COMBINATION ANTI CANCER THERAPY WITH AN IAP ANTAGONIST AND AN ANTI PD-1 MOLECULE

Abstract

Disclosed is the use of an IAP antagonist for pretreating a human subject diagnosed with a cancer to enhance the likelihood that a subsequent treatment with an anti-PD-1 molecule results in an anti-cancer response or to enhance the responsiveness of the subject's cancer to the subsequent treatment with the anti-PD-1 molecule. Also encompassed are methods of treatment of a subject's cancer, the methods comprising pretreatment of the subject with an IAP antagonist and subsequent treatment of the subject with an anti-PD-1 molecule.

Claims

1. A method of treating cancer in a human subject, the method comprising: (i) administering an inhibitor of apoptosis protein (IAP) antagonist during an induction period, wherein the duration of the induction period is selected from the range of 1 to 48 days before first administration of an anti-PD-1 molecule; followed by (ii) administering an anti-PD-1 molecule after the end of the induction period.

2. The method according to claim 1, wherein the human subject is administered with the IAP antagonist during an induction period of 1 to 28 days, followed by the administration of the anti-PD-1 molecule.

3. The method according to claim 1, wherein the human subject is administered with the IAP antagonist during an induction period of 5 to 28 days, followed by the administration of the anti-PD-1 molecule.

4. The method according to claim 1, wherein the IAP antagonist is not administered on one or more days during the induction period.

5. The method according to claim 1, wherein the administration of the IAP antagonist is continued after the administration with the anti-PD-1 molecule has started; or another IAP antagonist is administered concurrently with the anti-PD-1 molecule.

6. The method according to claim 1, wherein the cancer is a cancer that is known to be responsive to treatment with an anti-PD-1 molecule in 10% or more of treated patients.

7. The method according to claim 1, wherein the cancer is head & neck cancer, melanoma, urothelial cancer, non-small cell lung cancer, microsatellite instability (MSI) high tumors from agnostic primary site or kidney cancer.

8. The method according to claim 1, wherein the cancer is a cancer with a response rate to treatment with an anti-PD-1 molecule of 10% or less, preferably 5% or less.

9. The method according to claim 1, wherein the cancer is pancreatic cancer, colorectal cancer, multiple myeloma, small cell lung cancer, hepatocarcinoma or ovarian cancer.

10. The method according to claim 1, wherein the cancer has been assessed to be poorly immunogenic.

11. The method according to claim 10, wherein said assessment consists of an analysis of a marker of immunogenicity in a patient's biological sample taken prior to the induction period and a finding that the marker's presence, expression level or derived score fails a predetermined threshold.

12. The method according to claim 11, wherein the marker is PD-L1 expressed on cancer cells and/or immune cells.

13. The method according to claim 11, wherein the marker is tumor-infiltrating lymphocytes, preferably CD8+ cells, or tumor mutation burden.

14. The method according to claim 1, wherein the administering the IAP antagonist during an induction period is continued until the cancer is assessed to be of high immunogenicity.

15. The method according to claim 14, wherein said assessment consists of an analysis of a marker of immunogenicity in a patient's biological sample taken after the induction period and a finding that the marker's presence, expression level or derived score exceeds a predetermined threshold.

16. The method according to claim 15, wherein the marker is PD-L1 expressed on cancer cells and/or immune cells.

17. The method according to claim 15, wherein the marker is tumor-infiltrating lymphocytes, preferably CD8+ cells, or tumor mutation burden.

18. The method according to claim 1, wherein the biological sample is a tumor or liquid biopsy.

19. The method according to claim 1, wherein the anti-PD-1 molecule is Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, PDR001, IBI-308, Cemiplimab, Camrelizumab, BGB-A317, BCD-100, JS-001, JNJ-3283, MEDI0680, AGEN-2034, TSR-042, Sym-021, PF-06801591, MGD-013, MGA-012, LZM-009, GLS-010, Genolimzumab, BI 754091, AK-104, CX-072, WBP3155, SHR-1316, PD-L1 Inhibitor millamolecule, BMS-936559, M-7824, LY-3300054, KN-035, FAZ-053, CK-301, or CA-170.

20. The method according to claim 19, wherein the anti-PD-1 molecule is Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, PDR001, or BI 754091.

21. The method according to claim 1, wherein the anti-PD-1 molecule is an antibody against PD-1 or PD-L1.

22. The method according to claim 1, wherein the administration of the anti-PD-1 molecule is combined with one or more other cancer therapies, including another immunotherapy, radiotherapy, chemotherapy, chemioradiotherapy, oncolytic viruses, anti-angiogenic therapies, and/or targeted cancer therapies.

23. The method according to claim 1, wherein the IAP antagonist is a second mitochondrial-derived activator of caspases (SMAC) mimetic.

24. The method according to claim 1, wherein the IAP antagonist administered during the induction period is Debio 1143, GDC-917/CUDC-427, LCL161, GDC-0152, TL-32711/Birinapant, HGS-1029/AEG-40826, BI 891065, ASTX-660 or APG-1387, preferably, the IAP antagonist is Debio 1143, LCL161 or Biranapant.

25. The method according to claim 24, wherein the IAP antagonist is Debio 1143.

Description

BRIEF DESCRIPTION OF FIGURES

[0040] FIG. 1 is a graph showing that Debio 1143 treatment induces the degradation of cIAP1 in tumors of human head & neck cancer patients (n=12 patients), as per Example 1. Statistical analysis used a paired t-test and P-value=0.045.

[0041] FIG. 2 is a graph showing that Debio 1143 treatment increases the number of CD4+ (A) and CD8+ (B) T-lymphocytes in the tumor of head & neck cancer patients (n=12 patients), as per Example 1. Statistical analysis used a paired t-test. P-value for FIG. 2(A)=0.511 and P-value for FIG. 2(B)=0.020.

[0042] FIG. 3 is a graph showing that Debio 1143 increases the number of PD-1+ immune cells (A) and PD-L1+ immune (B) and tumor (C) cells in the tumor of head & neck cancer patients (n=12 patients), as per Example 1. Statistical analysis used a paired t-test. P-value for FIG. 3(A)=0.002, P-value for FIG. 3(B)=0.004 and P-value for FIG. 3(C)=0.129.

