Patent Application
20230129258
The present invention relates to heterotandem bicyclic peptide complexes which comprise a first peptide ligand, which binds to a component present on a cancer cell, conjugated via a linker to two or more second peptide ligands, which bind to a component present on an immune cell. The invention also relates to the use of said heterotandem bicyclic peptide complexes in preventing, suppressing or treating cancer.
Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).
Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.
Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO 2006/078161.
Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene).
According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:
According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a heterotandem bicyclic peptide complex as defined herein in combination with one or more pharmaceutically acceptable excipients.
According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer.
According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:
According to one aspect of the invention which may be mentioned, there is provided a heterotandem bicyclic peptide complex comprising:
References herein to the term “cancer cell” includes any cell which is known to be involved in cancer. Cancer cells are created when the genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutation and epimutation of the genetic material of normal cells, which upsets the normal balance between proliferation and cell death. This results in uncontrolled cell division and the evolution of those cells by natural selection in the body. The uncontrolled and often rapid proliferation of cells can lead to benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to distant locations (metastasis) and become life-threatening.
In one embodiment, the cancer cell is selected from an HT1080, A549, SC-OV-3, PC3, HT1376, NCI-H292, LnCap, MC38, MC38 #13, 4T1-D02, H322, HT29, T47D and RKO tumor cell.
In one embodiment, the component present on a cancer cell is Nectin-4.
Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which comprises 4 members. Nectins are cell adhesion molecules that play a key role in various biological processes such as polarity, proliferation, differentiation and migration, for epithelial, endothelial, immune and neuronal cells, during development and adult life. They are involved in several pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus and measles virus. Mutations in the genes encoding Nectin-1 (PVRL1) or Nectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated with other abnormalities. Nectin-4 is expressed during foetal development. In adult tissues its expression is more restricted than that of other members of the family. Nectin-4 is a tumor-associated antigen in 50%, 49% and 86% of breast, ovarian and lung carcinomas, respectively, mostly on tumors of bad prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumors, Nectin-4 is expressed mainly in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, the detection of soluble forms of Nectin-4 is associated with a poor prognosis. Levels of serum Nectin-4 increase during metastatic progression and decrease after treatment. These results suggest that Nectin-4 could be a reliable target for the treatment of cancer. Accordingly, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and is currently clinically investigated for the treatment of patients suffering from solid tumors.
In one embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand.
Suitable examples of Nectin-4 binding bicyclic peptide ligands are disclosed in WO 2019/243832, the peptides of which are incorporated herein by reference.
In one embodiment, the Nectin-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from:
In a further embodiment, the Nectin-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from:
In a further embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises N-terminal modifications and comprises:
In a yet further embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises N-terminal modifications and comprises:
In a yet further embodiment, the Nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (herein referred to as BCY8116).
In an alternative embodiment, the component present on a cancer cell is EphA2.
Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosine residues. Ephs and their membrane bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur at higher ligand oligomerization states (Stein et al. (1998) Genes Dev 12, 667-678).
Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of the ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly-formed, adult micro-vessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).
The de-regulated re-emergence of some ephrins and their receptors in adults also has been observed to contribute to tumor invasion, metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph family members have been found to be over-expressed on tumor cells from a variety of human tumors (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).
EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene.
EphA2 is upregulated in multiple cancers in man, often correlating with disease progression, metastasis and poor prognosis e.g.: breast (Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung (Brannan et al (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al (2013) J Thorac Oncol. 8, 301-308), gastric (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417), pancreatic (Mudali et al (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), liver (Yang et al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumor Biol. 31, 477-488).
The full role of EphA2 in cancer progression is still not defined although there is evidence for interaction at numerous stages of cancer progression including tumor cell growth, survival, invasion and angiogenesis. Downregulation of EphA2 expression suppresses tumor cancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780), whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19, 1884-1886).
An antibody drug conjugate to EphA2 has been shown to significantly diminish tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374) and a similar approach has been tried in man although treatment had to be discontinued for treatment related adverse events (Annunziata et al (2013) Invest New drugs 31, 77-84).
