WO2022008890A1 - Cancer treatment - Google Patents

Cancer treatment Download PDF

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Publication number
WO2022008890A1
WO2022008890A1 PCT/GB2021/051706 GB2021051706W WO2022008890A1 WO 2022008890 A1 WO2022008890 A1 WO 2022008890A1 GB 2021051706 W GB2021051706 W GB 2021051706W WO 2022008890 A1 WO2022008890 A1 WO 2022008890A1
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Prior art keywords
cancer cell
interferon
cancer
treatment
hybrid
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PCT/GB2021/051706
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French (fr)
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William Stimson
Chris Mckenzie
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ILC Therapeutics Limited
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Publication of WO2022008890A1 publication Critical patent/WO2022008890A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the field of cancer therapy in general and the treatment of a cancer cell(s) wherein the cancer cell comprises a decrease in expression of an interferon.
  • the Type I IFN receptor consists of two chains, IFNR1 and IFNR2. There is a range of binding affinities for each of the 12 IFN-a subtypes with the different receptor chains.
  • the present inventors have considered ways in which defects in the interferon signalling pathway which result in immuno-resistance can be mitigated. This would be advantageous to allow improved treatment of cancer, in particular to cancer cells that have become insensitive or escaped conventional cancer therapies.
  • the inventor has made the surprising discovery that the administration of a specific interferon alpha (IFN-a) subtype which is a hybrid of IFN-a10 and IFN-a14, preferably wherein the hybrid includes the primary interferon receptor binding sites of IFN-a10 and IFN-a14 can be advantageously used to treat cancer, in particularly glial cancer, wherein the cancer cells comprise a decrease in expression of an interferon.
  • the decrease in expression may be due to a deletion, insertion, or mutation of the interferon gene.
  • a decrease may be considered relative to a non-tumour cell of corresponding or similar cell type.
  • a decrease may be considered relative to a tumour cell of corresponding or similar cell type without a deletion, insertion, or mutation of the interferon gene.
  • a decrease may be at least 5% of a level provided in a non-tumour cell of corresponding cell type, at least 10% of a level provided in a non-tumour cell of corresponding cell type, at least 20% of a level provided in a non-tumour cell of corresponding cell type, at least 30% of a level provided in a non-tumour cell of corresponding cell type.
  • a decrease may be at least 5%, at least 10% of a level provided in a tumour cell of corresponding cell type without a deletion, insertion, or mutation of the interferon gene, at least 20% of a level provided in a tumour cell of corresponding cell type without deletion, insertion, or mutation of the interferon gene, at least 30% of a level provided in a tumour cell of corresponding cell type without deletion, insertion, or mutation of the interferon gene.
  • Interferon subtypes IFN-alpha10 and IFN- alpha14 and hybrids thereof are discussed in PCT Publication Number WO2014/037717 and PCT Application Number PCT/GB2015/050717.
  • IFN-alpha10 and IFN-alpha14 hybrids are disclosed that contain sequences characteristic of the IFN-alpha10 and IFN-alpha14 subtype binding sites based on a consensus backbone sequence of all 12 alpha- interferons, for example CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVL HEMMQQTFNLFSTENSSAAWEQTLLEKFSIELFQQMNDLEACVIQEVGVEETPLMN EDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO: 3). It would be advantageous to provide alternative hybrids and further compositions and methods that provide alternative immunotherapeutic approaches.
  • composition comprising an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
  • a method for the treatment and / or prophylaxis of a cancer wherein the cancer cell comprises a decrease in expression of an interferon comprising the step of administering to a subject in need thereof an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
  • SEQ ID NO: 1 may comprise several amino acid substitutions of protein comprising an IFN-a10 amino acid sequence with amino acids of IFN-a14 determined to be involved in binding to interferon receptor 1. Suitably this may enhance the binding of the protein to interferon receptor 1.
  • an amino acid substitution of protein comprising an IFN-a14 amino acid sequence with amino acids of IFN-a10 determined to be involved in binding to interferon receptor 2 may enhance the binding of the protein to interferon receptor 2.
  • the hybrid sequence may include at least one, at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 modifications of SEQ ID NO: 1 , suitably modifications of SEQ ID NO: 1 such that the portions of the hybrid sequence based on the IFN-a10 sequence are changed to the corresponding residues of the IFN-a14 sequence.
  • IFN-a14 can be utilised as a backbone structure of the hybrid and the residues which differ between the IFN-a10 and IFN-a14 sequences at the N and C terminal regions of the sequences can be provided in the hybrid sequence as those present in the IFN-a10 sequence.
  • At least 1, at least 2, or 3 substitutions are provided at the IFN-a14 C terminal sequence to provide residues from IFN-a10 to the hybrid sequence SEQ ID NO: 1 at those amino acid positions which are not shared/common between IFN-a10 and IFN-a14.
  • at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 substitutions from the N-terminal sequence and at least 1 , at least 2, or 3 substitutions from the C-terminal sequence of the IFN-a14 are made to provide residues from IFN-a10 to the hybrid SEQ ID NO: 1 at those amino acid positions which have amino acids that are not shared/common between IFN-a10 and IFN-014.
  • the hybrid comprises or consists of an amino acid sequence SEQ ID NO: 1 or a functionally active fragment or variant thereof.
  • the cancer cell comprises a decrease in expression of interferon due to the cancer cell having a mutation to the interferon gene which renders it decreases the expression rate or decreases the amount of functional interferon produced, such as a deletion or insertion (to cause a codon shift), or a mutation to a stop codon, or mutation of key residues involved in structure or activity (e.g. a cysteine bridge).
  • the cancer cell comprises a decrease in expression of a type I interferon, suitably an alpha interferon.
  • the term “functionally active” is construed to mean an IFN-a10 IFN-a14 hybrid peptide comprising the primary interferon binding sites of IFN-a10 and IFN-a14 wherein the administration of peptide to a subject or expression of peptide in a subject promotes enhancement of Th1 mediated immune response and suppression of a Th2/Th17 mediated immune response.
  • a fragment can comprise at least 50, preferably 100 and more preferably 150 or greater contiguous amino acids from SEQ ID NO: 1 and which is functionally active.
  • a fragment may be determined using, for example, C-terminal serial deletion of cDNA such as SEQ ID NO: 2 or SEQ ID NO: 3. Said deletion constructs may then be cloned into suitable plasmids. The activity of these deletion mutants may then be tested for biological activity as described herein.
  • variant is meant an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, even more preferably with at least 95% sequence identity to SEQ ID NO: 1, even more preferably with at least 96% sequence identity to SEQ ID NO: 1, even more preferably with at least 97% sequence identity to SEQ ID NO: 1 and most preferably with at least 98% sequence identity, at least 99% sequence identity with SEQ ID NO: 1.