[0043] FIG. 4 is a graph showing that pretreatment with Debio 1143 sensitizes MC38 tumors to a subsequent treatment with an anti-PD-L1 antibody, as measured by median tumor volume. At day of optimal T/C (day 18): p<0.05 (*) for Debio 1143 pretreatment only versus vehicles; p<0.0001 (**) for Debio 1143 pretreatment then PD-L1 versus vehicles; p<0.0001 (**) for Debio 1143 pretreatment then combo versus vehicles; as determined by student t-test (two-tailed, unpaired, equal variance). N=8 mice per group, except n=6 for vehicles on day 18. Note: combo=Debio 1143+ anti-PD-L1.

[0044] FIG. 5 is a graph showing that pretreatment with birinapant sensitizes MC38 tumors to a subsequent treatment with an anti-PD-L1 antibody, as measured by median tumor volume. At day of optimal T/C (day 15): p>0.05 for birinapant pretreatment only versus vehicles; p<0.05 (*) for birinapant pretreatment then PD-L1 versus vehicles; p<0.001 (**) for birinapant pretreatment then combo versus vehicles; as determined by student t-test (two-tailed, unpaired, equal variance). N=8 mice per group. Note: combo=birinapant+ anti-PD-L1.

[0045] FIG. 6 is a graph showing that pretreatment with LCL161 sensitizes MC38 tumors to a subsequent treatment with an anti-PD-L1 antibody, as measured by median tumor volume. At day of optimal T/C (day 15): p<0.05 (*) for LCL161 pretreatment only versus vehicles; p<0.05 (*) for LCL161 pretreatment then PD-L1 versus vehicles; p<0.001 (**) for LCL161 pretreatment then combo versus vehicles; as determined by student t-test (two-tailed, unpaired, equal variance). N=8 mice per group. Note: combo=LCL161+ anti-PD-L1.

[0046] FIG. 7 is a graph showing that pretreatment with Debio 1143 sensitizes CT26 tumors to a subsequent treatment with an anti-PD-1 antibody, as measured by median tumor volume. At day of optimal T/C (day 17): p>0.05 for Debio 1143 pretreatment only versus vehicles; p<0.05 (*) for Debio 1143 pretreatment then PD-1 versus vehicles; p<0.0001 (**) for Debio 1143 pretreatment then combo versus vehicles; as determined by student t-test (two-tailed, unpaired, equal variance). N=8 mice per group, except n=7 for vehicles on day 17. Note: combo=Debio 1143+ anti-PD-1.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0047] The terms “antagonist” and “inhibitor” are used interchangeably and refers to a substance which interferes with or inhibits the physiological action of another. In some embodiments, the terms “antagonist” and “inhibitor” have the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0048] The term “antibody” refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular antigen. The term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof. The term “antibody” in particular includes bispecific antibodies.

[0049] A typical type of antibody comprises at least two heavy chains (“HC”) and two light chains (“LC”) interconnected by disulfide bonds.

[0050] Each “heavy chain” comprises a “heavy chain variable domain” (abbreviated herein as “VH”) and a “heavy chain constant domain” (abbreviated herein as “CH”). The heavy chain constant domain typically comprises three constants domains, CH1, CH2, and CH3.

[0051] Each “light chain” comprises a “light chain variable domain” (abbreviated herein as “VL”) and a “light chain constant domain” (“CL”). The light chain constant domain (CL) can be of the kappa type or of the lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FW”).

[0052] Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure inter alia presents VH and VL sequences as well as the subsequences corresponding to CDR1, CDR2, and CDR3.

[0053] Accordingly, a person skilled in the art would understand that the sequences of FW1, FW2, FW3 and FW4 are equally disclosed. For a particular VH, FW1 is the subsequence between the N-terminus of the VH and the N-terminus of H-CDR1, FW2 is the subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is the subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3, and FW4 is the subsequence between the C-terminus of H-CDR3 and the C-terminus of the VH. Similarly, for a particular VL, FW1 is the subsequence between the N-terminus of the VL and the N-terminus of L-CDR1, FW2 is the subsequence between the C-terminus of L-CDR1 and the N-terminus of L-CDR2. FW3 is the subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3, and FW4 is the subsequence between the C-terminus of L-CDR3 and the C-terminus of the VL.

[0054] The variable domains of the heavy and light chains contain a region that interacts with an antigen, and this region interacting with an antigen is also referred to as an “antigen-binding site” or “antigen binding site” herein. The constant domains of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Exemplary antibodies of the present disclosure include typical antibodies, but also fragments and variations thereof such as scFvs, and combinations thereof where, for example, an scFv is covalently linked (for example, via peptidic bonds or via a chemical linker) to the N-terminus of either the heavy chain and/or the light chain of a typical antibody, or intercalated in the heavy chain and/or the light chain of a typical antibody. Further, exemplary antibodies of the present disclosure include bispecific antibodies.

[0055] As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain variable fragment (scFv), disulfide stabilized scFvs, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site.

[0056] An antibody can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates. In some embodiments, the term “antibody” has the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0057] The terms “anti-cancer response”, “response” or “responsiveness” relate to objective radiological and clinical improvements assessed using RECIST v1.1 criteria (Eur. J. Cancer 45; 2009: 228-247). RECIST is a set of published rules that define objectively when cancer patients improve (“respond”), stay the same (“stable”) or worsen (“progression”) during treatments. RECIST 1.1 has recently been adapted for evaluation of immunotherapeutic agents iRECIST 1.1. (Seymour, L., et al., iRECIST: Guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol, 2017. 18(3): p. e143-e152). In the present invention, a patient is considered to respond to a given treatment if there is any clinical benefit for the patient as per RECIST v 1.1, assessed as complete response (CR), partial response (PR) or stable disease (SD) or as having an increased duration of the response or disease stabilization as measured by progression free survival or overall survival status.

[0058] The term “anti-PD-1 molecule” refers to PD-1 inhibitors and PD-L1 inhibitors. These inhibitors include but are not limited to antibodies targeting PD-1 or PD-L1. The anti-PD-1 molecule may be a small molecule such as CA-170 (AUPM-170, Curis, Aurigene, described e.g. in J. J. Lee et al., Journal of Clinical Oncology 35, no. 15_suppl, DOI: 10.1200/JCO.2017.35.15_suppl.TPS3099). Further small molecule inhibitors of the PD-1/PD-L1 interaction, which are useful for the present invention, are described in WO 2018/195321 A.

