Patent Application
20210322545
The instant application contains a Sequence listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 23, 2021, is named 51333-002001_Sequence_Listing_6_23_21_ST25.txt and is 987 bytes in size.
The death of cells by apoptosis (or programmed cell death), and other cell death pathways, is regulated by various cellular mechanisms. Inhibitor of apoptosis (IAP) proteins, such as X-linked IAP (XIAP) or cellular IAP proteins 1 and 2 (cIAP1 and 2), are regulators of programmed cell death, including (but not limited to) apoptosis pathways, e.g., in cancer cells. Other forms of cell death could include, but are not limited to, necroptosis, necrosis, pyroptosis, and immunogenic cell death. In addition, these IAPs regulate various cell signaling pathways through their ubiquitin E3 ligase activity, which may or may not be related to cell survival. Another regulator of apoptosis is the polypeptide Smac. Smac is a proapoptotic protein released from mitochondria in conjunction with cell death. Smac can bind to the IAPs, antagonizing their function. Smac mimetic compounds (SMCs) are non-endogenous proapoptotic compounds capable of carrying out one or more of the functions or activities of endogenous Smac.
The prototypical XIAP protein directly inhibits key initiator and executioner caspase proteins within apoptosis cascades. XIAP can thereby thwart the completion of apoptotic programs. Cellular IAP proteins 1 and 2 are E3 ubiquitin ligases that regulate apoptotic signaling pathways engaged by immune cytokines. The dual loss of cIAP1 and 2 can cause TNFα, TRAIL, and/or IL-1β to become toxic to, e.g., the majority of cancer cells. SMCs may inhibit XIAP, cIAP1, cIAP2, or other IAPs, and/or contribute to other proapoptotic mechanisms.
Treatment of cancer by the administration of SMCs has been proposed. However, SMCs alone may be insufficient to treat certain cancers. There exists a need for methods of treating cancer that improve the efficacy of SMC treatment in one or more types of cancer.
The present invention includes compositions and methods for the treatment of cancer by the administration of an SMC and an immunostimulatory, or immunomodulatory, agent. SMCs and agents are described herein, including, without limitation, the SMCs of Table 1 and the agents of Table 2, Table 3, and Table 4.
One aspect of the present invention is a composition including an SMC from Table 1, and one or more (e.g., two, three, four, five, or more) agents, wherein each agent is independently an immune checkpoint inhibitor (ICI) or is an agent from Table 2 or angent from Table 3 or is a STING agonist. In some embodiments, the ICI is an ICI from Table 4. The SMC and the agent(s) are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In some embodiments, the two, three, or four agents are from different categories (i.e., one agent is an ICI, one agent is from Table 2, one agent is from Table 3, and/or one agent is a STING agonist).
Another aspect of the present invention is a method for treating a patient diagnosed with cancer, the method including administering to the patient an SMC from Table 1 and one or more (e.g., two, three, four, five, or more) agents, wherein each agent is independently an ICI or is an agent from Table 2 or angent from Table 3 or is a STING agonist. In some embodiments, the ICI is an ICI from Table 4, such that the SMC and the agent are administered. In some embodiments, the two, three, or four agents are from different categories (i.e., one agent is an ICI, one agent is from Table 2, one agent is from Table 3, and/or one agent is a STING agonist). simultaneously or within 28 days of each other in amounts that together are sufficient to treat the cancer.
In some embodiments, the SMC and the agent(s) are administered within 14 days of each other, within 10 days of each other, within 5 days of each other, within 24 hours of each other, within 6 hours of each other, or simultaneously.
In particular embodiments, the SMC is a monovalent SMC, such as LCL161, SM-122, GDC-0152/RG7419, GDC-0917/CUDC-427, or SM-406/AT-406/Debio1143. In other embodiments, the SMC is a bivalent SMC, such as AEG40826/HGS1049, OICR720, TL32711/Birinapant, SM-1387/APG-1387, or SM-164.
In particular embodiments, one of the agents is a TLR agonist from Table 2. In certain embodiments, the agent is a lipopolysaccharide, peptidoglycan, or lipopeptide. In other embodiments, the agent is a CpG oligodeoxynucleotide, such as CpG-ODN 2216. In still other embodiments, the agent is imiquimod or poly(I:C).
In particular embodiments, one of the agents is a virus from Table 3. In certain embodiments, the agent is a vesicular stomatitis virus (VSV), such as VSV-M51R, VSV-MΔ51, VSV-IFNβ, or VSV-IFNβ-NIS. In other embodiments, the agent is an adenovirus, maraba vesiculovirus, reovirus, rhabdovirus, or vaccinia virus, or a variant thereof. In some embodiments, the agent is a Talimogene laherparepvec, a variant herpes simplex virus.
In particular embodiments, one of the agents is an ICI. In certain embodiments, the agent is Ipilimumab, Tremelimumab, Pembrolizumab, Nivolumab, Pidilizumab, AMP-224, AMP-514, AUNP 12, PDR001, BGB-A317, REGN2810, Avelumab, BMS-935559, Atezolizumab, Durvalumab, BMS-986016, LAG525, IMP321, MBG453, Lirilumab, or MGA271.
In some embodiments, a composition or method of the present invention includes a plurality of immunostimulatory or immunomodulatory agents, including but not limited to interferons, and/or a plurality of SMCs.
In some embodiments, a composition or method of the present invention includes one or more interferon agents, such as an interferon type 1 agent, an interferon type 2 agent, and/or an interferon type 3 agent.
In any method of the present invention, the cancer can be a cancer that is refractory to treatment by an SMC in the absence of an immunostimulatory or immunomodulatory agent. In any method of the present invention, the treatment can further include administration of a therapeutic agent including an interferon.
In any method of the present invention, the cancer can be a cancer that is selected from adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, cancer of the head and neck, hepatocellular carcinoma, intra-epithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cavity cancer, ovarian cancer, paediatric cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, cancer of the respiratory system, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and cancer of the urinary system.
The invention further includes a composition including an SMC from Table 1 and one or more (e.g., two, three, four, or more) agents described above. One of the agents may include a killed virus, an inactivated virus, or a viral vaccine, such that the SMC and the agent are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In particular embodiments, the said agent is a NRRP or a rabies vaccine. In other embodiments, the invention includes a composition including an SMC from Table 1, a first agent that primes an immune response, and a second agent that boosts the immune response, such that the SMC and the agents are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In certain embodiments, one or both of the first agent and the second agent is an oncolytic virus vaccine. In other particular embodiments, the first agent is an adenovirus carrying a tumor antigen and the second agent is a vesiculovirus, such as a Maraba-MG1 carrying the same tumor antigen as the adenovirus or a Maraba-MG1 that does not carry a tumor antigen.
“Neighboring” cell means a cell sufficiently proximal to a reference cell to directly or indirectly receive an immune, inflammatory, or proapoptotic signal from the reference cell.
“Potentiating apoptosis or cell death” means to increase the likelihood that one or more cells will apoptose or die. A treatment may potentiate cell death by increasing the likelihood that one or more treated cells will apoptose, and/or by increasing the likelihood that one or more cells neighboring a treated cell will apoptose or die.
“Endogenous Smac activity” means one or more biological functions of Smac that result in the potentiation of apoptosis, including at least the inhibition of cIAP1 and cIAP2. It is not required that the biological function occur or be possible in all cells under all conditions, only that Smac is capable of the biological function in some cells under certain naturally occurring in vivo conditions.
“Smac mimetic compound” or “SMC” means a composition of one or more components, e.g., a small molecule, compound, polypeptide, protein, or any complex thereof, capable of inhibiting cIAP1 and/or inhibiting cIAP2. Smac mimetic compounds include the compounds listed in Table 1.
To “induce an apoptotic program” means to cause a change in the proteins or protein profiles of one or more cells such that the amount, availability, or activity of one or more proteins capable of participating in an IAP-mediated apoptotic pathway is increased, or such that one or more proteins capable of participating in an IAP-mediated apoptotic pathway are primed for participation in the activity of such a pathway. Inducing an apoptotic program does not require the initiation of cell death per se: induction of a program of apoptosis in a manner that does not result in cell death may synergize with treatment with an SMC that potentiates apoptosis, leading to cell death.
“Agent” means a composition of one or more components cumulatively capable of inducing an apoptotic or inflammatory program in one or more cells of a subject, and cell death downstream of this program being inhibited by at least cIAP1 and cIAP2. An agent may be, e.g., a TLR agonist (e.g., a compound listed in Table 2), a virus (e.g., a virus listed in Table 3), such as an oncolytic virus, or an immune checkpoint inhibitor (e.g., one listed in Table 4).
“Treating cancer” means to induce the death of one or more cancer cells in a subject, or to provoke an immune response which could lead to tumor regression and block tumor spread (metastasis). Treating cancer may completely or partially abolish some or all of the signs and symptoms of cancer in a subject, decrease the severity of one or more symptoms of cancer in a subject, lessen the progression of one or more symptoms of cancer in a subject, or mediate the progression or severity of one or more subsequently developed symptoms.
“Prodrug” means a therapeutic agent that is prepared in an inactive form that may be converted to an active form within the body of a subject, e.g. within the cells of a subject, by the action of one or more enzymes, chemicals, or conditions present within the subject.
By a “low dosage” or “low concentration” is meant at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage or lowest standard recommended concentration of a particular compound formulated for a given route of administration for treatment of any human disease or condition.
By a “high dosage” is meant at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition.
“Immune checkpoint inhibitor” means a cancer treatment drug that prevents immune cells from being turned off by cancer cells by antagonistically blocking respective receptors or binding their ligands thus re-establishing the immune system's capacity to attack a tumor.
Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison (compared to method of implantation): *, p<0.05; **, p<0.01; ***, p<0.001. Numbers in parentheses represent number of mice per group.
The present invention includes methods and compositions for enhancing the efficacy of Smac mimetic compounds (SMCs) in the treatment of cancer. In particular, the present invention includes methods and compositions for combination therapies that include an SMC and a second agent that stimulates one or more cell death pathways that are inhibited by cIAP1 and/or cIAP2. The second agent may be, e.g., a TLR agonist a virus, such as an oncolytic virus, or an interferon or related agent.
The data provided herein demonstrates that treatment with an agent and an SMC results in tumor regression and durable cures in vivo (see, e.g., Example 1). These combination therapies were well tolerated by mice, with body weight returning to pre-treatment levels shortly after the cessation of therapy. Tested combination therapies were able to treat several treatment refractory, aggressive mouse models of cancer. One of skill in the art will recognize, based on the disclosure and data provided herein, that any one or more of a variety of SMCs and any one or more of a variety of agents, such as a TLR agonist, pathogen, or pathogen mimetic, may be combined in one or more embodiments of the present invention to potentiate apoptosis and treat cancer.
While other approaches to improve SMC therapy have been attempted, very rarely have complete responses been observed, particularly in aggressive immunocompetent model systems. Some embodiments of the present invention, including treatment of cancer with a pathogen mimetic, e.g., a pathogen mimetic having a mechanism of action partially dependent on TRAIL, can have certain advantages. First, this approach can evoke TNFα-mediated apoptosis and necroptosis: given the plasticity and heterogeneity of some advanced cancers, treatments that simultaneously induce multiple distinct cell death mechanisms may have greater efficacy than those that do not. Second, pathogen mimetics can elicit an integrated innate immune response that includes layers of negative feedback. These feedback mechanisms may act to temper the cytokine response in a manner difficult to replicate using recombinant proteins, and thus act as a safeguard to this combination therapy strategy.
Multiple myeloma (MM) is an incurable cancer that is characterized by rapid expansion of plasma cells in the bone marrow. MM is the second most common haematological malignancy and has a median survival of only three to five years after diagnosis. The MM cells cause bone resorption leading to fractures and immune suppression as they populate the bone marrow compartment. MM cells can disseminate to other tissues to form plasmacytomas, and the disease can have an aggressive leukemic phase. Current therapies can prolong survival and mitigate symptoms, but they are no curative treatments. New therapies are desperately needed to combat treatment resistance and inevitable relapse.