In one embodiment, the first peptide ligand comprises an EphA2 binding bicyclic peptide ligand.
Suitable examples of EphA2 binding bicyclic peptide ligands are disclosed in WO 2019/122860, WO 2019/122861 and WO 2019/122863, the peptides of which are incorporated herein by reference.
In one embodiment, the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence selected from:
[K(PYA-(Palmitoyl-Glu-LysN3)] represents [K(PYA(Palmitoyl-Glu-LysN3))], Nle represents norleucine, MerPro represents 3-mercaptopropionic acid and Cysam represents cysteamine, or a pharmaceutically acceptable salt thereof.
In one particular embodiment, the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence which is:
In one alternative particular embodiment, the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence which is:
In a further embodiment, the EphA2 binding bicyclic peptide ligand optionally comprises N-terminal and/or C-terminal modifications and comprises:
or a pharmaceutically acceptable salt thereof.
In one particular embodiment, the EphA2 binding bicyclic peptide ligand optionally comprises N-terminal and/or C-terminal modifications and comprises:
In one alternative particular embodiment, the EphA2 binding bicyclic peptide ligand optionally comprises N-terminal and/or C-terminal modifications and comprises:
In an alternative embodiment, the component present on a cancer cell is PD-L1.
Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in evasion of immune responses involved in chronic infection, e.g., chronic viral infection (including, for example, HIV, HBV, HCV and HTLV, among others), chronic bacterial infection (including, for example, Helicobacter pylori, among others), and chronic parasitic infection (including, for example, Schistosoma mansoni). PD-L1 expression has been detected in a number of tissues and cell types including T-cells, B-cells, macrophages, dendritic cells, and nonhaematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.
PD-L1 expression is also involved in suppression of anti-tumor immune activity. Tumors express antigens that can be recognised by host T-cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor microenvironment. PD-L1 expression on many tumors is a component of this suppressive milieu and acts in concert with other immunosuppressive signals. PD-L1 expression has been shown in situ on a wide variety of solid tumors including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown J A et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J, et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome S E et al. 2003 Cancer Res. 63:6501-5; Inman B A et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). In addition, the expression of the receptor for PD-L1, Programmed cell death protein 1 (also known as PD-1 and CD279) is upregulated on tumor infiltrating lymphocytes, and this also contributes to tumor immunosuppression (Blank C et al. 2003 Immunol. 171:4574-81). Most importantly, studies relating PD-L1 expression on tumors to disease outcome show that PD-L1 expression strongly correlates with unfavourable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman B A et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). In addition, these studies suggest that higher levels of PD-L1 expression on tumors may facilitate advancement of tumor stage and invasion into deeper tissue structures.
The PD-1 pathway can also play a role in haematologic malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al. 2007 Blood 110:296-304). PD-L1 is expressed on some primary T-cell lymphomas, particularly anaplastic large cell T lymphomas (Brown J A et al, 2003 Immunol. 170:1257-66). PD-1 is highly expressed on the T-cells of angioimmunoblastic lymphomas, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman D M et al. 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, the T-cells associated with lymphocytic or histiocytic (L&H) cells express PD-1. Microarray analysis using a readout of genes induced by PD-1 ligation suggests that tumor-associated T-cells are responding to PD-1 signals in situ in Hodgkin lymphoma (Chemnitz J M et al. 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T-cells in HTLV-1-mediated adult T-cell leukaemia and lymphoma (Shimauchi T et al. 2007 Int. J. Cancer 121: 2585-90). These tumor cells are hyporesponsive to TCR signals.
Studies in animal models demonstrate that PD-L1 on tumors inhibits T-cell activation and lysis of tumor cells and in some cases leads to increased tumor-specific T-cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also utilise the PD-1:PD-L1 pathway to control antitumor T-cell responses. PD-L1 expression on a population of tumor-associated myeloid DCs is upregulated by tumor environmental factors (Curiel T J et al. 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the tumor-draining lymph node of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T-cells. The suppressive activity of IDO-treated regulatory T-cells required cell contact with IDO-expressing DCs (Sharma M D et al. 2007 Clin. Invest. 117:2570-82).
