Patent Application
20240343794
The present invention relates to the treatment of cancer of the intestine. The invention extends to inhibitors of the IL-25:IL-25R signalling pathway for use in methods of treating, preventing, or ameliorating cancer of the intestine.
Intestinal cancer is a common cause of cancer-related death. For example, colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide, and is expected to increase by a further 60% by 2030. The aim is to diagnose early and allow curative treatments, usually involving surgery, and in some cases in combination with adjuvant and neoadjuvant treatments. For patients intolerant to surgery, or with unresectable lesions, the goal is to maximally shrink the tumours and suppress further tumour growth. Unfortunately, current checkpoint inhibitor therapies are ineffectual in CRC. Approximately 80% of human CRC have mutations in the adenomatous polyposis coli (APC) tumour suppressor gene, and follow a well-defined pathway of loss of heterozygosity (LOH) of APC in intestinal epithelial cells, aberrant activation of Wnt signalling and polyp formation.
Despite the recent advances in immune checkpoint inhibitor therapy, showing efficacy in various different types of cancers, the response in CRC patients is largely limited to mismatch-repair-deficient (dMMR) CRC, comprising only a small proportion (15%) of total CRC cases. By contrast, the vast majority of CRC, following the conventional pathway initiated by mutations in the APC gene, are mismatch-repair-proficient (pMMR), and rarely respond to immune checkpoint inhibitors. Furthermore, in the past decade, efforts to develop novel immunotherapies for CRC, such as tumour vaccines and adoptive T cell transfer, have shown little efficacy. Recently, the phase III clinical trial IMblaze370 using the anti-PD-L1 checkpoint inhibitor atezolizumab to treat CRC patients (>98% had mismatch-repair-proficient CRC) failed to meet the primary end point.
Thus, there is a need for new drugs to treat intestinal cancer.
Interleukin-25 (IL-25) is a cytokine that is expressed by epithelial cells in the intestine, and studies have implied that IL-25 may play an inhibitory role in the growth and development of colonic tumours. Thelen et al. reported that acute blockade of IL-25 in a colitis associated colon cancer model leads to increased tumour burden (Thelen, T., Green, R. & Ziegler, S. Acute blockade of IL-25 in a colitis associated colon cancer model leads to increased tumor burden. Sci Rep 6, 25643 (2016)).
Benatar et al. reported that injection of recombinant IL-25 resulted in significant antitumour activity for melanoma, lung, breast, colon and pancreatic cancer in human tumour xenograft models (Benatar, T., Cao, M. Y., Lee, Y. et al. IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo. Cancer Immunol Immunother 59, 805-817 (2010)).
Thus, in light of these studies, it might be expected that agonists of the IL-25:IL-25R signalling pathway would be useful in the treatment of intestinal cancer.
Group 2 innate lymphoid cells (ILC2s) are a type of immune cell that can help to defend the host against intestinal parasitic helminth infection, and are associated with inappropriate allergic reactions. Moral et al. reported that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity, and suggest that ILC2s are anti-cancer immune cells for PDAC (Moral, J. A., Leung, J.,
Rojas, L. A. et al. ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature 579, 130-135 (2020)).
Thus, in light of this study, it might be expected that activation of ILC2s would be useful in the treatment of cancer.
WO2017/194554 discloses experiments relating to the effects of IL-25 on triple-negative breast cancer (TNBC) cell lines. The authors suggest that IL-25 might contribute to TNBC tumour resistance to EGRF inhibitors.
In an aspect of the invention, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the inhibitor specifically binds to IL-25 or to IL-25R.
The cancer may be colorectal cancer. The cancer may comprise a mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The cancer may comprise a tumour that has an increased frequency of group 2 innate lymphoid cells (ILC2s) compared to the ILC2 frequency in adjacent normal intestine
The inhibitor may specifically bind to IL-25. The inhibitor may bind to one or more amino acid sequences selected from the group consisting of amino acid residues 46-63 of SEQ ID NO: 48, amino acid residues 66-84 of SEQ ID NO: 48, and amino acid residues 129-135 of SEQ ID NO: 48.
The inhibitor may be or may comprise an anti-IL-25 antibody, or functional fragment or derivative thereof. Thus, there is provided an anti-IL-25 antibody, or functional fragment or derivative thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine.
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The anti-IL-25 antibody, or functional fragment or derivative thereof, may comprise:
The inhibitor may specifically bind to IL-25R. The inhibitor may be or may comprise an anti-IL-25R antibody, or functional fragment or derivative thereof. Thus, there is provided an anti-IL-25R antibody, or functional fragment or derivative thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine.
The anti-IL-25R antibody, or functional fragment or derivative thereof may comprise:
The anti-IL-25R antibody, or functional fragment or derivative thereof may comprise:
The anti-IL-25R antibody, or functional fragment or derivative thereof may comprise:
The anti-IL-25R antibody, or functional fragment or derivative thereof may comprise:
The anti-IL-25R antibody, or functional fragment or derivative thereof may comprise:
The anti-IL-25 antibodies or the anti-IL-25R antibodies disclosed herein may optionally comprise at least one CDR sequence that comprises one, two, or three amino acid substitutions compared to a CDR recited sequence recited herein. In other embodiments, the anti-IL-25 antibodies or the anti-IL-25R antibodies disclosed herein may optionally comprise at least two, three, four, five, or six CDR sequences that comprise one, two, or three amino acid substitutions compared to a CDR recited sequence recited herein
The inhibitor of the IL-25:IL-25R signalling pathway may be an agent that competes for binding with any one of the inhibitors disclosed herein.
The inhibitor of the IL-25:IL-25R signalling pathway may be an antibody mimetic, or a functional fragment or derivative thereof.
The methods of treating, preventing, or ameliorating cancer of the intestine disclosed herein, may further comprise the administration of an immune checkpoint inhibitor. The immune checkpoint inhibitor may be an inhibitor of the PD-1: PD-L1 pathway.
The inventors provide data herein that demonstrate a role for IL-25-activated ILC2s in the intestine where they create a cancer-permissive microenvironment.
The inventors show that in a mouse model of spontaneous intestinal tumorigenesis exogenous IL-25 increases tumours, whilst ablation of IL-25-signalling reduces tumour burden and virtually doubles life-expectancy, while reducing IL-25-responsive tumour
ILC2s and shifting the balance towards cytotoxic CD8 T cell infiltration. Without being bound to a particular theory, the inventors provide evidence herein to show that blockade of IL-25:IL25R signalling has a therapeutic effect due to effects mediated by ILC2S.
Thus, in a first aspect of the invention, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the inhibitor specifically binds to IL-25 or to IL-25R.
Previous reports addressing the role of IL-25:IL-25R signalling in intestinal cancer have reached the opposite conclusions to the present inventors. As discussed in the background section, Thelen et al. and Benatar et al. suggest that IL-25 has an anti-tumour effect, and thus suggest that the blockade of IL-25 activity would not be a suitable therapeutic strategy for the treatment of colonic tumours. The inventors believe that previous studies may have failed to discover the present invention due to deficiencies of the previously examined models of intestinal cancer. For instance, several studies have shown that chemical-induced mouse models of colitis-associated cancer (CAC), such as those that rely on the induction of cancer by treatment with azoxymethane (AOM) and dextran sodium sulphate (DSS), do not accurately reflect the genomic alterations found in sporadic human CRC. Thelen et al. makes use of a model involving AOM and DSS. In addition, xenograft models featuring immunodeficient mice, such as those used in Benatar et al. may not accurately recreate the tumour microenvironment, particularly with respect to immune cell infiltration.
The present inventors make use of the Apc1322T/+ mouse model of intestinal cancer. Intestinal tumours in APC-mutant mouse models are driven by the loss of the APC tumour suppressor gene. Specifically, the Apc1322T/+ model very closely mimics the mutations found in human cancer (Jackstadt, R. & Sansom, O. J. Mouse models of intestinal cancer. J Pathol 238, 141-151, doi: 10.1002/path.4645 (2016)).
A previous report has suggested that ILC2s are anti-cancer immune cells for PDAC (Moral et al; discussed in the background section), and hence play the opposite role in PDAC. The inventors conclude that the roles of the epithelium-derived cytokines IL-25 and IL-33, and ILC2s in tumorigenesis are likely to be location-dependent and organ specific.
Thus, the present invention is particularly surprising in light of previous studies.
The inhibitor of the IL-25:IL-25R signalling pathway may be any which, upon administration or application to a patient, is capable of binding to IL-25 or IL-25R and blocking signalling resulting from the binding of IL-25 to its receptor. The inhibitor may inhibit the functional interaction of IL-25R and IL-25.