  • a variant encompasses a polypeptide sequence of SEQ ID NO: 1 which includes substitution of amino acids, especially a substitution(s) which is/are known for having a high probability of not leading to any significant modification of the biological activity or configuration, or folding, of the protein. These substitutions, typically known as conserved substitutions, are known in the art.
  • amino acids of the same charge, size or hydrophobicity may be substituted with each other.
  • any substitution may be selected based on analysis of amino acid sequence alignments of interferon alpha subtypes to provide amino acid substitutions to amino acids which are present in other alpha subtypes at similar or identical positions when the sequences are aligned.
  • Hybrids, and variants and fragments thereof may be generated using suitable molecular biology methods as known in the art. The skilled person would be aware of the way sequence identity can be determined using methods and suitable computer programs as known in the art. Suitably default parameters where appropriate for such computer programs could be utilised.
  • the cancer cell has a homozygous deletion of an interferon alpha gene.
  • the cancer cell has a deletion of or within the interferon gene cluster located at the loci 9p21.
  • the cancer cell has a homozygous deletion of or with the interferon gene cluster at the loci 9p21.
  • the 16 type I interferon genes are located on 9p21, which includes 13 IFN-a genes, 1 IFN-b, 1 IFN-e and 1 IFN-w gene.
  • the cancer cell may be a cell from a cancer selected from melanoma, glioma, suitably a brain lower grade glioma, a glioblastoma, in particular glioblastoma multiforme, prostrate adenocarcinoma, colorectal cancer, lymphoid neoplasias, acute lymphoblastic leukaemia and non-Hodgkin lymphoma, bladder cancer, breast cancer, prostate cancer, or lung cancer.
  • the cancer may be a cancer selected from a glioma, in particular a brain lower grade glioma, a glioblastoma, in particular glioblastoma multiforme, bladder cancer or lung cancer.
  • the hybrid interferon of the invention may be provided in combination with an immunotherapy treatment for a cancer.
  • an immunotherapy treatment may be a monoclonal antibody with binding specificity to a checkpoint inhibitor, for example Pembrolizumab, an immune checkpoint inhibitor.
  • a checkpoint inhibitor for example Pembrolizumab, an immune checkpoint inhibitor.
  • an immunotherapy may be Nivolumab.
  • a combination treatment may be provided with Galunisertib which is considered to enhance anti-TGF-beta effects.
  • the hybrid interferon of the invention may be provided in combination with a monoclonal antibody cancer treatment, for example a monoclonal antibody targeting CTLA4.
  • a combination treatment may comprise a monoclonal antibody targeting CTLA4, for example Ipilimumab and a hybrid interferon the present invention.
  • a combination treatment for use in the treatment of melanoma may comprise a monoclonal antibody targeting CTLA4, for example Ipilimumab and a hybrid interferon the present invention.
  • the hybrid interferon of the invention may be provided in combination with NK and / or T cell therapies.
  • the hybrid interferon of the invention may be provided in combination with treatments selected from, for example Temozolomide or Bevacizumab, Cetuximab, Panitumumab, Ramucirumab, Monamulizumab, Obinutuzumab, Rituximab.
  • the hybrid interferon of the invention may be provided in combination with chemotherapeutics.
  • treatment is used herein to refer to any regimen that can benefit a human or non-human animal.
  • the treatment may be in respect of an existing condition and the treatment may be prophylactic (preventative treatment).
  • Treatment may include curative or alleviative effects.
  • Reference herein to "therapeutic” and “prophylactic” treatment is to be considered in its broadest context.
  • the term “therapeutic” does not necessarily imply that a subject is treated until total recovery.
  • “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition.
  • therapeutic and/or prophylactic treatment includes amelioration of the symptoms of a particular allergic condition or preventing or otherwise reducing the risk of developing a particular allergic condition.
  • the term “prophylactic” may be considered as reducing the severity or the onset of a particular condition.
  • “Therapeutic” may also reduce the severity of an existing condition.
  • a hybrid IFN-a10 and IFN-a14 subtype, for example SEQ ID NO: 1, as described herein can be administered separately to the same subject, optionally sequentially, or can be co-administered simultaneously with another agent, for example a chemotherapeutic or immunotherapy or a NK or T cell as a pharmaceutical, immunogenic composition.
  • the pharmaceutical composition will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration.
  • the active ingredients can be administered to a patient in need of treatment via any suitable route.
  • the precise dose will depend upon a number of factors, as is discussed below in more detail.
  • Suitably administration may be daily, for example by cannula or implanted as a PEG slow release formulation at a time of surgery.
  • One suitable route of administration is parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch).
  • Other suitable routes of administration include (but, are not limited to) oral, ocular, nasal, topical (including buccal and sublingual), infusion, intradermal or administration via oral or nasal inhalation, by means of, for example, a nebuliser or inhaler, or by an implant.
  • Preferable routes of administration include (but, are not limited to) oral, buccal and sublingual.
  • the compositions of the invention may also be administered in such a manner that they are directed to, or released in, specific areas of the gut intestinal tract (such as the small intestine/duodenum). Typically such release will occur after passage through the stomach, this targeted release being achievable through the use of coatings and the like.
  • the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection, Lactated Ringer’s injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions of the present invention for oral administration may be in tablet, capsule, lozenge, powder or liquid form. Oral administration may involve placing a lozenge under the tongue of the patient.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • compositions of the present invention may also be administered via microspheres, liposomes, other micro-particulate delivery systems or sustained release formulations placed in certain tissues including blood.
  • sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington’s Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition (December 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7 th Edition ISBN 0-683305-72-7, the entire disclosures of which are herein incorporated by reference.
  • compositions according to the present invention may comprise, in addition to an active ingredient, a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation.
  • the formulation may be a liquid, for example, a physiologic salt solution containing non phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze-dried powder.
  • the composition is preferably administered to an individual in a “therapeutically effective amount” or a “desired amount”, this being sufficient to show benefit to the individual.
  • an "effective amount” means an amount necessary to at least partly obtain the desired response, or to delay the onset or inhibit progression or halt altogether the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation and other relevant factors. It is expected that the amount will fall in a relatively broad range, which may be determined through routine trials. Prescription of treatment, e.g.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
  • a broad range of doses may be applicable.
  • Considering oral administration to a human patient for example, from about 10 pg to about 1000 pg of agent may be administered per human dose, optionally for 3 to 4 doses.
  • Dosage regimes may be adjusted to provide the optimum therapeutic response and reduce side effects. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.