[0059] “Cancer” generally refers to malignant neoplasm, which may be metastatic or non-metastatic. For instance, non-limiting examples of cancer that develops from epithelial tissues such as gastrointestinal tract and skin include non-melanoma skin cancer, head and neck cancer, esophageal cancer, lung cancer, stomach cancer, duodenal cancer, breast cancer, prostate cancer, cervical cancer, cancer of endometrial uterine body, pancreatic cancer, liver cancer, cholangiocarcinoma, gallbladder cancer, colorectal cancer, colon cancer, bladder cancer, and ovarian cancer. Non-limiting examples of sarcoma that develops from non-epithelial tissues having mesodermal origin (stroma) such as muscles include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, gastrointestinal stromal tumors (GIST) and angiosarcoma. Non-limiting examples of tumors from an ectodermal (neural crest ontogeny) include brain tumors, neuroendocrine tumors, etc. Furthermore, non-limiting examples of hematological cancer derived from hematopoietic organs include malignant lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, leukemia including acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphatic leukemia, chronic lymphatic leukemia, and multiple myeloma. The latter examples of cancer are also referred to herein as types of cancer.

[0060] The terms “cancer” and “tumor” (meaning malignant tumor) are used interchangeably herein.

[0061] The term “concurrent therapy”, “concurrent treatment” or “co-therapy” refers to the contemporaneous or simultaneous administration of both the IAP antagonist and the anti-PD-1 molecule. In some embodiment, the term “concurrent therapy” or “concurrent treatment” refers to a treatment wherein the IAP antagonist is not given sufficient time to enhance the immunogenic potency of a tumor's microenvironment before the anti-PD-1 molecule is administered. In some embodiments, the terms “concurrent treatment”, “co-therapy” and “concurrent therapy” has the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0062] “Effective amount” of an IAP antagonist or an anti-PD-1 molecule means the amount of compound that will elicit the biological or medical anti-cancer response sought by the clinician.

[0063] The phrase “to enhance the immunogenic potency of a tumor's microenvironment” refers to a stimulation of the immune system in the tumor microenvironment which results in an increased immune response in comparison to an unstimulated immune system. In the present case, the immune system may be stimulated by an IAP antagonist. The stimulation may increase the immunogenicity of the cancer, the stimulation may increase the amount of effector cells at the tumor microenvironment, and/or the stimulation may increase the sensitivity of immune effector cells present in the tumor microenvironment towards the cancerous cells. In some embodiments, the phrase “to enhance the immunogenic potency of a tumor's microenvironment” has the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0064] The term “first administration” of an anti-PD-1 molecule, as used herein, specifies that the anti-PD-1 molecule is administered for the first time to a patient. In some embodiments, the patient has never been previously treated with an anti-PD-1 molecule. In some embodiments, the patient has been treated with an anti-PD-1 molecule but the patient has relapsed or the anti-PD-1 molecule therapy was ineffective. In these embodiments, the previously administered anti-PD-1 molecule level in the serum has been sufficiently reduced, e.g. by 95%, before the induction therapy of the present invention is started. In some embodiments, the time between the last administration of the previously administered anti-PD-1 molecule and the start of the induction therapy of the present invention represents at least one or two dosing interval (time between repeated administration) as approved by regulatory agencies or accepted by the medical community. In some embodiments, the subject has not been administered with an anti-PD-1 molecule for at least, 1, 2, 3, 4 or even 6 weeks before the start of the induction period.

[0065] The terms “immunogenic” and “immunogenicity” as used herein in relation to the tumor microenvironment means causing or producing an immune response. In some embodiments, immunogenicity is assessed by determining the expression level of PD-L1 revealed by immunostaining on the patient's cancer cells.

[0066] In some embodiments, immunogenicity is assessed by considering the level of CD8+ cells in the cancer sample as a marker. This assessment may be carried out using the materials and methods of Example 1 below.

[0067] In some embodiments, cancer samples may be assessed and classified as being of low and high immunogenicity by considering the above-mentioned markers in combination. Hence, in some embodiments, immunogenicity is assessed by considering a combination of the PD-L1 marker expression levels together with the level of CD8+ cells in the cancer sample. If, in some embodiments, the treatment with IAP antagonist during the induction period increases the expression level of PD-L1 on the patient's cancer cells, for example by at least 1, 2, 3 or 4% in terms of the fraction of cells of a cancer sample exhibiting staining for PD-L1 (at any intensity) in an immunohistochemistry assay using a suitable antibody such as, for example, antibody 22c3 pharmDx (Dako, Inc.), the treatment is with IAP antagonist is considered to enhance the immunogenic potency of the tumor's microenvironment. Similarly, an enhancement in immunogenic potency may be identified in some embodiments by means of an increase in the level of CD8+ cells in the cancer sample by at least 1, 2, 3 or 4%, when determined using the materials and methods of Example 1 below.

[0068] IAP antagonist or inhibitor as used herein means a compound having affinity for inhibitor of apoptosis proteins (abbreviated as IAP). The compound is an inhibitor or antagonist of IAPs. In some embodiments, the IAP antagonist shows the characteristic that an interaction between the IAP antagonist and cIAP1 and/or cIAP2 leads to degradation of these proteins and subsequent NF-κB modulation. In some embodiments, this effect can be used for testing a compound for IAP inhibitory activity: when contacting the potential IAP antagonist with cIAP1 and/or cIAP2 in vitro and analyzing the effect with a suitable technique including but not limited to western blot analysis, for an IAP inhibitor, an effect on cIAP1 should be observed at concentrations below 10 μM, preferably, <1 μM. In some embodiments, the term “IAP inhibitor” and “IAP antagonist” has the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0069] In general, the term “induction therapy” refers to a type of treatment wherein a drug is administered to a patient to induce a response in the patient that potentiates the effectiveness of another drug that is administered afterwards. In the context of the present invention, the induction therapy involves a “pretreatment”. The “pretreatment” or “induction” refers to the administration of an IAP antagonist for a certain amount of time before the first administration of the anti-PD-1 molecule. The period in which the IAP antagonist is administered is referred to as the “induction period” or “pretreatment period”. The induction period is not particularly limited as long as the immunogenic potency of a tumor's microenvironment is enhanced. In some embodiments, the induction period has a duration selected from the range of 1 to 48 days, preferably 1 to 28 days, more preferably 5 to 28 days. In some embodiments, the induction period is sufficiently long to enhance the immunogenic potency of a tumor's microenvironment. In some embodiments, the efficacy of the anti-PD-1 molecule treatment is increased in comparison with a concurrent treatment without induction therapy with an IAP antagonist. The anti-PD-1 molecule is then administered after the induction period, i.e. after the immunogenic potency of a tumor's microenvironment has been enhanced. This results in an increased potency of the anti-PD-1 molecule because the immune system has been primed by the IAP antagonist.