Whether an agent is capable of inhibiting the IL-25:IL-25R signalling pathway may be measured by any technique known in the art. For instance, an in vitro assay may be used to measure whether the inhibitor is able to reduce or abolish IL-13 release from non B/non T (NB/NT) cells isolated from the mesenteric lymph nodes of mice. In this assay cells are incubated with or without IL-25 and varying concentrations of inhibitor or control. The IL-25 may be recombinant IL-25 at a concentration of 10 ng/ml. As a readout, IL-13 levels may be measured post stimulation, for instance three days post stimulation. IL-13 levels may be measured by ELISA. In some embodiments, the agent is considered to be an inhibitor of the IL-25:IL-25R signalling pathway if it is capable of reducing IL-13 release to the same extent as an anti-IL-25 or anti-IL-25R antibody disclosed herein. In an embodiment, the agent is considered to be an inhibitor of the IL-25:IL-25R signalling pathway if it is capable of reducing IL-13 release to the same extent as the antibody 2C3 or the antibody D9.2 disclosed herein, or their humanised versions. The humanised versions may include RH2.5_R71V, RH2.5_S30T, RH2.5, or M6 (discussed herein). In some embodiments, an agent or an antibody is considered to be an inhibitor of the IL-25:IL-25R signalling pathway if it is capable of ablating IL-13 production when tested at a concentration of 4 μg/ml, 2 μg/ml, 0.5 μg/ml, 0.25 μg/ml, or 0.1 μg/ml in the in vitro assay. For potential inhibitors with a different molecular weight to antibodies, the concentrations may be adjusted accordingly.
In another embodiment, whether an agent is an inhibitor of the IL-25:IL-25R signalling pathway is measured by determining whether the agent is able to reduce the activation of STAT, e.g. determined by measuring p-STAT5 levels, in a IL-25R+ cell exposed to IL-25 (Wu et al.; J Immunol. 2015 May 1; 194 (9): 4528-4534; herein incorporated by reference).
In other embodiments, whether an agent is an inhibitor of the IL-25:IL-25R signalling pathway is measured by determining whether the agent is able to reduce the incidence of tumours in young-adult Apc1322T/+ mice treated with said inhibitor, but has no effect or a reduced effect in young-adult Apc1322T/+ mice that are deficient in IL-25R and/or IL-25. Treatment of a young-adult mouse may be treatment from three weeks of age for a further two, three, four, five, six, seven, or more weeks. In a particular embodiment, the treatment is for seven weeks. The treatment may be one, twice, thrice, four times, five times, six times, or seven times a week. In a particular embodiment, the treatment is twice a week. In another embodiment, whether an agent is an inhibitor of the IL-25:IL-25R signalling pathway is measured by determining whether the agent is able to reduce the incidence of tumours in adult Apc1322T/+ mice treated with said inhibitor, but has no effect or a reduced effect in adult Apc1322T/+ mice that are deficient in IL-25R and/or IL-25. In an embodiment, the adult Apc1322T/+ mice may have already developed tumours at the time of the treatment, for instance the mice may be seven weeks old. Treatment of an adult mouse may be treatment from seven weeks of age for a further two, four, seven, 14, 21, 28, or more days. In a particular embodiment, the treatment is for four weeks. The treatment may be one, twice, thrice, four times, five times, six times, or seven times a week. In a particular embodiment, the treatment is twice a week. Further information is disclosed in the Examples herein.
In some embodiments, a saturating amount of the inhibitor of the IL-25:IL-25R signalling pathway can reduce IL-25:IL-25R signalling by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, about 100%, or 100%. In a particular embodiment, a saturating amount of the inhibitor of the IL-25:IL-25R signalling pathway abolishes IL-25:IL-25R signalling.
For the avoidance of doubt, an inhibitor of the IL-25:IL-25R signalling pathway is not an agent that acts globally or generically. For instance, an agent that acts to reduce IL-25:IL-25R signalling due to general cytotoxic effects or due to global effects on cell signalling is not an inhibitor of the IL-25:IL-25R signalling pathway.
The IL-25 may be human IL-25, and the sequence may be as set out below.
The IL-25R may be human IL-25R, and the sequence may be as set out below. This sequence is isoform 1 (identifier Q9NRM6-1).
In another embodiment, the IL-25R may be isoform 2 (identifier: Q9NRM6-2), and may have the sequence set out below.
Alleles of IL-25 and IL-25R are also encompassed by the present disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridisation, amplification and/or database sequence comparison).
The inhibitor may act by binding to IL-25 and sterically blocking, preventing, or reducing the interaction with IL-25R. The inhibitor may act by binding to IL-25R and sterically blocking, preventing, or reducing the interaction with IL-25. For instance, the inhibitor may bind to a region of IL-25 that interacts with IL-25R, or the inhibitor May bind to a region of IL-25R that interacts with IL-25. Alternatively, the inhibitor may bind to either IL-25 or IL-25R in any position, and may lead to a conformational change that prevents or reduces the IL-25:IL-25R interaction.
The IL-25 and/or IL-25R upon which the inhibitor acts may be human IL-25 and/or human IL-25R. In some embodiments, the inhibitor is able to act upon cynomolgus monkey IL-25 and/or murine IL-25, as well as human IL-25. In some embodiments, the inhibitor is able to act upon cynomolgus monkey IL-25R and/or murine IL-25R, as well as human IL-25R. In other embodiments, the inhibitor acts upon the IL-25 and/or IL-25R found within the subject to be treated.
The inhibitor of the IL-25:IL-25R signalling pathway may bind one or more amino acid sequences selected from the group consisting of amino acid residues 46-63 of SEQ ID NO: 48, amino acid residues 66-84 of SEQ ID NO: 48, and amino acid residues 129-135 of SEQ ID NO: 48. In particular embodiment, the inhibitor may bind to amino acid residues 56-63 of SEQ ID NO: 48 and amino acid residues 66-74 of SEQ ID NO: 48.
In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway competes for binding with any IL-25 or IL-25R binding molecule disclosed herein. The inhibitor may bind to the same epitope, or bind to an overlapping epitope, compared to any IL-25 or IL-25R binding molecule disclosed herein. In particular embodiments, the inhibitor of the IL-25:IL-25R signalling pathway competes for binding with, binds to the same epitope as, or binds to an overlapping epitope with any one of the antibodies: 2C3, any humanised 2C3, RH2.5_R71V, RH2.5_S30T, RH2.5, M6, D9.2, or any humanised D9.2. Two binding molecules may be considered to “compete” when the binding of one to a target is capable of detectably decreasing the binding of the other to the same target.
The inhibitor may be or may comprise an anti-IL-25 antibody, derivative, or functional fragment thereof. The inhibitor may be or may comprise an anti-IL-25R antibody, derivative, or functional fragment thereof. An anti-IL-25 or anti-IL-25R antibody is an antibody capable of specifically binding to its target. “Specifically binding” may mean that the antibody does not bind to the target due to only non-specific interactions, and this property can be determined by comparison to an isotype control or similar. Specific binding does not necessarily require, although it may include, exclusive binding to a single target.
Thus, in an aspect of the invention, there is provided an anti-IL-25 antibody, derivative, or functional fragment thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine.
In another aspect of the invention, there is provided an anti-IL-25R antibody, derivative, or functional fragment thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine.
The anti-IL-25 antibody or anti-IL-25R antibody may be a monoclonal or a polyclonal antibody. The anti-IL-25 antibody or anti-IL-25R antibody may be referred to as a neutralising or blocking antibody. The inhibitor of the IL-25:IL-25R signalling pathway may be an antibody that is: human, i.e. having an amino acid sequence corresponding to that of an antibody produced by a human and/or which have been made using any of the techniques for making human antibodies; humanised, i.e. derived from a parent antibody generated using a non-human system and modified to contain minimal human sequences; or chimeric, i.e. a fusion including regions from more than one species.
A functional fragment or derivative of any agent or antibody disclosed herein may be a part of said agent or antibody that retains the ability to specifically bind to a given target, and so is an antigen-binding fragment. Functionality does not necessarily require, although it may include, retention of the same binding affinity. An antibody fragment may retain the six CDRs of the parent antibody. A fragment or derivative of any antibody disclosed herein may, for instance, be a Fab fragment (e.g. Fab, Fab′, or F(ab′)2), an Fv fragment, an Fd fragment, a single chain variable fragment (scFv), a minibody (e.g. VL-VH-CH3), a diabody (e.g. (scFv)2 or sc (Fv)2), a single-domain antibody (e.g. VH or VL), triabody, a tetrabody, or any suitable arrangement or fragment. The inhibitor may also have specificity for another antigen, and hence bispecific antibodies, functional fragments, and derivatives are envisaged. The term derivative encompasses antibodies that have undergone humanisation, further humanisation, affinity maturation, fusion to a modified constant domain, or fusion to an antibody constant domain of a different antibody subtype or of a different species. Thus, the inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise any of the fragments disclosed herein, and may be any derivative of an antibody disclosed herein.
The affinity of an antibody, functional fragment, or derivative described herein is the extent or strength of binding to an epitope. Affinity may be expressed as the dissociation constant, KD. The KD may be measured under standard conditions, for instance in a Biacore assay.
An inhibitor of the IL-25:IL-25R signalling pathway may bind to its target with an equilibrium dissociation constant or KD of less than 5×106 M, less than 1×106 M, less than 5×107 M, less than 1×107 M, less than 5×108 M, less than 1×108 M, less than 5×109 M, less than 1×109 M, less than 5×1010 M, less than 1×1010 M, less than 5×1011 M, less than 1×1011 M, less than 5×1012 M, or less than 1×1012 M.
An inhibitor of the IL-25:IL-25R signalling pathway that binds to IL-25, such as an anti-IL-25 antibody, may have a KD that is similar to that of any anti-IL-25 antibody disclosed herein, including 2C3, humanised 2C3, RH2.5_R71V, RH2.5_S30T, RH2.5, or M6. A similar KD may be the same order of magnitude, or may be 10% higher or lower.