The malignant cells are reliant on the bone marrow microenvironment in early stages of the disease, specifically TNFα and interleukin-6 (IL-6) from cells within the bone marrow microenvironment. As the disease progresses, the cells become independent of their environment, surviving on high autocrine production of TNFα. Throughout all stages the cells have high levels of NF-κB signalling that enhance their survival, in part due to common mutations in key components of the pathway Targeting the NF-κB pathway in MM contributes to the increase in efficacy of many standard therapeutics used in MM, such as the proteasome inhibitor bortezomib, immunomodulatory agents (IMiDs) thalidomide and lenalidomide and the synthetic glucocorticoid dexamethasone.
TNFα-mediated NF-κB signalling can be switched from a pro-survival signal to an apoptotic signal with the removal of the cellular inhibitors of apoptosis (cIAPs); this process appears to be selective to cancer cells. cIAP1 and cIAP2 act interchangeably as E3 ligases in all members of the TNFα receptor superfamily, either ubiquitinating specific proteins to form a scaffold for signalling complexes, or targeting them for degradation. Examples of this can been seen in both arms of the NF-κB pathway: RIP1 is ubiquitinated via K63 linkages to form a scaffolding signalling complex that is required for the activation of the classical pathway whereas NIK receives a K48 linked ubiquitination targeting it for degradation, and keeping the alternative pathway inactive (
SMCs have been shown to have strong synergy with TNFα to induce NF-κB-mediated apoptosis in many cancer lines. SMCs also have synergistic cancer cell killing in combination other inflammatory cytokines such as IFNs, which can be induced by TLR agonists or oncolytic viruses. SMCs can even standardize therapeutics used for MM to enhance apoptosis of cancer cells. Several clinical trials that are currently being conducted for assessing the the efficacy of SMCs with chemotherapeutics in MM as well as other cancers have shown great therapeutic potential.
Activating the immune system increases cytokine production, which is advantageous for SMC-mediated MM cell killing. However, this cytokine production may have undesirable consequences on the MM cells. Many innate immune stimulants, such as IFNs and TLR agonists, have been shown to upregulate ligands of the immune checkpoint PD-1. PD-1 is expressed on the surface of T cells and NK cells. When PD-1 binds its ligands, PD-L1 and PD-L2, it acts as a co-inhibitory signal for the T cell receptor to supress the cytotoxic ability of T cells. PD-L1 is expressed constitutively at low levels in many tissues and can be upregulated, presumably to prevent autoimmune reactions. However, PD-L1 is upregulated on cancer cells, leading to the cells evading detection by the adaptive immune system. In particular, PD-L1 can be upregulated in MM in response to IFNγ and TLR agonists such as LPS. PD-L2 has a much more selective expression compared to PD-L1. It is present in a subset of B cells and upregulated on select cells in response to strong NF-κB or STAT6 signalling.
SMCs can also affect the function of T cells of SMC-treated mice both in vitro and in vivo, e.g., increased proliferation, increased cytokine production of activated T cells extracted from mouse spleens after exposure to SMCs, and higher cytokine production from NKT and NK cells. Additionally, mice treated with SMC exhibit hyperresponsive T cells upon antigen stimulation. Therefore a SMC-based combination therapy could not only increase the apoptosis of MM cells but may also stimulate a selective adaptive response. Combining SMCs with innate immune stimulants or immune checkpoint inhibitors (ICIs) may be the best approach to overcome the strong pro-survival signals the MM cells receive.
Cancer cells are able to manipulate many of the pro-survival strategies healthy cells utilize in order to make them resistant to death-inducing signals. MM cells specifically are able to further amplify the constitutive NF-κB signalling used in plasma cells to make them resistant to apoptotic stimuli. This is accomplished by increased expression of pro-survival NF-κB target genes such as IL-6 and TNFα.
Additionally, MM cells are able to enhance expression of checkpoint inhibitors, which are presumably used to protect cells from inflammatory and cytotoxic environments; this helps them evade detection by T cells and NK cells. Targeting both apoptotic resistance and immune evasion in MM has the potential to overcome two of the major aspects of treatment resistance in this disease.
PD-1 blockade is effective at delaying MM disease progression and improving the survival time of mice significantly as shown using the syngeneic murine MM model. Using a monoclonal antibody against PD-1 has several advantages compared to alternative approaches for immune checkpoint blockade. Firstly, it is able to block binding of both PD-I ligands, PD-L1 and PD-L2. Many cancers are able to upregulate PD-L1 in response to interferon treatment, and PD-1/PD-L1 are upregulated in MM patients after treatment. Additionally, a subset of immature B cells, called B1 cells, which secrete non-specific antibodies, have shown high expression of PD-L2. Furthermore, PD-L2 expression can increase in response to certain stimuli, such as NF-κB and STAT6 activation demonstrating the importance of examining expression levels of both ligands on MM cells. Human MM cells are able to upregulate both PD-1 ligands, making them unique in comparison to solid cancers. Although this suggests monoclonal antibody therapy targeting only PD-L1 (such as Bristol-Myers Squibb's BMS-936559/MDX-1105, Genentech's MPDL3280A, MedImmune's MED1473, and EMD Serono's avelumab) would be less effective than treatments targeting PD-1(such as Bristol-Myers Squibb's nivolumab. Merck's pembrolizumab, and Curetech's pidilizumab), it shows the value of using anti-PD-1 antibodies in MM.
Secondly, PD-1 targeted approaches have the potential to have a more robust response against the cancer in comparison to other ICIs such as anti-CTLA-4. The differences in activity may be due to the particular roles of these molecules in T cell regulation. PD-1 is often found on CD8+ T cells and engagement with its ligand inhibits the cytotoxic response activated by TCR signalling. In contrast, CTLA-4 has a more prominent role in secondary lymphoid tissues on regulatory T cells. CTLA-4 engagement with its receptor, CD28, outcompetes and even down regulates the activating ligands for CD28, and causes dampening of T cell secondary clonal expansion. It is entirely possible that the lack of efficacy of anti-CTLA-4 treatment in Example 3 indicates MM invasion into secondary lymphoid organs. This could compromise anti-CTLA4 efficacy either by the CD4+ T cell population being proportionately lower within the germinal centres or T cell infiltration to the secondary lymphoid organs being hampered. In extramedullary MM, the cells can form plasmacytomas in the spleen and lymph nodes, which is often seen in late stages of the MM mouse model discussed in Example 3. Therefore, it is evident the germinal centres are compromised by the MPC-11 cells
An SMC of the present invention may be any small molecule, compound, polypeptide, protein, or any complex thereof, capable, or predicted of being capable, of inhibiting cIAP1, cIAP2 and/or XIAP, and, optionally, one or more additional endogenous Smac activities. An SMC of the present invention is capable of potentiating apoptosis by mimicking one or more activities of endogenous Smac, including but not limited to, the inhibition of cIAP1 and the inhibition of cIAP2. An endogenous Smac activity may be, e.g., interaction with a particular protein, inhibition of a particular protein's function, or inhibition of a particular IAP. In particular embodiments, the SMC inhibits both cIAP1 and cIAP2. In some embodiments, the SMC inhibits one or more other IAPs in addition to cIAP1 and cIAP2, such as XIAP or Livin/ML-IAP, the single BIR-containing IAP. In particular embodiments, the SMC inhibits cIAP1, cIAP2, and XIAP. In any embodiment including an SMC and an immune stimulant, an SMC having particular activities may be selected for combination with one or more particular immune stimulants. In any embodiment of the present invention, the SMC may be capable of activities of which Smac is not capable. In some instances, these additional activities may contribute to the efficacy of the methods or compositions of the present invention.
Treatment with SMCs can deplete cells of cIAP1 and cIAP2, through, e.g., the induction of auto- or trans-ubiquitination and proteasomal-mediated degradation. SMCs can also de-repress XIAP's inhibition of caspases. SMCs may primarily function by targeting cIAP1 and 2, and by converting TNFα, and other cytokines or death ligands, from a survival signal to a death signal, e.g., for cancer cells.
Certain SMCs inhibit at least XIAP and the cIAPs. Such “pan-IAP” SMCs can intervene at multiple distinct yet interrelated stages of programmed cell death inhibition. This characteristic minimizes opportunities for cancers to develop resistance to treatment with a pan-IAP SMC, as multiple death pathways are affected by such an SMC, and allows synergy with existing and emerging cancer therapeutics that activate various apoptotic pathways in which SMCs can intervene.
One or more inflammatory cytokines or death ligands, such as TNFα, TRAIL, and IL-1β, potently synergize with SMC therapy in many tumor-derived cell lines. Strategies to increase death ligand concentrations in SMC-treated tumors, in particular using approaches that would limit the toxicities commonly associated with recombinant cytokine therapy, are thus very attractive. TNFα, TRAIL, and dozens of other cytokines and chemokines can be upregulated in response to pathogen recognition by the innate immune system of a subject. Importantly, this ancient response to microbial pathogens is usually self-limiting and safe for the subject, due to stringent negative regulation that limits the strength and duration of its activity.
SMCs may be rationally designed based on Smac. The ability of a compound to potentiate apoptosis by mimicking one or more functions or activities of endogenous Smac can be predicted based on similarity to endogenous Smac or known SMCs. An SMC may be a compound, polypeptide, protein, or a complex of two or more compounds, polypeptides, or proteins.
In some instances, SMCs are small molecule IAP antagonists based on an N-terminal tetrapeptide sequence (revealed after processing) of the polypeptide Smac. In some instances, an SMC is a monomer (monovalent) or dimer (bivalent). In particular instances, an SMC includes 1 or 2 moieties that mimic the tetrapeptide sequence of AVPI (SEQ ID NO: 2) from Smac/DIABLO, the second mitochondrial activator of caspases, or other similar IBMs (e.g., IAP-binding motifs from other proteins like casp9). A dimeric SMC of the present invention may be a homodimer or a heterodimer. In certain embodiments, the dimer subunits are tethered by various linkers. The linkers may be in the same defined spot of either subunit, but could also be located at different anchor points (which may be ‘aa’ position, P1, P2, P3 or P4, with sometimes a P5 group available). In various arrangements, the dimer subunits may be in different orientations, e.g., head to tail, head to head, or tail to tail. The heterodimers can include two different monomers with differing affinities for different BIR domains or different IAPs. Alternatively, a heterodimer can include a Smac monomer and a ligand for another receptor or target which is not an IAP. In some instances, an SMC can be cyclic. In some instances, an SMC can be trimeric or multimeric. A multimerized SMC can exhibit a fold increase in activity of 7,000-fold or more, such as 10-, 20-, 30-, 40-, 50-, 100-, 200-, 1,000-, 5,000-, 7,000-fold, or more (measured, e.g., by EC50 in vitro) over one or more corresponding monomers. This may occur, in some instances, e.g., because the tethering enhances the ubiquitination between IAPs or because the dual BIR binding enhances the stability of the interaction. Although multimers, such as dimers, may exhibit increased activity, monomers may be preferable in some embodiments. For example, in some instances, a low molecular weight SMC may be preferable, e.g., for reasons related to bioavailability.
In some instances of the present invention, an agent capable of inhibiting cIAP1/2 is a bestatin or Me-bestatin analog. Bestatin or Me-bestatin analogs may induce cIAP1/2 autoubiquitination, mimicking the biological activity of Smac.
In certain embodiments of the present invention, an SMC combination treatment includes one or more SMCs and one or more interferon agents, such as an interferon type 1 agent, an interferon type 2 agent, and an interferon type 3 agent. Combination treatments including an interferon agent may be useful in the treatment of cancer, such as multiple myeloma.
In some embodiments, a VSV expressing IFN, and optionally expressing a gene that enables imaging, such as NIS, the sodium-iodide symporter, is used in combination with an SMC. For instance, such a VSV may be used in combination with an SMC, such as the Ascentage Smac mimetic SM-1387/APG-1387, the Novartis Smac mimetic LCL161, or Birinapant. Such combinations may be useful in the treatment of cancer, such as hepatocellular carcinoma or liver metastases.