[0070] In some embodiments, the terms “induction therapy”, “pretreatment”, “induction”, “induction period” and “pretreatment period” have the same meaning as understood by the person skilled in the art at the first priority date, i.e. Dec. 21, 2017, bearing in mind the skilled person's common general knowledge at the first priority date.

[0071] “SMAC mimetic” means a small-molecule inhibitor for therapeutic inhibition of IAP which small-molecule inhibitor mimics the N-terminal four-amino acid stretch of the endogenous SMAC sequence and is at least partly comprised of non-peptidic elements. The N-terminal sequence of endogenous SMAC is Ala-Val-Pro-Ile (AVPI) and is required for binding to IAP.

[0072] The term “subject” relates to a mammalian animal and, preferably, to a human person. A human subject is also referred to as a “patient”.

[0073] Induction Therapy

[0074] Inventors propose that a patient having a tumor can be pretreated with an IAP antagonist, such as a SMAC mimetic to enhance the immunogenicity of the patient's tumor microenvironment. Subsequently, the patient is treated with an anti-PD-1 molecule. The pretreatment increases the likelihood that a patient's tumor will respond to a treatment with an anti-PD-1 molecule and/or enhances the effectiveness of the tumor's response to an anti-PD-1 molecule. The IAP antagonist may be selected among those that are already (as at Dec. 21, 2017) approved or are currently in clinical development, in particular among the following ones: Debio 1143 (Debiopharm, CAS RN: 1071992-99-8), GDC-917/CUDC-427 (Curis/Genentech, CAS RN: 1446182-94-0), LCL161 (Novartis, CAS RN: 1005342-46-0), GDC-0152 (Genentech, CAS RN: 873652-48-3), TL-32711/Birinapant (Medivir, CAS RN: 1260251-31-7), HGS-1029/AEG-408268 (Aegera, CAS RN: 1107664-44-7), BI 891065 (Boehringer Ingelheim), ASTX-660 (Astex/Otsuka, CAS RN: 1605584-14-2), APG-1387 (Ascentage, CAS RN: 1802293-83-9), or any of their pharmaceutical acceptable salts. Preferably, the IAP antagonist is a SMAC mimetic, the most preferred one being Debio 1143.

[0075] Pretreatment with an IAP antagonist may be made dependent on a finding that the patient's tumor microenvironment is poorly immunogenic. Immunogenicity may be assessed in a patient's biological sample, such as a tumor biopsy (including liquid biopsy) taken prior to pretreatment. Criteria for immunogenicity that may be employed include the level of PD-L1 expressed in the cancerous cells or in all cells present in the cancer biopsy. It may also be the percentage of tumor cells and/or immune cells expressing detectable amounts of PD-L1. The threshold for immunogenicity may be defined by the medical community, the manufacturer/distributor of the anti-PD-1 molecule to be used for analysis or the treating physician. For example, the threshold level for treatment with Pembrolizumab has been defined by the manufacturer (Merck) as more than 50% of cells of the cancer staining for PD-L1 (at any intensity) in an immunohistochemistry assay using antibody 22c3 pharmDx (Dako, Inc.) for first line therapy, and more than 1% of cells staining for PD-L1 for second line therapy. Hence, in this example, patients with cancers with lower frequencies of PD-L1-expressing cells would be considered eligible for pretreatment with an IAP antagonist. In some embodiments, pretreatment with an IAP antagonist may be carried out until the frequency of PD-L1-expressing cells and/or CD8+ cells exceeds the above-mentioned threshold levels for high immunogenicity. Additional criteria may include the percentage of lymphocytes, or CD8+ T cells, or CD4+ T cells present in the baseline biopsy or sample. Other suitable criteria of immunogenicity may gain acceptance by the medical community (e.g., number/percentage of dendritic cells, ratio of CD8+ T cells to regulatory T cells, tumor mutation burden, etc.). Eligibility may also be assessed based on multiple criteria.

[0076] Without being bound to a particular theory, an increase in the expression of the PD-L1 marker on cancer cells after the induction period is believed to be a sign that the immunogenic potency of the tumor microenvironment has been enhanced. This is because an increased immunogenic potency should be associated with an increased need to circumvent the immune system for the cancer cell to survive. Overexpression of PD-L1 is thought to be a mechanism with which the cancer cell can hide from the immune system. Thus, an increased level of PD-L1 expression is a sign that the tumor cell is being confronted with an enhanced immune system at the tumor microenvironment.

[0077] The method may also be adapted to select patients for treatment with an anti-PD-1 molecule based on the immunogenicity of their cancer microenvironment at the end of a pretreatment with an IAP antagonist. Immunogenicity may be assessed in a patient's biological sample, such as a tumor biopsy (including liquid biopsy) taken at the end of the pretreatment. Criteria for assessing immunogenicity and for defining thresholds may be similar to those that have been described in the previous section. Patients with cancers for which the selected marker of immunogenicity surpasses a predetermined threshold may be selected for treatment with an anti-PD-1 molecule.

[0078] Methods for Assessing Immunogenicity in Cancer Biopsies

[0079] In principle, any suitable method may be employed. Most often used are procedures based on immunohistochemistry and flow cytometry.

[0080] Immunohistochemistry: Immunohistochemistry (IHC) is a method capable of demonstrating the presence and location of proteins in tissue sections. It enables the observation of processes in the context of intact tissue. The basic steps of the IHC protocol are as follows: fixing and embedding the tissue, cutting and mounting the section, deparaffinizing and rehydrating the section, applying antigen retrieval process, immunohistochemical staining and viewing the staining under the microscope. In an example protocol, immunostaining was performed on 4-μm paraffin-embedded tissue sections. Briefly, slides were deparaffinized in xylene and dehydrated utilizing a graded ethanol series, and endogenous peroxidase was blocked with 3% hydrogen peroxide. After epitope retrieval, the slides were washed with and blocked with TRIS-buffered saline with 0.1% (vol.) Tween 20/5% (vol.) normal goat serum. Incubation with a primary antibody was performed overnight at 4° C. followed by incubation with a secondary antibody for 30 min at room temperature. Sections were washed three times with TRIS-buffered saline with 0.1% (vol.) Tween 20, stained with diaminobenzidine (DAB) and counterstained with hematoxylin. Guancial et al. (2014); Redler, A. et al. (2013) PLoS One. 8: e72224. Procedures may be carried out manually or may be partially or completely automated. A specific IHC method is also described in the example section below.