An inhibitor of the IL-25:IL-25R signalling pathway that binds to IL-25, such as an anti-IL-25R antibody, may have a KD that is similar to that of any anti-IL25R antibody disclosed herein, including D9.2 and humanised variants. A similar KD may be the same order of magnitude, or may be 10% higher or lower.
An antibody, functional fragment, or derivative described herein may be modified so as to be more suitable for the purposes disclosed herein. For instance, a constant region may be chosen or modified so as to minimise effects such as antibody-dependent cell-mediated cytotoxicity, complement fixation, or binding to inhibitory and/or activating Fc receptors. An antibody, functional fragment, or derivative described herein may be modified so as to have an improved half-life, or improved stability in a particular tissue or environment such as the intestine.
The inhibitor of the IL-25:IL-25R signalling pathway may be an antibody of any class, such as: IgA, IgD, IgE, IgG, or IgM, or subclass (isotype), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. The relevant constant regions may be combined with any VH or VL domains disclosed herein, including VH or VL domains defined only by the complementarity determining regions (CDRs).
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise a monobody or functional fragment or derivative thereof, and hence may be based upon a fibronectin type III domain scaffold.
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise any antibody mimetic, or a functional fragment or derivative thereof. The antibody mimetic may, for example, be any described in Yu et al. including adnectins (monobodies), affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, armadillo repeat protein-based scaffolds, atrimers, avimers, darpins, fynomers, knottins, kunitz domain peptides, or nanofitins (Yu, Yang, Dikici, Deo, and Daunert; Annu Rev Anal Chem (Palo Alto Calif). 2017 Jun. 12; 10 (1): 293-320, incorporated herein by reference in its entirety).
Thus, in another aspect of the invention, there is provided an antibody mimetic, or a functional fragment or derivative thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the antibody mimetic, functional fragment, or derivative thereof specifically binds to IL-25.
Thus, in another aspect of the invention, there is provided an antibody mimetic, or a functional fragment or derivative thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the antibody mimetic, functional fragment, or derivative thereof specifically binds to IL-25R.
The inhibitor of the IL-25:IL-25R signalling pathway may be based upon IL-25R itself. For instance, a soluble IL-25R capable of binding, and hence sequestering, IL-25. Such soluble IL-25R molecules may comprise an Fc domain.
In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway does not have a direct cytotoxic effect on IL-25R+ cancer cells or IL-25R+ cancer stem cells. For instance, in some embodiments the inhibitor is not an antibody-drug conjugate capable of inducing cell death in IL-25R+ colorectal cancer stem cells. In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway has little to no effect on Tuft cell numbers. In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway reduces Tuft cell numbers by no more than 5%, 10%, 20%, 40%, or 50%.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises the antibody “2C3” or any antibody or binding molecule, or functional fragment or derivative thereof, as defined in WO2008/129263, EP2144934B1, U.S. Pat. No. 8,206,717B2, or any member of said patent family (each herein incorporated by reference in its entirety).
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or functional fragment or derivative thereof, that comprises the CDRs of 2C3. The CDRs may be determined by any technique or method, or a combination of such methods. The CDRs may be determined by any method discussed herein.
The CDRs of 2C3, determined according to Kabat, are as follows:
The VH domain of 2C3 may have a sequence as follows:
The VL domain of 2C3 may have a sequence as follows:
Thus, in an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises:
Therefore, in an aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises:
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 22, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In some embodiments, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 11, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 22, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 11.
Thus, in a particular aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 22 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 11.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway may comprise a target binding member that binds IL-25 and which comprises an antibody VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO: 4. In more specific embodiments, the target binding member is a VH domain which comprises a VH CDR3 of SEQ ID NO: 4 together with a CDR1 of SEQ ID NO: 1 and a CDR2 of SEQ ID NO: 3. The VH domain may have human framework regions, or the framework regions shown in SEQ ID NO: 22. The VH domains may be paired with a VL domain of the invention, e.g. a VL domain with a CDR1 of SEQ ID NO: 5, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 8. These CDRs may be in a VL domain having human framework regions or may be the VL domain of SEQ ID NO: 11. Thus in a particular embodiment, the target binding member may bind IL-25 and comprise the 2C3 VH domain (SEQ ID NO: 22) and/or the 2C3 VL domain (SEQ ID NO: 11).
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or fragment or derivative thereof, that binds IL-25 and which comprises an antibody VH domain or a substantial portion thereof comprising a VH CDR1 having an amino acid sequence as set out in SEQ ID NO: 1; a VH CDR2 having an amino acid sequence as set out in SEQ ID NO: 3; and a VH CDR3 having an amino acid sequence as set out in SEQ ID NO: 4, and which further comprises a VL domain or a substantial portion thereof comprising a CDR1 having an amino acid sequence as set out in SEQ ID NO: 5, a CDR2 having an amino acid sequence as set out in SEQ ID NO: 7; and a CDR3 having an amino acid sequence as set out in SEQ ID NO: 8. Optionally, the VH domain or substantial portion thereof comprises a human framework region or the 2C3 amino acid sequence as set out in SEQ ID NO: 22. Optionally, the VL domain comprises a human framework region or the 2C3 amino acid sequence as set out in SEQ ID NO: 11. The 2C3 antibody may, in some embodiments, comprise an IgG1 or IgG4 constant region.
In another particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a humanised form of the antibody 2C3 or any antibody or binding molecule, or functional fragment or derivative thereof, as defined in WO2010/038155, EP2344538B1, U.S. Pat. No. 8,658,169B2, or any member of said patent family (each herein incorporated by reference in its entirety). In particular, the inhibitor may be, or may comprise, the antibodies RH2.5_R71V, RH2.5_S30T, or RH2.5, or a functional fragment or derivative thereof.
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or functional fragment or derivative thereof, that comprises the CDRs of a humanised 2C3 as defined in the WO2010/038155 patent family, for instance RH2.5_R71V, RH2.5_S30T, or RH2.5. The CDRs may be determined by any technique or method, or a combination of such methods. The CDRs may be determined by any method discussed herein.
The CDRs of humanised 2C3, according to one method of determination, may have any of the following sequences.
The VH domain of humanised 2C3, or of a 2C3 derivative, may have any of the following sequences:
wherein:
The VL domain of humanised 2C3, or of a 2C3 derivative, may have any of the following sequences:
Thus, in an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises:
Therefore, in an aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises:
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises:
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOS: 10, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 41, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 24, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 41.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 25, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 41.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 26, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 41.
Thus, in a particular aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 24, 25, or 26, and an antibody VL domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 11 or 41.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway may be a target binding member that binds IL-25, and comprises an antibody VH domain which comprises SEQ ID NO:24:
wherein:
In some embodiments:
In some embodiments, the residues Xa1-Xa5 are in the following combinations:
In some embodiments:
The humanised 2C3 antibody may comprise a constant region, for instance an IgG1 or IgG4 constant region.
In another particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a humanised form of the antibody 2C3 or any antibody or binding molecule, or functional fragment or derivative thereof, as defined in WO2011/123507, EP2552961B1, U.S. Pat. No. 8,785,605B2, or any member of said patent family (each herein incorporated by reference in its entirety). In a particular embodiment, the inhibitor is or comprises the antibody M6, or a functional fragment or derivative thereof.
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or functional fragment or derivative thereof, that comprises the CDRs of a humanised 2C3, for instance M6, as defined in the WO2011/123507 patent family. The CDRs may be determined by any technique or method, or a combination of such methods. The CDRs may be determined by any method discussed herein.
The CDRs of M6, according to one method of determination, may have any of the following sequences.
The VH domain of M6 may the following sequence:
The heavy chain of M6 may have the following sequence:
The VL domain of M6 may the following sequence:
The light chain of M6 may have the following sequence:
Thus, in an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises:
Therefore, in an aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises:
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOS: 25, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 46, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 25, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 46.
Thus, in a particular aspect of the invention, there is provided an anti-IL-25 antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25 antibody comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 25, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 46.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 45, and polypeptide comprising the amino acid sequence shown in SEQ ID NO: 47.
In one embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a target binding member that binds IL-25, wherein the target binding member binds one or more amino acid sequences selected from the group consisting of amino acid residues 46-63 of SEQ ID NO: 48, amino acid residues 66-84 of SEQ ID NO: 48 and amino acid residues 129-135 of SEQ ID NO: 48. In a particular embodiment, the target binding member of the invention binds amino acid residues 56-63 of SEQ ID NO: 48 and amino acid residues 66-74 of SEQ ID NO: 48. In another embodiment, the target binding member comprises an antibody VL domain comprising a CDR3 having the amino acid sequence QQYLAFPYTF (SEQ ID NO: 79).