  • “About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.
  • Figure 1 Indicates an IFN-a Hybrid of the present invention induced secretion of Granzyme B from Leukocytes
  • FIG. 1 Indicates an IFN-a Hybrid of the present invention inhibited production of TGB-beta from endothelial cells
  • Figure 3 Indicates inhibition of b-catenin signaling by an IFN-a Hybrid of the present invention in the Ovm1 ovarian cancer cell line.
  • Figure 4 Indicates suppression of IL-17A synthesis from human leucocytes by an IFN-a Hybrid of the present invention.
  • Figure 5 Indicates suppression of IL-17F synthesis by an IFN-a Hybrid of the present invention.
  • Figure 6. Indicates enhanced suppression of CXCL-10 synthesis from human leucocytes by an IFN-a Hybrid of the present invention, compared to rIFN-a 2a or rlFN-b 1a.
  • Figure 7 Indicates enhanced suppression of CXCL-10 synthesis from human leucocytes by an IFN-a Hybrid of the present invention, compared rlFN-b 1a.
  • Figure 8 Indicates the enhanced induction of lnterferon-g production from human leucocytes with IFN-a Hybrid of present invention in the presence and absence of lipopolysaccharide - only the IFN-a Hybrid induces IFN-y directly without the need for LPS stimulation.
  • FIG. 9 Indicates that the IFN-a Hybrid of the present invention induces significant, dose-dependent secretion of TFN-a without LPS stimulation.
  • SK-OV-3 NucLight Red cells which are resistant to tumour necrosis factor
  • IL-2 and IL-12 (10 ng/ml of each)
  • IFN-a Hybrid of the present invention (3 x 10 5 lU/ml) or PBS (vehicle), indicating that both IFN-a Hybrid IL-2 and IL-12 recruit the natural killer cells to kill the SK-OV-3 cells.
  • the IFN-a Hybrid of present invention induces apoptosis and reduces target cell number in a co-culture model of NK cell killing.
  • Area under the curve (AUC) values were calculated for green and red object counts using GraphPad Prism. Data is shown as the mean of three wells ⁇ standard deviation. Curve fitting was carried out using non-linear regression (four parameters) with GraphPad Prism. The top of the apoptosis curve was constrained using data from the positive controls.
  • An EC50 value of 1.5 x 10 5 was derived for apoptosis (95% confidence interval 1.1 x 10 5 to 2.0 x 10 5 ), while an IC50 value of 1.3 x 10 5 was calculated for cell number (95% confidence interval 8.1 x 10 4 to 2.2 x 10 5 ).
  • Figure 12 Natural killer cell clustering is indicative of activation.
  • Figure 12 shows natural killer cell clustering in response to treatment with IFN-a Hybrid of present invention in a natural killer cell monoculture model, which is considered to be highly significant when compared to the positive control (P0.001).
  • Hybrid 1 is the IFN-a Hybrid as disclosed in WO2015136287, Alfacyte Ltd., which is herein incorporated by reference.
  • Figure 14 Shows the induction of Granzyme B in natural killer cells by IFN-a Hybrid of present invention. Natural killer cells were seeded on a 96 well culture plate (10K and 50K cells/well), IFN-a Hybrid (1 x 10 6 or 3 x 10 6 units) or IL-2 (10 ng/mL) were then added and the cells were cultured. After 4 or 5 days, when NK cells begin to aggregate, media was removed and Granzyme B was measured using the LEGEND MAXTM Human Granzyme B ELISA.
  • Figure 15 A) Real time monitoring of the cytotoxic effects of IFN-a Hybrid of present invention in an NK Cells/IFN-a Hybrid co-cultured model indicates that IFN-a Hybrid is not cytotoxic. B) Shows a proliferation timelapse graph of SK-OV-3 ovarian cancer cells and Flo-1 oesophageal adenocarcinoma cells in a co-culture model.
  • Figure 16 Shows the effect of IFN-a Hybrid of present invention on the amount of GM-CSF secreted by whole human leucocytes stimulated with PHA.
  • FIG. 17 Shows the inhibition of Neutrophil Chemotactic/Activation Cytokines by IFN-a Hybrid of present invention, showing that Hybrid (SEQ ID NO: 1) inhibits the release of IL-8, GM-CSF, G-CSF and CXCL-1 by over 70% at 1 IU.
  • Figure 18 Shows the effect of various stimulants on the production of IL-10.
  • Cancer cells that co-operate in tumour progression and metastasis. Cancer cells recruit neutrophils to the tumour microenvironment through several signals (green arrows). In the tumour microenvironment TANS are activated to release NETs (blue arrows) to promote tumour growth, tumour progression, metastasis, and tumour- associated thrombosis.
  • TANs tumour-associated neutrophils
  • NETs Neutrophil Extracellular Traps
  • the IFN-a Hybrids of the present invention show a significantly different characteristic to the natural Interferon-alpha.
  • IFN-a Hybrids characteristics are suitable towards the treatment of malignant cancer: IFN-y, TNF-a, Granzyme B and CXCL10 concentrations increase without any additional stimulation, IL-17A and F production is inhibited, natural killer cells are activated, cancer cell killing and clustering are all boosted and active b- catenin and TGF-b are inhibited.
  • Fresh human whole blood (Source: Tissue Solutions Ltd.) was diluted 1:10 with fresh RPMI 1640 culture medium containing 1% Penicillin/Streptomycin and 1% L- Glutamine - 10 pi was added to 10 mI of 0.4% Trypan Blue solution and mixed. A further 10 mI was placed onto a LunaTM cell counting slide. The slide was then placed into the Luna-llTM Automated Cell Counter (Logos Biosystems) to determine the percentage viability of the cells by Trypan Blue exclusion.
  • ELISA reagents and supernatants were allowed to heat to room temperature, and the pre-coated ELISA plate was washed four times with wash buffer (1x phosphate buffer saline + 0.05% Tween 20), and 200 pi assay diluent was added to each well to prevent non-specific antibody binding, and incubated at room temperature for 1 hour, with shaking (300-500 rpm, depending on protocol).
  • the plate was washed 4 times, and 100 mI of diluted standards and samples were added to the appropriate wells. The plate was then sealed and incubated at room temperature for 2 hours with shaking.
  • the plate was washed a further 4 times, and diluted Avidin-HRP solution added to each well, followed by a 30 minute incubation at room temperature, with shaking.
  • the plate was then washed a total of 5 times, allowing for 30 seconds to 1 minute of soaking between washes, before adding 100 mI Substrate Solution for 15 minutes in darkness. After 15 minutes, or when the standard wells reach the desired colour, 100 pi of stop solution 1M Sulphuric Acid and the plate was read via spectrophotometry, typically at wavelengths 450 and 570 nm.