[0081] Flow Cytometry: Flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, involving suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Using antibody specific of protein, flow cytometry can provide information regarding the expression of cell surface and, in some cases, cytoplasmic or nuclear markers that are used to understand complex cellular populations or processes. Yan, D. et al. (2011) Arthritis Res. Ther. 13: R130.

[0082] Pharmaceutical Compositions Comprising an IAP Antagonist and their Administration

[0083] Pharmaceutical compositions comprising an IAP antagonist may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. However, it is noted that dimeric SMAC mimetics are typically administered intravenously. The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the active agent or its delivery form. Standard pharmaceutical carriers and their formulations are described, in a non-limiting fashion, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 19th ed. 1995. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0084] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to active agent (IAP antagonist, such as a SMAC mimetic), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other emulsifiers, solubilizing agents and solvents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0085] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride and dextrose solutions. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0086] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, ionizing radiation, or by incorporating active agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Depending on the chemical nature of the particular IAP antagonist employed, sterilization may also be by autoclaving or dry heat.

[0087] In order to prolong the effect of the active agent, it is often desirable to slow the absorption of the active agent from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the active agent then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the active agent in an oil vehicle. Injectable depot forms are made by microencapsulating the active agent in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of release of the active agent can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the active agent in liposomes or microemulsions that are compatible with body tissues.

[0088] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, cellulose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, croscarmellose, crospovidone, carboxymethylcellulose, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution-retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol, sodium lauryl sulfate and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and/or i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0089] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0090] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active agent only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0091] The amount of active agent that may be combined with pharmaceutically acceptable excipients or carriers to produce a single dosage form will vary depending on the particular IAP antagonist chosen, the particular mode of administration and, possibly, the subject treated. A typical preparation will contain from 1% to 95% active agent (w/w). Alternatively, such preparations may contain from 20% to 80% active agent. Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the age, body weight, body surface area, general health status, sex, diet, time of administration, rate of excretion, IAP antagonist, drug combination, the severity and course of the disease, condition or symptoms, the subject's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0092] Pharmaceutical Compositions Comprising an Anti-PD-1 Molecule and their Administration

[0093] Anti-PD-1 molecules are administered typically by intravenous infusion.

[0094] Nivolumab is being distributed under the brand “OPDIVO”. It comes as a 10 mg/ml solution that comprises the Nivolumab antibody, mannitol, pentetic acid, polysorbate 80, sodium chloride, sodium citrate dihydrate and water. For administration, it is diluted into 0.9% sodium chloride or 5% dextrose. Pembrolizumab is being distributed under the brand “KEYTRUDA”. It is furnished as a solid composition comprising 50 mg antibody and inactive ingredients L-histidine, polysorbate-80 and sucrose. For administration, the composition is suspended in 0.9% sodium chloride. Atezolizumab (brand name: “TECENTRIQ”) is provided as an IV solution (1200 mg active/20 ml) containing glacial acetic acid, histidine, sucrose and polysorbate 20. For administration, the solution is diluted with 0.9% NaCl. Durvalumab (“IMFINZI”) comes as 500 mg/10 ml or 120 mg/2.4 ml solutions in L-histidine, L-histidine hydrochloride monohydrate, α,α-trehalose dihydrate, polysorbate 80, and water for injection, USP. Avelumab (“BAVENCIO”) is marketed as a 200 mg (active)/10 ml solution for injection that contains mannitol, acetic acid, polysorbate 20, sodium hydroxide and water. After dilution in 0.45% or 0.9% NaCl, an appropriate dose is administered by infusion during 60 min.

[0095] Suitable doses of checkpoint inhibitors are those used in the clinic. A suitable dose of Nivolumab is 3 mg/kg body weight. This dose is administered by intravenous infusion during a period of 60 min. A suitable dose of Pembrolizumab is 2 mg/kg body weight. This dose is administered by intravenous infusion during a period of 30 min. The adult dose of Atezolizumab is 1200 mg infused over a period of 60 min. The recommended dose for Durvalumab is 10 mg/kg body weight administered by intravenous infusion over 60 min. A suitable dose for Avelumab is 10 mg/kg body weight. These doses may be adapted in parallel with adaptations accepted in clinical practice. Dosing of Nivolumab is typically repeated every two weeks, Pembrolizumab every three weeks, Atezolizumab every three weeks, Durvalumab every two weeks and Avelumab every two weeks.

[0096] Dose amounts and schedules (including dosing intervals) of administration of anti-PD-1 molecules will be as approved by regulatory agencies. Any modification of doses and schedules accepted by the medical community will also be applied to the presently described therapy.

[0097] In one aspect, the present invention comprises the items listed below. These items may be combined with any of the above aspects or embodiments

[0098] 1. IAP antagonist for pretreating a human subject attained with a cancer to enhance the likelihood that a subsequent treatment with an anti-PD-1 molecule results in an anti-cancer response or to enhance the responsiveness of the subject's cancer to the subsequent treatment with the anti-PD-1 molecule.

[0099] 2. IAP antagonist according to item 1, wherein the human subject is pretreated with the IAP antagonist during a pretreatment period of 1 to 28 days, preferably 5 to 28 days, said pretreatment period being followed by the initiation of said subsequent anti-PD-1 molecule treatment.

[0100] 3. IAP antagonist according to item 2, wherein said pretreatment period comprises one or more days without administration of the IAP antagonist.

[0101] 4. IAP antagonist according to any one of the preceding items, wherein said IAP antagonist or a different IAP antagonist is also administered during said subsequent treatment with the anti-PD-1 molecule.

[0102] 5. IAP antagonist according to item 4, wherein administration of said IAP antagonist is continued during the entire period of said subsequent treatment with the anti-PD-1 molecule, or is ended prior to the completion of said subsequent treatment with the anti-PD-1 molecule, or is continued beyond of the completion of said subsequent treatment with the anti-PD-1 molecule.

[0103] 6. IAP antagonist according to any one of the preceding items, wherein said cancer is of a type that is known to be responsive to treatment with an anti-PD-1 molecule in a substantial fraction of treated patients.

[0104] 7. IAP antagonist according to item 6, wherein said cancer is head & neck cancer, melanoma, urothelial cancer, non-small cell lung cancer, microsatellite instability (MSI) high tumors from agnostic primary site or kidney cancer.

[0105] 8. IAP antagonist according to any one of items 1 to 5, wherein said cancer is of a type for which a low percentage of patients (e.g. 5% or less) have been shown to respond to treatment with an anti-PD-1 molecule.

