Patent Application
20240181049
This application includes as part of its disclosure a Biological Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 30, 2020, is named “1159583o000001.txt” and is 6,261 bytes in size.
The present disclosure relates generally to methods and compositions for treating cancer, and in a specific aspect colorectal cancer. In another aspect the disclosure relates to novel strains of bacteria, as well as compositions and uses thereof.
Colorectal cancer (CRC) is the second and third most common malignancy in Western countries in women and men, respectively (Ferlay et al., 2015). In addition to genetic aberrations, which are essential for the development of CRC, other disease-contributing factors have been identified. These include the microbiota and inflammation, whereby inflammation can drive or inhibit CRC development. Interferon (IFN)-γ producing T helper type 1 (Th1) cells are known to be protective (Mager et al., 2016; Mlecnik et al., 2016; Wang et al., 2015), whereas interleukin (IL)-17-producing Th17 cells promote CRC development (Galon et al., 2006; Grivennikov et al., 2012; Le Gouvello et al., 2008). In fact, the impact of the immune system is so potent that immune cell infiltration in the tumor is a superior prognostic factor compared to the classical tumor-lymph nodes-metastasis (TNM) system in CRC (Anitei et al., 2014; Mlecnik et al., 2016). Similarly, the microbiota also impacts on CRC progression (Arthur et al., 2012; Dejea et al., 2018) and may even alter the efficacy of chemotherapeutics (Iida et al., 2013; Viaud et al., 2013).
Immune checkpoint blockade (ICB) therapy is an efficient anti-cancer strategy that utilizes the therapeutic potential of the immune system. Most notably, ICB inhibitors targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), or its ligand (PD-L1) have shown great success in the treatment of various cancers, including melanoma, renal cell carcinoma, and non-small cell lung cancer (Brahmer et al., 2012; Hodi et al., 2010). More recently, seminal work has shown that the efficacy of ICB therapy is dependent on the presence of certain ICB-promoting gut bacteria (Routy et al., 2018; Sivan et al., 2015; Vetizou et al., 2015).
Despite these exciting advances, ICB therapy efficacy in CRC has been disappointing (Brahmer et al., 2012), with only 5-10% of all CRC patients responding (Le et al., 2017). Moreover, the detailed molecular mechanisms through which bacteria enhance the efficacy of ICB therapies remains unclear. Here, we identified three bacterial species that promote ICB efficacy in CRC and identified inosine as a critical bacterial metabolite that promoted differentiation of Th1-mediated anti-tumor immunity.
In one aspect there is provided a method of treating a subject having a cancer or suspected of having a cancer, comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli or a combination thereof.
In one aspect there is provided a method of treating a subject having a cancer or suspected of having a cancer, comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, or Olsenella sp. or a combination thereof.
In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a method of treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli, such as the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a method of treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum (B.p.), Lactobacillus johnsonii (L.j), or Olsenella sp. (O.sp.), such as the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a method of treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium sp. (B.sp.), Lactobacillus sp. (L.sp.), or Olsenella sp. (O.sp.), such as the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a use of an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli, or a combination thereof, for treating a subject having a cancer or suspected of having a cancer. Said bacterium may comprise Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a use of an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, or Olsenella sp., or a combination thereof, for treating a subject having a cancer or suspected of having a cancer. Said bacterium may comprise Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a use of an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli, or a combination thereof, for treating a subject having or suspected of having colorectal cancer (CRC). Said bacterium may comprise Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a use of an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum (B.p.), Lactobacillus johnsonii (L.j), or Olsenella sp. (O.sp.), or a combination thereof, for treating a subject having or suspected of having colorectal cancer (CRC). Said bacterium may comprise Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a use of an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium sp. (B.sp.), Lactobacillus sp. (L.sp.), or Olsenella sp. (O.sp.), or a combination thereof, for treating a subject having or suspected of having colorectal cancer (CRC). Said bacterium may comprise Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a kit for treating a subject having a cancer or suspected of having a cancer, comprising or consisting of, an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli, or a combination thereof and optionally a container. In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a kit for treating a subject having a cancer or suspected of having a cancer, comprising or consisting of an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, or Olsenella sp., or a combination thereof, and optionally a container. In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a kit for treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, an immune checkpoint inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli or a combination thereof and optionally a container. In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a kit for treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum (B.p.), Lactobacillus johnsonii (L.j), or Olsenella sp. (O.sp.) and optionally a container. In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a kit for treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering an immune checkpoint inhibitor and one or more bacteria selected from Bifidobacterium sp. (B.sp.), Lactobacillus sp. (L.sp.), or Olsenella sp. (O.sp.) and optionally a container. In an exemplary embodiment the bacterium is selected from the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01, Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02, or Olsenella sp. strain deposited as IDAC Deposit No. 231020-03, or a combination thereof.
In one aspect there is provided a method of treating a subject having a cancer or suspected of having a cancer, comprising or consisting of, administering: an immune checkpoint inhibitor; inosine, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine; and a co-stimulant.
In one aspect there is provided a method of treating a subject having or suspected of having colorectal cancer (CRC), comprising or consisting of, administering: an immune checkpoint inhibitor; inosine, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine; and a co-stimulant.
In one aspect there is provided a use of an immune checkpoint inhibitor; inosine, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine; and a co-stimulant, for treating a subject having a cancer or suspected of having a cancer.
In one aspect there is provided a use of an immune checkpoint inhibitor; inosine, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine; and a co-stimulant, for treating a subject having a cancer or suspected of having a cancer.
In one aspect there is provided a kit for treating a subject having a cancer or suspected of having a cancer, comprising or consisting of an immune checkpoint inhibitor; inosine, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine; and a co-stimulant, and optionally a container.
In one example, the cancer is colorectal cancer (CRC), lung cancer, melanoma, bladder cancer, kidney cancer, breast cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, cervical cancer, ovarian cancer, thyroid cancer, lip cancer, oral cancer, larynx cancer, nasopharynx cancer, or uterine cancer.
In an exemplary embodiment the cancer is a solid cancer. In an exemplary embodiment the cancer is a blood cancer (e.g., a leukemia or a lymphoma).
In another example, the cancer is selected from non-small cell lung cancer, small cell lung cancer, gastric carcinoma, testicular cancer, mesothelioma, head and neck cancers, glioblastoma, thymic carcinoma, or Merkel cell cancer. In another example, the cancer is selected from leukemias, myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL), myelodysplastic syndrome (MDS), Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL), multiple myeloma (MM), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic eosinophilic leukemia, or mycosis fungoides.
In one example the cancer is mismatch repair deficient, such as an MMRD colorectal cancer, gastrointestinal cancer, endometrial cancer, breast cancer, prostate cancer, bladder cancer, or thyroid cancer, and/or in a subject having Lynch syndrome. In one example, the cancer is a CRC that is mismatch repair deficient (MMRD) CRC or inflammation-associated CRC. In exemplary embodiments the MMRD is determined based on a lack of functional expression of one or more mismatch repair proteins, e.g., MLH1, MSH2, MSH6 and PMS2 gene. MMRD may result from a loss of function in or decreased expression of at least one of mismatch repair protein, such as due to gene methylation, e.g., in the MLH1 gene. MMRD deficiency can be determined by immunohistochemical analysis of mismatch repair proteins. Said MMRD may be determined based on cancer histological features, e.g., increased tumor infiltrating lymphocytes, medullary or micro-glandular morphology, and/or mucinous or signet ring cell morphology in 50% or more of the tumor. MMRD may also be identified by the presence of microsatellite instability (MSI).
In one example, said ICB inhibitor is an anti-CTLA4 antibody, or an anti-PD-L1 antibody, or an anti-PD-1 antibody.
In one example said ICB inhibitor is an antagonist of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, VISTA, IDO, IDO1 IDO2, TIGIT, BTLA, HVEM, CD226 (DNAM-1), CD96 (Tactile), TIM-3, LAIR1, CD160 (BY55), CD244 (2B4), VTCN1 (B7-H4), KIR, A2AR, or B7-H3.
In one example said ICB inhibitor is a small molecule antagonist of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, VISTA, IDO, IDO1 IDO2, TIGIT, BTLA, HVEM, CD226 (DNAM-1), CD96 (Tactile), TIM-3, LAIR1, CD160 (BY55), CD244 (2B4), VTCN1 (B7-H4), KIR, A2AR, or B7-H3.
In one example said ICB inhibitor comprises an antagonist antibody that specifically binds to CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, VISTA, IDO, IDO1 IDO2, TIGIT, BTLA, HVEM, CD226 (DNAM-1), CD96 (Tactile), TIM-3, LAIR1, CD160 (BY55), CD244 (2B4), VTCN1 (B7-H4), KIR, A2AR, or B7-H3.