In one embodiment, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand.
Suitable examples of PD-L1 binding bicyclic peptide ligands are disclosed in WO 2020/128526 and WO 2020/128527, the peptides of which are incorporated herein by reference.
In one embodiment, the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from:
In a further embodiment, the PD-L1 binding bicyclic peptide ligand optionally comprises N-terminal and/or C-terminal modifications and comprises:
In an alternative embodiment, the component present on a cancer cell is prostate-specific membrane antigen (PSMA).
Prostate-specific membrane antigen (PSMA) (also known as Glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) and NAAG peptidase) is an enzyme that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.
Human PSMA is highly expressed in the prostate, roughly a hundred times greater than in most other tissues. In some prostate cancers, PSMA is the second-most upregulated gene product, with an 8- to 12-fold increase over levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as potential biomarker for therapy and imaging of some cancers. In human prostate cancer, the higher expressing tumors are associated with quicker time to progression and a greater percentage of patients suffering relapse.
In one embodiment, the first peptide ligand comprises a PSMA binding bicyclic peptide ligand.
Suitable examples of PSMA binding bicyclic peptide ligands are disclosed in WO 2019/243455 and WO 2020/120980, the peptides of which are incorporated herein by reference
References herein to the term “immune cell” includes any cell within the immune system. Suitable examples include white blood cells, such as lymphocytes (e.g. T lymphocytes or T cells, B cells or natural killer cells). In one embodiment, the T cell is CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells, follicular dendritic cells and granulocytes.
In one embodiment, the component present on an immune cell is CD137.
CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4- IBB and induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but to a larger extent on CD8+ than on CD4+ T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and cells of blood vessel walls at sites of inflammation. One characterized activity of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival and cytolytic activity. Further, it can enhance immune activity to eliminate tumors in mice.
CD137 is a T-cell costimulatory receptor induced on TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev, Immunol., 23:23-68 (2005)). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen-presenting cells including B cells, monocyte/macrophages, and dendritic cells (Watts et al. Annu. Rev. Immunol, 23:23-68 (2005)). On interaction with its ligand, CD137 leads to increased TCR-induced T-cell proliferation, cytokine production, functional maturation, and prolonged CD8+ T-cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et d-l., Annu. Rev. Immunol, 23:23-68 (2005)).
Signalling through CD137 by either CD137L or agonistic monoclonal antibodies (mAbs) against CD137 leads to increased TCR-induced T cell proliferation, cytokine production and functional maturation, and prolonged CD8+ T cell survival. These effects result from: (1) the activation of the NF-κB, c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) signalling pathways, and (2) the control of anti-apoptotic and cell cycle-related gene expression.
Experiments performed in both CD137 and CD137L-deficient mice have additionally demonstrated the importance of CD137 costimulation in the generation of a fully competent T cell response.
IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137 by agonistic mAbs stimulates NK cell proliferation and IFN-γ secretion, but not their cytolytic activity.
Furthermore, CD137-stimulated NK cells promote the expansion of activated T cells in vitro.
In accordance with their costimulatory function, agonist mAbs against CD137 have been shown to promote rejection of cardiac and skin allografts, eradicate established tumors, broaden primary antiviral CD8+ T cell responses, and increase T cell cytolytic potential. These studies support the view that CD137 signalling promotes T cell function which may enhance immunity against tumors and infection.
In one embodiment, the two or more second peptide ligands comprise a CD137 binding bicyclic peptide ligand.
Suitable examples of CD137 binding bicyclic peptide ligands are disclosed in WO 2019/025811, the peptides of which are incorporated herein by reference.