[0106] 9. IAP antagonist according to item 8, wherein said cancer is pancreas cancer, colorectal cancer, multiple myeloma, small cell lung cancer, hepatocarcinoma or ovarian cancer.

[0107] 10. IAP antagonist according to any one of the preceding items, wherein said pretreatment is conditional on an assessment that the cancer is poorly immunogenic.

[0108] 11. IAP antagonist according to item 10, wherein said assessment consists of an analysis of a marker of immunogenicity in a patient's biological sample taken prior to pretreatment and a finding that the marker's presence, expression level or derived score fails a predetermined threshold.

[0109] 12. IAP antagonist according to item 11, wherein said marker is PD-L1 expressed on cancer cells and/or immune cells.

[0110] 13. IAP antagonist according to items 11, wherein said marker is tumor-infiltrating lymphocytes or tumor mutation burden.

[0111] 14. IAP antagonist according to any of the preceding items, wherein initiation of said subsequent treatment with an anti-PD-1 molecule is conditional on an assessment that the cancer is immunogenic at the end of the pretreatment.

[0112] 15. IAP antagonist according to item 14, wherein said assessment consists of an analysis of a marker of immunogenicity in a patient's biological sample taken after the pretreatment with a IAP inhibitor and a finding that the marker's presence, expression level or derived score exceeds a predetermined threshold.

[0113] 16. IAP antagonist according to item 15, wherein said marker is PD-L1 expressed on cancer cells and/or immune cells.

[0114] 17. IAP antagonist according to item 15, wherein said marker is tumor-infiltrating lymphocytes or tumor mutation burden.

[0115] 18. IAP antagonist according to any one of items 11-13 and 15 to 17, wherein said patient's biological sample is a tumor or liquid biopsy.

[0116] 19. IAP antagonist according to any one of the preceding items, wherein said anti-PD-1 molecule is Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, PDR001, IBI-308, Cemiplimab, Camrelizumab, BGB-A317, BCD-100, JS-001, JNJ-3283, MEDI0680, AGEN-2034, TSR-042, Sym-021, PF-06801591, MGD-013, MGA-012, LZM-009, GLS-010, Genolimzumab, BI 754091, AK-104, CX-072, WBP3155, SHR-1316, PD-L1 Inhibitor millamolecule, BMS-936559, M-7824, LY-3300054, KN-035, FAZ-053, CK-301, or CA-170.

[0117] 20. IAP antagonist according to item 19, wherein the anti-PD-1 molecule is Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, PDR001, or BI 754091.

[0118] 21. IAP antagonist according to any one of items 1 to 19, wherein said anti-PD-1 molecule is an antibody against PD-1 or PD-L1.

[0119] 22. IAP antagonist according to any one of the preceding items, wherein said subsequent treatment with the anti-PD-1 molecule is combined with one or more other cancer therapies, including another immunotherapy, radiotherapy, chemotherapy, chemoradiotherapy, oncolytic viruses, anti-angiogenic therapies, targeted cancer therapies.

[0120] 23. IAP antagonist according to any one of the preceding items, wherein one or more other cancer therapies is used during said pretreatment period, to the exclusion of a treatment with an anti-PD-1 molecule.

[0121] 24. IAP antagonist according to any one of the preceding items, wherein said IAP antagonist is Debio 1143, GDC-917/CUDC-427, LCL161, GDC-0152, TL-32711/Birinapant, HGS-1029/AEG-40826, BI 891065, ASTX-660 or APG-1387.

[0122] 25. IAP antagonist according to item 24, wherein said IAP antagonist is a SMAC mimetic.

[0123] 26. IAP antagonist according to item 25, wherein said IAP antagonist is Debio 1143.

EXAMPLES

Example 1: Pre-Operative Window-of-Opportunity Study of Debio 1143 with or without Cisplatin (CDDP) in Patients with Resectable Squamous Cell Carcinoma of the Head and Neck (EUDRACT 2014-004655-31)

[0124] For this clinical trial, Debio 1143 was used under its free base and formulated with starch and filed within hard gelatin capsules.

[0125] The main objective of this clinical trial was to investigate the pharmacodynamic activity of Debio 1143, alone or in combination with cisplatin, in patients with squamous cell carcinoma of the head and neck. Among the numerous secondary objectives, potential effects on immune signaling were also examined.

[0126] The study enrolled adult patients with newly diagnosed histologically proven squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx or larynx. During a screening period of two weeks (days −14 to −1), a tumor biopsy was taken and analyzed. Treatment was from day 1 to day 15 (+/−2 days) and consisted (in one arm) of daily administration p.o. of 200 mg Debio 1143. At the end of this treatment period, a second tumor biopsy was taken and analyzed, and the patients underwent surgery.

[0127] Biopsies were analyzed by immunohistochemical methods. Staining for cIAP1 was carried out using a Dako autostainer automaton (Agilent). The EPR4673 mouse mAb (Abcam) was utilized at a 1/100 dilution, and tissue slides were exposed to the antibody for 20 min. Pretreatment of the slides was with EnVision FLEX Target Retrieval Solution, Low pH; the EnVision FLEX system (chromogen: DAB) was employed for visualization of the signal. EnVision Flex system and reagent were from Agilent. The same protocol was applied for PD-L1 staining. The E1 L3N rabbit mAb (Cell Signaling Technology) was used at a 1/500 dilution.

[0128] T cells were identified using CD3 rabbit mAb 2GV6 from Ventana Roche (provided as a ready-to-use solution). Slides were processed on a Ventana Benchmark Ultra automaton. Exposure to antibody was 20 min. Pretreatment of the slides (64 min) was with cell conditioning solution CC1 (Ventana); the Optiview system (Ventana) (chromogen: DAB) was employed for visualization of the signal. Staining of CD8 and CD4 T cells was by the same protocol. The CD8 antibody was the SP57 rabbit mAb, and the CD4 antibody was the SP35 rabbit mAb. Both antibodies were from Ventana Roche and were provided as ready-to-use solutions. The antibody selected for PD-1 detection was the NAT105 mouse mAb that was also provided as a ready-to-use solution (Cell Marque). The protocol for PD-1 detection was the same as that used for CD3 staining, except that antibody exposure and pretreatment times were each 16 min.