Various SMCs are known in the art. Non-limiting examples of SMCs are provided in Table 1. While Table 1 includes suggested mechanisms by which various SMCs may function, methods and compositions of the present invention are not limited by or to these mechanisms.
An immunostimulatory or immunomodulatory agent of the present invention may be any agent capable of inducing a receptor-mediated apoptotic program that is inhibited by cIAP1 and cIAP2 in one or more cells of a subject. An immune stimulant of the present invention may induce an apoptotic program regulated by cIAP1(BIRC2), cIAP2 (BIRC3 or API2), and optionally, one or more additional IAPs, e.g., one or more of the human IAP proteins NAIP (BIRC1), XIAP (BIRC4), survivin (BIRCS), Apollon/Bruce (BIRC6), ML-IAP (BIRC7 or livin), and ILP-2 (BIRC8). It is additionally known that various immunomodulatory or agents, such as CpGs or IAP antagonists, can change immune cell contexts.
In some instances, an immune stimulant may be a TLR agonist, such as a TLR ligand. A TLR agonist of the present invention may be an agonist of one or more of TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10 in humans or related proteins in other species (e.g., murine TLR-1 to TLR-9 and TLR-11 to TLR-13). TLRs can recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens, as well as danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells. PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipopeptides, as well as flagellin, bacterial DNA, and viral double-stranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix. Agonists of the present invention further include, for example, CpG oligodeoxynucleotides (CpG ODNs), such as Class A, B, and C CpG ODN's, base analogs, nucleic acids such as dsRNA or pathogen DNA, or pathogen or pathogen-like cells or virions. In certain embodiments, the agent is an agent that mimics a virus or bacteria or is a synthetic TLR agonist.
Various TLR agonists are known in the art. Non-limiting examples of TLR agonists are provided in Table 2. While Table 2 includes suggested mechanisms, uses, or TLR targets by which various TLR agonists may function, methods and compositions of the present invention are not limited by or to these mechanisms, uses, or targets.
In other instances, an immune stimulant may be a virus, e.g., an oncolytic virus. An oncolytic virus is a virus that selectively infects, replicates, and/or selectively kills cancer cells. Viruses of the present invention include, without limitation, adenoviruses, Herpes simplex viruses, measles viruses, Newcastle disease viruses, parvoviruses, polioviruses, reoviruses, Seneca Valley viruses, retroviruses, Vaccinia viruses, vesicular stomatitis viruses, lentiviruses, rhabdoviruses, sindvis viruses, coxsackieviruses, poxviruses, and others. In particular embodiments of the present invention, the agent is a rhabodvirus, e.g., VSV. Rhabdoviruses can replicate quickly with high IFN production. In other particular embodiments, the agent is a feral member, such as Maraba virus, with the MG1 double mutation, Farmington virus, Carajas virus. Viral agents of the present invention include mutant viruses (e.g., VSV with a Δ51 mutation in the Matrix, or M, protein), transgene-modified viruses (e.g., VSV-hIFNβ), viruses carrying -TNFα, -LTα/TNFβ, -TRAIL, FasL, -TL1α, chimeric viruses (eg rabies), or pseudotyped viruses (e.g., viruses pseudotyped with G proteins from LCMV or other viruses). In some instances, the virus of the present invention will be selected to reduce neurotoxicity. Viruses in general, and in particular oncolytic viruses, are known in the art.
In certain embodiments, the agent is a killed VSV NRRP particle or a prime-and-boost tumor vaccine. NRRPs are wild type VSV that have been modified to produce an infectious vector that can no longer replicate or spread, but that retains oncolytic and immunostimulatory properties. NRRPs may be produced using gamma irradiation, UV, or busulfan. Particular combination therapies include prime-and-boost with adeno-MAGE3 (melanoma antigen) and/or Maraba-MG1-MAGE3. Other particular combination therapies include UV-killed or gamma irradiation-killed wild-type VSV NRRPs. NRRPs may demonstrate low or absent neurotixicity. NRRPs may be useful, e.g., in the treatment of glioma, hematological (liquid) tumors, or multiple myeloma.
In some instances, the agent of the present invention is a vaccine strain, attenuated virus or microorganism, or killed virus or microorganism. In some instances, the agent may be, e.g., BCG, live or dead Rabies vaccines, or an influenza vaccine.
Non-limiting examples of viruses of the present invention, e.g., oncolytic viruses, are provided in Table 3. While Table 3 includes suggested mechanisms or uses for the provided viruses, methods and compositions of the present invention are not limited by or to these mechanisms or uses.
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Cancer J. 2012 January-February; 18(1): 69-81; Chiocca E A, Abbed K M,
Cancer J. 2012 January-February; 18(1): 69-81
The methods and compositions of the present invention may be used to treat a wide variety of cancer types. One of skill in the art will appreciate that, since cells of many if not all cancers are capable of receptor-mediated apoptosis, the methods and compositions of the present invention are broadly applicable to many if not all cancers. The combinatorial approach of the present invention is efficacious in various aggressive, treatment refractory tumor models. In particular embodiments, for example, the cancer treated by a method of the present invention may be adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and other central nervous system (CNS) cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, cancer of the head and neck, hepatocellular carcinoma, intra-epithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphomas including Hodgkin's and non-Hodgkin's lymphomas, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cavity cancer (e.g. lip, tongue, mouth, and pharynx), ovarian cancer, paediatric cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenomathymoma, prostate cancer, renal cell carcinoma, cancer of the respiratory system, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, cancer of the urinary system, and other carcinomas and sarcomas. Other cancers are known in the art.
The cancer may be a cancer that is refractory to treatment by SMCs alone. The methods and compositions of the present invention may be particularly useful in cancers that are refractory to treatment by SMCs alone. Typically, a cancer refractory to treatment with SMCs alone may be a cancer in which IAP-mediated apoptotic pathways are not significantly induced. In particular embodiments, a cancer of the present invention is a cancer in which one or more apoptotic pathways are not significantly induced, i.e., is not activated in a manner such that treatment with SMCs alone is sufficient to effectively treat the cancer. For instance, a cancer of the present invention can be a cancer in which a cIAP1/2-mediated apoptotic pathway is not significantly induced.
A cancer of the present invention may be a cancer refractory to treatment by one or more agents. In particular embodiments, a cancer of the present invention may be a cancer refractory to treatment by one or more agents (absent an SMC) and also refractory to treatment by one or more SMCs (absent an agent).
In some instances, delivery of a naked, i.e. native form, of an SMC and/or agent may be sufficient to potentiate apoptosis and/or treat cancer. SMCs and/or agents may be administered in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitably pharmacologically effective, e.g., capable of potentiating apoptosis and/or treating cancer.
Salts, esters, amides, prodrugs and other derivatives of an SMC or agent can be prepared using standard procedures known in the art of synthetic organic chemistry. For example, an acid salt of SMCs and/or agents may be prepared from a free base form of the SMC or agent using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the SMC or agent is dissolved in a polar organic solvent, such as methanol or ethanol, and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to, both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain typical acid addition salts of SMCs and/or agents, for example, halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of SMCs and/or agents of the present invention may be prepared in a similar manner using a pharmaceutically acceptable base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Certain typical basic salts include, but are not limited to, alkali metal salts, e.g., sodium salt, and copper salts.
Preparation of esters may involve functionalization of, e.g., hydroxyl and/or carboxyl groups that are present within the molecular structure of SMCs and/or agents. In certain embodiments, the esters are acyl-substituted derivatives of free alcohol groups, i.e., moieties derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters may be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.
Amides may also be prepared using techniques known in the art. For example, an amide may be prepared from an ester using suitable amine reactants or prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.
An SMC or agent of the present invention may be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, e.g., to stabilize the composition, increase or decrease the absorption of the SMC or agent, or improve penetration of the blood brain barrier (where appropriate). Physiologically acceptable compounds may include, e.g., carbohydrates (e.g., glucose, sucrose, or dextrans), antioxidants (e.g. ascorbic acid or glutathione), chelating agents, low molecular weight proteins, protection and uptake enhancers (e.g., lipids), compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds, particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, suspending agents, and the like. In certain embodiments, a pharmaceutical formulation may enhance delivery or efficacy of an SMC or agent.
In various embodiments, an SMC or agent of the present invention may be prepared for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration. Administration may occur, for example, transdermally, prophylactically, or by aerosol.
A pharmaceutical composition of the present invention may be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, and lipid complexes.
In certain embodiments, an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrator (e.g. calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone, etc.), a binder (e.g. alpha-starch, gum arabic, microcrystalline cellulose, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), or an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.) may be added to an SMC or agent and the resulting composition may be compressed to manufacture an oral dosage form (e.g., a tablet). In particular embodiments, a compressed product may be coated, e.g., to mask the taste of the compressed product, to promote enteric dissolution of the compressed product, or to promote sustained release of the SMC or agent. Suitable coating materials include, but are not limited to, ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).
Other physiologically acceptable compounds that may be included in a pharmaceutical composition including an SMC or agent may include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound, depends, e.g., on the route of administration of the SMC or agent and on the particular physio-chemical characteristics of the SMC or agent.
In certain embodiments, one or more excipients for use in a pharmaceutical composition including an SMC or agent may be sterile and/or substantially free of undesirable matter. Such compositions may be sterilized by conventional techniques known in the art. For various oral dosage form excipients, such as tablets and capsules, sterility is not required. Standards are known in the art, e.g., the USP/NF standard.
An SMC or agent pharmaceutical composition of the present invention may be administered in a single or in multiple administrations depending on the dosage, the required frequency of administration, and the known or anticipated tolerance of the subject for the pharmaceutical composition with respect to dosages and frequency of administration. In various embodiments, the composition may provide a sufficient quantity of an SMC or agent of the present invention to effectively treat cancer.
The amount and/or concentration of an SMC or agent to be administered to a subject may vary widely, and will typically be selected primarily based on activity of the SMC or agent and the characteristics of the subject, e.g., species and body weight, as well as the particular mode of administration and the needs of the subject, e.g., with respect to a type of cancer. Dosages may be varied to optimize a therapeutic and/or prophylactic regimen in a particular subject or group of subjects.
In certain embodiments, an SMC or agent of the present invention is administered to the oral cavity, e.g., by the use of a lozenge, aersol spray, mouthwash, coated swab, or other mechanism known in the art.
In certain embodiments, an SMC or agent of the present invention is administered using a slow-release solid wafer inserted in the brain cavity left upon tumor resection at the time of surgery. The wafer may be a biodegradable polyanhydride wafer containing an SMC or poly(I:C). The number of wafers placed may depend on the size of the resection cavity following surgical excision of the primary brain tumor. Delivery of drug from a slow-release wafer directly to brain tissue bypasses the problem of delivering systemic treatment across the blood-brain barrier. The polymer matrix may be comprised of a copolymer of 1,3-bis-(p-carboxyphenoxy) propane and sebacic acid (PCPP-SA; 80:20 molar ratio) that is dissolved in an organic solvent with drug, spraydried into microparticles ranging from 1-20 μm, and compression molded into wafers. In certain embodiments, the rigid wafers degrade in a two-step process wherein water penetration hydrolyzes the anyhydride bonds during the first 10 hours followed by erosion of the copolymer into the surrounding aqueous environment.
In certain embodiments, an SMC or agent of the present invention may be administered systemically (e.g., orally or as an injectable) in accordance with standard methods known in the art. In certain embodiments, the SMC or agent may be delivered through the skin using a transdermal drug delivery systems, i.e., transdermal “patches,” wherein the SMCs or agents are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer or reservoir underlying an upper backing layer. The reservoir of a transdermal patch includes a quantity of an SMC or agent that is ultimately available for delivery to the surface of the skin. Thus, the reservoir may include, e.g., an SMC or agent of the present invention in an adhesive on a backing layer of the patch or in any of a variety of different matrix formulations known in the art. The patch may contain a single reservoir or multiple reservoirs.
In particular transdermal patch embodiments, a reservoir may comprise a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes. Alternatively, the SMC and/or agent-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, a liquid or hydrogel reservoir, or another form of reservoir known in the art. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the patch and provides the device with a substantial portion of flexibility. The material selected for the backing layer is preferably substantially impermeable to the SMC and/or agent and to any other materials that are present.