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a target binding member that binds IL-25, wherein the target binding member comprises:
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a target binding member comprises a VL domain comprising SEQ ID NO: 47 and a VH domain comprising SEQ ID NO: 45. In a further embodiment, the target binding member comprises a whole antibody.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a target binding member that competes for binding to IL-25 with a target binding member that binds one or more amino acid sequences selected from the group consisting of amino acid residues 46-63 of SEQ ID NO: 48, amino acid residues 66-84 of SEQ ID NO: 48 and amino acid residues 129-135 of SEQ ID NO: 48. In a particular embodiment, the target binding member has a binding affinity for human IL-25 that is less than or equal to about 50 pM.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a target binding member of the invention comprising:
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a humanized antibody or antigen binding fragment thereof, that binds IL-25, wherein the antibody or antigen binding fragment thereof comprises:
the antibody or antigen binding fragment thereof has a binding affinity for IL-25 that is less than or equal to 53 pM as determined in a Biacore assay. The binding affinity for IL-25 may be less than or equal to about 50 pM, about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, or about 20 pM. The humanized antibody or antigen binding fragment thereof may comprise the VL domain of the antibody light chain of SEQ ID NO: 47. The humanized antibody or antigen binding fragment thereof of may comprise the VH domain of the antibody heavy chain of SEQ ID NO: 45.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25, and comprises
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOS: 10, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38, and an antibody VL domain comprising the amino acid sequence shown in any one of SEQ ID NOS: 11, 41, or 47. Optionally the VH and/or VL domain comprises at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
The leader sequence for the VH domain of any 2C3 antibody, variant, or derivative may be any suitable sequence, for instance any sequence as follows:
The leader sequence for the VL domain of any 2C3 antibody, variant, or derivative may be any suitable sequence, for instance any sequence as follows:
In another particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises the antibody “D9.2” or any antibody or binding molecule as defined in WO2010/116123, EP2417161B1, U.S. Pat. No. 8,586,037B2, U.S. Pat. No. 8,852,589, or any member of said patent family (herein incorporated by reference in its entirety), or a functional fragment or derivative thereof.
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or fragment or derivative thereof, that comprises the CDRs of D9.2. The CDRs may be determined by any technique or method, or a combination of such methods. The CDRs may be determined by any method discussed herein.
The CDRs of D9.2, according to one method of determination, are as follows:
The VH domain of D9.2 may have a sequence as follows:
The VL domain of D9.2 may have a sequence as follows:
Thus, in an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25R, and comprises:
Therefore, in an aspect of the invention, there is provided an anti-IL-25R antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25R antibody comprises:
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In some embodiments, the antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 55, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In some embodiments, the antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 56, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25R, and comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 55, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 56.
Thus, in a particular aspect of the invention, there is provided an anti-IL-25R antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25R antibody comprises an antibody VH domain comprising the amino acid sequence shown in SEQ ID NO: 55, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 56.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises an antibody molecule which binds IL-25R and which comprises an antibody VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO: 51.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises an antibody molecule that comprises a VH domain which comprises a VH CDR3 of SEQ ID NO: 51 together with a CDR1 of SEQ ID NO: 49 and a CDR2 of SEQ ID NO: 50. The VH domain may be paired with a VL domain, for example a VL domain with a CDR1 of SEQ ID NO: 52, a CDR2 of SEQ ID NO: 53 and a CDR3 of SEQ ID NO: 54. In some embodiments, a VH domain may be paired with a VL domain of SEQ ID NO: 55.
In an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises an antibody molecule that comprises a VH domain which comprises a VH CDR1 of SEQ ID NO: 49, a VH CDR2 of SEQ ID NO: 50 and a VH CDR3 of SEQ ID NO: 51 and a VL domain which comprises a VL CDR1 of SEQ ID NO: 52, a VL CDR2 of SEQ ID NO: 53 and a VL CDR3 of SEQ ID NO: 54.
The VH domain may further comprise human or non-human framework regions, for example the framework regions shown in SEQ ID NO: 55. In some embodiments, the antibody molecule may comprise the VH domain of SEQ ID NO: 55.
The VL domain may further comprise human or non-human framework regions, for example the framework region shown in SEQ ID NO: 56. In some embodiments, the antibody molecule may comprise the VL domain of SEQ ID NO: 56.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises an antibody molecule that comprises the VH domain of SEQ ID NO: 55 and the VL domain of SEQ ID NO: 56.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises an antibody molecule that specifically binds mouse and/or human IL-17BR (also known as IL-25R) and which comprises:
The VH domain may comprise a human framework region or SEQ ID NO: 55.
The antibody may be cross reactive with both human IL-17BR and mouse IL-17BR.
The VL domain may comprise a human framework region or SEQ ID NO: 56.
The D9.2 antibody may comprise a constant region, for instance an IgG1 or IgG4 constant region.
In another particular embodiment, the inhibitor is or comprises a humanised form of the antibody D9.2 or any antibody or binding molecule as defined in WO2020/115319 (herein incorporated by reference in its entirety), or a functional fragment or derivative thereof.
The inhibitor of the IL-25:IL-25R signalling pathway may be or may comprise an antibody, or fragment or derivative thereof, that comprises the CDRs of humanised
D9.2. The CDRs may be determined by any technique or method, or a combination of such methods. Such methods include use of the Kabat numbering system, Chothia numbering system, IMGT numbering system, and/or the extended definition. The CDRs may be determined by modelling software (such as AbM or WAM), or by structural or crystallographic techniques to determine contact residues.
The CDRs of humanised D9.2, according to one method of determination, may have any of the following sequences.
The VH domain of humanised D9.2 may have a sequence as follows:
The VL domain of humanised D9.2 may have a sequence as follows:
The humanised D9.2 antibody may comprise a constant region, for instance an IgG1 or IgG4 constant region. The constant region may be a hinge stabilised IgG4 constant region according to the following sequence:
The heavy chain of the humanised D9.2 antibody may have the following sequence:
Thus, in an embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25R, and comprises
Therefore, in an aspect of the invention, there is provided an anti-IL-25R antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25R antibody comprises:
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25R, and comprises
an antibody VH domain, or functional fragment or derivative thereof, comprising:
an antibody a VL domain, or functional fragment or derivative thereof, comprising:
Therefore, in an aspect of the invention, there is provided an anti-IL-25R antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25R antibody comprises:
an antibody VH domain, or functional fragment or derivative thereof, comprising:
an antibody a VL domain, or functional fragment or derivative thereof, comprising:
a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 65.
The above recited CDRs may comprise one, two, or three amino acid substitutions compared to the recited sequence, which may be conservative substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 66, 67, 68, 69, or 70, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 66, 67, 68, 69, 70, 80, 81, 82, 83, 84, 85, or 86, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 71 or SEQ ID NO: 72, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway comprises an antibody VL domain comprising the amino acid sequence shown in any one of SEQ ID NOS: 71, 72, 87, 88, 89, 90, or 91, optionally comprising at least one, from one to ten, from one to five, from one to three, or one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. The substitutions may be in residues that are not within a CDR. In some embodiments, said amino acid substitutions are conservative amino acid substitutions. The substitutions may be such that the properties of the inhibitor are not affected, or are not negatively affected. For instance, binding affinity may be retained or improved. In a particular embodiment, the domain comprises from one to three conservative amino acid substitutions.
In a particular embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is capable of binding to IL-25R, and comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 68, 69, or 70, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 72.
Thus, in a particular aspect of the invention, there is provided an anti-IL-25R antibody for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the anti-IL-25R antibody comprises an antibody VH domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 68, 69, or 70, and an antibody VL domain comprising the amino acid sequence shown in SEQ ID NO: 72.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a humanised antibody molecule comprising a heavy chain variable domain and a light chain variable domain, wherein a) the heavy chain variable domain (VH) comprises a VHCDR1 of SEQ ID NO: 49, a VHCDR2 of SEQ ID NO: 57, and a VHCDR3 of SEQ ID NO: 51, and b) the light chain variable domain (VL domain) comprises a VLCDR1 of SEQ ID NO: 52, a VLCDR2 of SEQ ID NO: 53, and a VLCDR3 of SEQ ID NO: 65. The antibody molecule may specifically bind to interleukin-17 receptor B (IL17BR), preferably the extracellular domain of IL17BR. Optionally the antibody may bind to the interleukin-17 receptor with a binding affinity of at least 75% of the binding affinity of the murine antibody D9.2 to interleukin-17 receptor B, as measured by ELISA. The expression level of the antibody may be at least 40% of the expression level of the murine antibody D9.2 in mammalian cells.
In another embodiment, the inhibitor of the IL-25:IL-25R signalling pathway is or comprises a humanised form of the antibody D9.2 that comprises the VH domain of SEQ ID NO: 67, optionally with up to four additional framework substitutions. For example, the antibody may comprise a VH domain of SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, optionally with up to four additional framework substitutions. The antibody may comprise the VL domain of SEQ ID NO: 71, optionally with up to four additional framework substitutions. In some embodiments, the antibody molecule of may comprise the VH domain of SEQ ID NO: 68 and the VL domain of SEQ ID NO: 72.
The heavy chain variable domain (VH domain) of the humanised form of the antibody D9.2 may comprise SEQ ID NO: 67 wherein X3 is N. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X4is K. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X5 is D. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X1 is Q. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X2 is A.
The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X6 is M.
The heavy chain variable domain (VH domain) of the humanised form of the antibody D9.2 may comprise SEQ ID NO: 67 wherein X, is E. . . . The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X2 is T. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X6 is L.
The heavy chain variable domain (VH domain) of the humanised form of the antibody D9.2 may comprise SEQ ID NO: 67 wherein X3 is A. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X4 is Q. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X5 is G. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X, is E. The heavy chain variable domain (VH domain) may comprise SEQ ID NO: 67 wherein X2 is T. The heavy chain variable domain (VH domain) comprises SEQ ID NO: 67 wherein X6 is L.