In one example said ICB inhibitor comprises a fragment of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, VISTA, IDO, IDO1 IDO2, TIGIT, BTLA, HVEM, CD226 (DNAM-1), CD96 (Tactile), TIM-3, LAIR1, CD160 (BY55), CD244 (2B4), VTCN1 (B7-H4), KIR, A2AR, or B7-H3, or comprises a fragment of a binding partner (e.g., receptor or ligand) of any of the foregoing.
In exemplary embodiments, said ICB inhibitor comprises an antibody, small molecule, or fusion protein, or a combination thereof. In exemplary embodiments, said ICB inhibitor is selected from ipilimumab (YERVOY®, anti-CDLA-4 antibody, Bristol-Myers Squibb), nivolumab (OPDIVO®, anti-PD-1 antibody, Bristol-Myers Squibb), pembrolizumab (KEYTRUDA®, anti-PD-1 antibody, Merck), atezolizumab (TECENTRIQ®, anti-PD-L1 antibody, Roche), avelumab (BAVENCIO®, anti-PD-L1 antibody, Merck KGaA/Pfizer), durvalumab (IMFINZI®, anti-PD-L1 antibody, Medimmune/AstraZeneca), cemiplimab (LIBTAYOR, anti-PD-1 antibody, Regeneron/Sanofi), lambrolizumab (anti-PD-1 antibody, Merck), pidilizumab (anti-PD-1 and anti-DLL antibody, Medivation), BMS-936559 (anti-PD-L1, Bristol-Myers Squibb), MEDI-0680 (anti-PD-1 antibody; AMP-514; AstraZeneca), REGN2810 (anti-PD-1 antibody, Regeneron), CA-170 (small molecule PD-1 and PD-L1 inhibitor; Curis), BMS-1166 (small molecule PD-L1 inhibitor, Bristol-Myers Squibb), AMP-224 (anti-PD-1 fusion protein, Medimmune), spartalizumab (anti-PD-1 antibody, Novartis), STI-A1110 (anti-PD1 antibody, Sorrento/Servier), Dostarlimab (anti-PD-1 antibody, TSR-042, Tesaro), RG-7446 (anti-PD-L1 antibody, Roche), AUR-012 (peptide antagonist of PD1, Aurigene), STI-A1010 (anti-PD-L1 antibody, Sorrento), or a combination thereof.
In one example, the Bifidobacterium sp. is presented in
In one example, the Lactobacillus sp. is presented in
In one example, the Olsenella sp. is presented in
In one example, the Bifidobacterium sp. comprises a 16S rDNA sequence having at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 1.
In one example, the Lactobacillus sp. comprises a 16S rDNA sequence having at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 2.
In one example, the Olsenella sp. comprises a 16S rDNA sequence having at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 3.
In one example, the method or use or kit or use of a kit further comprises administration of a chemotherapeutic agent, an immunotherapeutic agent, or a radiotherapy, or a combination thereof.
In one example, said subject is a human. Said human subject may be of any age, e.g., infant, child, adolescent, adult, or elderly.
In one example, said subject is a non-human animal, such as a non-human primate, a companion animal (e.g., a mammalian animal such as a dog, cat, ferret, horse, rabbit, guinea pig, gerbil, hamster, chinchilla, rat, mouse, or other small mammal; a bird; a reptile; a fish; an amphibian; an arthropod) or a livestock animal (e.g., a mammalian livestock animal such as a cow, pig, sheep, goat, alpaca, donkey, camel, water buffalo, or mink; or a chicken).
In exemplary embodiments, said bacteria may be a strain that raises the level of inosine, xanthine, hypoxanthine and/or xanthine monophosphate, preferably inosine or hypoxanthine, in vivo or in an in vitro secretion assay.
In exemplary embodiments, said bacteria may be administered in an effective amount to raise the level of inosine, xanthine, hypoxanthine and/or xanthine monophosphate in said subject.
In exemplary embodiments, said bacteria may be administered in an effective amount to sensitize said cancer to treatment with said immune checkpoint inhibitor.
In one example, the CRC is mismatch repair deficient (MMRD) CRC or inflammation-associated CRC. In exemplary embodiments the MMRD is determined based on a lack of functional expression of one or more mismatch repair proteins, e.g., MLH1, MSH2, MSH6 and PMS2 gene. MMRD may result from a loss of function in or decreased expression of at least one of mismatch repair protein, such as due to gene methylation, e.g., in the MLH1 gene. MMRD deficiency can be determined by immunohistochemical analysis of mismatch repair proteins. Said MMRD may be determined based on cancer histological features, e.g., increased tumor infiltrating lymphocytes, medullary or micro-glandular morphology, and/or mucinous or signet ring cell morphology in 50% or more of the tumor. MMRD may also be identified by the presence of microsatellite instability (MSI).
In one example, said co-stimulant is Toll like receptor (TLR) signals, CpG, LPS, Flagellin, Nucleotide-binding oligomerization domain-like receptors (NLRs), meso-diaminopimelic acid, muramyl dipeptide, ATP, extracellular glucose, crystals of monosodium urate, calcium pyrophosphate dihydrate, alum, cholesterol or environmental irritants; silica; asbestos; UV irradiation and skin irritants. RIG-I-like receptors (retinoic acid-inducible gene-I-like receptors), single- or double-stranded RNA (e.g., from viruses), C-type lectin receptors (CLR), repeated mannose units, C-type lectin domain, Cytokine receptor signalling, IL-12, IL-18, IL-33, IFN-g, Stimulation provided through antigen presenting cells or their counterpart on T-cells, CD80-CD28, CD86-CD28, CD40CD40L, OX-40L-OX40, -cGAS-STING pathway, for example, cytosolic DNA.
In another aspect, the disclosure provides an isolated bacterium comprising a 16S rDNA sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 1, preferably having at least 99.5%, or having 100% identity to SEQ ID NO: 1.
In another aspect, the disclosure provides an isolated bacterium comprising a 16S rDNA sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 2, preferably having at least 99.5%, or having 100% identity to SEQ ID NO: 2.
In another aspect, the disclosure provides an isolated bacterium comprising a 16S rDNA sequence having at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 3, preferably having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or having 100% identity to SEQ ID NO: 3.
In another aspect, the disclosure provides an isolated bacterium of the Bifidobacterium pseudolongum strain deposited as IDAC Deposit No. 231020-01.
In another aspect, the disclosure provides an isolated bacterium of the Lactobacillus johnsonii strain deposited as IDAC Deposit No. 231020-02.
In another aspect, the disclosure provides an isolated bacterium of the Olsenella sp. strain deposited as IDAC Deposit No. 231020-03.
In another aspect, the disclosure provides a composition comprising a bacterium of any of the aforementioned bacteria and a pharmaceutically acceptable carrier.
In another aspect, the disclosure provides a composition comprising an effective amount of any of the aforementioned bacteria for the treatment of a cancer and optionally further comprising a pharmaceutically acceptable carrier.
In another aspect, the disclosure provides a composition comprising a mixture of two or more of the aforementioned strains of bacteria and optionally further comprising a pharmaceutically acceptable carrier.
In another aspect, the disclosure provides a composition comprising an effective amount of a mixture of two or more of the aforementioned strains of bacteria for the treatment of a cancer and optionally further comprising a pharmaceutically acceptable carrier.
In another aspect, the disclosure provides a food, beverage, food supplement, probiotic, or nutraceutical comprising a bacterium of any of the aforementioned bacteria, which preferably is formulated for ingestion.
In exemplary embodiments, said bacteria produce elevated levels of inosine, xanthine, hypoxanthine, and/or inosine monophosphate, preferably inosine, in an in vitro or in vivo assay.
In exemplary embodiments, said bacterium or composition is lyophilized.
In exemplary embodiments, said bacterium or composition is adapted for administration to a subject, preferably a human subject. Said human subject may be of any age, e.g., infant, child, adolescent, adult, or elderly. Said subject may be a non-human animal, such as a non-human primate, a companion animal (e.g., a mammalian animal such as a dog, cat, ferret, horse, rabbit, guinea pig, gerbil, hamster, chinchilla, rat, mouse, or other small mammal; a bird; a reptile; a fish; an amphibian; an arthropod) or a livestock animal (e.g., a mammalian livestock animal such as a cow, pig, sheep, goat, alpaca, donkey, camel, water buffalo, or mink; or a chicken).
In exemplary embodiments, said bacterium or composition is adapted for use in any of the methods disclosed herein, e.g., methods of treating cancer as described above.