Additional formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams including an SMC or agent are typically viscous liquids or semisolid emulsions, e.g. oil-in-water or water-in-oil emulsions. Cream bases are typically water-washable and include an oil phase, an emulsifier, and an aqueous phase. The oil phase, also sometimes called the “internal” phase, of a cream base is generally comprised of petrolatum and a fatty alcohol, e.g., cetyl alcohol or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant. The specific ointment or cream base to be used may be selected to provide for optimum drug delivery according to the art. As with other carriers or vehicles, an ointment base may be inert, stable, non-irritating, and non-sensitizing.
Various buccal and sublingual formulations are also contemplated.
In certain embodiments, administration of an SMC or agent of the present invention may be parenteral. Parenteral administration may include intraspinal, epidural, intrathecal, subcutaneous, or intravenous administration. Means of parenteral administration are known in the art. In particular embodiments, parenteral administration may include a subcutaneously implanted device.
In certain embodiments, it may be desirable to deliver an SMC or agent to the brain. In embodiments including system administration, this could require that the SMC or agent cross the blood brain barrier. In various embodiments this may be facilitated by co-administering an SMC or agent with carrier molecules, such as cationic dendrimers or arginine-rich peptides, which may carry an SMC or agent over the blood brain barrier.
In certain embodiments, an SMC or agent may be delivered directly to the brain by administration through the implantation of a biocompatible release system (e.g., a reservoir), by direct administration through an implanted cannula, by administration through an implanted or partially implanted drug pump, or mechanisms of similar function known the art. In certain embodiments, an SMC or agent may be systemically administered (e.g., injected into a vein). In certain embodiments, it is expected that the SMC or agent will be transported across the blood brain barrier without the use of additional compounds included in a pharmaceutical composition to enhance transport across the blood brain barrier.
In certain embodiments, one or more an SMCs or agents of the present invention may be provided as a concentrate, e.g., in a storage container or soluble capsule ready for dilution or addition to a volume of water, alcohol, hydrogen peroxide, or other diluent. A concentrate of the present invention may be provided in a particular amount of an SMC or agent and/or a particular total volume. The concentrate may be formulated for dilution in a particular volume of diluents prior to administration.
An SMC or agent may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. The compound may also be administered topically in the form of foams, lotions, drops, creams, ointments, emollients, or gels. Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer, such as ethanol, can be applied. Other suitable formulations and modes of administration are known or may be derived from the art.
An SMC or agent of the present invention may be administered to a mammal in need thereof, such as a mammal diagnosed as having cancer. An SMC or agent of the present invention may be administered to potentiate apoptosis and/or treat cancer.
A therapeutically effective dose of a pharmaceutical composition of the present invention may depend upon the age of the subject, the gender of the subject, the species of the subject, the particular pathology, the severity of the symptoms, and the general state of the subject's health.
The present invention includes compositions and methods for the treatment of a human subject, such as a human subject having been diagnosed with cancer. In addition, a pharmaceutical composition of the present invention may be suitable for administration to an animal, e.g., for veterinary use. Certain embodiments of the present invention may include administration of a pharmaceutical composition of the present invention to non-human organisms, e.g., a non-human primates, canine, equine, feline, porcine, ungulate, or lagomorphs organism or other vertebrate species.
Therapy according to the invention may be performed alone or in conjunction with another therapy, e.g., another cancer therapy, and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment optionally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed or it may begin on an outpatient basis. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the subject, the stage and type of the subject's disease, and how the patient responds to the treatment.
In certain embodiments, the combination of therapy of the present invention further includes treatment with a recombinant interferon, such as IFN-α, IFN-β, IFN-γ, pegylated IFN, or liposomal interferon. In some embodiments, the combination of therapy of the present invention further includes treatment with recombinant TNF-α, e.g., for isolated-limb perfusion. In particular embodiments, the combination therapy of the present invention further includes treatment with one or more of a TNF-α or IFN-inducing compound, such as DMXAA, Ribavirin, or the like. Additional cancer immunotherapies that may be used in combination with present invention include antibodies, e.g., monoclonal antibodies, targeting CTLA-4, PD-1, PD-L1, PD-L2, or other checkpoint inhibitors. Cyclic dinucleotides (CDNs) [cyclic di-GMP (guanosine 5′-monophosphate) (CDG), cyclic di-AMP (adenosine 5′-monophosphate) (CDA), and cyclic GMP-AMP (cGAMP)] are a class of pathogen-associated molecular pattern molecules (PAMPs) that activate the TBK1/interferon regulatory factor 3 (IRF3)/type 1 interferon (IFN) signaling axis via the cytoplasmic pattern recognition receptor stimulator of interferon genes (STING). In certain embodiments, STING agonists can be combined with an SMC to treat cancer.
Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, nasal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, or oral administration). As used herein, “systemic administration” refers to all nondermal routes of administration, and specifically excludes topical and transdermal routes of administration.
In any of the above embodiments, the route of administration may be optimized based on the characteristics of the SMC or agent. In some instances, the SMC or agent is a small molecule or compound. In other instances, the SMC or agent is a nucleic acid. In still other instances, the agent may be a cell or virus. In any of these or other embodiments, appropriate formulations and routes of administration will be selected in accordance with the art.
In the embodiments of the present invention, an SMC and an agent are administered to a subject in need thereof, e.g., a subject having cancer. In some instances, the SMC and agent will be administered simultaneously. In some embodiments, the SMC and agent may be present in a single therapeutic dosage form. In other embodiments, the SMC and agent may be administered separately to the subject in need thereof. When administered separately, the SMC and agent may be administered simultaneously or at different times. In some instances, a subject will receive a single dosage of an SMC and a single dosage of an agent. In certain embodiments, one or more of the SMC and agent will be administered to a subject in two or more doses. In certain embodiments, the frequency of administration of an SMC and the frequency of administration of an agent are non-identical, i.e., the SMC is administered at a first frequence and the agent is administered at a second frequency.
In some embodiments, an SMC is administered within one week of the administration of an agent. In particular embodiments, an SMC is administered within 3 days (72 hours) of the administration of an agent. In still more particular embodiments, an SMC is administered within 1 day (24 hours) of the administration of an agent.
In particular embodiments of any of the methods of the present invention, the SMC and agent are administered within 28 days of each other or less, e.g., within 14 days of each other. In certain embodiments of any of the methods of the present invention, the SMC and agent are administered, e.g., simultaneously or within 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 4 days, 8 days, 10 days, 12 days, 16 days, 20 days, 24 days, or 28 days of each other. In any of these embodiments, the first administration of an SMC of the present invention may precede the first administration of an agent of the present invention. Alternatively, in any of these embodiments, the first administration of an SMC of the present invention may follow the first administration of an agent of the present invention. Because an SMC and/or agent of the present invention may be administered to a subject in two more doses, and because, in such instances, doses of the SMC and agent of the present invention may be administered at different frequencies, it is not required that the period of time between the administration of an SMC and the administration of an agent remain constant within a given course of treatment or for a given subject.
One or both of the SMC and the agent may be administered in a low dosage or in a high dosage. In embodiments in which the SMC and agent are formulated separately, the pharmacokinetic profiles for each agent can be suitably matched to the formulation, dosage, and route of administration, etc. In some instances, the SMC is administered at a standard or high dosage and the agent is administered at a low dosage. In some instances, the SMC is administered at a low dosage and the agent is administered at a standard or high dosage. In some instances, both of the SMC and the agent are administered at a standard or high dosage. In some instances, both of the SMC and the agent are administered at a low dosage.
The dosage and frequency of administration of each component of the combination can be controlled independently. For example, one component may be administered three times per day, while the second component may be administered once per day or one component may be administered once per week, while the second component may be administered once per two weeks. Combination therapy may be given in on-and-off cycles that include rest periods so that the subject's body has a chance to recover from effects of treatment.
In general, kits of the invention contain one or more SMCs and one or more agents. These can be provided in the kit as separate compositions, or combined into a single composition as described above. The kits of the invention can also contain instructions for the administration of one or more SMCs and one or more agents.
Kits of the invention can also contain instructions for administering an additional pharmacologically acceptable substance, such as an agent known to treat cancer that is not an SMC or agent of the present invention.
The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, ointments, foams etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dosage regimen or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.
The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the disease (e.g., a type of cancer) to be treated, the severity of the disease, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect the dosage regimen or other aspects of administration.
Smac mimetic compounds are a class of apoptosis sensitizing drugs that have proven safe in cancer patient Phase I trials. Stimulating an innate anti-pathogen response may generate a potent yet safe inflammatory “cytokine storm” that would trigger death of tumors treated with Smac mimetics. The present example demonstrates that activation of innate immune responses via oncolytic viruses and adjuvants, such as poly(I:C) and CpG, induces bystander death of cancer cells treated with Smac mimetics in a manner mediated by IFNβ, TNFα or TRAIL. This therapeutic strategy may lead to durable cures, e.g., in several aggressive mouse models of cancer. With these and other innate immune stimulants having demonstrated safety in human clinical trials, the data provided herein points strongly towards their combined use with Smac mimetics for treating cancer.
The present example examines whether stimulating the innate immune system using pathogen mimetics would be a safe and effective strategy to generate a cytokine milieu necessary to initiate apoptosis in tumors treated with an SMC. We report here that non-pathogenic oncolytic viruses, as well as mimetics of microbial RNA or DNA, such as poly (I:C) and CpG, induce bystander killing of cancer cells treated with an SMC that is dependent either upon IFNβ, TNFα, or TRAIL production. Importantly, this therapeutic strategy was tolerable in vivo and led to durable cures in several aggressive mouse models of cancer.