In one embodiment, the CD137 binding bicyclic peptide ligand comprises an amino acid sequence:
In a further embodiment, the CD137 binding bicyclic peptide ligand comprises an amino acid sequence:
In one embodiment, the bicyclic peptide ligand is other than the amino acid sequence [dCi][dI][dE][dE][K(PYA)][dQ][dY][dCii][dF][dA][dD][dP][dY][dNle][dCiii] (SEQ ID NO: 13), which has been demonstrated not to bind to CD137.
In one particular embodiment which may be mentioned, the CD137 binding bicyclic peptide ligand comprises an amino acid sequence:
In a further embodiment, the CD137 binding bicyclic peptide ligand comprises N- and C-terminal modifications and comprises:
In a yet further embodiment, the CD137 binding bicyclic peptide ligand comprises N- and C-terminal modifications and comprises:
In one embodiment, the bicyclic peptide ligand is other than BCY11506, which has been demonstrated not to bind to CD137.
In a further embodiment which may be mentioned, the CD137 binding bicyclic peptide ligand comprises N- and C-terminal modifications and comprises:
In an alternative embodiment, the component present on an immune cell is OX40.
The OX40 receptor (also known as Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4) and also known as CD134 receptor), is a member of the TNFR-superfamily of receptors which is not constitutively expressed on resting naïve T cells, unlike CD28. OX40 is a secondary co-stimulatory immune checkpoint molecule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their activation. Expression of OX40 is dependent on full activation of the T cell; without CD28, expression of OX40 is delayed and of fourfold lower levels.
OX40 has no effect on the proliferative abilities of CD4+ cells for the first three days, however after this time proliferation begins to slow and cells die at a greater rate, due to an inability to maintain a high level of PKB activity and expression of Bcl-2, Bcl-XL and survivin. OX40L binds to OX40 receptors on T-cells, preventing them from dying and subsequently increasing cytokine production. OX40 has a critical role in the maintenance of an immune response beyond the first few days and onwards to a memory response due to its ability to enhance survival. OX40 also plays a crucial role in both Th1 and Th2 mediated reactions in vivo.
OX40 binds TRAF2, 3 and 5 as well as PI3K by an unknown mechanism. TRAF2 is required for survival via NF-κB and memory cell generation whereas TRAF5 seems to have a more negative or modulatory role, as knockouts have higher levels of cytokines and are more susceptible to Th2-mediated inflammation. TRAF3 may play a critical role in OX40-mediated signal transduction. CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF3 DN defect was partially overcome by CTLA-4 blockade in vivo. TRAF3 may be linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.
In one embodiment, the OX40 is mammalian OX40. In a further embodiment, the mammalian OX40 is human OX40 (hOX40).
OX40 peptides will be primarily (but not exclusively) used to agonistically activate OX40, and consequently immune cells, to prevent, suppress or treat cancer such as early or late stage human malignancies, which includes solid tumors such as Non-Small Cell Lung Carcinomas (NSCLC), breast cancers, including triple negative breast cancers (TNBC), ovarian cancers, prostate cancers, bladder cancers, urothelial carcinomas, colorectal cancers, head and neck cancer, Squamous Cell Carcinoma of the Head and Neck (SCCHN), melanomas, pancreatic cancers, and other advanced solid tumors where immune suppression blocks anti-tumor immunity. Other solid and non-solid malignancies where OX40 peptides will be used as a therapeutic agent includes, but not limited to, B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma and Acute Myeloid Leukemia (AML).
In one embodiment, the two or more second peptide ligands comprise an OX40 binding bicyclic peptide ligand.
Suitable examples of OX40 binding bicyclic peptide ligands are disclosed in International Patent Application No. PCT/GB2020/051144, the peptides of which are incorporated herein by reference.
In one embodiment, the OX40 binding bicyclic peptide ligand comprises an amino acid sequence selected from:
wherein Ci, Cii and Ciii represent first, second and third cysteine residues, respectively, or a modified derivative, or a pharmaceutically acceptable salt thereof.