The VH domain of the humanised form of the antibody D9.2 may be fused to an antibody constant region comprising SEQ ID NO: 73. The humanised D9.2 may comprise an amino acid sequence of the VH domain and constant region which comprises SEQ ID NO: 74.
The humanised D9.2 antibody may further comprise a second heavy chain variable domain and a second light chain variable domain which form an antigen binding site for an antigen other than IL17BR.
The following sequences relate to the humanised D9.2 antibodies.
The following tables relate to the humanised D9.2 antibodies.
In the tables above, D9.2 VH is SEQ ID NO: 80, EF178110 is SEQ ID NO: 94. D9 HA is SEQ ID NO: 66, D9 HB is SEQ ID NO: 81, D9 HC is SEQ ID NO: 82, D9 HD is SEQ ID NO: 83, D9 HE is SEQ ID NO: 84, D9 HF is SEQ ID NO: 85, D9 HG is SEQ ID NO: 86, D9 HH is SEQ ID NO: 69, D9 HI is SEQ ID NO: 70, D9 HJ is SEQ ID NO: 68, D9.2 VK is SEQ ID NO: 87, Y14869 is SEQ ID NO: 95, D9_KA is SEQ ID NO: 71, D9_KB is SEQ ID NO: 88, D9_KC is SEQ ID NO: 89, D9_KD is SEQ ID NO: 90, KM is SEQ ID NO: 91, and D9_KE is SEQ ID NO: 72.
A CDR may be determined by technique or method, or a combination of such methods. Such methods include use of the Kabat numbering system, Chothia numbering system, the accumulation of both Kabat and Chothia, and/or the IMGT numbering system. The CDRs may be determined by modelling software (such as AbM, Accelrys, or WAM), or by structural or crystallographic techniques to determine contact residues. The CDRs may be determined by the contact definition which is based on observed antigen contacts and is described in MacCallum et al. (J. Mol. Biol., 262:732-745, 1996). The CDRs may be determined by the conformational definition, which identified residues that make enthalpic contributions to antigen binding (Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008). Thus, while examples of CDRs are given herein, the inhibitor of the IL-25:IL-25R signalling pathway may comprise CDRs that are determined from any VH or VL sequence disclosed herein according to any suitable method.
As used herein, a conservative substitution is a replacement of an amino acid residue with an amino acid residue of a different type that has similar properties to the original residue. For instance, a conservative substitution may be one that does not change the functional properties of the polypeptide in which the original residue is found. In an example, a conservative substitution may be any replacement within the group: G, A, V, L, or I; within the group: S, C, T, M; within the group: F, Y, W; within the group: H, K, R; or within the group: D, E, N, Q.
In embodiments of the invention, the cancer of the intestine may be any cancer involving abnormal cell growth in the intestine of a subject. The cancer may be a primary tumour arising in the intestine. The cancer of the intestine may be secondary tumour, which has spread from a primary tumour in another tissue. The cancer of the intestine may be colorectal cancer (CRC). CRC may also be referred to as bowel cancer, colon cancer, or rectal cancer. CRC is caused by the abnormal growth of cells within the large intestine, for instance the abnormal growth of cells within the colon or rectum. CRC may encompass: colorectal adenocarcinomas, such as mucinous adenocarcinoma or signet ring cell adenocarcinoma; gastrointestinal carcinoid tumours; primary colorectal lymphomas; gastrointestinal stromal tumours;
leiomyosarcomas; and colitis-associated cancer (CAC). The CRC may be mismatch-repair-deficient (dMMR) CRC or mismatch-repair-proficient (pMMR) CRC. The CRC may be consensus molecular subtype (CMS) 1, CMS2, CMS3, and/or CMS4.
In some embodiments, the cancer of the intestine does not include CAC. In some embodiments, the cancer of the intestine is pMMR CRC. In a particular embodiment, the CRC comprises intestinal epithelial cells with mutations in the APC tumour suppressor gene.
In some embodiments, the disease to be treated is IL25high CRC. In some embodiments, the disease to be treated has a high frequency of ILC2s. In an embodiment, the tumour has an increased frequency of ILC2s compared to the ILC2 frequency in adjacent normal intestine. This may be determined by comparison of a tumour biopsy to non-tumour intestinal tissue from the same subject, or to the expected ILC2 frequency in non-tumour intestinal tissue. “Adjacent normal intestine” may mean intestinal tissue that does not comprise tumour cells or tumour stroma, and which is taken from a part of the intestine in which the tumour is situated.
In some embodiments, a tumour biopsy from the subject is tested before treatment. The tumour biopsy may be tested in order to determine if the tumour comprises mutations in the APC tumour suppressor gene. Alternatively, or in addition, the tumour biopsy may be tested in order to determine if the tumour is IL25high. Alternatively, or in addition, the tumour biopsy may be tested in order to determine if the tumour has a high frequency of ILC2s.
The inventors have noted that response to immune checkpoint inhibitor therapy in CRC patients is largely limited to mismatch-repair-deficient (dMMR) CRC, comprising only a small proportion (15%) of total CRC cases. By contrast, the vast majority of CRC, following the conventional pathway initiated by mutations in the APC gene, are mismatch-repair-proficient (pMMR), and rarely respond to immune checkpoint inhibitors. Furthermore, in the past decade, efforts to develop novel immunotherapies for CRC, such as tumour vaccines and adoptive T cell transfer, have shown little efficacy. Recently, the phase III clinical trial IMblaze370 using the anti-PD-L1 checkpoint inhibitor atezolizumab to treat CRC patients (>98% had mismatch-repair-proficient CRC) failed to meet the primary end point.
However, the inventors demonstrate herein that, when treating intestinal cancer, inhibitors of the IL-25:IL-25R signalling pathway shift the balance of the immune response away from MDSCs and towards infiltration by anti-tumoural CD8 T cell infiltration. As such, the blockade of the IL-25:IL-25R signalling pathway may increase the efficacy of immune checkpoint inhibitor therapy.
As such, in an embodiment, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method further comprises immune checkpoint inhibitor therapy.
The inhibitor of the IL-25:IL-25R signalling pathway may be any disclosed herein. In a particular embodiment, there is provided an anti-IL-25 antibody, derivative, or functional fragment thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method further comprises immune checkpoint inhibitor therapy. In another embodiment, there is provided there is provided an anti-IL-25R antibody, derivative, or functional fragment thereof for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method further comprises immune checkpoint inhibitor therapy.
The cancer of the intestine may be any disclosed herein. In a particular embodiment, the cancer of the intestine is pMMR CRC. The pMMR CRC may comprise intestinal epithelial cells with mutations in the APC tumour suppressor gene.
The immune checkpoint inhibitor therapy may be administered before, at the time of, or after the administration of the inhibitor of the IL-25:IL-25R signalling pathway. The immune checkpoint inhibitor may be physically separate from, or combined with, the inhibitor of the IL-25:IL-25R signalling pathway.
The immune checkpoint inhibitor therapy may comprise the administration of an inhibitor of the PD-1: PD-L1 pathway. The inhibitor of the PD-1: PD-L1 pathway may specifically bind to PD-1 or specifically bind to PD-L1. For instance, the inhibitor of the PD-1: PD-L1 pathway may be an anti-PD-1 antibody or an anti-PD-L1 antibody. Also encompassed are compositions comprising functional fragments or derivatives of such antibodies.
The immune checkpoint inhibitor therapy may comprise the administration of an inhibitor of the CTLA-4:CD80/CD86 pathway. The inhibitor of the CTLA-4:CD80/CD86 pathway may specifically bind to CTLA-4. For instance, the inhibitor of the CTLA-4:CD80/CD86 pathway may be an anti-CTLA-4 antibody. Also encompassed are compositions comprising functional fragments or derivatives of such antibodies.
The immune checkpoint inhibitor therapy may target LAG-3, TIM-3, TIGIT, VISTA, B7-H4, BTLA, and/or Siglec-15. Inhibitors targeting any of these molecules may specifically bind to said molecule, for instance the inhibitor may be an antibody, functional fragment, or derivative thereof, capable of specifically binding to any of said molecules.
Thus, in an embodiment, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method further comprises an immune checkpoint inhibitor therapy targeted towards a molecule selected from the group: PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H4, BTLA, Siglec-15, and any combination thereof. In a particular embodiment, the immune checkpoint inhibitor therapy is a combination targeted towards CTLA-4 and PD-1/PD-L1.
In an aspect of the invention, there is provided a kit comprising an immune checkpoint inhibitor and an inhibitor of the IL-25:IL-25R signalling pathway. In another aspect of the invention, there is provided a pharmaceutical composition comprising an immune checkpoint inhibitor and an inhibitor of the IL-25:IL-25R signalling pathway.
In an embodiment, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method does not comprise the administration of a HER inhibitor. In an embodiment, there is provided an inhibitor of the IL-25:IL-25R signalling pathway for use in a method of treating, preventing, or ameliorating cancer of the intestine, wherein the method does not comprise the administration of an EGFR (HER1) inhibitor. The HER inhibitor or EGFR inhibitor may be any as disclosed in WO2017/194554 (herein incorporated by reference).