  • TGF-b Transforming Growth Factor-Beta (TGF-b) Secretion by Endothelial Cells.
  • the human ovarian cystadenocarcinoma line (OvM1) was propagated in DM EM (Gibco Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Gibco Invitrogen). The cells were used at approximately 80% confluence.
  • an IFN-a Hybrid of the present invention induces secretion of Granzyme B from Leucocytes. This is considered to promote the killing mechanism of NK, Tc, NKT and some neutrophils against cancer cells etc.
  • TGF-beta has been implicated in the characteristics of malignant glioma - excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumour surveillance.
  • Astrocytes which are thought to represent the precursor of the majority of gliomas, are a major source of TGF-beta in vivo and have been suggested to release a large amount of TGB-beta in response to pathogenic stimuli, and tissue trauma.
  • TGF-beta is responsible for Glioma (and the tumours) growth, inhibition of innate and adaptive immunity against the tumour and induction of MMP2 and 9 to solubilise tissues and enhance metastatic spread.
  • an IFN-a Hybrid (Hybrid 2 discussed herein) of the present invention inhibits production of TGF-beta.
  • Wnt / b-catenin signalling is a highly conserved pathway through evolution. WNT- triggered gene transcription is thought to function via accumulation of b-catenin, wherein the b-catenin translocates to the nucleus where it interacts with TCF/Lef1 and activates TCF/LEF1 transcription complex.
  • Wnt / b-catenin signalling is considered to be involved in several cancers, in particular more aggressive cancer biologies b-catenin is considered involved in stem cell maintenance, immune cell inhibition, DNA repair mechanisms and is intimately active in glioma growth and metastases Specifically, components of the WNT pathway are usually overexpressed in glioblastoma multiforme tumours and have been associated to metastasis in colorectal, breast and prostate cancer. Wnt / b-catenin signalling has also been implicated in inhibiting immune invasion, wherein a T cell inflamed tumour microenvironment is characterised by infiltration of CD8+ T cells, chemokines and interferon signature. A T cell inflamed tumour microenvironment correlates with improved survival and improved response to immunotherapy. As indicated in Figure 3, an IFN-a Hybrid of the present invention is considered to provide inhibition of b-catenin signalling.

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Abstract

The present invention relates to a composition comprising an interferon alpha (IFN- α) subtype which is a hybrid of IFN-α10 and IFN-α14 for use in the treatment of cancer wherein the cancer cell comprises a decrease in expression of an interferon, as well as a method of treatment of cancer wherein the cancer cell comprises a decrease in expression of an interferon using said IFN-α hybrid.

Description

Cancer Treatment
Field of the Invention
The present invention relates to the field of cancer therapy in general and the treatment of a cancer cell(s) wherein the cancer cell comprises a decrease in expression of an interferon.
Background
Although immunotherapy treatments are increasingly used to treat cancer, the ability of cancer cells to evade immune destruction is of concern and has been an area of considerable study over the recent years. Several mechanisms and molecules known to have important roles in recognising tumour cells and / or activating an immune response have been considered. Interferon is one such molecule that has been considered to be important for activating natural immune responses against tumours. Defensins have also been considered to induce anti-tumour effects such as tumour cytolysis. Whilst immunotherapies, such as targeting of the PD-1:PD-L-1 interaction have been utilised, immune resistance has been observed. Defects in signalling pathways have been suggested as a mechanism by which tumours can acquire immuno-resistance. It has been observed that Type I interferon genes are frequently deleted in 14 tumour types (l-type and C-type). There have also been suggestions that homozygous deletion could confer a growth / survival advantage to tumour cells after tumorigenesis.
Different IFN-a subtypes have different anti-proliferative and immunomodulatory activities. The pleiotropic effects appear to be due to differential interaction with the receptor chains and signaling through different intracellular pathways to an array of effector molecules. The Type I IFN receptor consists of two chains, IFNR1 and IFNR2. There is a range of binding affinities for each of the 12 IFN-a subtypes with the different receptor chains.
The limited efficacy of monotherapy for treating cancer has highlighted the need to use different and potentially multiple therapeutic modalities to achieve a beneficial effect. Summary of the Invention
The present inventors have considered ways in which defects in the interferon signalling pathway which result in immuno-resistance can be mitigated. This would be advantageous to allow improved treatment of cancer, in particular to cancer cells that have become insensitive or escaped conventional cancer therapies.
The inventor has made the surprising discovery that the administration of a specific interferon alpha (IFN-a) subtype which is a hybrid of IFN-a10 and IFN-a14, preferably wherein the hybrid includes the primary interferon receptor binding sites of IFN-a10 and IFN-a14 can be advantageously used to treat cancer, in particularly glial cancer, wherein the cancer cells comprise a decrease in expression of an interferon. Suitably, the decrease in expression may be due to a deletion, insertion, or mutation of the interferon gene. Suitably a decrease may be considered relative to a non-tumour cell of corresponding or similar cell type. Suitably a decrease may be considered relative to a tumour cell of corresponding or similar cell type without a deletion, insertion, or mutation of the interferon gene. Suitably a decrease may be at least 5% of a level provided in a non-tumour cell of corresponding cell type, at least 10% of a level provided in a non-tumour cell of corresponding cell type, at least 20% of a level provided in a non-tumour cell of corresponding cell type, at least 30% of a level provided in a non-tumour cell of corresponding cell type. Suitably a decrease may be at least 5%, at least 10% of a level provided in a tumour cell of corresponding cell type without a deletion, insertion, or mutation of the interferon gene, at least 20% of a level provided in a tumour cell of corresponding cell type without deletion, insertion, or mutation of the interferon gene, at least 30% of a level provided in a tumour cell of corresponding cell type without deletion, insertion, or mutation of the interferon gene.
Interferon subtypes IFN-alpha10 and IFN- alpha14 and hybrids thereof are discussed in PCT Publication Number WO2014/037717 and PCT Application Number PCT/GB2015/050717. In particular IFN-alpha10 and IFN-alpha14 hybrids are disclosed that contain sequences characteristic of the IFN-alpha10 and IFN-alpha14 subtype binding sites based on a consensus backbone sequence of all 12 alpha- interferons, for example CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVL HEMMQQTFNLFSTENSSAAWEQTLLEKFSIELFQQMNDLEACVIQEVGVEETPLMN EDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO: 3). It would be advantageous to provide alternative hybrids and further compositions and methods that provide alternative immunotherapeutic approaches.