Oncolytic viruses (OVs) are emerging biotherapies for cancer currently in phase I-III clinical evaluation. One barrier to OV therapy may be the induction of type I IFN- and NFκB-responsive cytokines by the host, which orchestrate an antiviral state in tumors. It was examined whether we could harness those innate immune cytokines to induce apoptosis in cancer cells pretreated with an SMC. To begin, a small panel of tumor-derived and normal cell lines (n=30) was screened for responsiveness to the SMC LCL161 and the oncolytic rhabdovirus VSVΔ51. We chose LCL161 because this compound is the most clinically advanced drug in the SMC class, and VSVΔ51 because it is known to induce a robust antiviral cytokine response. In 15 of the 28 cancer cell lines tested (54%), SMC treatment enhanced sensitivity the EC50 of VSVΔ51 by 10-10,000 fold (
To determine if VSVΔ51 elicits bystander cell death in IAP-depleted neighbouring cells not infected by the virus, cells were treated with SMCs prior to infection with a low dose of VSVΔ51 (MOI=0.01 infectious particles per cell). We assessed whether conditioned media derived from cells infected with VSVΔ51 (which was subsequently inactivated by UV light) could induce death when transferred to a plate of virus naïve cancer cells treated with an SMC. The conditioned media induced cell death only when the cells were co-treated with an SMC (
The cellular innate immune response to an RNA virus infection in mammalian tumor cells can be initiated by members of a family of cytosolic (RIG-I-like receptors, RLRs) and endosomal (toll-like receptors, TLRs) viral RNA sensors. Once triggered, these receptors can seed parallel IFN-response factor (IRF) 3/7 and nuclear-factor kappa B (NF-κB) cell signalling cascades. These signals can culminate in the production of IFNs and their responsive genes as well as an array of inflammatory chemokines and cytokines. This prompts neighboring cells to preemptively express an armament of antiviral genes and also aids in the recruitment and activation of cells within the innate and adaptive immune systems to ultimately clear the virus infection. The cIAP proteins have recently been implicated in numerous signalling pathways downstream of pathogen recognition, including those emanating from RLRs and TLRs. Accordingly, it was examined whether SMC therapy alters the antiviral response to oncolytic VSV infection in tumor cells and in mice. To begin, the effect of SMC therapy on VSVΔ51 productivity and spread was evaluated. Single-step and multi-step growth curves of VSVΔ51 productivity revealed that SMC treatment does not affect the growth kinetics of VSVΔ51 in EMT6 or SNB75 cells in vitro (
To probe deeper, IFNβ production was measured in EMT6 and SNB75 cells treated with VSVΔ51 and SMCs. This experiment revealed that the SMC treated cancer cells respond to VSVΔ51 by secreting IFNβ (
SMCs sensitize a number of cancer cell lines towards caspase 8-dependant apoptosis induced by TNFα, TRAIL, and IL-1β. As RNA viruses can trigger the production of these cytokines as part of the cellular antiviral response, the involvement of cytokine signaling in SMC and OV induced cell death was investigated. To start, the TNF receptor (TNF-R1) and/or the TRAIL receptor (DR5) were silenced and synergy between SMC and VSVΔ51 was assayed. This experiment revealed that TNFα and TRAIL are not only involved, but collectively are indispensable for bystander cell death (
Next, the type I IFN receptor (IFNAR1) was silenced and it was found, unexpectedly, that IFNAR1 knockdown prevented the synergy between SMC therapy and oncolytic VSV (
To explore the non-canonical induction of TNFα further, the mRNA expression levels of TRAIL and TNFα in SNB75 cells treated with recombinant IFNβ were measured. Both cytokines were induced by IFNβ treatment (
To evaluate SMC and oncolytic VSV co-therapy in vivo, the EMT6 mammary carcinoma was used as a syngeneic, orthotopic model. Preliminary safety and pharmacodynamic experiments revealed that a dose of 50 mg/kg LCL161 delivered by oral gavage was well tolerated and induced cIAP1/2 knockdown in tumors for at least 24 hrs, and up to 48-72 hours in some cases (
To confirm these in vivo data in another model system, the human HT-29 colorectal adenocarcinoma xenograft model was tested in nude (athymic) mice. HT-29 is a cell line that is highly responsive to bystander killing by SMC and VSVΔ51 co-treatment in vitro (
It was next determined whether oncolytic VSV infection coupled with SMC treatment leads to TNFα- or IFNβ-mediated cell death in vivo. It was investigated whether blocking TNFα signalling via neutralizing antibodies would affect SMC and VSVΔ51 synergy in the EMT6 tumor model. Compared to isotype matched antibody controls, the application of TNFα neutralizing antibodies reverted the tumor regression and decreased the survival rate to values close to the control and single treatment groups (
To investigate the role of IFNβ signaling in the SMC and OV combination paradigm, Balb/c mice bearing EMT6 tumors were treated with IFNAR1 blocking antibodies. Mice treated with the IFNAR1 blocking antibody succumbed to viremia within 24-48 hours post infection. Prior to death, tumors were collected at 18-20 hours after virus infection, and the tumors were analyzed for caspase activity. Even though these animals with defective type I IFN signaling were ill due to a large viral burden, the excised tumors did not demonstrate signs of caspase-8 activity and only showed minimal signs of caspase-3 activity (
To assess the contribution of innate immune cells or other immune mediators to the efficacy of OV/SMC combination therapy, treating EMT6 tumors was first attempted in immunodeficient NOD-scid or NSG (NOD-scid-IL2Rgammanull) mice. However, similar to the IFNAR1 depletion signaling studies, these mice also died rapidly due to viremia. Therefore, the contribution of innate immune cells was addressed by employing an ex vivo splenocyte culture system as a surrogate model. Innate immune populations that have the capacity to produce TNFα were positively selected and further sorted from naïve splenocytes. Macrophages (CD11b+ F4/80+), neutrophils (CD11b+ Gr1+), NK cells (CD11b− CD49b+) and myeloid-negative (lymphoid) population (CD11b− CD49−) were stimulated with VSVΔ51, and the conditioned medium was transferred to EMT6 cells to measure cytotoxicity in the presence of SMC. These results show that VSVΔ51-stimulated macrophages and neutrophils, but not NK cells, are capable of producing factors that lead to cancer cell death in the presence of SMCs (
It was next investigated whether synthetic TLR agonists, which are known to induce an innate proinflammatory response, would synergize with SMC therapy. EMT6 cells were co-cultured with mouse splenocytes in a transwell insert system, and the splenocytes were treated with SMC and agonists of TLR 3, 4, 7 or 9. All of the tested TLR agonists were found to induce the bystander death of SMC treated EMT6 cells (
The use of current cancer immunotherapies, such as BCG (Bacillus Calmette-Guerin), recombinant interferon (e.g. IFNα), and recombinant Tumor Necrosis Factor (e.g. TNFα used in isolated limb perfusion for example), and the recent clinical use of biologics (e.g. blocking antibodies) to immune checkpoint inhibitors that overcome tumor-mediated suppression of the immune system (such as anti-CTLA-4 and anti-PD-1 or PDL-1 monoclonal antibodies) highlight the potential of ‘cancer immunotherapy’ as an effective treatment modality. As shown in Example 1, we have demonstrated the robust potential of non-viral immune stimulants to synergize with SMCs (
Our success in finding synergy between SMCs and live or inactivated single-stranded RNA oncolytic rhabdoviruses (e.g., VSVΔ51, Maraba-MG1, and NRRPs) suggested that a clinic approved attenuated vaccine may be able to synergize with SMCs. To test this possibility, we assessed the ability to synergize with SMCs of the cancer biologic, the vaccine for tuberculosis mycobacterium, BCG, which is typically used to treat bladder cancer in situ due to the high local production of TNFα. Indeed, the combination of SMC and BCG potently synergises to kill EMT6 cells in vitro (
Type I IFN Synergizes with SMCs In Vivo
The effects of viruses, and likely other TLR agonists and vaccines, appear to be mediated, in part, by type I IFN production, which is controlled by various signaling mechanism, including mRNA translation. Our findings raised the distinct possibility of combining SMC treatment with existing immunotherapies, such as recombinant IFN, as an effective approach to treat cancer. To explore the potential of this combination, we conducted two treatment regimens of SMC and either intraperitoneal or intratumoral injections of recombinant IFNα in the syngeneic orthotopic EMT6 mammary carcinoma model. While treatment of IFNα had no effect on EMT6 tumor growth or overall survival, SMC treatment slightly extended mouse survival and had a cure rate of 17% (
Assessment of Additional Oncolytic Rhabdoviruses for the Potential of Synergy with SMCs
While VSVΔ51 is a preclinical candidate, the oncolytic rhabdoviruses VSV-IFNβ and Maraba-MG1 are currently undergoing clinical testing in cancer patients. As shown in Example 1, we have demonstrated that Maraba-MG1 synergizes with SMCs in vitro (
As shown in Example 1, we documented that a form of VSVΔ51 that was engineered to express full-length TNFα can enhance oncolytic virus induced death in the presence of SMC (
The combination of SMCs with immune stimulatory agents is applicable to many different types of cancer, including brain malignancies for which effective therapies are lacking and for which immunotherapies hold promise. As a first step, we determined whether SMCs can cross the blood-brain-barrier (BBB) in a mouse model of brain tumors, as the BBB is a significant barrier to drug entry into the brain. We observed the SMC-induced degradation of cIAP1/2 proteins in intracranial CT-2A tumors several hours after drug administration, indicative that SMCs are capable of crossing the BBB to antagonize cIAP1/2 and potentially XIAP within brain tumors (
Novartis provided LCL161 (Houghton, P. J. et al. Initial testing (stage 1) of LCL161, a SMAC mimetic, by the Pediatric Preclinical Testing Program. Pediatr Blood Cancer 58: 636-639 (2012); Chen, K. F. et al. Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells. Biochemical Pharmacology 84: 268-277 (2012)). SM-122 and SM-164 were provided by Dr. Shaomeng Wang (University of Michigan, USA) (Sun, H. et al. Design, synthesis, and characterization of a potent, nonpeptide, cellpermeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP. J Am Chem Soc 129: 15279-15294 (2007)). AEG40730 (Bertrand, M. J. et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell 30: 689-700 (2008)) was synthesized by Vibrant Pharma Inc (Brantford, Canada). OICR720 was synthesized by the Ontario Institute for Cancer Research (Toronto, Canada) (Enwere, E. K. et al. TWEAK and cIAP1 regulate myoblast fusion through the noncanonical NF-kappaB signalling pathway. Sci Signal 5: ra75 (2013)). IFNα, IFNβ, IL28 and IL29 were obtained from PBL Interferonsource (Piscataway, USA). All siRNAs were obtained from Dharmacon (Ottawa, Canada; ON TARGETplus SMARTpool). CpG-ODN 2216 was synthesized by IDT (5′-gggGGACGATCGTCgggggg-3′ (SEQ ID NO: 1), lowercase indicates phosphorothioate linkages between these nucleotides, while italics identify three CpG motifs with phosphodiester linkages). Imiquimod was purchased from BioVision Inc. (Milpitas, USA). poly(I:C) was obtained from InvivoGen (San Diego, USA). LPS was from Sigma (Oakville, Canada).
Cells were maintained at 37° C. and 5% CO2 in DMEM media supplemented with 10% heat inactivated fetal calf serum, penicillin, streptomycin, and 1% non-essential amino acids (Invitrogen, Burlington, USA). All of the cell lines were obtained from ATCC, with the following exceptions: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Cell lines were regularly tested for mycoplasma contamination. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) or DharmaFECT I (Dharmacon) for 48 hours as per the manufacturer's protocol.
The Indiana serotype of VSVΔ51 (Stojdl, D. F. et al. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4(4), 263-275 (2003)) was used in this study and was propagated in Vero cells. VSVΔ51-GFP is a recombinant derivative of VSVΔ51 expressing jellyfish green fluorescent protein. VSVΔ51-Fluc expresses firefly luciferase. VSVΔ51 with the deletion of the gene encoding for glycoprotein (VSVΔ51AG) was propagated in HEK293T cells that were transfected with pMD2-G using Lipofectamine2000 (Invitrogen). To generate the VSVΔ51-TNFα construct, full-length human TNFα gene was inserted between the G and L viral genes. All VSVΔ51 viruses were purified on a sucrose cushion. Maraba-MG1, VVDD-B18R-, Reovirus and HSV1 ICP34.5 were generated as previously described (Brun, J. et al. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol Ther 18, 1440-1449 (2010); Le Boeuf, F. et al. Synergistic interaction between oncolytic viruses augments tumor killing. Mol Ther 18, 888-895 (2011); Lun, X. et al. Efficacy and safety/toxicity study of recombinant vaccinia virus JX-594 in two immunocompetent animal models of glioma. Mol Ther 18, 1927-1936 (2010)). Generation of adenoviral vectors expressing GFP or co-expressing GFP and dominant negative IKKβ was as previously described16.
Cell lines were seeded in 96-well plates and incubated overnight. Cells were treated with vehicle (0.05% DMSO) or 5 μM LCL161 and infected with the indicated MOI of OV or treated with 250 U/mL IFNβ, 500 U/mL IFNα, 500 U/mL IFNγ, 10 ng/mL IL28, or 10 ng/mL IL29 for 48 hours. Cell viability was determined by Alamar blue (Resazurin sodium salt (Sigma)) and data was normalized to vehicle treatment. The chosen sample size is consistent with previous reports that used similar analyses for viability assays. For combination indices, cells were seeded overnight, treated with serial dilutions of a fixed combination mixture of VSVΔ51 and LCL161 (5000:1, 1000:1 and 400:1 ratios of PFU VSVΔ51: μM LCL161) for 48 hours and cell viability was assessed by Alamar blue. Combination indices (CI) were calculated according to the method of Chou and Talalay using Calcusyn (Chou, T. C. & Talaly, P. A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 252, 6438-6442 (1977)). An n=3 of biological replicates was used to determine statistical measures (mean with standard deviation or standard error).
A confluent monolayer of 786-0 cells was overlaid with 0.7% agarose in complete media. A small hole was made with a pipette in the agarose overlay in the middle of the well where 5×103 PFU of VSVΔ51-GFP was administered. Media containing vehicle or 5 μM LCL161 was added on top of the overlay, cells were incubated for 4 days, fluorescent images were acquired, and cells were stained with crystal violet.