In exemplary embodiments, said bacterium or composition contains an effective amount of said bacteria for treating a subject having a cancer or suspected of having a cancer according to the method disclosed herein.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Generally, the present disclosure provides a compound(s) and/or a compositions for use in treating a subject having cancer, or suspected of having cancer.
In some examples, the cancer may be colorectal cancer (CRC), lung cancer, melanoma, bladder cancer, or kidney cancer. In other examples, the cancer may be breast cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, cervical cancer, ovarian cancer, thyroid cancer, lip cancer, oral cancer, larynx cancer, nasopharynx cancer, uterine cancer, or other cancer as disclosed herein.
In a specific aspect, the present disclosure provides a compound(s) and/or a compositions for use in treating a subject having Colorectal cancer (CRC), or suspected of having CRC.
In one aspect, there is described a method of treating a subject having a cancer, or suspect of having a cancer, comprising or consisting of: administering an ICB inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, or Olsenella species.
In one aspect, there is described a method of treating a subject having a cancer, or suspect of having a cancer, comprising or consisting of: administering an ICB inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli
In a specific example the cancer may be colorectal cancer (CRC), lung cancer, melanoma, bladder cancer, or kidney cancer. In other examples, the cancer may be breast cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, cervical cancer, ovarian cancer, thyroid cancer, lip cancer, oral cancer, larynx cancer, nasopharynx cancer, uterine cancer.
In one aspect, there is described a method of treating a subject having CRC, or suspected of having CRC, comprising or consisting of: administering an ICB inhibitor and one or more bacteria selected from Bifidobacterium pseudolongum, Lactobacillus johnsonii, Olsenella profuse, Olsenella umbonata, or Olsenella uli.
In one aspect, there is described a method of treating a subject having CRC, or suspected of having CRC, comprising or consisting of: administering an ICB inhibitor and one or more bacterium selected from Bifidobacterium pseudolongum (B.p.), Lactobacillus johnsonii (L.j), or Olsenella sp. (O.sp.).
In one aspect, there is described a method of treating a subject having CRC, or suspected of having CRC, comprising or consisting of: administering an ICB inhibitor and one or more bacterium selected from Bifidobacterium sp. (B.sp.) listed in
In one aspect, there is described a method of treating a subject having a cancer, or suspected of having a cancer, comprising or consisting of: administering an ICB inhibitor and inosine, a derivative of inosine, functional derivative of inosine, or a physiologically functional derivative of inosine.
In a specific example the cancer may be colorectal cancer (CRC), lung cancer, melanoma, bladder cancer, or kidney cancer. In other examples, the cancer may be breast cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, cervical cancer, ovarian cancer, thyroid cancer, lip cancer, oral cancer, larynx cancer, nasopharynx cancer, uterine cancer.
In one aspect, there is described a method of treating a subject having CRC, or suspected of having CRC, comprising or consisting of: administering an ICB inhibitor and inosine, a derivative of inosine, functional derivative of inosine, or a physiologically functional derivative of inosine.
As used herein, the terms “immune checkpoint,” “checkpoint pathway,” and “immune checkpoint pathway” refer to a pathway by which the binding of an immune checkpoint ligand to an immune checkpoint receptor modulates the amplitude and quality of the activation of immune cells.
Immune checkpoint proteins include, but are not limited to, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), also known as CD152, programmed cell death protein 1 (PD-1), also known as CD279, PD-1 ligands (PD-L1 or CD274, PD-L2 or CD274), lymphocyte-activation gene 3 (LAG-3), also known as CD223, B7-H3 (CD276), V-domain Ig suppressor of T cell activation (VISTA), therapies targeting indoleamine 2′3′ dioxygenase (IDO, IDO1 and IDO2), TIGIT (also called T cell immunoreceptor with Ig and ITIM domains), B and T Lymphocyte Attenuator (BTLA), Herpes virus entry mediator (HVEM), CD226 (DNAM-1) and CD96 (Tactile), T cell immunoglobulin mucin (TIM-3), also known as HAVcr2, LAIR1 (Leukocyte Associated Immunoglobulin Like Receptor 1; CD305), CD160 (BY55), CD244 (2B4), VTCN1 (B7-H4), KIR, A2AR, or B7-H3.
The term “immune checkpoint blockade” or “ICB,” as used herein, refers to the administration of one or more inhibitors of one or more immune checkpoint proteins or their ligand(s). Thus, the term “immune checkpoint blockade” refers to the inhibition of an immune checkpoint pathway by the administration or expression of a “blockade agent” or “inhibitor”. Typically, the “blockade agent” prevents the interaction of the immune checkpoint receptor and ligand, thereby inhibiting the checkpoint pathway. A blockade agent may be a small molecule, peptide, antibody or fragment thereof, etc. that binds to an immune checkpoint ligand or immune checkpoint receptor and inhibits the formation of the ICR/ICL complex. A blockade agent may also function by preventing signaling by the ICR/ICL complex. Exemplary ICB agents include antibodies, fusion proteins, and small molecules, such as ipilimumab (YERVOY®, anti-CDLA-4 antibody, Bristol-Myers Squibb), nivolumab (OPDIVO®, anti-PD-1 antibody, Bristol-Myers Squibb), pembrolizumab (KEYTRUDA®, anti-PD-1 antibody, Merck), atezolizumab (TECENTRIQ®, anti-PD-L1 antibody, Roche), avelumab (BAVENCIO®, anti-PD-L1 antibody, Merck KGaA/Pfizer), durvalumab (IMFINZI®, anti-PD-L1 antibody, Medimmune/AstraZeneca), cemiplimab (LIBTAYO®, anti-PD-1 antibody, Regeneron/Sanofi), lambrolizumab (anti-PD-1 antibody, Merck), pidilizumab (anti-PD-1 and anti-DLL antibody, Medivation), BMS-936559 (anti-PD-L1, Bristol-Myers Squibb), MEDI-0680 (anti-PD-1 antibody; AMP-514; AstraZeneca), REGN2810 (anti-PD-1 antibody, Regeneron), CA-170 (small molecule PD-1 and PD-L1 inhibitor; Curis), BMS-1166 (small molecule PD-L1 inhibitor, Bristol-Myers Squibb), AMP-224 (anti-PD-1 fusion protein, Medimmune), and spartalizumab (anti-PD-1 antibody, Novartis).
As used herein, the term “immune checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. These proteins are responsible for co stimulatory or inhibitory interactions of T-cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. In some embodiments, the subject can be administered an additional agent that can enhance or boost the immune response, e.g., immune response effected by the binding molecules (e.g., BCMA-binding molecules), recombinant receptors, cells and/or compositions provided herein, against a disease or condition, e.g., a cancer, such as any described herein.
Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors, ligands and/or receptor-ligand interaction. In some embodiments, modulation, enhancement and/or stimulation of particular receptors can overcome immune checkpoint pathway components.
The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in biological activity, including full blocking of the activity.
An “inhibitor” is an active agent that inhibits, blocks, or suppresses biological activity in vitro or in vivo.
Inhibitors include but are not limited to small molecule compounds; nucleic acids, such as siRNA and shRNA; polypeptides, such as antibodies or antigen-binding fragments thereof, dominant-negative polypeptides, inhibitory peptides, and fusion proteins; and oligonucleotide or peptide aptamers.
In a specific example, the ICB inhibitor is an anti-CTLA4 antibody, or an anti-PD-L1 antibody, or an anti-PD-1 antibody.
Non-limiting examples of co-stimulants include: Toll like receptor (TLR) signals, for example CpG, LPS, Flagellin; Nucleotide-binding oligomerization domain-like receptors (NLRs), for example, meso-diaminopimelic acid, muramyl dipeptide, ATP, extracellular glucose, crystals of monosodium urate, calcium pyrophosphate dihydrate, alum, cholesterol or environmental irritants; silica; asbestos; UV irradiation and skin irritants; RIG-I-like receptors (retinoic acid-inducible gene-I-like receptors), for example, single- or double-stranded RNA (e.g., from viruses); C-type lectin receptors (CLR), for example, repeated mannose units, C-type lectin domain; Cytokine receptor signalling, for example, IL-12, IL-18, IL-33, IFN-g; Stimulation provided through antigen presenting cells or their counterpart on T-cells, for example, CD80-CD28, CD86-CD28, CD40CD40L, OX-40L-OX40; -cGAS-STING pathway; for example, cytosolic DNA.
A “standard dose” of ICB therapy is known by a person of skill in the art for each medication, and may be the dose that is indicated in the prescribing information and/or the dose that is most frequently administered under particular clinical circumstances (for example for the particular PD-1 inhibitor and/or CTLA-4 inhibitor being used, the particular route of administration being used, the particular stage of the CRC being treated, the age, weight, and/or sex of the particular patient, etc.).