In another aspect of the invention, there is provided a method of treating, preventing, or ameliorating cancer of the intestine in a subject in need thereof, comprising administering an effective amount of an inhibitor of the IL-25:IL-25R signalling pathway. The inhibitor of the IL-25:IL-25R signalling pathway any be any such inhibitor, including any disclosed herein. The cancer of the intestine may be any cancer of the intestine, including any disclosed herein. The subject of the treatment may be any subject, including any disclosed herein.
Also provided is a method of reducing ILC2s, M-MDSCs, and/or G-MDSCs in a tumour in the intestine, comprising administering an effective amount of an inhibitor of the IL-25:IL-25R signalling pathway. The inhibitor of the IL-25:IL-25R signalling pathway any be any such inhibitor, including any disclosed herein. The tumour of the intestine may be the result of any cancer of the intestine, including any disclosed herein. The subject of the treatment may be any subject, including any disclosed herein. The method may result in the treatment, prevention, or amelioration of the cancer.
Also provided is a method of altering the immune composition in the microenvironment of in a tumour in the intestine, comprising administering an effective amount of an inhibitor of the IL-25:IL-25R signalling pathway. The immune microenvironment may be altered to reduce ILC2s, M-MDSCs, and/or G-MDSCs and to increase ILC1s and/or CD8 T cells. The inhibitor of the IL-25:IL-25R signalling pathway any be any such inhibitor, including any disclosed herein. The tumour of the intestine may be the result of any cancer of the intestine, including any disclosed herein. The subject of the treatment may be any subject, including any disclosed herein. The method may result in the treatment, prevention, or amelioration of the cancer.
In another aspect of the invention, there is provided use of an inhibitor of the IL-25:IL-25R signalling pathway for the manufacture of a medicament for the treatment, prevention, or amelioration of cancer of the intestine.
As used herein, a therapeutically effective amount is the amount sufficient to effect any one or more beneficial or desired results.
As used herein, ameliorating means a lessening or improvement in one or more symptoms as compared to not administering the inhibitor of the IL-25:IL-25R signalling pathway.
In an embodiment, the subject of the treatments disclosed herein is an animal subject, for instance a jawed-vertebrate, mammal, or domestic animal. Hence, the inhibitor of the IL-25:IL-25R signalling pathway may be used to treat any mammal, for example livestock (for example, a horse), pets, or may be used in other veterinary applications. In a particular embodiment, the subject is a human. In some embodiments, the subject has been diagnosed with cancer of the intestine.
The inhibitors disclosed herein may be present as part of a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable vehicle, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable stabilizer, or a pharmaceutically acceptable preservative, or any combination thereof.
To be pharmaceutically acceptable, a substance or combination of substances must be suitable for the formulation of pharmaceutical compositions or a medicament.
The pharmaceutical compositions of the invention may be manufactured, in a further aspect, using a process comprising contacting a therapeutically effective amount of any of inhibitors disclosed herein and a pharmaceutically acceptable vehicle, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable stabilizer, or pharmaceutically acceptable preservative, or any combination thereof.
A composition comprising any one of inhibitors disclosed herein may take any number of forms known in the art depending on the manner in which it is to be used. For example, in the form of a liquid, powder, suspension, tablet, capsule, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, or any other suitable form for storage, shipping, or administration.
In some embodiments, the composition may be in a format suitable for storage or shipping. For instance, as part of lyophilized compositions which may include other components if necessary for lyophilization or storage, such as stabilizers. Alternatively, the composition may be in solution with a diluent, and may be present at a concentration appropriate for storage or shipping.
In some embodiments, the composition may be in a format suitable for administration to a patient. For instance, in the form of a liquid suitable for injection, or in a concentrated form suitable for administration after a preparatory step. The composition may be in association with carriers for administration, and may be sterile. The composition may be in a format suitable for injection into the patient, for instance intravenous injection or subcutaneous injection. The composition may be in a format suitable for topical administration to the intestine, rectum, or bowel.
In some embodiments, the composition may be present in slow-release compositions.
It will be appreciated that the amount of the composition that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the composition, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject to be treated. Optimal dosages to be administered may be determined by those skilled in the art and will vary depending on the context. Additional factors may depend on the particular subject being treated, the advancement of the disease, disorder, or condition, subject age, subject weight, subject sex, subject diet, and time of administration.
In some examples, the composition may be administered as a single dose, yearly dose, monthly dose, weekly dose, or daily dose. The dose may be administered two or more times a day. In an example, the dose may be from 0.001 μg to 10 mg per kg of body weight, 0.01 μg to 1 mg per kg of body weight, 0.05 μg to 100 μg per kg of body weight, or 0.1 μg to 10 μg per kg of body weight. In an example, the dose may be 0.07 μg to 700 mg, 0.7 μg to 70 mg, 3.5 μg to 7 mg, or 7 μg to 0.7 mg.
It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Methods and materials similar to or equivalent to those described herein can be used in the practice and testing of the invention, and suitable methods and materials are described below.
FIG. 10: IL-25 does not induce tumours in ILC2-deficient mice. a, Gut length in vehicle or rIL-25 treated ILC2-deficient (Roraf/fIl7rCre/+) Apc1322T/+ mice (vehicle, n=9; rIL25, n=9). b, Representative FACS plot showing lack-of ILC2s in vehicle and rIL-25 treated ILC2-deficient Apc1322T/+ mice. c-j, Frequency of tumour M-MDSCs and G-MDSCs (c), macrophages (d), conventional dendritic cells (CDC) (e), plasmacytoid dendritic cells (pDC) (f), mast cells and basophils (g), Tregs (h), γδ T cells (i) and tuft cells (j), in vehicle and rIL-25 treated ILC2-deficient Apc1322T/+ mice (vehicle, n=7; rIL25, n=7). Data collected from female mice between 77 to 83 days of age; each dot indicates an individual mouse. Data pooled from two or more independent experiments and error bars show mean±SEM. Statistical significance determined by unpaired two-tailed t-test.
IL-25 and ILC2s help defend the host against intestinal parasitic helminth infection, and are associated with inappropriate allergic reactions. Recently, it was reported that IL-33-activated ILC2s augment protective tissue-specific cancer immunity. Here the inventors show a diametrically opposite role for IL-25-activated ILC2s in the intestine where they create a cancer-permissive microenvironment. Higher tumour IL25 gene expression is associated with reduced survival of colorectal cancer (CRC) patients, and IL-25R-expressing ILC2s are elevated in human CRC tumours. In a mouse model of spontaneous intestinal tumorigenesis, exogenous IL-25 increases tumours, whilst ablation of IL-25-signalling reduces tumour burden and virtually doubles life-expectancy, while reducing IL-25-responsive tumour ILC2s and shifting the balance towards cytotoxic CD8 T cell infiltration. Mice genetically lacking ILC2s are also more resistant to tumour development and showed prolonged survival, even when treated with exogenous IL-25. Like IL-25-deficient mice, ILC2-deficient mice had fewer tumour-resident immunosuppressive myeloid-derived suppressor cells (MDSCs), and showed improved anti-tumour innate and adaptive immunity. Thus, the roles of the innate epithelium-derived cytokines IL-25 and IL-33, and ILC2s in cancer cannot be generalised and may take pro-or anti-cancer phenotypes dependent on local microenvironmental cues within specific tissues or organs. Notably, IL-25-signalling blockade decreased ILC2s, MDSCs and tumorigenesis suggesting immunotherapeutic studies may be warranted in CRC.
ILC2s are known to act as sentinels within barrier tissues, responding rapidly to acute immune perturbation by releasing effector cytokines. However, the tissue specific microenvironmental cues that differentially regulate innate ILC2-mediated immunity, in the context of a chronic cancer-permissive tissue niche, are poorly understood. Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide, and is expected to increase by a further 60% by 2030. Analysis of CRC tumour gene expression on large publicly available databases, stratified by IL25-high or IL25-low gene expression, identified significantly decreased overall, and disease-free, patient survival in the IL25-high group (
Approximately 80% of human CRC have mutations in the adenomatous polyposis coli (APC) tumour suppressor gene. Consequently, the inventors investigated IL-25 expression in the Apc1322T/+ model of spontaneous intestinal tumorigenesis that produces a mutant APC protein that retains one β-catenin binding degradation repeat—a scenario that closely mirrors human APC protein truncations. Analysis of IL25-tomato reporter mice (IL25tom/+Apc1322T/+) identified EpCAM+DCLK+ tuft cells as the main source of IL25-tomato in mouse tumours (
Collectively, these results demonstrate that IL-25 expression is associated with both human and mouse intestinal cancer, along with an increase in IL-25R-expressing ILC2s and immunosuppressive MDSCs, but fewer anti-tumour-associated T cells.
To investigate the role of IL-25 in intestinal tumorigenesis the inventors injected Apc1322T/+ mice with recombinant IL-25 (rIL-25) (
The inventors next generated IL-25-deficient (Il25tom/tom) Apc1322T/+ mice and compared their tumorigenesis to the IL-25-replete Apc1322T/+ mice. Il25tom/tom Apc1322T/+ mice developed considerably fewer and smaller tumours (
Although the inventors did not observe a change in tumour Th1 or CD8 T cell frequency (
To determine whether ILC2s could be responsible for IL-25-dependent intestinal tumorigenesis, the inventors generated ILC2-deficient (Roraf/fIl7rCre/+) Apc1322T/+ mice (
To confirm that the pro-tumoural effect of exogenous IL-25 is ILC2 dependent, the inventors injected ILC2-deficient Apc1322T/+ mice with rIL-25 using the same protocol as above.