In a further embodiment, the OX40 binding bicyclic peptide ligand additionally comprises N- and/or C-terminal modifications and comprises an amino acid sequence selected from:
In one embodiment, the two or more second peptides are specific for the same immune cell. In a further embodiment, each of said two or more second peptides are specific for the same binding site or target on the same immune cell. In an alternative embodiment, each of said two or more second peptides are specific for a different binding site or target on the same immune cell. In an alternative embodiment, the two or more second peptides are specific for two differing immune cells (i.e. CD137 and OX40). In a further embodiment, each of said two or more second peptides are specific for the same binding site or target on two differing immune cells. In an alternative embodiment, each of said two or more second peptides are specific for a different binding site or target on two differing immune cells.
In one embodiment, each of said two or more second peptides has the same peptide sequence.
In one embodiment, said heterotandem bicyclic peptide complex comprises two second peptide ligands. Thus, according to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:
According to a further aspect of the invention which may be mentioned, there is provided a heterotandem bicyclic peptide complex comprising:
In an alternative embodiment, said heterotandem bicyclic peptide complex comprises three second peptide ligands. Thus, according to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:
According to a further aspect of the invention which may be mentioned, there is provided a heterotandem bicyclic peptide complex comprising:
In a further embodiment, each of said two or more second peptides has the same peptide sequence and said peptide sequence comprises Ac-(SEQ ID NO: 11)-A (herein referred to as BCY8928), wherein Ac represents an acetyl group, or a pharmaceutically acceptable salt thereof.
In a yet further embodiment, said heterotandem bicyclic peptide complex comprises two second peptide ligands and both of said two second peptides have the same peptide sequence which comprises Ac-(SEQ ID NO: 11)-A (herein referred to as BCY8928), wherein Ac represents an acetyl group, or a pharmaceutically acceptable salt thereof.
It will be appreciated that the first peptide ligand may be conjugated to the two or more second peptide ligands via any suitable linker. Typically, the design of said linker will be such that the three Bicyclic peptides are presented in such a manner that they can bind unencumbered to their respective targets either alone or while simultaneously binding to both target receptors. Additionally, the linker should permit binding to both targets simultaneously while maintaining an appropriate distance between the target cells that would lead to the desired functional outcome. The properties of the linker may be modulated to increase length, rigidity or solubility to optimise the desired functional outcome. The linker may also be designed to permit the attachment of more than one Bicycle to the same target. Increasing the valency of either binding peptide may serve to increase the affinity of the heterotandem for the target cells or may help to induce oligomerisation of one or both of the target receptors.
In one embodiment, the linker is a branched linker to allow one first peptide at one end and the two or more second peptides at the other end.
In a further embodiment, the branched linker is selected from:
In on particular embodiment, the branched linker is:
In one specific embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold, the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands attached to a TATA scaffold and said heterotandem complex is selected from the complexes listed in Table A:
In one embodiment, the heterotandem bicyclic peptide complex is selected from: BCY11027, BCY11863 and BCY11864. In a further embodiment, the heterotandem bicyclic peptide complex is selected from: BCY11863 and BCY11864.
The heterotandem bicyclic peptide complex BCY11863 consists of a Nectin-4 specific peptide BCY8116 linked to two CD137 specific peptides (both of which are BCY8928) via a N-(acid-PEG3)-N-bis(PEG3-azide) linker, shown pictorially as:
CD137 is a homotrimeric protein and the natural ligand CD137L exists as a homotrimer either expressed on immune cells or secreted. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is through cellular cross-linking of the CD137 specific agonist through interaction with a specific receptor present on another cell. The advantage of the heterotandem complexes of the present invention is that the presence of two or more peptide ligands specific for an immune cell component, such as CD137, provides a more effective clustering of CD137. For example, data is presented herein in
The heterotandem bicyclic peptide complex BCY11027 consists of a Nectin-4 specific peptide BCY11015 linked to two CD137 specific peptides (both of which are BCY8928) via a TCA-[Peg10]3 linker, shown pictorially as:
Data shown in
In an alternative specific embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold, the two or more second peptide ligands comprise three CD137 binding bicyclic peptide ligands attached to a TATA scaffold and said heterotandem complex is selected from the complexes listed in Table B:
In one specific embodiment, the first peptide ligand comprises an EphA2 binding bicyclic peptide ligand attached to a TATA scaffold, the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands attached to a TATA scaffold and said heterotandem complex is selected from the complexes listed in Table C:
In one embodiment, the heterotandem bicyclic peptide complex is selected from: BCY12491, BCY12730, BCY13048, BCY13050, BCY13053 and BCY13272.
