three weeks for n weeks, where n is an integer between 6 and 36; A CPM agent for use according to claim 3.

5. - in said second predictive biomarker assay determined as defined in claim 4, - the patient is assigned a "second mismatch" status if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%; or - the patient is assigned a "second match" status if the percentage of activated Treg cells in the sample is 20% or higher; - after the status of "second mismatch" is assigned and / or if the patient exceeds a threshold time of median progression-free survival from the start of induction chemotherapy without a diagnosis of disease progression, a TLR9 agonist is administered twice weekly, once weekly, or every two weeks for n weeks, where n is an integer between 6 and 24; or - after a "second match" status is assigned, the CPM agent is administered once every 2 weeks or once every 3 weeks for n weeks, where n is an integer between 6 and 36, and the TLR9 agonist is lefitolimod; A CPM agent for use according to claim 4.

6. - said second predictive biomarker assay is performed every 6 to 15 weeks; - if the patient has received a CPM maintenance immunomodulatory regimen as defined in claim 3 during the preceding 3 weeks, and thereafter after a "second mismatch" status is assigned and / or if the patient exceeds a threshold time of median progression-free survival from the start of induction chemotherapy without a diagnosis of disease progression, administering the TLR9 agonist twice weekly, once weekly, or every two weeks for the subsequent n weeks, where n is an integer between 6 and 24; or - after a "second-match" status is assigned, continue to administer said CPM once every two or three weeks for the next n weeks, where n is an integer between 6 and 24; or - said patient has received a TLR9 maintenance immunomodulatory regimen as defined in claim 1 during the preceding 3 weeks; after a "second non-match" status is assigned, continue to administer the TLR9 agonist twice weekly, once weekly, or every two weeks for the subsequent n weeks, where n is an integer between 6 and 24; or After "second-matched" status is assigned and / or if the patient exceeds a threshold time of median progression-free survival from the start of induction chemotherapy without a diagnosis of disease progression, the CPM agent is administered once every 2 weeks or once every 3 weeks for the subsequent n weeks, where n is an integer between 6 and 24; A TLR9 agonist for use according to claim 1 or 2, or a CPM agent for use according to claim 4 or 5.

7. The TLR9 agonist or CPM modulator for use according to any one of claims 1 to 6, wherein said patient does not receive simultaneous administration of said TLR9 agonist and said CPM modulator.

8. 1. A method of classifying a cancer patient according to the likelihood of responding favorably to an individualized maintenance cancer therapy, the cancer patient receiving induction chemotherapy consisting of a first set of treatment cycles, The method includes a first predictive biomarker assay, The first predictive biomarker assay comprises: - determining the percentage of activated B cells in a blood sample obtained from said patient; assigning a "first match" status to said cancer patient if the percentage of activated B cells in the immune subset is equal to or greater than the median percentage of activated B cells, wherein said cancer patient assigned a status of "first match" is assigned a likelihood of responding favorably to a CPM-induced immunomodulatory cycle; The CPM-induced immunomodulatory cycle is defined as follows: a chemotherapy agent and a CPM agent are administered once during the first week of said CPM induction immunomodulatory cycle; and administering said CPM agent as part of a CPM maintenance immunomodulatory regimen after sets 1 to 5 of said CPM induction immunomodulatory cycles; - administering the CPM agent once every two or three weeks of the CPM maintenance immunomodulatory regimen for n weeks, where n is an integer between 6 and 36; or assigning the cancer patient a status of "first mismatch" if the percentage of activated B cells in the immune subset is less than the median percentage of activated cells, wherein said cancer patients assigned a status of "1st mismatch" are assigned a likelihood of responding well to a cycle of TLR9 agonist-induced immunomodulation followed by a cycle of TLR9 agonist maintenance regimen, said cycle of TLR9 agonist-induced immunomodulation followed by a cycle of TLR9 agonist maintenance regimen defined as follows: a chemotherapeutic agent is administered during week 1 of said TLR9 agonist-induced immunomodulatory cycle; and - administering the TLR9 agonist once or twice weekly during weeks 2 and 3 of the TLR9 agonist-induced immunomodulatory cycle; and administering said TLR9 agonist as part of a TLR9 agonist maintenance immunomodulatory regimen after set 1 to 4 of said TLR9 agonist induction immunomodulatory cycles; and administering the TLR9 agonist twice weekly, once weekly, or every two weeks for n weeks of the TLR9 agonist maintenance immunomodulatory regimen, where n is an integer between 5 and 35; and The method further comprises a second predictive biomarker assay administered 6-15 weeks following initiation of the TLR9 agonist maintenance immunomodulatory regimen or the CPM maintenance immunomodulatory regimen; wherein said second predictive biomarker assay comprises: - determining the percentage of Treg cells among CD4+ T cells in a blood sample obtained from said patient; assigning the cancer patient a "second match" status if the percentage of Treg cells among CD4+ T cells in the sample is 20% or more, Here, patients assigned a status of “second match” are assigned a likelihood of responding well to a CPM maintenance immunomodulatory regimen, which is defined as follows: the CPM agent is administered once every two or three weeks for n weeks, where n is an integer between 3 and 15; or - assigning the cancer patient a "second mismatch" status if the percentage of Treg cells among CD4+ T cells in the sample is less than 20%, Here, patients assigned a status of "first mismatch" are assigned a likelihood of responding well to a subsequent administration of a TLR9 agonist maintenance immunomodulatory regimen, which is defined as follows: - administering the TLR9 agonist twice weekly, once weekly, or every two weeks for n weeks of the TLR9 agonist switching maintenance immunomodulatory regimen, where n is an integer between 6 and 24; wherein the TLR9 agonist is lefitolimod, and the CPM agent is an antibody specific to PD-1 or PD-L1; The method comprising:

9. the patient exceeds a median progression-free survival threshold time from the start of induction chemotherapy of about 188 days without diagnosis of disease progression; And here - Patients who have received a TLR9 agonist maintenance immunomodulatory regimen in the previous 2 weeks are assigned a likelihood of responding well to a subsequent administration of a CPM maintenance immunomodulatory regimen, or - Patients who have received a CPM maintenance immunomodulatory regimen in the previous 2 weeks are assigned a likelihood of responding well to a subsequent administration of a TLR9 agonist maintenance immunomodulatory regimen; The method according to claim 8.

10. The TLR9 agonist is administered by subcutaneous injection: - a dose of 50-80 mg twice a week, - once a week at a dose of 30-60 mg, - once weekly at a dose of 100-150 mg, or - a dose of 100-150 mg once every 2 weeks, Administered at A TLR9 agonist for use according to any one of claims 1, 2, 6, 7 or 10 to 13, or a CPM agent for use according to any one of claims 5 to 7 or 10 to 13, or a method according to any one of claims 8 to 13.

11. 13. The TLR9 agonist for use according to any one of claims 1, 2, 6, 7 or 10, or the CPM agent for use according to any one of claims 3-7 or 10, or the method according to any one of claims 8-10, wherein the cancer is selected from breast cancer, cervical cancer, endometrial cancer, colorectal cancer, metastatic colorectal cancer, or lung cancer.

12. To the patient: - if the proportion of activated B cells is less than or equal to 16.9% of B cells, the patient is assigned a "first mismatch" status; and / or - if the proportion of activated B cells is greater than 15.42% of B cells, the patient is assigned a "first mismatch" status; A TLR9 agonist for use according to any one of claims 1, 2, 6, 7, 10 or 11, or a CPM agent for use according to any one of claims 3 to 7, 10 or 11, or a method according to any one of claims 8 to 11.

13. The induction chemotherapy comprises: - a first set of treatment cycles, including 2, 3, 4, or 5 cycles, each cycle consisting of 3 weeks; Consists of, and - a chemotherapy agent or a combination of chemotherapy agents is administered during the first week of said induction chemotherapy cycle, A TLR9 agonist for use according to any one of claims 1, 2, 6, 7 or 10-12, or a CPM agent for use according to any one of claims 3-7 or 10-12, or a method according to any one of claims 8-12.

14. The induction chemotherapy comprises: - Anticancer platinum complex drugs, and -Antineoplastic alkaloid drugs, comprising or consisting of treatment with A TLR9 agonist for use according to any one of claims 1, 2, 6, 7 or 10-13, or a CPM agent for use according to any one of claims 3-7 or 10-13, or a method according to any one of claims 1-13.

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