Critically, there was no increase in tumour burden or tumour size in ILC2-deficient mice treated with rIL-25 (
ILC2s are defined by their expression of IL-13, and several studies have shown that IL-13, and more recently ILC2s, can promote MDSCs to suppress anti-tumour T cells in both mouse and human cancers. IL-13, and another ILC2 cytokine IL-4, signal through STAT6, and STAT6 deletion reduces MDSCs and tumours, while restoring anti-tumour CD8 immunity in Apcmin/+ mice. The inventors found that M-MDSCs from Apc1322T/+ intestinal tumours express and upregulate the receptors IL-4RA and IL-13RA1 subunits as compared to the few M-MDSCs in the normal gut (
The results presented herein demonstrate that the innate IL-25:ILC2s axis supports intestinal tumour progression at least in part through promoting MDSCs and suppressing anti-tumoural T cells. To investigate whether these mechanisms are conserved in humans, the inventors analysed immune cells in paired unselected human CRC and normal colonic tissue samples. Th1 and CD8 T cell infiltration is amongst the strongest positive prognostic factors for survival of human CRC patients across all stages of disease. Notably, correlation analysis showed a significant negative correlation between tumour CD8 T cells and ILC2s in human CRC (
Furthermore, human CRCs had increased CD11b+CD14−CD15+CD33int G-MDSCs (
The similar immune cell representation in human CRC and Apc1322T/+ mouse tumours suggest potential mechanistic conservation. The inventors assessed whether interrupting the innate IL-25:ILC2 immune axis could be targeted therapeutically, given the need for new drugs to treat CRC patients and the disappointing outcome of the recent phase III clinical trial IMblaze370 using the anti-PD-L1 checkpoint inhibitor atezolizumab to treat CRC patients (>98% had mismatch-repair-proficient CRC), which failed to meet the primary end point. The inventors confirmed expression of several checkpoint ligands on CRC MDSCs including CD86 (ligand for CTLA-4), PD-L1 (ligand for PD-1), HVEM (ligand for BTLA), CD155 and CD112 (ligand for TIGIT) on M-MDSCs, and PD-L1 and CD112 on G-MDSCs (
The inventors treated young-adult Apc1322T/+ mice with neutralising anti-IL25R antibody (clone D9.2) or control (
Similar results were obtained when the inventors treated Apc1322T/+ mice with neutralising anti-IL25 antibody (RH2.5_R71V—with the variable domains expressed within the mouse IgG1 framework to enable in vivo studies in mice). Anti-IL-25 significantly reduced intestinal tumour numbers and size in the Apc-induced model of intestinal tumourigenesis as compared to isotype control (
Our results demonstrate critical roles for IL-25 and ILC2s in maintaining the chronic cancer-permissive microenvironment that prevents the ascendency of anti-tumoural immunity in intestinal cancer. Notably, this immune-suppressive niche was unlikely to be dependent on adaptive Th2 cells, which did not change numerically in intestinal tumours in response to IL-25-deficiency, rIL-25 treatment, or ILC2-deficiency, but was dependent on ILC2s. A recent study reported that tissue-specific IL-33 can activate ILC2s to induce anti-tumour immunity in mouse models of orthotopic pancreatic cancer, but not in subcutaneous grafts of pancreatic cancer (Moral, J. A. et al. ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature 579, 130-135, doi: 10.1038/s41586-020-2015-4 (2020)). Strikingly, the data presented herein now demonstrate that tissue specialisation of IL-25-responsive intestinal ILC2s have diametrically opposite function, instead leading to them sustaining a tumorigenic niche in the intestine. Thus, the roles of the epithelium-derived cytokines IL-25 and IL-33, and ILC2s in tumorigenesis are likely to be location-dependent and organ specific, and not easily generalisable. Although it has been suggested that a small proportion of tuft cells may act as tumour stem cells, ILC2-deficiency decreased tumour burden but not tuft cell frequency, emphasising the critical role of ILC2s in this process. Notably, serum IL-25 is elevated in advanced CRC, and CRC is largely resistant to current immunotherapy. The results presented herein identify the IL-25-ILC2-MDSC axis as a novel immunotherapeutic target, and indicate that equivalent mechanisms may operate in human CRC. Since the anti-IL25R monoclonal antibody (clone D9.2) and the anti-IL25 monoclonal antibody (RH2.5_R71V) also inhibit human IL-25R signalling, this raises the possibility for future human immunotherapeutic studies in treating CRC. Furthermore, as anti-IL25R and anti-IL25 shift the balance away from MDSCs and towards anti-tumoural CD8 T cell infiltration, the beneficial effects may be potentiated by combined therapies with currently ineffectual checkpoint inhibitors.
All wild-type and Apc1322T/+ mice (Pollard, P. et al. The Apc 1322T mouse develops severe polyposis associated with submaximal nuclear beta-catenin expression. Gastroenterology 136, 2204-2213.e2201-473 2213, doi: 10.1053/j.gastro.2009.02.058 (2009)) in this study were on a C57BL/6 background and bred in-house. Mice were maintained at the Medical Research Council ARES animal facility under specific pathogen-free conditions. Breeders were placed on breeding diet (ENVIGO, 2919), while all experimental animals were on standard diet (SDS, 801722). All mice used in this study were females and were euthanised between 11 to 12 weeks of age, unless stated otherwise. For survival studies, mice were culled when showing clinical signs of pale feet, piloerection, hunching and weight loss, and the age recorded. No statistical tests were used to predetermine sample sizes. All animal experiments performed were under the approval of the UK Home Office.
The Il25-knockout-tomato reporter mice were generated through recombineering (Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13, 476-484, doi: 10.1101/gr.749203 (2003); Warming, S., Costantino, N., Court, D. L., Jenkins, N. A. & Copeland, N. G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33, e36, doi: 10.1093/nar/gni035 (2005)).
Neomycin-negative and Cre-recombinase-negative mice were backcrossed onto C57BL/6 background at least six times. Genotyping was through using PCR primers (forward, GACAGAATTGCAGATGCTATTACTACGACC; SEQ ID NO: 75) and (reverse, ACCTCCTCGCCCTTGCTCACCAT; SEQ ID NO: 76) for the targeted product (380bp), and (forward, GGCTAGGCTTCCAGGCTTCCAG; SEQ ID NO: 77) and (reverse, GGGGTTCTTGCTCTTTGCTGGG; SEQ ID NO: 78) for the wild-type product (200bp).
Mice were injected intraperitoneally with 0.04 mg/kg of recombinant mouse IL-25 (Janssen) in PBS, three times per week for eight weeks, starting from weaning at three weeks of age. For IL-25R (IL-17BR) blockade, mice were injected intraperitoneally with 12.5 mg/kg of anti-IL25R (clone D9.2) 284 or control mIgG1 (LifeArc), twice a week for seven weeks, starting from three weeks of age. After the final injection, mice were euthanised and samples collected.
Gender, background and age-matched mice, as described above, were euthanised and tissues harvested. Small intestines were flushed with cold PBS+10 mM HEPES (Gibco), opened longitudinally, and fixed in methacarn (60% methanol, 30% chloroform and 10% acetic acid glacial) for 24 hours and subsequently washed with PBS. Tumour numbers were counted, and tumour size measured with a ruler under a light microscope. For tumour scoring, mouse genotypes and/or treatment conditions were blind to the examiner whenever possible, except in occasional unavoidable cases where only one genotype was present at an age-matched timepoint. Spleens were collected and weighed immediately.
All tumour samples were surgically resected primary CRCs performed at Cambridge University Hospitals NHS Trust and processed on the same day for analysis. All patients gave written consent to participate in this study. The study was approved by the Wales Research Ethics Committee 7 (15/WA/0131). No statistical tests were used to predetermine sample sizes.
To obtain single cell suspensions of mouse tumours and adjacent normal gut, tumours and 2 cm pieces of adjacent normal gut were collected separately, followed by mechanical dissociation in RPMI-1640 containing 10 mM HEPES 309 (Gibco) and 2% foetal calf serum (FCS). Samples were digested with 62.5 μg/mL Liberase TL (Roche) and 0.125 KU/mL DNase I (Sigma) for 30 minutes at 37° C. whilst shaking.
Subsequently, cells were passed through a 50 μm filter and washed at least twice with PBS+2% FCS before antibody staining for flow cytometry. In experiments where adjacent normal epithelial fraction and lamina propria were analysed separately, tumours were removed and the remaining intestine was cut into 2 cm pieces and incubated with 1 mM dithiothreitol (Melford) and 5 mM EDTA in RPMI+2% FCS+10 mM HEPES for 2×20 minutes, shaking at 37° C., to collect the epithelial fraction.