In one embodiment, the heterotandem bicyclic peptide complex is selected from: BCY12491, BCY12730, BCY13048, BCY13050 and BCY13053.
In a further embodiment, the heterotandem bicyclic peptide complex is BCY12491.
The heterotandem bicyclic peptide complex BCY12491 consists of a EphA2 specific peptide BCY9594 linked to two CD137 specific peptides (both of which are BCY8928) via a N-(acid-PEG3)-N-bis(PEG3-azide) linker, shown pictorially as:
Data is presented here in
In an alternative embodiment, the heterotandem bicyclic peptide complex is BCY13272.
The heterotandem bicyclic peptide complex BCY13272 consists of a EphA2 specific peptide BCY13118 linked to two CD137 specific peptides (both of which are BCY8928) via a N-(acid-PEG3)-N-bis(PEG3-azide) linker, shown pictorially as:
Data is presented here in
In one specific embodiment, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand attached to a TATA scaffold, the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands attached to a TATA scaffold and said heterotandem complex is selected from the complexes listed in Table D:
In one specific embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold, the two or more second peptide ligands comprise two OX40 binding bicyclic peptide ligands attached to a TATA scaffold and said heterotandem complex is the complex listed in Table E:
In one specific embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold, one of the two or more second peptide ligands comprises an OX40 binding bicyclic peptide ligand attached to a TATA scaffold and the other of the two or more second peptide ligands comprises a CD137 binding bicyclic peptide ligand attached to a TATA scaffold and said heterotandem complex is the complex listed in Table F:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.
When referring to amino acid residue positions within compounds of the invention, cysteine residues (Ci, Cii and Ciii) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within SEQ ID NO: 1 is referred to as below:
For the purpose of this description, all bicyclic peptides are assumed to be cyclised with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and yielding a tri-substituted structure. Cyclisation with TBMB and TATA occurs on Ci, Cii and Ciii.
N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail would be denoted as:
In light of the disclosure in Nair et al (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa). For the avoidance of doubt, references to amino acids either as their full name or as their amino acid single or three letter codes are intended to be represented herein as L-amino acids unless otherwise stated. If such an amino acid is intended to be represented as a D-amino acid then the amino acid will be prefaced with a lower case d within square parentheses, for example [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.
Certain heterotandem bicyclic peptide complexes of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:
A peptide ligand, as referred to herein, refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide is bound to the scaffold. In the present case, the peptides comprise at least three reactive groups selected from cysteine, 3-mercaptopropionic acid and/or cysteamine and form at least two loops on the scaffold.
The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a lysine side chain, or an N-terminal amine group or any other suitable reactive group, such as penicillamine. Details of suitable reactive groups may be found in WO 2009/098450.
Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold/molecular core.
The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.
In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.
In another embodiment, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand.
In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core. Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.
In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state.
In some embodiments, amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid. Excluded from these exchangeable amino acids are the ones harbouring functional groups for cross-linking the polypeptides to a molecular core, such that the loop sequences alone are exchangeable. The exchangeable polypeptide sequences have either random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are either located in defined positions within the polypeptide, since the position of these amino acids determines loop size.
In one embodiment, a polypeptide with three reactive groups has the sequence (X)lY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 3 and 6 defining the length of intervening polypeptide segments, which may be the same or different, and l and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.
Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention—in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection/isolation phase. Further details can be found in WO 2009/098450 or Heinis et al., Nat Chem Biol 2009, 5 (7), 502-7.