Accordingly there is provided a composition comprising an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
SEQ ID NO: 1
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVL HEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMN EDSI LAVKKYFQRITLYLI ERKYSPCAWEVVRAEIM RSLSFSTN LQKRLRRKD (SEQ ID NO: 1) for use the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon.
According to a second aspect of the present invention there is provided a method for the treatment and / or prophylaxis of a cancer wherein the cancer cell comprises a decrease in expression of an interferon, comprising the step of administering to a subject in need thereof an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVL HEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMN EDSI LAVKKYFQRITLYLI ERKYSPCAWEVVRAEIM RSLSFSTN LQKRLRRKD
SEQ ID NO: 1
Suitably the reverse translation of the protein sequence of SEQ ID NO: 1 when an E. coli codon usage table is used is provided below AT GT GT GATCTGCCGCAGACCCAT AGCCTGGGT AATCGTCGTGCACT GATTCT G CTGGGTCAGATGGGTCGTATTAGCCCGTTTAGCTGTCTGAAAGATCGTCATGAT TTTCGTATTCCGCAAGAGGAATTTGATGGCAACCAGTTTCAGAAAGCACAGGCA ATT AGCGTTCT GCAT GAAAT GAT GCAGCAGACCTTT AACCT GTTT AGCACCAAAA AT AGCAGCGCAGCATGGGAT GAAACCCT GCT GGAAAAATTCT AT ATCGAACT GT TT CAGCAG AT G AACG AT CT GG AAGCAT GT GTT ATT CAAGAAGTTGGCGTT G AAG AAACACCGCT GAT GAAT GAAGAT AGCATTCTGGCAGT GAAAAAAT ACTTTCAGC GCATT ACCCT GT ATCT GATCGAACGT AAAT AT AGCCCGT GTGCAT GGGAAGTT G TTCGTGCAGAAATT ATGCGT AGCCT GAGCTTT AGCACCAATCTGCAAAAACGTC TGCGTCGC AAAG ATT AAT AA
(SEQ ID NO: 2)
Suitably, SEQ ID NO: 1 may comprise several amino acid substitutions of protein comprising an IFN-a10 amino acid sequence with amino acids of IFN-a14 determined to be involved in binding to interferon receptor 1. Suitably this may enhance the binding of the protein to interferon receptor 1. Suitably, an amino acid substitution of protein comprising an IFN-a14 amino acid sequence with amino acids of IFN-a10 determined to be involved in binding to interferon receptor 2 may enhance the binding of the protein to interferon receptor 2.
In particular, the hybrid sequence may include at least one, at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 modifications of SEQ ID NO: 1 , suitably modifications of SEQ ID NO: 1 such that the portions of the hybrid sequence based on the IFN-a10 sequence are changed to the corresponding residues of the IFN-a14 sequence. In alternative embodiments, IFN-a14 can be utilised as a backbone structure of the hybrid and the residues which differ between the IFN-a10 and IFN-a14 sequences at the N and C terminal regions of the sequences can be provided in the hybrid sequence as those present in the IFN-a10 sequence. Suitably, at least 1, at least 2, or 3 substitutions are provided at the IFN-a14 C terminal sequence to provide residues from IFN-a10 to the hybrid sequence SEQ ID NO: 1 at those amino acid positions which are not shared/common between IFN-a10 and IFN-a14. In embodiments at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 substitutions from the N-terminal sequence and at least 1 , at least 2, or 3 substitutions from the C-terminal sequence of the IFN-a14 are made to provide residues from IFN-a10 to the hybrid SEQ ID NO: 1 at those amino acid positions which have amino acids that are not shared/common between IFN-a10 and IFN-014.
In embodiments, the hybrid comprises or consists of an amino acid sequence SEQ ID NO: 1 or a functionally active fragment or variant thereof.
Suitably the cancer cell comprises a decrease in expression of interferon due to the cancer cell having a mutation to the interferon gene which renders it decreases the expression rate or decreases the amount of functional interferon produced, such as a deletion or insertion (to cause a codon shift), or a mutation to a stop codon, or mutation of key residues involved in structure or activity (e.g. a cysteine bridge). Suitably the cancer cell comprises a decrease in expression of a type I interferon, suitably an alpha interferon.
The term “functionally active” is construed to mean an IFN-a10 IFN-a14 hybrid peptide comprising the primary interferon binding sites of IFN-a10 and IFN-a14 wherein the administration of peptide to a subject or expression of peptide in a subject promotes enhancement of Th1 mediated immune response and suppression of a Th2/Th17 mediated immune response.
A fragment can comprise at least 50, preferably 100 and more preferably 150 or greater contiguous amino acids from SEQ ID NO: 1 and which is functionally active. Suitably, a fragment may be determined using, for example, C-terminal serial deletion of cDNA such as SEQ ID NO: 2 or SEQ ID NO: 3. Said deletion constructs may then be cloned into suitable plasmids. The activity of these deletion mutants may then be tested for biological activity as described herein.
By variant is meant an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, even more preferably with at least 95% sequence identity to SEQ ID NO: 1, even more preferably with at least 96% sequence identity to SEQ ID NO: 1, even more preferably with at least 97% sequence identity to SEQ ID NO: 1 and most preferably with at least 98% sequence identity, at least 99% sequence identity with SEQ ID NO: 1. A variant encompasses a polypeptide sequence of SEQ ID NO: 1 which includes substitution of amino acids, especially a substitution(s) which is/are known for having a high probability of not leading to any significant modification of the biological activity or configuration, or folding, of the protein. These substitutions, typically known as conserved substitutions, are known in the art. For example the group of arginine, lysine and histidine are known interchangeable basic amino acids. Suitably, in embodiments amino acids of the same charge, size or hydrophobicity may be substituted with each other. Suitably, any substitution may be selected based on analysis of amino acid sequence alignments of interferon alpha subtypes to provide amino acid substitutions to amino acids which are present in other alpha subtypes at similar or identical positions when the sequences are aligned. Hybrids, and variants and fragments thereof may be generated using suitable molecular biology methods as known in the art. The skilled person would be aware of the way sequence identity can be determined using methods and suitable computer programs as known in the art. Suitably default parameters where appropriate for such computer programs could be utilised.
Suitably the cancer cell has a homozygous deletion of an interferon alpha gene.
Suitably the cancer cell has a deletion of or within the interferon gene cluster located at the loci 9p21. Suitably the cancer cell has a homozygous deletion of or with the interferon gene cluster at the loci 9p21. The 16 type I interferon genes are located on 9p21, which includes 13 IFN-a genes, 1 IFN-b, 1 IFN-e and 1 IFN-w gene.