The term “subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. Additional examples of domesticated animals include a ferret, horse, rabbit, guinea pig, gerbil, hamster, chinchilla, rat, mouse, or other small mammal; a bird; a reptile; a fish; an amphibian; an arthropod such as a tarantula or hermit crab. Additional livestock animals include donkey, alpaca, camel, water buffalo, mink, or chicken.
In a specific example, the subject is a human.
The terms “colorectal cancer” or “CRC”, used interchangeably herein, are used in the broadest sense and refer to (1) all stages and all forms of cancer arising from epithelial cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, and rectum), and/or (2) all stages and all forms of cancer affecting the lining of the large intestine and/or rectum.
In some examples, CRC is mismatch repair deficient (MMRD) CRC or inflammation-associated CRC.
Typically, in the staging systems used for classification of colorectal cancer, the colon and rectum are treated as one organ.
Additionally, as used herein, the term “colorectal cancer” also includes medical conditions which are characterized by cancer of cells of the duodenum and small intestine (jejunum and ileum).
The staging of CRC is known.
In some examples, CRC may be staged according to the Dukes system, the Astler-Coller system or the TNM system (tumors/nodes/metastases), whereby the latter is most commonly used. The TNM system of the American Joint Committee of Cancer (AJCC) describes the size of the primary tumor (T), the degree of lymph node involvement (N) and whether the cancer has already formed distant metastasis (M), i.e., spread to other parts of the body. Here, stages 0, IA, IB, IIA, IIB, III and IV are defined based on the determined T-, N- and M-values. A corresponding staging scheme can be derived from the Cancer Staging Manual of the AJCC. Another system for staging of colorectal cancer is the Dukes system, defining cancer stages A, B, C and D. This system was adapted by Astler and Coller, who further subdivided stages B and C (“modified Astler-Coller classification”).
As used herein, a CRC patient includes patients staged according to any staging system used and irrespective of the stage diagnosed.
As use herein “a patient suffering from colorectal cancer” or “a subject suffering from colorectal cancer” refers to any mammalian, in particular human, patient having developed atypical and/or malignant cells in the lining and/or the epithelium of the large intestine and/or rectum. This includes CRC patients independent of the stage and form of the CRC.
Patients suffering from colorectal cancer also include patients which are recurrent with colorectal cancer, i.e., patients wherein after surgical treatment the tumor could no longer be detected for a certain time span, but wherein the cancer has returned in the same or different part of the large intestine, and/or rectum and/or wherein metastases have developed at different sites of the patient's body such as in the liver, lung, peritoneum, lymph nodes, brain and/or bones.
In another example, the patient suffering from CRC is a patient wherein the initial tumor has already been treated surgically and the CRC is non-metastatic.
In some examples, a derivative of inosine, functional derivative of inosine, a prodrug of inosine, or a physiologically functional derivative of inosine, may be used.
The term “derivative”, “functional derivative” and “physiologically functional derivative” as used herein means an active compound with equivalent or near equivalent physiological functionality to the named active compound when used and/or administered as described herein. As used herein, the term “physiologically functional derivative” includes any pharmaceutically acceptable salts, solvates, esters, prodrugs derivatives, enantiomers, or polymorphs.
The term “prodrug” used herein refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutical active form in vivo, for example in the subject to which the compound is administered.
The term “therapeutically effective amount” or “effective amount”, as used herein, refers to an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject. The amount of a given compound or composition that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
The term “treatment” or “treat” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treating” and “treatment” as used herein also include prophylactic treatment. For example, a subject in the early stage of disease can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent progression.
“Prevent” or “prevention” refers to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those at risk of or susceptible to developing the disorder. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
In some examples, treatment results in prevention or delay of onset or amelioration of symptoms of a disease in a subject or an attainment of a desired biological outcome.
In some examples, treatment methods comprise administering to a subject a therapeutically effective amount of a compound or composition described herein and optionally consists of a single administration or application, or alternatively comprises a series of administrations or applications.
The term “pharmaceutically effective amount” as used herein refers to the amount of a compound, composition, drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician, for example, the treatment of colorectal cancer. This amount can be a therapeutically effective amount.
The compounds and compositions may be provided in a pharmaceutically acceptable form.
The term “pharmaceutically acceptable” as used herein includes compounds, materials, compositions, and/or dosage forms (such as unit dosages) which are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. is also “acceptable” in the sense of being compatible with the other ingredients of the formulation.
The term “functional derivative” and “physiologically functional derivative” as used herein means an active compound with equivalent or near equivalent physiological functionality to the named active compound when used and/or administered as described herein. As used herein, the term “physiologically functional derivative” includes any pharmaceutically acceptable salts, solvates, esters, prodrugs derivatives, enantiomers, or polymorphs.
In some examples the compounds are prodrugs.
The formulation(s) may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intratumoral, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot/for example, subcutaneously or intramuscularly. Preferably compositions comprising bacteria are delivered to the gastrointestinal system, eig., by oral (such as ingestion) or rectal route.
Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.
The compounds and/or compositions described herein may be administered either simultaneously (or substantially simultaneously) or sequentially, dependent upon the condition to be treated, and may be administered in combination with other treatment(s). The other treatment(s) may be administered either simultaneously (or substantially simultaneously) or sequentially.
As used herein the term ‘reduces at least one symptom of CRC’ refers to a qualitative or quantitative reduction in detectable symptoms, including but not limited to a detectable impact on the rate of recovery from disease or the rate of disease progression or severity.
As used herein, the term ‘at risk of developing CRC′’ in reference to a subject is understood as referring to a subject predisposed to the development of CRC by virtue of the subject's medical status.
In some example, a subject having CRC′ is a subject having been “diagnosed with CRC′”.
The term ‘diagnosed with CRC′’ refers to a subject demonstrating one or more symptoms of CRC′. Methods of diagnosing CRC′, are known in the art.
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges.
The pharmaceutical compositions described herein may be useful for parenteral administration, such as intravenous administration, intraperitoneal administration, or administration into a body cavity or lumen of an organ or joint.
The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Pharmaceutical compositions can be administered in any of numerous dosage forms, for example, tablet, capsule, liquid, solution, softgel, suspension, emulsion, syrup, elixir, tincture, film, powder, hydrogel, ointment, paste, cream, lotion, gel, mousse, foam, lacquer, spray, aerosol, inhaler, nebulizer, ophthalmic drops, patch, suppository, and/or enema. Pharmaceutical compositions typically comprise a pharmaceutically acceptable carrier, and can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. benzyl alcohol), a penetration enhancer, an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents. Choice of dosage form and excipients depends upon the active agent to be delivered and the disease or disorder to be treated or prevented, and is routine to one of ordinary skill in the art.
In some examples, the compositions for administration will commonly comprise a solution of the binding agent of the present disclosure dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of binding agents of the present disclosure in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
Optionally the treatment is combined with another moiety useful for treating CRC.
According to at least some embodiments of the present invention, there is provided use of a combination of the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, that can be combined with standard of care or novel treatments for CRC.
For example, treatment methods for a patient suffering from colorectal cancer, in particular after removal of the primary tumor, may include chemotherapy, radiotherapy, targeted therapy, and immunotherapy.
As used herein, the term “chemotherapy” relates to treatment of a subject with an antineoplastic drug.
The terms “radiation therapy” and “radiotherapy” relate to the use of ionizing radiation to treat or control a cancer such as CRC.
The term “targeted therapy”, as used herein, relates to application to a patient a chemical substance known to block growth of cancer cells by interfering with specific molecules known to be necessary for tumorigenesis or cancer or cancer cell growth
The term “immunotherapy” as used herein relates to the treatment of cancer by modulation of the immune response of a subject. Said modulation may be inducing, enhancing, or suppressing said immune response, e.g. by administration of at least one cytokine, and/or of at least one antibody specifically recognizing cancer cells. The term “cell-based immunotherapy” relates to a cancer therapy comprising application of immune cells, e.g. T-cells, preferably tumor-specific NK cells, to a subject.
Whether a patient or a tumor is “responsive,” as used herein with respect to a clinical response to treatment, can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction or shrinkage in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment. Responsiveness may also be expressed in terms of various measures of clinical outcome. Positive clinical outcome can also be considered in the context of an individual's outcome relative to an outcome of a population of patients having a comparable clinical diagnosis. In one example, an increase in the likelihood of positive clinical response corresponds to a decrease in the likelihood of cancer recurrence.