Splenocyte Co-Culture
EMT6 cells were cultured in multiwell plates and overlaid with cell culture inserts containing unfractionated splenocytes. Briefly, single-cell suspensions were obtained by passing mouse spleens through 70 μm nylon mesh and red blood cells were lysed with ACK lysis buffer. Splenocytes were treated for 24 hr with either 0.1 MOI of VSVΔ51ΔG, 1 μg/mL poly(I:C), 1 μg/mL LPS, 2 μM imiquimod, or 0.25 μM CpG prior in the presence of 1 μM LCL161. EMT6 cell viability was determined by crystal violet staining. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation).
Cells were infected with the indicated MOI of VSVΔ51 for 24 hours and the cell culture supernatant was exposed to UV light for 1 hour to inactive VSVΔ51 particles. Subsequently, the UV-inactivated supernatant was applied to naive cells in the presence of 5 μM LCL161 for 48 hours. Cell viability was assessed by Alamar blue. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation).
To measure caspase-3/7 activation, 5 μM LCL161, the indicated MOI of VSVΔ51, and 5 μM CellPlayer Apoptosis Caspase-3/7 reagent (Essen Bioscience, Ann Arbor, USA) were added to the cells. Cells were placed in an incubator outfitted with an IncuCyte Zoom microscope with a 10× objective and phase-contrast and fluorescence images were acquired over a span of 48 hours. Alternatively, cells were treated with 5 μM LCL161 and 0.1 MOI of VSVΔ51-GFP and SMC for 36 hours and labeled with the Magic Red Caspase-3/7 Assay Kit (ImmunoChemsitry Technologies, Bloomington, USA). To measure the proportion of apoptotic cells, 1 μg/mL Annexin V-CF594 (Biotium, Hayward, USA) and 0.2 μM YOYO-1 (Invitrogen) was added to SMC and VSVΔ51 treated cells. Images were acquired 24 hours post-treatment using the IncuCyte Zoom. Enumeration of fluorescence signals was processed using the integrated object counting algorithm within the IncuCyte Zoom software. An n=12 (caspase-3/7) or n=9 (Annexin V, YOYO-1) of biological replicates was used to determine statistical measures (mean, standard deviation).
Cells were treated with vehicle or 5 μM LCL161 for 2 hours and subsequently infected at the indicated MOI of VSVΔ51 for 1 hour. Cells were washed with PBS, and cells were replenished with vehicle or 5 μM LCL161 and incubated at 37° C. Aliquots were obtained at the indicated times and viral titers assessed by a standard plaque assay using African green monkey VERO cells.
Cells were scraped, collected by centrifugation and lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche, Laval, Canada). Equal amounts of soluble protein were separated on polyacrylamide gels followed by transfer to nitrocellulose membranes. Individual proteins were detected by western immunoblotting using the following antibodies: pSTAT1 (9171), caspase-3 (9661), caspase-8 (9746), caspase-9 (9508), DR5 (3696), TNF-R1 (3736), cFLIP (3210), and PARP (9541) from Cell Signalling Technology (Danvers, USA); caspase-8 (1612) from Enzo Life Sciences (Farmingdale, USA); IFNAR1 (EP899) and TNF-R1 (19139) from Abcam (Cambridge, USA); caspase-8 (AHZ0502) from Invitrogen; cFLIP (clone NF6) from Alexis Biochemicals (Lausen, Switzerland); RIP1 (clone 38) from BD Biosciences (Franklin Lakes, USA); and E7 from Developmental Studies Hybridoma Bank (Iowa City, USA). Our rabbit anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect human and mouse cIAP1/2 and XIAP, respectively. AlexaFluor680 (Invitrogen) or IRDye800 (Li-Cor, Lincoln, USA) were used to detect the primary antibodies, and infrared fluorescent signals were detected using the Odyssey Infrared Imaging System (Li-Cor).
Total RNA was isolated from cells using the RNAEasy Mini Plus kit (Qiagen, Toronto, Canada). Two-step RT-qPCR was performed using Superscript III (Invitrogen) and SsoAdvanced SYBR Green supermix (BioRad, Mississauga, Canada) on a Mastercycler ep realplex (Eppendorf, Mississauga, Canada). All primers were obtained from realtimeprimers.com. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation).
Cells were infected with virus at the indicated MOI or treated with IFNβ for 24 hours and clarified cell culture supernatants were concentrated using Amicon Ultra filtration units. Cytokines were measured with the TNFα Quantikine high sensitivity, TNFα DuoSet, TRAIL DuoSet (R&D Systems, Minneapolis, USA) and VeriKine IFNβ (PBL Interferonsource) assay kits. An n=3 of biological replicates was used to determine statistical analysis.
Mammary tumors were established by injecting 1×105 wild-type EMT6 or firefly luciferase-tagged EMT6 (EMT6-Fluc) cells in the mammary fat pad of 6-week old female BALB/c mice. Mice with palpable tumors (˜100 mm3) were co-treated with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 50 mg/kg LCL161 per os and either i.v. injections of either PBS or 5×108 PFU of VSVΔ51 twice weekly for two weeks. For poly(I:C) 25 and SMC treatments, animals were treated with LCL161 twice a week and either BSA (i.t.), 20 ug poly(I:C) i.t. or 2.5 mg/kg poly(I:C) i.p. four times a week. The SMC and CpG group was injected with 2 mg/kg CpG (i.p.) and the next day was followed with CpG and SMC treatments. The CpG and SMC treatments were repeated 4 days later. Treatment groups were assigned by cages and each group had min n=4-8 for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment. Blinding was not possible. Animals were euthanized when tumors metastasized intraperitoneally or when the tumor burden exceeded 2000 mm3. Tumor volume was calculated using (π)(W)2(L)/4 where W=tumor width and L=tumor length. Tumor bioluminescence imaging was captured with a Xenogen 2000 IVIS CCD-camera system (Caliper Life Sciences Massachusetts, USA) following i.p. injection of 4 mg luciferin (Gold Biotechnology, St. Louis, USA).
Subcutaneous tumors were established by injecting 3×106 HT-29 cells in the right flank of 6-week old female CD-1 nude mice. Palpable tumors (˜200 mm3) were treated with five intratumoral injections (i.t.) of PBS or 1×108 PFU of VSVΔ51. Four hours later, mice were administered vehicle or 50 mg/kg LCL161 per os. Treatment groups were assigned by cages and each group had min n=5-7 for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment. Blinding was not possible. Animals were euthanized when tumor burden exceeded 2000 mm3. Tumor volume was calculated using (π)(W)2(L)/4 where W=tumor width and L=tumor length.
All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service in concordance with guidelines established by the Canadian Council on Animal Care.
For neutralizing TNFα signaling in vitro, 25 μg/mL of α-TNFα (XT3.11) or isotype control (HRPN) was added to EMT6 cells for 1 hour prior to LCL161 and VSVΔ51 or IFNβ co-treatment for 48 hours. Viability was assessed by Alamar blue. For neutralizing TNFα in the EMT6-Fluc tumor model, 0.5 mg of α-TNFα or α-HRPN was administered 8, 10 and 12 days post-implantation. Mice were treated with 50 mg/kg LCL161 (p.o.) on 8, 10 and 12 days post-implantation and were infected with 5×108 PFU VSVΔ51 i.v. on days 9, 11 and 13. For neutralization of type I IFN signalling, 2.5 mg of α-IFNAR1 (MAR1-5A3) or isotype control (MOPC-21) were injected into EMT6-tumor bearing mice and treated with 50 mg/kg LCL161 (p.o.) for 20 hours. Mice were infected with 5×108 PFU VSVΔ51 (i.v.) for 18-20 hours and tumors were processed for Western blotting. All antibodies were from BioXCell (West Lebanon, USA).
EMT6 cells were co-treated with 0.1 MOI of VSVΔ51-GFP and 5 μM LCL161 for 20 hours. Cells were trypsinized, permeabilized with the CytoFix/CytoPerm kit (BD Biosciences) and stained with APC-TNFα (MP6-XT22) (BD Biosciences). Cells were analyzed on a Cyan ADP 9 flow cytometer (Beckman Coulter, Mississauga, Canada) and data was analyzed with FlowJo (Tree Star, Ashland, USA).
Splenocytes were enriched for CD11b using the EasySep CD11b positive selection kit (StemCell Technologies, Vancouver, Canada). CD49+ cells were enriched using the EasySep CD49b positive selection kit (StemCell Technologies) from the CD11b− fraction. CD11b+ cells were stained with F4/80− PE-Cy5 (BM8, eBioscience) and Gr1-FITC (RB6-8C5, BD Biosciences) and further sorted with MoFlo Astrios (Beckman Coulter). Flow cytometry data was analyzed using Kaluza (Beckman Coulter). Isolated cells were infected with VSVΔ51 for 24 hours and clarified cell culture supernatants were applied to EMT6 cells for 24 hours in the presence of 5 μM LCL161.
Mouse femurs and radius were removed and flushed to remove bone marrow. Cells were cultured in RPMI with 8% FBS and 5 ng/ml of M-CSF for 7 days. Flow cytometry was used to confirm the purity of macrophages (F4/80+ CD11b+).
Excised tumors were fixed in 4% PFA, embedded in a 1:1 mixture of OCT compound and 30% sucrose, and sectioned on a cryostat at 12 μm. Sections were permeablized with 0.1% Triton X-100 in blocking solution (50 mM Tris-HCl pH 7.4, 100 mM L-lysine, 145 mM NaCl and 1% BSA, 10% goat serum). α-cleaved caspase 3 (C92-605, BD Pharmingen, Mississauga, Canada) and polyclonal antiserum VSV (Dr. Earl Brown, University of Ottawa, Canada) were incubated overnight followed by secondary incubation with AlexaFluor-coupled secondary antibodies (Invitrogen).
Comparison of Kaplan-Meier survival plots was conducted by log-rank analysis and subsequent pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot, San Jose, USA). Calculation of EC50 values was performed in GraphPad Prism using normalized nonlinear regression analysis. The EC50 shift was calculated by subtracting the log10 EC50 of SMC-treated and VSVΔ51-infected cells from log10 EC50 of vehicle treated cells infected by VSVΔ51. To normalize the degree of SMC synergy, the EC50 value was normalized to 100% to compensate for cell death induced by SMC treatment alone.
Immune Checkpoint Blockade Synergizes with SMC Treatment to Delay Disease Progression in MM
MPC-11 cells stably expressing a luciferase transgene were implanted via intravenous injection in to BALB/c mice. This in vivo MM model mimics the human disease well and follows predictable disease progression. MPC-11 cells are obtained from a murine plasmacytoma. Following two rounds of treatment with SMC and monoclonal antibodies against either PD-1 or CTLA-4, only anti-PD-1 based treatments showed response in terms of delayed disease progression. Mice treated with the combination of anti-PD-1 and SMC showed the best response, with almost no tumour burden as determined by luminescence signal (
Type 1 Interferons Synergize with SMCs to Cause MM Cell Death
In vitro work examining the effects of various cytokines in combination with SMC highlighted the potential of type 1 IFNs. Specifically, IFNα and IFNβ showed very strong synergistic killing of MM cells with SMC in most cell lines tested (
Oncolytic Viruses Synergize with SMCs to Cause MM Cell Death
An oncolytic virus derived from vesicular somatic virus, VSVΔ51, synergizes well with SMC in vitro to cause cell death in MPC-11 cells (
SMC Synergizes with Standard MM Therapeutics
In vitro viability assays showed synergistic cell killing of MM cells in a SMC-based combination with the synthetic glucocorticoid dexamethasone (Dex) (
SMC treatment effectively caused rapid degradation of cIAP1 and cIAP2 (
Combining a SMC with either IFNβ (
Responsiveness to SMC-mediated cell death varies drastically between the related human MM cell lines MM1R and MM1S, which are derived from the same parent line and differ only in expression of GCR. MM1R, which has no detectable expression of GCR (
Human MM cell lines U266, MM1R and MM1S strongly upregulated PD-L1 in response to IFNβ treatment. Comparable upregulation was also seen with a combination of SMC and IFNβ. The other ligand for PD-1, PD-L2, was similarly upregulated with IFNβ-based treatments. This effect was noticeable at both early and late time points for both proteins (
Combination of SMCs and Immunomodulatory Agents Leads to Cancer Cell Death that Also Involves CD8+ T Cells
SMCs Synergize with Immune Checkpoint Inhibitors in Orthotopic Mouse Models of Cancer
Cell lines RPMI-8226, U266, MM1R, MM1S, MPC-11 were acquired from ATCC. MPC-11 was cultured in DMEM (Hyclone) with 10% FBS (Hyclone), U266 was cultured in RPMI-1640 (Hyclone) with 15% FBS, all other lines were cultured in RPMI-1640 with 10% FBS.