Suitably the cancer cell may be a cell from a cancer selected from melanoma, glioma, suitably a brain lower grade glioma, a glioblastoma, in particular glioblastoma multiforme, prostrate adenocarcinoma, colorectal cancer, lymphoid neoplasias, acute lymphoblastic leukaemia and non-Hodgkin lymphoma, bladder cancer, breast cancer, prostate cancer, or lung cancer. Suitably the cancer may be a cancer selected from a glioma, in particular a brain lower grade glioma, a glioblastoma, in particular glioblastoma multiforme, bladder cancer or lung cancer.
Suitably the hybrid interferon of the invention may be provided in combination with an immunotherapy treatment for a cancer. Suitably an immunotherapy treatment may be a monoclonal antibody with binding specificity to a checkpoint inhibitor, for example Pembrolizumab, an immune checkpoint inhibitor. Suitably an immunotherapy may be Nivolumab. Suitably a combination treatment may be provided with Galunisertib which is considered to enhance anti-TGF-beta effects.
Suitably the hybrid interferon of the invention may be provided in combination with a monoclonal antibody cancer treatment, for example a monoclonal antibody targeting CTLA4. For example, a combination treatment may comprise a monoclonal antibody targeting CTLA4, for example Ipilimumab and a hybrid interferon the present invention. For example, a combination treatment for use in the treatment of melanoma may comprise a monoclonal antibody targeting CTLA4, for example Ipilimumab and a hybrid interferon the present invention.
In embodiments the hybrid interferon of the invention may be provided in combination with NK and / or T cell therapies. In embodiments the hybrid interferon of the invention may be provided in combination with treatments selected from, for example Temozolomide or Bevacizumab, Cetuximab, Panitumumab, Ramucirumab, Monamulizumab, Obinutuzumab, Rituximab. In embodiments the hybrid interferon of the invention may be provided in combination with chemotherapeutics.
T reatment / Therapy
The term “treatment” is used herein to refer to any regimen that can benefit a human or non-human animal. The treatment may be in respect of an existing condition and the treatment may be prophylactic (preventative treatment). Treatment may include curative or alleviative effects. Reference herein to "therapeutic" and "prophylactic" treatment is to be considered in its broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, therapeutic and/or prophylactic treatment includes amelioration of the symptoms of a particular allergic condition or preventing or otherwise reducing the risk of developing a particular allergic condition. The term "prophylactic" may be considered as reducing the severity or the onset of a particular condition. "Therapeutic" may also reduce the severity of an existing condition.
Administration
A hybrid IFN-a10 and IFN-a14 subtype, for example SEQ ID NO: 1, as described herein can be administered separately to the same subject, optionally sequentially, or can be co-administered simultaneously with another agent, for example a chemotherapeutic or immunotherapy or a NK or T cell as a pharmaceutical, immunogenic composition. The pharmaceutical composition will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration.
The active ingredients can be administered to a patient in need of treatment via any suitable route. The precise dose will depend upon a number of factors, as is discussed below in more detail.
Suitably administration may be daily, for example by cannula or implanted as a PEG slow release formulation at a time of surgery.
One suitable route of administration is parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch). Other suitable routes of administration include (but, are not limited to) oral, ocular, nasal, topical (including buccal and sublingual), infusion, intradermal or administration via oral or nasal inhalation, by means of, for example, a nebuliser or inhaler, or by an implant. Preferable routes of administration include (but, are not limited to) oral, buccal and sublingual. The compositions of the invention may also be administered in such a manner that they are directed to, or released in, specific areas of the gut intestinal tract (such as the small intestine/duodenum). Typically such release will occur after passage through the stomach, this targeted release being achievable through the use of coatings and the like.
For intravenous injection, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection, Lactated Ringer’s injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
The compositions of the present invention for oral administration may be in tablet, capsule, lozenge, powder or liquid form. Oral administration may involve placing a lozenge under the tongue of the patient. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
The compositions of the present invention may also be administered via microspheres, liposomes, other micro-particulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington’s Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition (December 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7th Edition ISBN 0-683305-72-7, the entire disclosures of which are herein incorporated by reference.
Pharmaceutical Compositions
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to an active ingredient, a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation. The formulation may be a liquid, for example, a physiologic salt solution containing non phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze-dried powder.
Dose
The composition is preferably administered to an individual in a “therapeutically effective amount” or a “desired amount”, this being sufficient to show benefit to the individual. As defined herein, the term an "effective amount" means an amount necessary to at least partly obtain the desired response, or to delay the onset or inhibit progression or halt altogether the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation and other relevant factors. It is expected that the amount will fall in a relatively broad range, which may be determined through routine trials. Prescription of treatment, e.g. decisions on dosage etc., is ultimately within the responsibility and at the discretion of general practitioners, physicians or other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration. A broad range of doses may be applicable. Considering oral administration to a human patient, for example, from about 10 pg to about 1000 pg of agent may be administered per human dose, optionally for 3 to 4 doses. Dosage regimes may be adjusted to provide the optimum therapeutic response and reduce side effects. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.
Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.
As used herein, the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.
“About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.
Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
An embodiment of the present invention will now be described by way of example only, with reference to the accompanying figures in which:
Figure 1. Indicates an IFN-a Hybrid of the present invention induced secretion of Granzyme B from Leukocytes
Figure 2. Indicates an IFN-a Hybrid of the present invention inhibited production of TGB-beta from endothelial cells
Figure 3. Indicates inhibition of b-catenin signaling by an IFN-a Hybrid of the present invention in the Ovm1 ovarian cancer cell line.
Figure 4. Indicates suppression of IL-17A synthesis from human leucocytes by an IFN-a Hybrid of the present invention.
Figure 5. Indicates suppression of IL-17F synthesis by an IFN-a Hybrid of the present invention. Figure 6. Indicates enhanced suppression of CXCL-10 synthesis from human leucocytes by an IFN-a Hybrid of the present invention, compared to rIFN-a 2a or rlFN-b 1a.
Figure 7. Indicates enhanced suppression of CXCL-10 synthesis from human leucocytes by an IFN-a Hybrid of the present invention, compared rlFN-b 1a.
Figure 8. Indicates the enhanced induction of lnterferon-g production from human leucocytes with IFN-a Hybrid of present invention in the presence and absence of lipopolysaccharide - only the IFN-a Hybrid induces IFN-y directly without the need for LPS stimulation.
Figure 9. Indicates that the IFN-a Hybrid of the present invention induces significant, dose-dependent secretion of TFN-a without LPS stimulation.