[0129] Data obtained from 12 evaluable patients are discussed. As can be seen in FIG. 1, treatment with Debio 1143 reduced levels of cIAP1 in the tumors of most patients (p-value of 0.045 using paired t-test), demonstrating that an effective tumor concentration of the SMAC mimetic had been reached. The treatment also resulted in substantial increases in tumor-infiltrating lymphocytes as evidenced by the findings that numbers of CD4+ and CD8+ T cells in the tumor microenvironment were elevated as a consequence of the treatment (FIG. 2). Statistical analysis of the data revealed that mean CD8+ and CD4+ T cell numbers were both increased, the increase in CD8+ T cell number being significant (p-value of 0.020 with paired t-test) (FIG. 2(B)). The percentages of immune cells expressing PD-1 or PD-L1 increased significantly in treated tumors (FIG. 3(A), p-value of 0.002 and (B), p-value of 0.004). In most tumors, the frequency of PD-L1-expressing cells was also increased (FIG. 3(C)). Overall, the data strongly suggest that treatment with Debio 1143 enhances the immunogenicity of the tumor microenvironment in the human patients.

Example 2: Animal Studies with IAP Inhibitor Debio 1143

[0130] Five groups (n=8) of adult female C57BL/6J mice (obtained from Shanghai Lingchang Bio-Technology Co.) were inoculated in the right lower flank with 1×10.sup.6 cells of the syngeneic colon carcinoma cell line MC38. When average tumor size reached about 50 mm.sup.3 (day 1), animals received either pretreatment consisting of p.o. SMAC mimetic Debio 1143 (Debiopharm) at a dose of 100 mg/kg or vehicle as indicated in Table 1. The dosing was repeated on each day for 7 days (day 1-7). On the subsequent day (day 8), animals of a vehicle-treated group and a Debio 1143-pretreated group were given i.p. 10 mg/kg of control antibody rlgG2b (Clone: LTF-2, BioXcell). Control antibody was administered twice weekly until the end of the study. Another set of two groups (vehicle- and Debio 1143-pretreated animals) received i.p. 10 mg/kg of anti-PD-L1 antibody (Mouse surrogate antibody, anti-mouse PD-L1, Clone: 10F.9G2, BioXcell). Administration was repeated twice weekly as for the control antibody. A final group of Debio 1143-pretreated animals received both anti-PD-L1 antibody as well as was continued on daily Debio 1143. Tumor volumes and body weights were assessed trice weekly. Tumor size was measured in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=0.5 a×b.sup.2 where a and b are the long and short diameters of the tumor, respectively.

[0131] The results of the experiment are shown in FIG. 4. Pretreatment with Debio 1143 alone (i.e., followed by administration of control antibody) had a modest anti-cancer effect (group 2). Treatment with PD-L1 antibody in the absence of a pretreatment with Debio 1143 essentially failed to retard tumor growth (group 3). The combination of a pretreatment with Debio 1143 followed by a treatment with PD-L1 antibody had a profound anti-cancer effect (group 4). Continuation of Debio 1143 during the treatment period appeared to provide a small additional benefit (group 5).

TABLE-US-00001 TABLE 1 Experimental Design Treatment Stage 1 (when mean TV @~50 mm.sup.3, dosing from day 1 to day 7) Stage 2 (from day 8 to study end) Dose Dosing Dose Dosing Group n Articles (mg/kg) Route Schedule Articles (mg/kg) Route Schedule 1 8 Vehicle of — p.o. QD rlgG2b 10 i.p. BIW × Debio1143 3 wks 2 8 Debio1143 100 p.o. QD rlgG2b 10 i.p. BIW × 3 wks 3 8 Vehicle of — p.o. QD anti-PD-L1 10 i.p. BIW × Debio1143 3 wks 4 8 Debio1143 100 p.o. QD anti-PD-L1 10 i.p. BIW × 3 wks 5 8 Debio1143 100 p.o. QD anti-PD-L1 10 i.p. BIW × 3 wks Debio1143 100 p.o. QD × 21 days p.o.: orally; i.p.: intraperitoneally; QD: daily; BIW: twice weekly

[0132] These animal studies provide direct evidence of the effectiveness of a pretreatment with an IAP antagonist to enhance the likelihood and/or the magnitude of an anti-tumor response to a subsequent treatment with an anti-PD-1 molecule.

Example 3: IAP Inhibitors Birinapant and LCL161 Pretreatment Enhance Efficacy of Anti-PD-L1 in the MC38 Model

[0133] 72 adult female C57BL/6J mice (obtained from Shanghai Lingchang Bio-Technology Co.) were inoculated subcutaneously at the right lower flank with 1×10.sup.6 cells of the syngeneic colon carcinoma cell line MC-38 in 0.1 ml of PBS. Tumor volumes were measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=(L×W×W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). All animals were randomly allocated to the 9 different study groups with a mean tumor size of 52 mm.sup.3 based on the “Matched distribution” randomization method (StudyDirector™ software, version 3.1.399.19) and treatments started (denoted as day 1). Dosing as well as tumor and body weight measurement were conducted in a Laminar Flow Cabinet.

[0134] On day 1, part of the animals received either a 1 week pretreatment consisting of i.p. SMAC mimetic birinapant at a dose of 30 mg/kg, or its vehicle, in a biweekly schedule as indicated in Table 2. The other part of the animals received either a 1 week pretreatment consisting of p.o. SMAC mimetic LCL161 at a dose of 75 mg/kg, or its vehicle, in a biweekly schedule as indicated in Table 2.

[0135] On day 8, vehicle or SMAC mimetic pretreated animals were then further treated until study end with either biweekly i.p. 10 mg/kg of control antibody rlgG2b (Clone: LTF-2, BioXcell), or biweekly i.p. 10 mg/kg of anti-PD-L1 antibody (Mouse surrogate antibody, anti-mouse PD-L1, Clone: 10F.9G2, BioXcell). 1 group of animals that had received 1 week of birinapant pretreatment, and 1 group of animals that had received 1 week of LCL161 pretreatment, were each continued on the respective SMAC mimetic during the period of anti-PD-L1 treatment until study end.

[0136] The results of the experiment are shown in FIG. 5 for birinapant, and FIG. 6 for LCL161.

[0137] Pretreatment with birinapant alone (i.e., followed by administration of control antibody) had a modest anti-cancer effect (group 2). Treatment with anti-PD-L1 antibody in the absence of a pretreatment with birinapant essentially failed to retard tumor growth (group 3). The combination of a pretreatment with birinapant followed by a treatment with anti-PD-L1 antibody had a singificant anti-cancer effect (group 4). Continuation of birinapant during the treatment period appeared to provide a small additional benefit (group 5).