Cells were maintained at 37° C. and 5% CO2 in DMEM media supplemented with 10% heat-inactivated fetal calf serum and 1% non-essential amino acids (Invitrogen). All of the cell lines were obtained from ATCC, with the following exceptions: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Primary NF1−/+p53−/+ cells were derived from C57BI/6J p53+/−/NF1+/− mice. Cell lines were regularly tested for mycoplasma contamination. BTICs were cultured in serum-free culture medium supplemented with EGF and FGF-250. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 h as per the manufacturer's protocol. Cell lines were regularly tested for mycoplasma contamination. BTICs were cultured in serum-free culture medium supplemented with EGF and FGF-2. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 h as per the manufacturer's protocol.
In vivo: LCL161 was a generous gift from Novartis. Anti-PD-1 (clone J43) was purchased from BioXcell. Poly(I:C) (HMW vaccigrade, Invivogen). IFNα (for in vivo use) was a generous gift from Dr Peter Staeheli in Germany. Tetralogic Pharmaceuticals provided Birinapant.
In vitro: IFNs were obtained from PBL assay science; Dexamethasone and RU486 were purchased from Sigma Aldrich.
Antibodies used include RIAP1 (in house), PD-L1 (Abcam), PD-L2 (R&D Systems), GCR (Santa Cruz), P100 (Cell Signalling), P65 (cell signalling), p-P65 (cell signalling), IFNAR1 (Abcam), PARP (Cell Signalling), tubulin (Developmental Studies Hybridoma Bank), RIP1 (R&D Systems), capsase 8 (R&D Systems).
AT-406, GDC-0917, and AZD-5582 were purchased from Active Biochem. TNF-α was purchased from Enzo. IFN-β was obtained from PBL Assay Science. Broad host range IFN-αB/D was produced in yeast and purified by affinity immunochromatography. Nontargeting siRNA or siRNA targeting cFLIP were obtained from Dharmacon (ON-TARGETplus SMARTpool). High molecular weight poly(I:C) was obtained from Invivogen.
4-5 week old BALB/c mice were purchased from Charles River and injected IV with 1×106 MPC-11 Fluc cells stably expressing a firefly luciferase (Fluc) transgene. Treatments include 50 mg/kg LCL161, 250 μg anti-PD-1, 250 μg anti-CTLA4, 25 μg poly(I:C), 5×108 pfu VSVΔ51, 1 ug IFNα. Imaging of mice was done with the in vivo imaging system IVIS, after IP injection of 200 μL of luciferin to measure luminescence.
The Indiana serotype of VSV was used in this study. VSV-EGFP, VSVΔ51 (lacking amino acid 51 in the M gene) and Maraba-MG1 were propagated in Vero cells and purified on an OptiPrep gradient. VSVΔ51 with the deletion of the gene encoding for glycoprotein (VSVΔ51AG) was propagated in HEK293T-cells that were transfected with pMD2-G using Lipofectamine2000 (Invitrogen), and purified on a sucrose cushion. NRRPs were generated by exposing VSV-EGFP to UV (250 mJ cm-2) using a XL-1000 UV crosslinker (Spectrolinker).
Cell lines were seeded in 96-well plates and incubated overnight. Cells were treated with vehicle (0.05% DMSO) or LCL161 and infected with the indicated MOI of virus or treated with 1 μg mL−1 IFN-αB/D, 0.1 ng mL−1 TNF-α, or the indicated of NRRPs for 48 h. Cell viability was determined by Alannar blue (Resazurin sodium salt (Sigma)), and data were normalized to vehicle treatment. The chosen sample size is consistent with previous reports that used similar analyses for viability assays, but no statistical methods were used to determine sample size.
Cells were scraped, collected by centrifugation, and lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche). Tumors were excised, minced, and lysed as above. Equal amounts of soluble protein were separated on polyacrylamide gels followed by transfer to nitrocellulose membranes. Individual proteins were detected by Western blotting using for cFLIP (7F10, 1:500, from Alexis Biochemicals) and β-tubulin (1:1000, E7 from Developmental Studies Hybridoma Bank). Rabbit anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect human and mouse cIAP1/2 and XIAP, respectively (1:5000; Cyclex Co.). AlexaFluor680 (Invitrogen) or IRDye800 (Li-Cor) (1:2500) were used to detect the primary antibodies, and infrared fluorescent signals were detected using the Odyssey Infrared Imaging System (Li-Cor). Full-length blots are shown in
For detection of TNF-α in vivo, mice were treated with 50 μg poly(I:C) intraperitoneally (i.p.) or 5×108 PFU of VSVΔ51 intravenously (i.v.). Brains were homogenized in 20 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 10% glycerol and 1 mM MgCl2, supplemented with EDTA-free protease inhibitor cocktail (Roche). NP-40 was added to final concentration of 0.1% and clarified through centrifugation. Equal amounts were processed for the detection of TNF-α with the TNF-α Quantikine assay kits (R&D Systems).
To assess the specificity of the adaptive immune response, mice cured of CT-2A tumors by SMC and anti-PD-1 treatment and age-matched control (naïve) C57BL/6 female mice were injected subcutaneously with 1×106 CT-2A cells. After seven days, splenocytes were isolated and cocultured with CT-2A cells for 48 hours (20:1 ratio of splenocytes to cancer cells) in the presence of vehicle or 5 μM SMC or 20 μg mL−1 of the indicated antibodies. The secretion of IFN-γ, GrzB, TNF-α, IL-17, IL-6, and IL-10 was determined by ELISA (kits are from R&D Systems).
Female 5-week old C57BL/6 or CD-1 nude mice were anesthetized with isofluorane and the surgical site was shaved and prepared with 70% ethanol. 5×104 cells were stereotactically injected in a 10-μL volume into the left striatum over 1 minute into the following coordinates: 0.5 mm anterior, 2 mm lateral from bregma, and 3.5 mm deep. The skin was closed using surgical glue. Mice were treated with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 75 mg kg-1 LCL161 orally and intratumorally (i.t.) in 10 μL with 50 μg poly(I:C), intravenously (i.v.) with 5×108 VSVΔ51 or intraperitoneally (i.p.) with 250 μg of anti-CD4 (GK1.5), anti-CD8 (YTS169.4), anti-PD1 (J43), or CTLA-4 (9H10).
For treatment with birinapant, mice were treated with vehicle (12.5% Captisol) or 30 mg kg−1 birinapant (i.p.). In some cases, animals were treated with anti-IFNAR1 (MAR1-5A3), anti-IFN-γ (R4-6A2) or anti-TNF-α (XT3.11). Isotype control IgG antibodies were used as appropriately: BE0091, 13E0087, BP0090, MOPC-21, or HPRN. All neutralizing and control antibodies were from BioXCell. For intracranial cotreatment of SMC and type I IFN, mice were injected 10 μL i.t. with combinations of vehicle (0.5% DMSO), 100 μM LCL161, 0.01% BSA, or 1 μg IFN-αB/D. Alternatively, mice were treated orally with vehicle or 75 mg kg-1 LCL161 and 1 μg IFN-α B/D (i.p.). Animals were euthanized when they showed predetermined signs of neurologic deficits (failure to ambulate, weight loss >20% body mass, lethargy, hunched posture). Treatment groups were assigned by cages and each group had 5 to 9 mice for statistical measures (Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.
The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment but no statistical methods were used to determine sample size.
Live mouse brain MRI was performed at the University of Ottawa pre-clinical imaging core using a 7 Tesla GE/Agilent MR 901. Mice were anaesthetized for the MRI procedure using isoflurane. A 2D fast spin echo sequence (FSE) pulse sequence was used for the imaging, with the following parameters: 15 prescribed slices, slice thickness=0.7 mm, spacing=0 mm, field of view=2 cm, matrix=256×256, echo time=25 ms, repetition time=3,000 ms, echo train length=8, bandwidth=16 kHz, 1 average, and fat saturation. The FSE sequence was performed in both transverse and coronal planes, for a total imaging time of about 5 minutes.
Mammary tumors were established by injecting 1×105 EMT6 cells in the mammary fat pad of 5-week old female BALB/c mice. Mice with palpable tumors (˜100 mm3) were cotreated with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 50 mg kg-1 LCL161 orally and either i.t. injections of 5×108 PFU of VSVΔ51 or i.p. injections of control IgG (BE0091) or anti-PD-1 (J43). Animals were euthanized when tumors metastasized intraperitoneally or when the tumor burden exceeded 2,000 mm3. Tumor volume was calculated using (π)(W)2(L)/4 where W=tumor width and L=tumor length. Treatment groups were assigned by cages and each group had 4 to 5 mice for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.
A mouse model of multiple myeloma and plasmacytoma was established by injecting 1×108 luciferase-tagged MPC-11 cells (i.v.) into female 4-5 week old BALB/c mice. Mice were treated with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 75 mg kg-1 LCL161 orally and with 250 μg of control IgG or α-PD-1 antibodies (i.p). Bioluminescence imaging was captured with a Xenogen2000 IVIS CCD-camera system (Caliper Life Sciences) following i.p. injection of 4 mg luciferin (Gold Biotechnology). Treatment groups were assigned by cages and each group had 3 to 4 mice for statistical measures (Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.
Naïve age-matched female C57BL/6 mice or mice previously cured of intracranial CT-2A tumors by SMC-based combination treatment with immunostimulants (minimum of 180 days post-implantation) were reinjected with CT-2A cells i.c. as described above or with 5×105 cells subcutaneously. Naïve BALB/c or mice previously cured of luciferase-tagged EMT6 mammary tumors with SMC and VSVΔ51 combination treatment (90 to 120 d post-implantation) were reinjected with 5×105 untagged EMT6 cells in the fat pad. Animals were euthanized as described above. Blinding or randomization was not possible. All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service in accordance with guidelines established by the Canadian Council on Animal Care.
For in vitro analysis, cells were treated with vehicle (0.01% DMSO) or 5 μM LCL161 and 0.01% BSA, 1 ng mL-1 TNF-α, 250 U mL-1 IFN-β or 0.1 MOI of VSVΔ51 for 24 hr. Cells were released from plates with enzyme-free dissociation buffer (Gibco) and stained with Zombie Green and the indicated antibodies. For analysis of tumor immune infiltrates, intracranial CT-2A tumors were mechanically dissociated, RBCs lysed in ACK lysis buffer and stained with Zombie Green and the indicated antibodies. In some cases, cells were stimulated with 5 ng/ml PMA and 500 ng/ml Ionomycin in the presence of Brefeldin A for 5 h, and intracellular antigens were processed using BD Cytofix/Cytoperm kit. Antibodies include Fc Block (101319, 1:500), PD-L1(10F.9G2, 1:250), PD-L2 (TY25, 1:100), I-A/I-E (M5/114.15.2, 1:200) and H-2Kd/H-2Dd- (34-1-2S, 1:200), CD45 (30-F11, 1:300), CD3 (17A2, 1:500), CD4 (GK1.5, 1:500), CD8 (53-6.7,1:500), PD-1 (29.1A12, 1:200), CD25 (PC61, 1:150), Gr1 (RB6-AC5, 1:200), F4/80 (BM8, 1:200), GrzB (GB11, 1:150) and IFN-γ (XMG1.2, 1:200). All antibodies were from BioLegend except for TNF-α (MP6-XT22, 1:200) and CD11b (M1/70, 1:100) where from BD Biosciences. Cells were analyzed on a Cyan ADP 9 (Beckman Coulter) or BD Fortessa (BD Biosciences) and data was analyzed with FlowJo (Tree Star).