Figure 10. SK-OV-3 NucLight Red cells (which are resistant to tumour necrosis factor) alone or in co-culture with natural killer cells were treated with IL-2 and IL-12 (10 ng/ml of each), IFN-a Hybrid of the present invention (3 x 105 lU/ml) or PBS (vehicle), indicating that both IFN-a Hybrid IL-2 and IL-12 recruit the natural killer cells to kill the SK-OV-3 cells.
Figure 11. The IFN-a Hybrid of present invention induces apoptosis and reduces target cell number in a co-culture model of NK cell killing. Area under the curve (AUC) values were calculated for green and red object counts using GraphPad Prism. Data is shown as the mean of three wells ± standard deviation. Curve fitting was carried out using non-linear regression (four parameters) with GraphPad Prism. The top of the apoptosis curve was constrained using data from the positive controls. An EC50 value of 1.5 x 105 was derived for apoptosis (95% confidence interval 1.1 x 105 to 2.0 x 105), while an IC50 value of 1.3 x 105 was calculated for cell number (95% confidence interval 8.1 x 104 to 2.2 x 105).
Figure 12. Natural killer cell clustering is indicative of activation. Figure 12 shows natural killer cell clustering in response to treatment with IFN-a Hybrid of present invention in a natural killer cell monoculture model, which is considered to be highly significant when compared to the positive control (P0.001).
Figure 13. Shows natural killer (NK) cell clustering in the presence of various stimulants, indicating that an IFN-a Hybrid of the present invention induces more aggregation of NK cells than other stimulants. NB: Hybrid 1 is the IFN-a Hybrid as disclosed in WO2015136287, Alfacyte Ltd., which is herein incorporated by reference.
Figure 14. Shows the induction of Granzyme B in natural killer cells by IFN-a Hybrid of present invention. Natural killer cells were seeded on a 96 well culture plate (10K and 50K cells/well), IFN-a Hybrid (1 x 106 or 3 x 106 units) or IL-2 (10 ng/mL) were then added and the cells were cultured. After 4 or 5 days, when NK cells begin to aggregate, media was removed and Granzyme B was measured using the LEGEND MAX™ Human Granzyme B ELISA.
Figure 15. A) Real time monitoring of the cytotoxic effects of IFN-a Hybrid of present invention in an NK Cells/IFN-a Hybrid co-cultured model indicates that IFN-a Hybrid is not cytotoxic. B) Shows a proliferation timelapse graph of SK-OV-3 ovarian cancer cells and Flo-1 oesophageal adenocarcinoma cells in a co-culture model.
Figure 16. Shows the effect of IFN-a Hybrid of present invention on the amount of GM-CSF secreted by whole human leucocytes stimulated with PHA.
Figure 17. Shows the inhibition of Neutrophil Chemotactic/Activation Cytokines by IFN-a Hybrid of present invention, showing that Hybrid (SEQ ID NO: 1) inhibits the release of IL-8, GM-CSF, G-CSF and CXCL-1 by over 70% at 1 IU.
Figure 18. Shows the effect of various stimulants on the production of IL-10.
Figure 19. Diagram to indicate how tumours proliferate:
A) Cancer cells, tumour-associated neutrophils (TANs), and Neutrophil Extracellular Traps (NETs) co-operate in tumour progression and metastasis. Cancer cells recruit neutrophils to the tumour microenvironment through several signals (green arrows). In the tumour microenvironment TANS are activated to release NETs (blue arrows) to promote tumour growth, tumour progression, metastasis, and tumour- associated thrombosis.
B) Indicates the pathways that are inhibited by IFN-a Hybrid of present invention, resulting in a chemotherapeutic effect.
Notably, the IFN-a Hybrids of the present invention show a significantly different characteristic to the natural Interferon-alpha. IFN-a Hybrids characteristics are suitable towards the treatment of malignant cancer: IFN-y, TNF-a, Granzyme B and CXCL10 concentrations increase without any additional stimulation, IL-17A and F production is inhibited, natural killer cells are activated, cancer cell killing and clustering are all boosted and active b- catenin and TGF-b are inhibited.
Examples
Human whole blood assay and Granzyme B ELISA protocol Determination of viability:
Fresh human whole blood (Source: Tissue Solutions Ltd.) was diluted 1:10 with fresh RPMI 1640 culture medium containing 1% Penicillin/Streptomycin and 1% L- Glutamine - 10 pi was added to 10 mI of 0.4% Trypan Blue solution and mixed. A further 10 mI was placed onto a Luna™ cell counting slide. The slide was then placed into the Luna-ll™ Automated Cell Counter (Logos Biosystems) to determine the percentage viability of the cells by Trypan Blue exclusion.
Whole blood assay protocol
Working within a Class II hood, whole human blood containing 50 U/ml Sodium Heparin, diluted 1/10 with RPMI 1640 culture medium containing 1% Penicillin/Streptomycin and 1% L-Glutamine, was incubated with either 100 pg/ml phytohemagglutinin-L (PHA), or 10 pg/ml lipopolysaccharide (LPS) from Salmonella enterica serotype abortus equi, in the presence of a range of concentrations of either human rlFN-a14, Hybrid IFN 1 or Hybrid IFN 2 (present invention) for 24 hours at 37 °C in an atmosphere of 5 % CO2 in air in a humidified incubator. IFN concentrations were 0, 1 , 5 10, 50, 100, 1,000, 10,000, 100,000 and 1,000,000 lU/ml. Each culture concentration was 1 ml in a 24 well plate.
After 24 hours incubation, the samples were harvested by centrifugation at 10,000 g for 5 minutes to remove cells, the supernatants were carefully removed without disturbing the pellet of cells, and stored at -20°C for future analysis. The supernatants were then assayed using the following ELISA (following the manufacturer’s instructions):
Human Granzyme B ELISA - BioLegend UK, London, United Kingdom.
ELISA procedure.
ELISA reagents and supernatants were allowed to heat to room temperature, and the pre-coated ELISA plate was washed four times with wash buffer (1x phosphate buffer saline + 0.05% Tween 20), and 200 pi assay diluent was added to each well to prevent non-specific antibody binding, and incubated at room temperature for 1 hour, with shaking (300-500 rpm, depending on protocol).
The plate was washed 4 times, and 100 mI of diluted standards and samples were added to the appropriate wells. The plate was then sealed and incubated at room temperature for 2 hours with shaking.
After a further 4 wash cycles, 100 mI of Detection Antibody was added to each well, followed by a further 1 hour incubation at room temperature, with shaking.
The plate was washed a further 4 times, and diluted Avidin-HRP solution added to each well, followed by a 30 minute incubation at room temperature, with shaking.