In another example, clinical response to treatment can be measured based on disease control (DC), wherein tumors displaying disease control include tumors whose response to treatment is a complete response (CR), partial response (PR) or stable disease (SD). In one example, tumors displaying disease control do not include tumors in a progressive disease (PD) state.
In another example, clinical response to treatment can be measured based on an objective tumor response, e.g., tumor shrinkage, wherein tumors undergoing an objective tumor response include tumors undergoing either a complete response (CR) or a partial response (PR). In one embodiment, tumors undergoing an objective tumor response do not include tumors that display stable disease (SD) or tumors in a progressive disease (PD) state.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
The recitation of a listing of chemical group(s) in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, “one or more” is understood as each value 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and any value greater than 10.
Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.
Cancer is a leading cause of death globally. Immune checkpoint blockade therapies offer a promising new therapeutic strategy for many cancers, but have been relatively ineffective for colorectal cancers. Previous studies have shown that the efficacy of immune checkpoint therapies is modulated by the microbiota. Consequently, we hypothesized that specific gut bacteria could be harnessed to promote immunotherapy for colorectal cancer. Herein, we identify three commensal bacterial species and a microbial metabolite, inosine, that enhance the efficacy of immune checkpoint blockade therapy in colorectal cancer. We show that inosine interacts with the adenosine A2A receptor on T cells resulting in intestinal Th1 cell differentiation. Decreased gut barrier function induced by immune checkpoint blockade increased the translocation of bacterial metabolites and promoted cancer protective Th1 cell activation. This novel microbial-metabolite-immune circuit may provide a mechanism for a new class of bacteria-elicited checkpoint blockade therapies. We show that the efficacy of this mechanism differs among the subtypes of colorectal cancer and highlight the strengths and potential limitations of this novel bacterial co-therapy for cancer.
DNA extraction and purification from feces and cancer epithelial cells was performed using QIAamp Fast DNA Stool extraction kit (Qiagen). The V4 region of the 16S rRNA gene was amplified with barcoded primers (Kozich et al., 2013) using KAPA HiFi polymerase (Roche) under the following cycling conditions: initial denaturation 98° C. for 2 min, 25 cycles of 98° C. for 30 sec, 55ºC for 30 sec, 72ºC for 20 sec and final elongation at 72ºC for 7 min. NucleoMag® NGS (Macherey-Nagel) was used for PCR clean-up and size selection followed by PCR product normalization with the SequalPrep™ Normalization Plate Kit (ThermoFisher) according to the manufacturer's protocols. Individual PCR libraries were pooled, then qualitatively and quantitatively assessed on a High Sensitivity D1000 ScreenTape station (Agilent) and on a Qubit fluorometer (ThermoFisher). 16S rRNA v4 gene amplicon sequencing was performed using a V2-500 cycle cartridge (Illumina) on the MiSeq platform (Illumina). Sequences were demultiplexed and processed using the dada2 pipeline (Callahan et al., 2016) within R. Forward and reverse reads were trimmed to 230 and 210 base pairs, respectively. Dereplicated sequences were merged, and chimeras were identified and removed using the removeBimeraDenovo function. Taxonomy was assigned using the Greengenes database (DeSantis et al., 2006). Differentially abundant taxa were identified using DEseq2 (Love et al., 2014), fit to the mean (base mean intensity threshold 20) and with Benjamini-Hochberg correction applied to calculate adjusted p-values. For weighted UniFrac analysis, PERMANOVA was used (9999 permutations).
AOM/DSS tumors were homogenized in sterile culture media (see below) using a steel bead and a TissueLyser II (Qiagen). Homogenized lysate was streaked on brain heart infusion agar (BHI) and Fastidious Anaerobic Agar (FAA), both supplemented with hemin (5 μg/ml), menadione (0.5 μg/ml), mucin (250 μg/ml), cysteine-HCL (250 μg/ml) and Sodium sulfide nonahydrate (250 μg/ml) (all reagents from SIGMA) in anaerobe conditions (Whitley, A95 Workstation). Single colonies were picked 48 hours later and cultivated in BHA or FAA medium containing the same supplements as the agar plates. 48 hours after liquid culture, bacteria were lysed and full-length 16S rRNA PCR was performed followed by standard Sanger sequencing. Bacteria were identified by using BLAST. 16S rRNA full length PCR was performed with the following primers: forward: AGA GTT TGA TCC TGG CTC AG, and a mix of both reverse1: AGA GTT TGA TCA TGG CTC AG, reverse2: ACG GTT ACC TTG TTA CGA CTT.
C57BL/6J and B6(Cg)-Zbtb46tm1(HBEGF)Mnz/J (CDC-DTR)(Meredith et al., 2012) and C; 129S-Adora2atm1Jfc/J mice were obtained from Jackson and then bred and maintained in house. Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre and Msh2LoxP/LoxP Villin-Cre were kindly provided by Dr. Kevin Haigis and Dr. Winfried Edelman. C57BL/6-Tg(TcraTcrb)1100Mjb/J(Hogquist et al., 1994) (OT-I) mice were bred in house. B6.Cg-Tg(TcraTcrb)425Cbn/J(Barnden et al., 1998) (OT-II) mice were kindly supplied by Dr. Markus Geuking. Global B.6-Adora2atm1Jfc/J (Allard et al., 2019) were obtained from Dr. John Stagg (Barnden et al., 1998). All animals were kept in a 12-hour light-dark cycle on standard 4% fat chow. Offsprings of different SPF (specific pathogen free) breeding pairs were housed together after weaning to minimize cage effects. Germ free C57BL/6J and Rag1−/− mice were bred and maintained in flexible film isolators at the IMC, University of Calgary, Canada. Germ-free status was routinely monitored by culture-dependent and -independent methods and all mice were independently confirmed to be pathogen-free. For experiments, germ-free and monocolonized mice were housed in HEPA filtered Isocages (Tecniplast). Male and female mice between 7-12 weeks were used for experiments. In each experiment mice were age and sex matched and randomly assigned to the different experimental groups. All experiments were performed in accordance with the ethical laws of Alberta and with protocols approved by the Health Sciences Animal Care Committee (AC17-0090 and AC17-0011) following the guidelines set forth by the Canadian Council for Animal Care.