Subsequently, samples were digested with 62.5 μg/mL Liberase TL (Roche) and 0.125 KU/mL DNase I (Sigma) in RPMI+2% FCS+10 mM HEPES at 37° C., shaking, for 45 minutes to collect lamina propria cells. After digestion, cells were passed through a 70 μm cell strainer and washed with PBS+2% FCS before antibody staining. Mesenteric lymph nodes were mechanically dissociated in RPMI, filtered through a 50 μm cell strainer, and washed with PBS+2% FCS before antibody staining. Fresh, surgically-resected human CRC and adjacent normal tissue were collected in sterile PBS and transported on ice before sample processing. Samples were mechanically dissociated into small pieces and digested with 100 μg/mL Liberase TM (Roche) and 0.2 KU/mL DNase I (Sigma) in RPMI, and incubated at 37° C. for 30 minutes. Subsequently, cells were filtered through a 70 μm cell strainer and washed with PBS+2% FCS before antibody staining for flow cytometry.
For surface staining, single cell suspensions were 333 blocked with anti-CD16/32 antibody (Fc block, clone 2.4G2) and Human TruStain FcX (Biolegend, 422302) for mouse and human samples in PBS+2% FCS respectively, and incubated with fluorochrome-conjugated or biotinylated antibodies on ice for 30 minutes in the dark. If necessary, samples were then incubated with fluorochrome-conjugated streptavidin on ice for a further 30 minutes.
For intracellular and nuclear staining of mouse samples, cells were fixed in 2% paraformaldehyde (ThermoFisher) for 45 minutes at room temperature in the dark, washed and subsequently stained with fluorochrome-conjugated antibodies for 45 minutes at room temperature in 1× Permeabilisation Buffer (eBioscience, 00-8333-56). For intracellular DCLK1 staining for tuft cell identification, fixed cells were incubated with primary anti-DCLK1 antibody (rabbit polyclonal, ab31704) for 45 minutes at room temperature, washed, and subsequently incubated with a secondary donkey-anti-rabbit AF647-conjugated antibody (ab150075) for another 45 minutes in 1× Permeabilisation Buffer. For intracellular and nuclear staining of human samples, cells were fixed using the True-Nuclear Transcription Factor Buffer Set (Biolegend, 424401) according to the manufacturer's protocol, and stained for intracellular and nuclear markers with fluorochrome-conjugated antibodies for 30 minutes, at room temperature and in the dark.
For analysis of IFNγ, perforin and granzyme expression by CD8 and γδ T cells, tumours and adjacent normal epithelium were processed into single cell suspensions, and stained with fluorochrome-conjugated antibodies as above. Subsequently, CD8 and γδ T cells were sorted using an iCyt Synergy cell sorter (Sony Biotechnology), and cultured with plate-coated anti-CD3e (Biolegend, 100360) and 10 ng/mL rm-IL-2 (Biolegend, 575406) for 16 hours. Protein Transport Inhibitor Cocktail (eBioscience, 00-4980-93) was added for the last 4 hours of culture, before antibody staining. Cells were blocked with anti-CD16/32 and stained for surface markers in PBS+2% FCS, on ice for 30 minutes and in the dark. Subsequently, cells were fixed using the Foxp3 Transcription Factor Staining Buffer Set (ebioscience, 00-5523-00) according to the manufacturer's protocol, and stained for intracellular markers with fluorochrome-conjugated antibodies for 30 minutes, at room temperature and in the dark.
Following intracellular staining, samples were washed serially with indicated 1× Permeabilisation Buffer and PBS+2% FCS, and resuspended in PBS+2% FCS for analyses through the BD LSRFortessa Flow Cytometer and FlowJo v10.2
For mouse experiments, antibodies used for surface staining include NK1.1 (PK136, BUV395), CD45 (30-F11, BV510), IL7Rα (SB/199, biotin), streptavidin BV605, CD8 (53-6.7, BV785), CD11b (M1/70, FITC), KLRG1 (2F1, PerCP-eFluor 710), CD3 (145-2C11, PE-Cy7), CD11c (N418, PE-Cy7), CD19 (eBio1D3, PE-Cy7), Gr1 (RB6-8C5, PE-Cy7), FcεRI (MAR-1, PE-Cy7), TER119 (TER-119, PE-Cy7), CD4 (GK1.5, AF700), CD3 (17A2, eF450), CD11b (M1/70, eF450), CD11c (N418, eF450), CD19 (eBio1D3, eF450), F4/80 (BM8, eF450), Gr1 (RB6-8C5, eF450), FcεRI (MAR-1, eF450), TCRβ (H57-597, eF450), TER119 (TER-119, eF450), ST2 (RMST2-2, AF488), streptavidin PE, CD11b (M1/70, BUV395), EpCAM (G8.8, BV421), Ly6C (HK1.4, BV711), Ly6G (1A8-Ly6g, PerCP-eFluor 710), MHC-II (M5/114.15.2, PE),
TCRγδ (GL3, BV605), FcεRI (MAR-1, FITC), CD11c (N418, 383 PerCP-Cy5.5), CD317 (eBio927, APC), MHC-II (M5/114.15.2, BV510), CD19 (6D5, BV605), CD45 (30-F11, BV785), Gr1 (RB6-8C5, PerCP-Cy5.5), Siglec-F (E50-2440, PE), CD11C (N418, AF700), CD3 (17A2, BV510), CD8 (53-6.7, PE-Cy7), CD45 (30-F11, AF700), CD4 (GK1.5, PerCP-Cy5.5), TCRγδ (GL3, APC), IL17BR (D9.2, AF647), IL13RA1 (13MOKA, PE), IL4RA (mIL4R-M1, PE), ST2 (D.J8, FITC), Fixable Viability Dye eFluor 780 (eBioscience) and appropriate isotype controls. Lineage includes CD3, CD4, CD8, CD11b, CD11c, CD19, Gr1, FceRI, NK1.1, and TER119, and in some cases individual lineage markers were analysed in a separate channel as indicated.
Antibodies used for intracellular and nuclear staining include GATA3 (L50-823, BV711), FOXP3 (FJK-16s, PE-Cy7), Tbet (eBio4B10, eF660), Ki67 (SolA15, PE-Cy7), IFNγ (XMG1.2, BV785), Granzyme (NGZB, PE), Perforin (S16009B, APC) and appropriate isotype controls.
For human samples, antibodies used for surface staining include IL17BR (D9.2, biotin), streptavidin BV421, CD56 (HCD56, BV650), CD4 (OKT4, BV785), CD45 (HI30, AF488), CRTH2 (BM16, PE), CD127 (A019D5, PE-Cy7), CD3 (OKT3, APC), FcεRI (AER-37 (CRA-1), APC), CD11b (ICRF44, APC), CD11c (Bu15, APC), CD14 (HCD14, APC), CD19 (HIB19, APC), CD8 (RPA-T8, APC), CD123 (6H6, APC), CD14 (HCD14, Pacific Blue), CD86 (IT2.2, BV711), CD15 (W6D3, PerCP-Cy5.5), CD33 (WM53, PE), HLA-DR (L243, PE-Cy7), CD56 (HCD56, APC), CD45 (H130, AF700), PD-L1 (29E.2A3, BV650), CD33 (WM53, BV785), CD155 (SKII.4, FITC), CD112 (TX31, PE), HVEM (122, PE-Cy7), CD8 (RPA-T8, BV510), CD3 (OKT3, Super Bright 645), PD1 (EH12.2H7, PerCP-Cy5.5), LAG3 (11C3C65, PE-Cy7), CD11b (ICRF44, FITC), Fixable Viability Dye eFluor 780 (eBioscience) and appropriate isotype controls. Lineage includes CD3, CD4, CD8, CD14, CD19,CD11b, CD11c, FcεRI, and CD123. Antibodies used for intracellular and nuclear staining include FOXP3 (206D, FITC), Tbet (eBio4B10, eF660), CTLA4 (L3D10, PE) and appropriate isotype controls.
The publicly available TCGA Firehose Legacy Colorectal Adenocarcinoma dataset was analysed using the online open-access cBioPortal resource (https://www.cbioportal.org/)5,6. All patient samples (222) with Agilent microarray mRNA data were included in the analysis, and the mRNA expression z-scores were calculated relative to all samples. Using the query “IL25: EXP>0”, CRC patients were separated into two groups based on tumour IL25 expression above (IL25high) or below (IL25low) the mean IL25 expression. The two groups were compared for available survival data, including overall and disease-free survival. The same method was repeated for tumour IL33 expression, and survival between IL33high and IL33low groups compared. Relapse-free survival and tumour IL25 expression data by Marisa, L. et al. (Gene Expression Classification of Colon Cancer into Molecular 454 Subtypes: Characterization, Validation, and Prognostic Value. PLOS Medicine 10, 455 e1001453, doi: 10.1371/journal.pmed.1001453 (2013)) were analysed using the online open-access R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). All patient samples (566) were included in the analysis, and segregated into two groups based on tumour IL25 expression above (IL25high) and below (IL25low) the mean tumour IL25 expression of all samples, and relapse-free survival compared.
Statistical significance for human survival analyses was calculated automatically by the built-in system on cBioPortal and the R2 platform, for analysis of the TCGA Firehose Legacy (Cerami, E. et al. The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Discovery 2, 401-404, doi: 10.1158/2159-8290.Cd-12-0095 (2012); Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 6, pl1, doi: 10.1126/scisignal.2004088 (2013)) and Marisa et al. 2013 CRC datasets respectively. All other statistical analyses in this study were performed using the GraphPad Prism 8 software.
Publicly available TCGA data of CRC patients were separated into the four CMS subtypes, and analysed for IL25 expression (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018015.4 | Nov 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/081750 | 11/15/2021 | WO |