Detection of mKate2-CT-2A cells was performed in an incubator outfitted with an Incucyte Zoom microscope equipped with a 10× objective. Enumeration of fluorescent signals from the Incucyte Zoom was processed using the integrated object counting algorithm within the Incucyte Zoom software.
The detection of serum proteins following combinatorial SMC and anti-PD-1 treatment was analyzed by a flow cytometry-based multiplex kit (LEGENDplex inflammation panel from Biolegend). Hierarchical analysis was determined using Morpheus (https://software.broadinstitute.org/morpheus).
Total RNA was extracted from cells using the RNeasy mini prep kit (Qiagen). Two step RT-qPCR was performed using iScript and SsoAdvanced SYBR Green supermix (BioRad) on a Mastercycler ep realplex (Eppendorf). qPCR was done with PD-L1 and PD-L2 primers (Qiagen) and SIBR green reagent (Bio-Rad). Relative expression was calculated as ΔΔCt using RPL13A as a control.
The library panel of cytokine and chemokine genes was from realtimeprimers.com. A n=4 was performed for each treatment conditioned and data was normalized to eight different reference genes and compared to each vehicle and IgG sample. The data was analyzed by hierarchical analysis using Morpheus.
CD8+ T-cells were enriched from splenocytes of female age-matched naïve mice or mice previously cured of intracranial CT-2A (180 days post-implantation) or mammary EMT6 tumors (120 days post-implantation) using a CD8 magnetic selection kit (Stemcell Technologies). CD8+ cells were co-cultured with cancer cells (1:20 for CT-2A, LLC, and 1:12.5 for EMT6 or 4T1 cells) and with 10 μg mL-1 IgG (BE0091) or anti-PD-1 (J43) for 48 h using the IFN-γ or Granzyme B ELISpot kits (R&D Systems).
For all animal studies, survival was calculated from the number of days post implantation of MM cells, and plotted as Kaplan Meier curves. From those, log rank test was used to determine significance. For in vitro viability assays, error is presented at standard deviation. Subsequent pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot). Comparison between multiple treatment groups was analyzed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test with adjustments for multiple comparison (GraphPad). Estimate of variation was analyzed with GraphPad. Comparison of treatment pairs was analyzed by two-sided t-tests (GraphPad).
We show here that cultured and primary glioblastoma cell lines are killed with SMC when combined with exogenous TNF-α, the oncolytic virus VSVΔ51, or with an infectious but non-replicating virus, VSVΔ51ΔG (
Since VSVΔ51 is neurotoxic, and since issues remain about the ‘immune privileged’ brain microenvironment and penetration of drugs across the blood-brain barrier (BBB), we set out to test the effects of systemic and intracranial immunotherapy agent delivery. Following the establishment of intracranial CT-2A tumors (
When mice bearing intracranial CT-2A glioblastoma were treated singly with SMC (oral gavage), VSVΔ51 (i.v.)m or poly(I:C) (intracranially, i.c.), the extension of mouse survival was minimal for this aggressive cancer (17% survival rate) (
The virus-induced immune effects are mediated in part by type I IFNs. We show here that CT-2A cells are partially sensitive to combined SMC and recombinant IFN-α in vitro (
The innate immune system is a key player in the SMC-mediated death of tumor cells. Nevertheless, fundamental questions remain as to the contributory role of the adaptive immune system in this SMC combination approach. Furthermore, a potential pitfall of the proposed use of oncolytic viruses or other immunostimulatory agents in combination with SMC treatment could be the increase in expression of checkpoint inhibitor ligands on cancer cells, thereby negating CTL-mediated attack of tumors. Flow cytometry analysis revealed that treatment of glioma cells with recombinant type I IFN or infection with VSVΔ51, but not treatment with TNF-α, resulted in the increased surface expression of PD-L1 and major histocompatibility complex (MHC) I markers. Moreover, there was no significant impact on the expression of these tumor surface molecules by SMC treatment (
Interestingly, mice previously cured of orthotopic EMT6 mammary carcinomas by combined SMC treatments were completely resistant to tumor engraftment when rechallenged with EMT6 cells (
We next investigated whether a current class of cancer immunotherapy, known as immune checkpoint inhibitors or ICIs, could enhance SMC efficacy. It has been recently reported that ICI treatment of glioblastoma in mice results in at least a partial extension of survival. We first sought to determine whether SMC treatment influences PD-1 expression in a subset of infiltrating immune cells within CT-2A brain tumors. While there was no statistical difference between the levels of infiltrating CD3+ or CD3+ CD8+ cells within intracranial CT-2A tumors, we observed a robust increase of CD3+ and CD3+ CD8+ cells expressing the immune checkpoint, PD-1 (
To determine whether the increased levels of PD-1+ CD8 T-cells may be a negative modulator for SMC efficacy, we assessed blocking the checkpoint target, PD-1, as well as CTLA-4, in combination with SMC using two mouse models of glioblastoma. The systemic administration of anti-PD-1 or anti-CTLA4 antibodies demonstrated no activity on their own (
There are two structural classes of SMCs: monomers and dimers. Monomeric SMCs consist of a single chemical molecule that binds to the BIR domains of the IAPs while dimeric SMCs consist of two SMC molecules connected by a linker allowing for cooperative binding and/or tethering of IAPs. A clinically advanced SMC, LCL161, is the focus of most of our studies, and is a potent monomer. We next sought to assess whether another clinically advanced dimeric SMC similarly synergizes with an ICI for the treatment of glioblastoma. We observed a significant increase in survival of mice bearing intracranial CT-2A tumors when treated with anti-PD-1 and the dimer SMC, Birinapant (
The synergistic effect between SMC and ICIs is not restricted to brain tumors. We also observed a significant extension of the survival of mice bearing a highly aggressive and treatment refractory model of multiple myeloma using MPC-11 cells (
To provide an initial insight into the cellular mechanism of action, we profiled the production of immune factors from CT-2A cells that were co-cultured with splenocytes derived from mice cured of intracranial CT-2A tumors using combined SMC and anti-PD-1 treatment. We observed a significant increase in the production of IFN-γ and GrzB from CT-2A cells co-incubated with splenocytes derived from surviving mice (
However, the expression of IFN-γ and IL-17 from splenocytes isolated from cured mice significantly increased with anti-PD-1 or PD-L1 treatment, suggesting that a T-cell-based immune response can be augmented upon checkpoint inhibition through the PD-1 axis. We next sought to determine whether this gene response is affected by SMC treatment. Among the previously analyzed cytokines, the inclusion of SMC in these cocultures along with ant-PD-1 blockade increased the secretion of IFN-γ, GrzB, IL-17, and TNF-α (
As there is an increase in the levels of GrzB, a cytotoxic factor that is partially blocked by XIAP31-33 and TNF-α, we next assessed whether co-cultures of glioblastoma cells with splenocytes from naïve mice or mice previously cured of CT-2A intracranial tumors would lead to death of CT-2A cells. Using various differently structured SMCs, we saw a statistically significant increase in the death of CT-2A cells in the presence of SMCs, and this response was increased with the inclusion of anti-PD-1 antibodies (
Collectively, these results indicate that a robust effector T-cell response is elicited with the combination treatment of ICI and SMC. To further elucidate the cellular mechanism of action, we undertook the depletion of immune cells using specific CD4 or CD8 targeting antibodies. We found that the 71% cure rate induced by the combination therapy is completely abrogated upon depletion of CD8+ T-cells (
To understand the immune cellular aspect of the synergy between SMC and ICI treatment, we evaluated the profiles of infiltrating CD45+ immune cells of mice bearing glioblastoma. In these studies, we evaluated the infiltrating immune cells in later stage glioblastoma tumors following anti-PD-1 and SMC cotreatment (
Next, we characterized the surface presentation of PD-1 in T-cells following single and combinatorial treatment. We noted a significant increase in CD8+ T-cells expressing PD-1 in mice treated with SMC alone, and the treatment of anti-PD-1 or combined treatment of SMC and anti-PD-1 resulted in less detectable surface presentation of PD-1 (
In addition to the observed T-cell infiltration of intracranial glioblastoma tumors, we next characterized the presence of myeloid-derived suppressor cells (MDSC) and astrocytes/microglia. In contrast to a previous report, we did not detect differences in the MDSC population (CD11b+ Gr1+) in any treatment cohorts (
We next characterized the tumoral cellular cytokine and chemokine profiles of mice bearing intracranial glioblastoma tumors treated with combinations of SMC and anti-PD-1. Flow cytometry analysis revealed that there was an increase of CD8+ cells expressing GrzB with the inclusion of anti-PD-1 antibodies. The ratio of cytotoxic CD8+ (
We also evaluated the effect of combined SMC treatment and anti-PD-1 blockade on serum concentration and gene expression levels of cytokines and chemokines in the intracranial CT-2A glioblastoma model. We detected statistically significant increases in the proinflammatory cytokines IFN-, IL-1-α, IL-1β, and IL-17 and the multifaceted cytokines IFN-γ, IL-27, and GM-CSF (
As we observed a consistent increase in the levels of IFN-β and IFN-γ, we next sought to characterize the functional role of these signaling molecules with the use of blocking/neutralizing antibodies in mice bearing intracranial CT-2A tumors and treated with SMC and anti-PD-1. Abrogation of type I IFN signaling by using an antibody that blocks the IFNAR1 receptor negated the synergistic effects towards increasing survival of mice bearing intracranial CT-2A tumors following combined SMC and anti-PD1 treatment (
Overall, our results indicate that the synergistic effects between SMC and ICI can be primarily attributed towards enhancing a CTL-mediated attack against glioblastoma cells, and this involves a proinflammatory response that includes type I IFN. The coculture of CT-2A cells and CD8+ Tcells isolated from mice previously cured of intracranial tumors resulted in an increase of GrzB positive CD8+ T-cells, which was not increased with SMC treatment alone (
The inclusion of SMC significantly increased the proportion of CD8+ T-cells expressing TNF-α, regardless of inclusion of antibodies targeting PD-1 (
These results indicate that cytotoxic T-cells, in response to SMC and anti-PD-1 treatment, may lead to enhanced tumor cell death due to the increased production of GrzB and TNF-α, pro-death factors that induce tumor cell death due to the antagonism of the IAPs. We functionally characterized the role of TNF-α by employing blocking antibodies targeting TNF-α. When systemic blockade of TNF-α was applied, we observed almost a complete reversal of the efficacy of combined SMC and ICI treatment (
The immunomodulatory anti-cancer effects of SMCs are multimodal (
Our current studies demonstrate that SMCs can cooperate and dramatically intensify the action of ICIs, including anti-PD-1 or anti-CTLA4 antibodies, allowing for durable cures of mice bearing aggressive intracranial tumors. The multiplicity and complexity of mechanisms involved with SMC therapy make it difficult to isolate the individual roles for the varied immunomodulatory actions in the combination synergy. However, it is clear that TNF-α cytotoxicity is involved. Moreover, the current study further demonstrates that CD8+ T-cells are also required for anti-cancer activity when an ICI is combined with an SMC.
In summary, we have shown for the first time that SMCs can potentiate the activity of ICIs in mouse tumor models. Furthermore, this combination effect depends on the presence of CD8+ T-cells with a concomitant decrease of immunosuppressive CD4+ T-cells, and type I and II IFN and TNF-α signaling pathways, clearly implicating the role of adaptive immunity for SMC-mediated cures in mice. Thus, SMC-mediated T-cell co-stimulatory signals provide the drive for adaptive immune responses that develop against the tumor and this is fully realized when the brakes imposed by co-inhibitory signals, such as PD-1 or PD-L1, are removed with ICIs.
All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.
While the invention has been described in connection with the specific embodiments, it will be understood that it is capable of further modifications. Therefore, this application is intended to cover any variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including departures from the present disclosure that come within known or customary practice within the art.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2017/050237 | 2/23/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62299288 | Feb 2016 | US |