The plate was then washed a total of 5 times, allowing for 30 seconds to 1 minute of soaking between washes, before adding 100 mI Substrate Solution for 15 minutes in darkness. After 15 minutes, or when the standard wells reach the desired colour, 100 pi of stop solution 1M Sulphuric Acid and the plate was read via spectrophotometry, typically at wavelengths 450 and 570 nm.
Transforming Growth Factor-Beta (TGF-b) Secretion by Endothelial Cells.
Primary endothelial cells were cultured in 24 well tissue culture plates until they became 75% confluent - human aortic endothelial cells (HAEC, Thermofisher) in Medium 200PRF containing 10% (v/v) human AB serum. They were then stimulated with E. coli lipopolysaccharide (10pg/ml Sigma) together with an IFN-a Hybrid of the present invention concentrations for 4 days and the supernatants assessed for the presence of TGF-b by ELISA (as above).
Active b-Catenin Assay.
The human ovarian cystadenocarcinoma line (OvM1) was propagated in DM EM (Gibco Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Gibco Invitrogen). The cells were used at approximately 80% confluence.
Flow Cytometry Analysis
Fluorescence was evaluated with a FACSCalibur Cytometer and data analyzed using FlowJo software (TreeStar, Ashland, OR). The OvM1 cell line was left untreated or cultured with I04 or 105 lU/ml Interferon-alpha 2a or the HYBRID for 24 h. prior to transfection with TOPflash luciferase and Renilla luciferase constructs. After 4h, the cells were cultured again with or without the 2 initial treatments. Dual luciferase activity was measured 24 h later. Data shown is normalised to Renilla activity and the expression of the hypophosphorylated/active b-catenin was measured by conventional intracellular flow cytometry. Data represent a minimum of 5 experiments and was significant only for the 105 lU/ml HYBRID sample at P<0.01. Granzyme B secretion
As indicated in Figure 1, an IFN-a Hybrid of the present invention induces secretion of Granzyme B from Leucocytes. This is considered to promote the killing mechanism of NK, Tc, NKT and some neutrophils against cancer cells etc.
TGF-beta
TGF-beta has been implicated in the characteristics of malignant glioma - excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumour surveillance. Astrocytes which are thought to represent the precursor of the majority of gliomas, are a major source of TGF-beta in vivo and have been suggested to release a large amount of TGB-beta in response to pathogenic stimuli, and tissue trauma. In particular, it is considered TGF-beta is responsible for Glioma (and the tumours) growth, inhibition of innate and adaptive immunity against the tumour and induction of MMP2 and 9 to solubilise tissues and enhance metastatic spread. As indicated in Figure 2, an IFN-a Hybrid (Hybrid 2 discussed herein) of the present invention inhibits production of TGF-beta.
Wnt / b-catenin signalling
Wnt / b-catenin signalling is a highly conserved pathway through evolution. WNT- triggered gene transcription is thought to function via accumulation of b-catenin, wherein the b-catenin translocates to the nucleus where it interacts with TCF/Lef1 and activates TCF/LEF1 transcription complex. Wnt / b-catenin signalling is considered to be involved in several cancers, in particular more aggressive cancer biologies b-catenin is considered involved in stem cell maintenance, immune cell inhibition, DNA repair mechanisms and is intimately active in glioma growth and metastases Specifically, components of the WNT pathway are usually overexpressed in glioblastoma multiforme tumours and have been associated to metastasis in colorectal, breast and prostate cancer. Wnt / b-catenin signalling has also been implicated in inhibiting immune invasion, wherein a T cell inflamed tumour microenvironment is characterised by infiltration of CD8+ T cells, chemokines and interferon signature. A T cell inflamed tumour microenvironment correlates with improved survival and improved response to immunotherapy. As indicated in Figure 3, an IFN-a Hybrid of the present invention is considered to provide inhibition of b-catenin signalling.
Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

Claims
1. A composition comprising an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
SEQ ID NO: 1
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKA QAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQE VGVEETPLM N EDSI LAVKKYFQRITLYLI ERKYSPCAWEVVRAEIM RSLSFST NLQKRLRRKD (SEQ ID NO: 1) for use the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon.
2. A method for the treatment and / or prophylaxis of a cancer wherein the cancer cell comprises a decrease in expression of an interferon, comprising the step of administering to a subject in need thereof an IFN-alpha hybrid comprising or consisting of a SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKA QAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQE VGVEETPLM N EDSI LAVKKYFQRITLYLI ERKYSPCAWEVVRAEIM RSLSFST NLQKRLRRKD
SEQ ID NO: 1
3. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or the method of claim 2 wherein the hybrid sequence SEQ ID NO: 1 includes at least 11 modifications of SEQ ID NO: 1.
4. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or the method of claim 2 wherein the hybrid sequence SEQ ID NO: 1 includes at least 5 modifications of SEQ ID NO: 1.
5. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 4 or the method of claim 2 to 4 wherein the hybrid comprises or consists of an amino acid sequence SEQ ID NO: 1.
6. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 5 or the method of claim 2 to 5 wherein the cancer cell has a deletion of an interferon gene.
7. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 6 or the method of claim 2 to 5 wherein the cancer cell has a deletion of or within the interferon gene cluster located at the loci 9p21.
8. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 7 or the method of claim 2 to 7 wherein the cancer cell is a cell from a cancer selected from melanoma, glioma, a brain lower grade glioma, a glioblastoma, glioblastoma multiforme, prostrate adenocarcinoma, colorectal cancer, lymphoid neoplasias, acute lymphoblastic leukaemia and non- Hodgkin lymphoma, bladder cancer, breast cancer, prostate cancer, or lung cancer.
9. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 8 or the method of claim 2 to 8 wherein the cancer cell is a cell from a cancer selected from a glioma, in particular a brain lower grade glioma, a glioblastoma, glioblastoma multiforme, bladder cancer or lung cancer.
10. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 9 or the method of claim 2 to 9 wherein the hybrid interferon is provided in combination with an immunotherapy treatment for a cancer.
11. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 10 or the method of claim 2 to 10 wherein the hybrid interferon is provided in combination with a monoclonal antibody cancer treatment.
12. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 11 or the method of claim 2 to 11 wherein the hybrid interferon is provided in combination with NK and / or T cell therapies.
13. The composition for use in the treatment of a cancer cell wherein the cancer cell comprises a decrease in expression of an interferon as claimed in claim 1 or 3 to 12 or the method of claim 2 to 12 wherein the hybrid interferon is provided in combination with treatments selected from a group consisting of Temozolomide, Bevacizumab, Cetuximab, Panitumumab, Ramucirumab,
Monamulizumab, Obinutuzumab and Rituximab.
PCT/GB2021/051706 2020-07-06 2021-07-05 Cancer treatment WO2022008890A1 (en)

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