[0138] Pretreatment with LCL161 alone (i.e., followed by administration of control antibody) had a modest anti-cancer effect (group 7). The combination of a pretreatment with LCL161 followed by a treatment with anti-PD-L1 antibody appeared to provide a small additional benefit to LCL161 pretreatment alone (group 8), whereas continuation of LCL161 during the treatment period provided a significant additional benefit (group 9).

[0139] These animal studies provide direct evidence of the effectiveness of a pretreatment with any IAP antagonist to enhance the likelihood and/or the magnitude of an anti-tumor response to a subsequent treatment with an anti-PD-L1 molecule.

TABLE-US-00002 TABLE 2 Experimental Design Treatment Stage 1 (when mean TV @~50 mm3, dosing start from day 1 to 7, one week) Dose Dosing Stage 2 (from day 8 to study end) Group N Articles (mg/kg) Route Schedule Articles Dose Dosing Schedule 1 8 Vehicle of — i.p. BIW rlgG2b 10 i.p. BIW × Birinapant 3 wks 2 8 Birinapant 30 i.p. BIW rlgG2b 10 i.p. BIW × 3 wks 3 8 Vehicle of — i.p. BIW anti-PD- 10 i.p. BIW × Birinapant L1 3 wks 4 8 Birinapant 30 i.p. BIW anti-PD- 10 i.p. BIW × L1 3 wks 5 8 Birinapant 30 i.p. BIW anti-PD- 10 i.p. BIW × L1 3 wks Birinapant 30 i.p. BIW × 3 wks 6 8 Vehicle of — p.o. BIW rlgG2b 10 i.p. BIW × LCL161 3 wks 7 8 LCL161 75 p.o. BIW rlgG2b 10 i.p. BIW × 3 wks 8 8 LCL161 75 p.o. BIW anti-PD- 10 i.p. BIW × L1 3 wks 9 8 LCL161 75 p.o. BIW anti-PD- 10 i.p. BIW × L1 3 wks LCL161 75 p.o. BIW × 3 wks p.o.: orally; i.p.: intraperitoneally; QD: daily BIW: twice weekly; wks: weeks

Example 4: 3.Debio 1143 Induction Enhances Efficacy of Anti-PD-1 in the CT26 Model

[0140] Five groups (n=8) of adult female BALB/c mice (obtained from Shanghai Lingchang Bio-Technology Co.) were inoculated in the right lower flank with 0.5×10.sup.6 cells of the syngeneic colon carcinoma cell line CT26. When average tumor size reached about 50 mm.sup.3 (day 1), animals received either pretreatment consisting of p.o. SMAC mimetic Debio 1143 (Debiopharm) at a dose of 100 mg/kg or vehicle as indicated in Table 3a. The dosing was repeated on each day for 7 days (day 1-7). As indicated in Table 3b, on the subsequent day (day 8) animals of a vehicle-treated group and a Debio 1143-pretreated group were given daily oral vehicle until study end. Another set of two groups (vehicle- and Debio 1143-pretreated animals) received biweekly i.p. 10 mg/kg of anti-PD-1 antibody (Mouse surrogate antibody, anti-mouse PD-1, Clone: RMP1-14, BioXcell). A final group of Debio 1143-pretreated animals received both anti-PD-1 antibody as well as was continued on daily Debio 1143. Tumor volumes and body weights were assessed trice weekly. Tumor size was measured in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=0.5 a×b.sup.2 where a and b are the long and short diameters of the tumor, respectively.

[0141] The results of the experiment are shown in FIG. 7. Treatment with anti-PD-1 antibody in the absence of a pretreatment with Debio 1143 essentially failed to retard tumor growth (group 2). Pretreatment with Debio 1143 alone (i.e., followed by administration of oral vehicle) had a modest anti-cancer effect (group 3). The combination of a pretreatment with Debio 1143 followed by a treatment with anti-PD-1 antibody appeared to provide a small additional benefit to Debio 1143 pretreatment alone (group 4). Continuation of Debio 1143 during the treatment period provided a significant additional benefit (group 5).

[0142] These animal studies provide direct evidence of the effectiveness of a pretreatment with an IAP antagonist to enhance the likelihood and/or the magnitude of an anti-tumor response to a subsequent treatment with an anti-PD-1 molecule.

[0143] These animal studies provide direct evidence of the effectiveness of a pretreatment with any IAP antagonist to enhance the likelihood and/or the magnitude of an anti-tumor response to a subsequent treatment with any ICI molecule, in particular anti-PD-1 molecules, or anti-PD-L1 molecules.

TABLE-US-00003 TABLE 3a Pre-Treatment plan of the subcutaneous CT26 Colon Cancer Syngeneic Model in Female BALB/c mice Dos- Dose Dosing ing Dos- Level So- Vol- ing Treat- (mg/ lution ume Dosing Freq- Group N ment kg) (μg/μL) (μL/g) route uency Schedule 1 8 Vehicle N/A N/A 10 p.o. QD Day 1-7 2 8 Vehicle N/A N/A 10 p.o. QD Day 1-7 3 8 Debio 100 10 10 p.o. QD Day 1-7 1143 4 8 Debio 100 10 10 p.o. QD Day 1-7 1143 5 8 Debio 100 10 10 p.o. QD Day 1-7 1143 p.o.: orally; i.p.: intraperitoneally; QD: daily; BIW: twice weekly; wks: weeks.

TABLE-US-00004 TABLE 3b Continued treatment plan of the subcutaneous CT26 Colon Cancer Syngeneic Model in female BALB/c mice Dose Dosing Dosing Level Solution Volume Dosing Dosing Group N Treatment (mg/kg) (μg/μL) (μL/g) route Frequency Schedule 1 8 Vehicle N/A N/A 10 p.o. QD From Day 8 2 8 Anti-PD-1 10 1 10 i.p. BIW From Day 8 3 8 Vehicle N/A N/A 10 p.o. QD From Day 8 4 8 Anti-PD-1 10 1 10 i.p. BIW From Day 8 Debio 1143 100 10 10 p.o. QD From Day 8 5 8 Anti-PD-1 10 1 10 i.p. BIW From Day 8 p.o.: orally; i.p.: intraperitoneally; QD: daily; BIW: twice weekly; wks: weeks.

[0144] Scope and Equivalence

[0145] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by “about,” where appropriate).

[0146] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated.

[0147] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents. The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of”, “consists essentially of” or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e. g., a composition described as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0148] This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

[0149] All publications and patent documents cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent document were specifically and individually indicated to be incorporated by reference.