Single cells were isolated from spleen, small intestine, mesenteric-, colon draining- and inguinal lymph nodes. Spleen and lymph nodes were cleaned of fat and connective tissue, minced and digested for 20 minutes at 37° C. in a shaking incubator (220 rpm) in RPMI-1640 supplemented with Collagenase type IA (Sigma) 1 mg/ml and DNase I (Roche) 10 IU/ml. Tissues was then filtered through a 40 μm cell strainer (Thermo Fisher) and resuspended in PBS with 2% heat-inactivated fetal bovine serum (FBS) and 2 mM EDTA. Fat, connective tissue and Peyer's patches were removed from small intestines, which was then cut into 0.5-1 cm small pieces. Tissue pieces were washed in pre-warmed calcium- and magnesium-free HBSS (Sigma) containing 5 mM EDTA (Sigma) at 37° C. in a shaking incubator (220 rpm) for 20 min, twice. Supernatant containing intestinal epithelial cells and intraepithelial lymphocytes was discarded. The remaining tissue pieces were then resuspended in in pre-warmed calcium- and magnesium-free HBSS containing Collagenase type VIII (Sigma) 1 mg/ml and digested for 20-25 min at 37° C. in a shaking incubator (220 rpm). Supernatant was filtered first through a 100 μm and then 40 μm cell strainer (Thermo Fisher). For intracellular staining, cells were plated in a 96-well U-bottom plate (Greiner Bio-One) in 200 μl IMDM supplemented with 10% FBS, 0.05 mM 2-Mercaptoethanol, 50 ng/ml Phorbol 12-Myristate 13-Acetate (PMA), 750 ng/ml lonomycin and 10 μg/ml Brefeldin-A (all Sigma) and incubated at 37° C., 5% CO2 for four hours. Cells were then incubated in Fcγ receptor blocking antibody (BD Biosciences) for 10 min at 4° C. followed by surface staining for 25 min at 4° C. For intracellular staining, cells were fixed and permeabilized using the eBioscience™ Foxp3 Fixation/Permeabilization kit (eBioscience) according to the manufacturer's protocol. Cells were then stained with intracellular markers overnight at 4° C. Prior to acquisition, cells were washed and flow cytometry was performed on a FACSCanto (BD Biosciences). Data was analyzed using Flowjo v10.5.3 (Treestar). The antibodies used are tabulated below:
AOM/DSS tumors were induced as previously described in C57BL/6J mice (Mager et al., 2017; Mertz et al., 2016). In brief, AOM (10 mg/kg/BW) (Sigma) was injected twice at day 0 and 19. A 1% DSS (mpbio) in water solution was given to the animals 3 times at day 7, 19 and 29 for 5 days followed by regular water. Isotype, anti-CTLA-4 or anti-PD-L1 antibodies (all Bio X Cell) were injected 5 times every 72 hours (100 μg/injection) intraperitoneally (i.p.), starting at day 122. Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre mice have been described previously (Haigis et al., 2008). In short, animals have a median survival of 70 days after birth. Isotype or anti-CTLA-4 antibodies were injected 5 times every 72 hours (100 μg/injection) i.p., starting at day 47. In case of microbial transfer co-therapy, antibiotics (ampicillin 1 mg/ml (Sigma), Colistin 1 mg/ml (Cayman Chemical) and streptomycin 5 mg/ml (Sigma), were mixed with water and given ad libidum for 7 days starting at day 40 post birth. Bacteria were given through oral and rectal gavage 5 times every 72 hours starting at day 47. Tumor development in Msh2LoxP/LoxP Villin-Cre mice has been described before (Kucherlapati et al., 2010). The median survival of Msh2LoxP/LoxP Villin-Cre animals is 365 days after birth. Therefore, we started treatment with isotype, anti-CTLA-4 or anti-IL12p40 (500 μg, Bio X Cell) antibodies 319 days after birth 5 times every 72 hours. In case of microbial transfer co-therapy antibiotics, same as described above, were given for 7 days starting at day 312 and bacteria were supplied orally through gavage 5 times every 72 hours starting on day 319. For heterotopic cancer models, 1×106 cancer cells were injected subcutaneously (s.c.) in the flank of germ-free, monocolonized or SPF mice. Once tumors were palpable (7-10 days post injection) isotype or anti-CTLA-4 antibodies were injected 5 times every 72 hours (100 μg/injection i.p.). For serum transfer experiments, germ-free mice received pooled serum from animals shown in
For DC depletion experiments, chimeric mouse generation was adapted from previous reports (Mager et al., 2015; Meredith et al., 2012). In short, C57BL/6J mice were lethally irradiated with 1100 cGy, split into two sessions of 550 cGy each 4 hours apart in a Gamma Cell Exactor 40 (Nordion). Mice were then injected i.v. with 1×107 whole bone marrow from cDC-DTR mice, followed by two weeks of antibiotic treatment in the drinking water (ampicillin 1 mg/ml, Colistin 1 mg/ml and streptomycin 5 mg/ml). Mice then received normal drinking water and were gavaged with a mixture of ICB-promoting bacteria (B.p., L.j., O.sp.). 1×106 cancer cells were injected s.c. in the flank 8 week and DC depletion was initiated with diphtheria toxin (100 ng every 48 hours i.p., Sigma) 9 weeks after irradiation and bone marrow reconstitution). Anti-CTLA-4 was started one day after the first diphtheria toxin injection and given 5 times every 72 hours. Tumors were measured every 72 hours using a caliper (length×width×height×π/6) and weighed at the end of the experiment on a fine scale (Mettler Toledo).
MC38 parental strain, MC38-EGFP and MC38-OVA colorectal cancer cells were kindly provided by Dr. Charles Drake. Cells were tested for contamination (Charles River) initially and thereafter screened for absence of Mycoplasma every 6-8 weeks (PCR Mycoplasma detection kit, Thermo Scientific). MC38 cell were maintained at 37° C. under 5% CO2 in IMDM supplemented with 10% heat-inactivated FBS (Sigma), 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids (all Thermo Fisher). MB49 and B16F10 cells were cultured in IMDM supplemented with 10% FBS (Sigma), 100 units/ml penicillin.
Bone marrow derived dendritic cells (BMDCs) were generated from flushed bone marrow cells, maintained in RPMI-1640 (Sigma) supplemented with 10% FBS, 50 μm 2-Mercaptoethanol (Sigma) 100 units/ml penicillin, 100 μg/ml streptomycin sulfate and 20 ng/ml rm GM-CSF (R&D). Medium was exchanged after 48 hours and 72 hours. 5 days after culture, a magnetic cell sorting step was performed to enrich for CD11c+ cells (Miltenyi Biotec). CD11c+ cells were seeded in 96 flat bottom wells and pulsed with 20 ng/ml OVA323-339 and 100 ng/ml LPS (both Sigma) for 18 hours. In some conditions BMDCs were also cultured with 10 ng/ml rmIFN-γ (R&D).
Negative selection magnetic cell sorting (Miltenyi Biotec) was used to enrich naïve OT-II CD4+ T cells. Naïve OT-II CD4+ T cells were then co-cultured with BMDCs at a ratio of 2:1 or stimulated with anti-CD3/anti-CD28 T cell activation beads (Thermofisher) at a ratio of 1:1 for 48 hours prior to restimulation with PMA/lonomycin in the presence of Brefeldin-A and analysis (see Single cell preparation and flow cytometry for details). In some conditions cells were additionally cultured with various combinations of 2 μg/ml anti-CTLA-4, 100 μM db-cAMP (Sigma), 5 μM ZM 241385 (Sigma), 300 μM Rp-8-CPT-CAMPS (Cayman Chemical) or 2 mM inosine (Sigma) as described previously (He et al., 2017; Yao et al., 2013).
Naïve CD4 T-cells were MACS-purified (Miltenyi) according to the manufacturer's protocol. RNA was purified using TRI-reagent (Sigma-Aldrich). RNA was transcribed into cDNA using iScript™ (BioRad). PerfeCTa SYBR Green (Quanta Bio) was used to detect the target genes Il2rb1, Ifng, and Gapdh (QIAGEN). Expression levels of genes were normalized to Gapdh mRNA, and medium versus inosine stimulated groups were compared applying the 2−ΔΔCT method.
Ussing chamber measurements were performed as previously described (Mager et al., 2017). In brief, one intestinal section (approximately 3 cm long) per mouse was collected from the middle of the small intestine, taking care to exclude Peyer's patches. Electrical resistance was measured in 37% oxygenized HBSS after approximately 10 to 15 min of equilibration time.
Anti-commensal serum antibodies were measured as described before (Mager et al., 2017). Briefly, B.p. or C.sp. were cultured in anaerobe conditions and then diluted to an O.D.600 of 0.07. Bacteria were then inactivated using sodium-azide. Serum from germ-free, B.p. or C.sp. monocolonized mice, treated with or without anti-CTLA-4 was heat-inactivated and incubated with bacterial pellets. Fluorescent secondary antibodies against IgG1 and IgG2b were then used to detect systemic antibodies against pure cultured bacteria. Serum cytokines were by Multiplexing LASER Bead Technology (Eve Technologies).
Metabolites in serum or bacterial cultures were extracted in 50% methanol, centrifuged, and the resulting supernatants were diluted into a linear range for mass spectrometry analysis (1:20 final dilution for microbial cultures and 1:50 total dilution for serum). Ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) data were then acquired on a Q Exactive™ HF Mass Spectrometer (Thermo Scientific) in negative ion full scan mode (50-750 m/z) at 240,000 resolution. Metabolites were separated via UHPLC using a binary solvent mixture of 20 mM ammonium formate at pH3.0 in LC-MS grade water (Solvent A) and 0.1% formic acid (% v/v) in LC-MS grade acetonitrile (Solvent B) in conjunction with a Syncronis™ column (Thermo Fisher Scientific 97502-102130). Samples were analyzed using a flow rate of 600 uL/min using the following gradient: 0-2 mins, 100% B; 2-7 mins, 100-80% B; 7-10 mins, 80-5% B; 10-12 mins, 5% B; 12-13 mins, 5-100% B; 13-15 mins, 100% B. For all runs the sample injection volume was 2 uL. Metabolite data were analyzed using the XCMS (Gowda et al., 2014; Tautenhahn et al., 2012) and MAVEN software packages (Clasquin et al., 2012; Melamud et al., 2010). Metabolites were identified by matching observed m/z signals (+/−10 ppm) and chromatographic retention times to those observed from commercial metabolite standards (Sigma). Inosine assignments, a key metabolite for this study, were confirmed via MS/MS fragmentation patterns using parallel reaction monitoring. These assignments were further validated by spiking inosine standards into microbial extracts to demonstrate co-retention and matching fragmentation patterns between the observed biomarker and a 50 UM inosine standard.
To evaluate the effect of inosine on Th1 activation, mice received 30 μg CpG, 100 mg EndoFit Ovalbumin (both Invivogen) and 2 μg OVA323-339 (Sigma) i.p. and 24 hours later mice received 300 mg/kg/BW inosine or PBS as a control through i.p. injection. T cell differentiation was assessed 48 hours later. To assess the impact of inosine on tumor development during ICB therapy 1×106 cancer cells were injected subcutaneously (s.c.) in the flank of germ-free mice. Once tumors were palpable (7-10 days post injection) 100 μg anti-CTLA-4 antibodies and 20 μg CpG were injected 5 times every 72 hours injection i.p. 24 hours following the first anti-CTLA-4/CpG treatment mice received 300 mg/kg/BW inosine daily orally (gavage) or systemically (200 μl i.p. and 50 μl s.c.) until the end of the experiment. WT or A2A deficient cells were isolated from spleens using CD4/CD8 (TIL) MicroBeads (Miltenyi). T-cell purity was >95% and 1×107 T-cells were transferred i.v.
SYTOX green nucleic acid stain (Thermo Fisher) was performed according to the manufacturer's instructions. In brief, homogenized feces or tumor tissue was fixed in a 4% paraformaldehyde solution (Sigma) for 30 minutes. Subsequently, samples were diluted in PBS at a ratio 1:5 and stained with SYTOX green nucleic acid stain for 60 minutes prior to picture acquisition (Leica DM2500). 16SrRNA full length PCR was performed as described in above (“In vitro culture of bacteria and full 16SrRNA gene sequencing”).
GraphPad Prism v.5.04 for Windows was used. If variance between groups was similar parametric tests were used, such as standard student t test or one-way ANOVA with Bonferroni post-test, in case of significantly different variance between groups non-parametric tests, such as Mann Whitney U test or Kruskal Wallis with Dunn's post-test, were applied. Two-way ANOVA with Bonferroni post-test was used for tumor growth curves. Survival was analyzed using the Mantel-Cox Log-rank test. Other tests are denoted in the corresponding figure or table legend. Only statistically significant differences are indicated in the figures. For all statistical analyses: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Exact P values and statistical tests used for each panel are reported in the source data.
We first questioned whether ICB therapy efficacy in CRC is dependent on the microbiota. Heterotopic MC38 colorectal cancers were implanted into germ-free (GF) and specific pathogen free (SPF) mice and, upon palpable tumor development, ICB therapy was initiated, which led to smaller tumors in SPF animals (
Clinically, ICB therapies are notoriously ineffective in most CRC cases (Le et al., 2015) and heterotopic tumors may not adequately model the spatially close interactions between the gut microbiota and local immunity in intestinal tumors. We therefore employed a more physiological model of CRC to investigate the interactions between the microbiota and immunity in the context of ICB therapy. Intestinal tumors were induced using azoxymethane (AOM) and dextran sulfate sodium (DSS) in SPF animals. Following tumor development, we evaluated the ability of ICB therapy to induce anti-tumor immunity (
Although no significant changes were observed in the overall fecal bacterial composition (β-diversity) between ICB-treated and control mice (
Interestingly, Akkermansia muciniphila, which was recently identified to enhance the efficacy of anti-PD-L1 and anti-PD-1 treatments in lung and kidney cancers (Routy et al., 2018), was one of the seven bacteria cultured only from ICB-treated tumors. We also performed 16S rRNA gene V4 amplicon sequencing of fecal samples from control and ICB groups but found no significant differences in microbiota composition (
To address whether the bacteria that were found to be enriched in the ICB-treated tumors were able to boost the efficacy of ICB therapy, we selected five of the isolated culturable bacterial species for monocolonization of GF mice. Monocolonized or GF mice were injected with MC38 tumor cells, treated with anti-CTLA-4 upon palpable tumor development and assessed for effects on tumor growth and anti-tumor immunity (
In addition, CD4+ and CD8+ T cell activation and proliferation were substantially increased in the tumors of B.p., L.j., and O.sp. monocolonized animals (
To investigate the mechanism by which the identified bacteria enhanced ICB therapy we selected B.p. as a representative of the beneficial bacteria since it appeared to have the strongest ICB-promoting effect. GF or C.sp. monocolonized served as negative controls. Previous studies revealed the ability of some bacteria to accumulate in the tumor environment where they locally stimulate the immune system and kill tumor cells through toxic metabolites (Zheng et al., 2018). Although bacteria were abundantly present in the feces of B.p. and C.sp. monocolonized mice, we were unable to detect bacteria or amplify 16S rDNA from heterotopic tumors of these mice (
Since B.p. alone promoted only local and not systemic Th1 differentiation during homeostasis, we next asked whether the combination of B.p. monocolonization and anti-CTLA-4 therapy (in the absence of a tumor) would induce systemic Th1 activation. Indeed, when combined with anti-CTLA-4, B.p. was able to significantly enhance splenic Th1 cell activation and effector function as evidenced by IFN-γ production compared to C.sp. monocolonized or GF animals (
We were intrigued by the ability of B.p. to induce Th1 transcriptional differentiation during homeostasis versus activation of effector function following ICB treatment. Gastrointestinal inflammation is a common immune-related adverse effect of anti-CTLA-4 treatment (Hodi et al., 2010) and we reasoned that this may be due to alterations in gut barrier integrity. Indeed, animals treated with anti-CTLA-4 had increased systemic serum anti-commensal antibodies, particularly Th1-associated IgG2b (Germann et al., 1995), and reduced small intestinal transepithelial electrical resistance compared to controls (
In order to identify putative metabolites that might be responsible for the anti-tumor effects of the transferred serum, we determined the metabolomic profile of the transferred serum samples and identified metabolites that were increased in the serum of mice monocolonized with B.p. compared to C.sp. or GF mice. Untargeted metabolomics analysis revealed increased levels of several metabolites in sera from B.p. compared to C.sp. monocolonized and GF mice (
We next investigated whether inosine could directly enhance anti-tumor Th1 cell differentiation. To test this, we co-cultured activated OVA323-339 peptide-pulsed bone marrow derived dendritic cells (BMDCs) with naïve OVA323-339-specific OT-II CD4+ T cells in the presence or absence of inosine. Intriguingly, inosine led to context-dependent induction or inhibition of CD4+ T cell differentiation. Specifically, in the presence of exogenous IFN-γ, inosine strongly boosted Th1 differentiation of naïve T cells (
We next wondered if inosine could directly affect tumor cell survival or susceptibility to T cell-mediated killing. Direct exposure of MC38 tumor cells to inosine in vitro did not exert any effects on tumor cell viability (
Combined, these data suggest that the effect of inosine on T cells required sufficient co-stimulation (likely by DCs), IL-12 receptor engagement for Th1 differentiation and IFN-γ production for efficient anti-tumor immunity. Classical dendritic cells (cDCs) were found to be the primary source of IL-12 compared to macrophages (
To confirm whether the inosine-mediated Th1 promoting effect in vitro also applied to in vivo conditions, GF mice were immunized with ovalbumin in combination with CpG as a co-stimulus. One day later mice received inosine or vehicle only intraperitoneally. Inosine increased the proportions of T-bet+, IFN-γ+ CD8+ and CD4+ T cells in the MLN (
We then confirmed that inosine-induced anti-tumor immunity was dependent on A2AR signaling in vivo. Germ-free Rag1-deficient animals were challenged with MC38 tumor cells and simultaneously received WT or A2AR-deficient T cells. Seven days later, inosine was given orally in combination with anti-CTLA-4 and CpG (
Since we detected A. muciniphila in ICB-treated tumors, that was previously shown to increase ICB therapy efficacy and to produce inosine in vitro, we further investigated whether A. muciniphila also relies on A2AR signaling to enhance ICB-therapy efficacy. We found that monocolonization with A. muciniphila in combination with anti-CTLA-4 led to smaller tumors and increased anti-tumor immunity and this was dependent on T cell expression of A2AR (
We next tested whether inosine could also promote the efficacy of anti-CTLA-4 therapy in the presence of a complex microbiota. We first utilized a gnotobiotic model where mice are stably colonized with a defined microbiota consisting of 12 bacterial species, referred to as Oligo-Mouse-Microbiota-12 (Oligo-MM12), which lacks B. pseudolongum. We found that inosine was able to promote the anti-tumor effects of anti-CTLA-4 with reduced tumor size and increased intra-tumoral IFN-γ+CD8+ and IFN-y+CD4+ T cells even in gnotobiotic Oligo-MM12 mice (
Taken together, inosine-A2AR signaling drives or inhibits anti-tumor immunity in vivo, depending on the amount of co-stimulation present.
Lastly, we examined the effect of the identified ICB-promoting bacteria in two distinct models of CRC that mimic different subtypes of human CRC. First, we tested the ICB-promoting effect of B.p., L.j., and O.sp. in Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre (Haigis et al., 2008) SPF mice, which have conditional Apc deficiency and activation of Kras specifically in colonocytes. In this model of CRC, anti-CTLA-4 treatment alone did not improve survival compared to isotype-treated animals (
As B. pseudolongum was enriched in AOM/DSS tumors of ICB-treated animals and Bifidobacteria were previously associated with improved ICB-therapy efficacy in cancer patients, we wondered whether Bifidobacteria were also enriched in Msh2LoxP/LoxP Villin-Cre tumors of ICB-treated mice. While the total amount of tumor-associated bacteria did not change with anti-CTLA-4 or anti-PD-L1 treatment (
In summary, our results reveal a novel bacterial-inosine-immune pathway that boosts a cDC-dependent Th1 T cell circuit to greatly enhance the effect of ICB therapies in CRC (
Next, we investigated whether ICB-promoting properties were a conserved feature among multiple Bifidobacterium species. To test this, we monocolonized GF mice with different Bifidobacterium species, challenged these mice with MC38 colorectal cancer cells and treated them with anti-CTLA-4. Intriguingly, some but not all Bifidobacterium species tested improved ICB therapy efficacy (
Moreover, we also tested if the Bifidobacterium pseudolongum strain isolated by us improved the efficacy of another ICB, anti-PD-1. Indeed, compared to our control bacterium (Colidextribacter sp.), B. pseudolongum together with anti-PD-1 improved anti-tumor immunity against MC38 tumor cells (
ICB therapy has yielded rather disappointing results in CRC, with an objective response only in 40% of patients with the mismatch repair deficient (MMRD) sub-type of CRC, which amounts to only 4% of all CRC (Le et al., 2015). We have now identified a novel microbial-metabolite-immune circuit that enhances ICB therapy in two mouse models of CRC. These data indicate that modification of the microbiota may provide a promising adjuvant therapy to ICB in CRC. Of note, compared to anti-PD-L1, anti-CTLA-4 induced stronger anti-tumor effects in the AOM/DSS and heterotopic tumor models when both antibodies were administered at the same dose. At this point it is difficult to know if this is due to differences in the biological effects of blocking CTLA-4 versus PD-L1 in these models, but it should be noted that other experimental studies routinely use anti-PD-1 mAb at much higher does than anti-CTLA4 mAb (Routy et al., 2018).
By isolating tumor-associated bacteria we have identified several bacterial species that were found to be associated exclusively with tumors following treatment with ICB, with three of these bacteria able to significantly enhance the efficacy of ICB therapy in CRC. This suggests that the isolation of bacterial species from intestinal tumor biopsies rather than from feces may be a better approach in a clinical setting for defining ICB-promoting bacteria in CRC. Although isolated from mice, all three ICB-promoting bacteria are also found in humans, indicating their potential for clinical translation (Dewhirst et al., 2001; Pridmore et al., 2008; Turroni et al., 2009). Furthermore, we analyzed published human fecal microbiome metagenomic datasets and found a trend, although not significant, where B. pseudolongum was enriched [up to 2.4-fold] in responders compared to nonresponding cancer patients (
Our findings demonstrate a critical role for the bacterial metabolite inosine in setting a baseline Th1 level in local mucosal tissues. Initially, this was surprising because previous reports have demonstrated an inhibitory effect of inosine, and A2AR engagement in general, on Th1 differentiation in vitro and anti-tumor immunity in vivo (Csoka et al., 2008; Hasko et al., 2000; He et al., 2017; Ohta et al., 2006). Indeed, the wealth of data supporting an immunosuppressive role for adenosine and A2AR signaling has led to the development of novel immune checkpoint inhibitor targets, such as mAb targeting CD73, CD39 and CD38, and pharmacological antagonists of A2AR, many of which are currently in clinical trials (reviewed in (Vigano et al., 2019)). However, a small body of literature has demonstrated that inosine can be pro-inflammatory and A2AR signaling can sustain Th1/anti-tumor immunity in mice (Cekic and Linden, 2014; Lasek et al., 2015; Lioux et al., 2016). Our findings reconcile these contrasting observations by revealing a context-dependent effect of inosine-A2A receptor signaling based on the amount of co-stimulation. Mechanistically, inosine engages the A2A receptor and activates the transcription factor CREB, through cAMP. CREB, together with co-factors and the formation of heterodimers with ATF-2 and/or c-Jun, modulates the transcription of key Th1 genes, including Il2rb2 and Ifng (Samten et al., 2008). It is worth noting that in addition to CAMP signaling, inosine (compared to adenosine) has a distinct A2AR-dependent signaling bias, with a 3.3-fold preference for ERK1/2 phosphorylation. In light of our findings, blockade of inosine-A2A receptor signaling in cancer immunotherapy could negate a positive effect provided by beneficial microbes. We suggest that A2A receptor signaling is likely an integral anti-tumor pathway for bacterial-ICB co-therapies. Indeed, Tanoue et al recently identified a consortium of eleven bacteria that improve ICB therapies (Tanoue et al., 2019), which are not related to the bacteria identified in this work. Remarkably though, two of the most elevated metabolites in the cecum and serum of mice colonized with the consortium of 11 bacteria were inosine monophosphate and hypoxanthine, a substrate and product of inosine respectively, which are both A2A receptor agonists like inosine (Welihinda et al., 2016). The identification of this context-dependent effect of inosine-A2A receptor signaling is particularly relevant as inosine is currently used as an intervention in clinical trials in various Th1-associated diseases (Clinical.trials.gov), including multiple sclerosis, amyotrophic lateral sclerosis and Parkinson's disease (Bettelli et al., 2004; Kustrimovic et al., 2018; Lovett-Racke et al., 2004; Saresella et al., 2013).
We identified cDCs and their production of IL-12 as essential components for efficient induction of anti-tumor T cell immunity elicited by ICB therapy in the presence of beneficial bacteria. The critical involvement of cDC and IL-12 has also been recently reported upon anti-PD-1 treatment (Garris et al., 2018).
Seminal work by Guinney et. al revealed four molecular consensus subtypes of CRC (Guinney et al., 2015); MMRD, canonical, metabolic and mesenchymal. In line with the positive results of ICB in MMRD patients in the clinical setting (Le et al., 2015), in our animal model of MMRD (Msh2LoxP/LoxP Villin-Cre) we indeed observed some efficacy of anti-CTLA-4 single therapy. However, co-therapy with ICB-promoting bacteria strongly enhanced the tumoricidal effect of anti-CTLA-4. Thus, bacterial co-therapy may optimize treatment regimens in MMRD CRC patients. Secondly, ICB therapy was efficacious and was associated with B. pseudolongum, L. johnsonii, and Olsenella sp. in the AOM/DSS model of CRC. AOM/DSS tumors have been used to model inflammation-associated CRC. AOM/DSS tumors also display characteristics of epithelial to mesenchymal transition (Lin et al., 2015), such as reduced E-Cadherin, increased N-Cadherin, Vimentin and SNAIL expression as well as inflammation and increased TGF-β expression (Becker et al., 2004; Mager et al., 2017), which are hallmarks of the mesenchymal consensus molecular CRC subtype (Guinney et al., 2015). Thus, our results indicate a benefit of bacterial co-therapy also in this subtype. Canonical and metabolic CRC subtypes are both characterized by inactivation of Apc, canonical additionally by Wnt pathway and metabolic by KRAS activation (Guinney et al., 2015). These hallmarks are well represented in the Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre animal model and intriguingly bacterial co-therapy did not improve anti-CTLA-4 treatment. The divergent effect of ICB-promoting bacteria in the Msh2LoxP/LoxP Villin-Cre- compared to the Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre model is intriguing and at this stage we can only speculate about the underlying reason(s). The mutational load and associated number of neoantigens, which is likely higher in Msh2LoxP/LoxP Villin-Cre tumors, certainly impacts on the efficacy of ICB therapies (Havel et al., 2019). Moreover, anti-CTLA-4 had no effect on its own in the Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre model and bacteria alone did not impact on heterotopic tumor development. We also showed that B.p. increased the Th1 cell pool and their anti-tumor effect was unleashed followed by effective ICB therapy. Thus, we reason that the discovery of novel checkpoint blockade targets or other therapies that have an effect of their own in the Apc2lox14/+; KrasLSL-G12D/+; Fabpl-Cre model are required to enable efficacious bacterial co-therapy to treat similar subtypes in CRC patients.
Together, this work paves the way for new approaches to treatment of cancers including CRC.
The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
Exemplary aspects of the invention are specified by the following embodiments.
Additional Exemplary aspects of the invention are specified by the following embodiments.
The present application claims the benefit of U.S. Provisional Application Ser. No. 62/929,340 filed Nov. 1, 2019, the disclosure of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051487 | 11/2/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62929340 | Nov 2019 | US |