In one embodiment, the reactive groups are selected from cysteine, 3-mercaptopropionic acid and/or cysteamine residues.
It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.
The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (±)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.
If the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO−), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+ or Zn+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from:
methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Where the compounds of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the invention.
It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.
In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.
In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (the group referred to herein as Ci) is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.
In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.
In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group (the group referred to herein as CHI) is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.
In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.
Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.
In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (Ci) and/or the C-terminal cysteine (Ciii).
In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.
In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).
In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise □-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).
In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).
It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:
Examples of modified heterotandem bicyclic peptide complexes of the invention include those listed in Tables G and H below:
The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.
Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as 2H (D) and 3H (T), carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I, 125I and 131I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, sulfur, such as 35S, copper, such as 64Cu, gallium, such as 67Ga or 68Ga, yttrium, such as 90Y and lutetium, such as 177Lu, and Bismuth, such as 213Bi.
Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the Nectin-4 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. 3H (T), and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e. 2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.
Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
Molecular scaffolds are described in, for example, WO 2009/098450 and references cited therein, particularly WO 2004/077062 and WO 2006/078161.
As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.
In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.
In one embodiment, the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.
The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.
In one embodiment, the molecular scaffold may comprise or may consist of hexahydro-1,3,5-triazine, especially 1,3,5-Triacryloylhexahydro-1,3,5-triazine (‘TATA’), or a derivative thereof.
The molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold. Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.
Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes).
Examples include bromomethylbenzene (the scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, α-unsaturated carbonyl containing compounds and α-halomethylcarbonyl containing compounds. Examples of maleimides which may be used as molecular scaffolds in the invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an αβ unsaturated carbonyl containing compound is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an α-halomethylcarbonyl containing compound is N,N′,N″-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine is also a natural amino acid which has a similar reactivity to cysteine and can be used for the same reactions. Thus, wherever cysteine is mentioned, it is typically acceptable to substitute selenocysteine unless the context suggests otherwise.
The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al (supra).
Thus, the invention also relates to manufacture of polypeptides or conjugates selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide/conjugate made by chemical synthesis.
Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex.
Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.
To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages 6000-6003).
Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptides, forming a disulfide-linked bicyclic peptide-peptide conjugate.
Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.
Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.
According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.
Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
The peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.
The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.
The compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a “therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.
A composition containing a peptide ligand according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer.
Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumors of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenousleukemia [AML], chronic myelogenousleukemia [CML], chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocyticleukemia); tumors of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcomaprotuberans; tumors of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (for example pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary carcinoma of the thyroid); ocular and adnexal tumors (for example retinoblastoma); germ cell and trophoblastic tumors (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumors (for example medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).
In a further embodiment, the cancer is selected from a hematopoietic malignancy such as selected from: non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).
References herein to the term “prevention” involves administration of the protective composition prior to the induction of the disease. “Suppression” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. “Treatment” involves administration of the protective composition after disease symptoms become manifest.
Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.
The invention is further described below with reference to the following examples.
In general, some of the heterotandem bicyclic peptide complexes of the invention may be prepared in accordance with the following general method:
All solvents are degassed and purged with N2 3 times. A solution of BP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF is mixed for 5 minutes, then Bicycle1 (1.2 eq.) is added. The reaction mixture is stirred at 40° C. for 16 hr. The reaction mixture is then concentrated under reduced pressure to remove solvent and purified by prep-HPLC to give intermediate 2.
A mixture of intermediate 2 (1.0 eq) and Bicycle2 (2.0 eq) are dissolved in t-BuOH/H2O (1:1), and then CuSO4 (1.0 eq), VcNa (4.0 eq), and THPTA (2.0 eq) are added. Finally, 0.2 M NH4HCO3 is added to adjust pH to 8. The reaction mixture is stirred at 40° C. for 16 hr under N2 atmosphere. The reaction mixture was directly purified by prep-HPLC.
Heterotandem bicyclic peptide complexes which were prepared using this method are listed below:
More detailed experimental for selected heterotandem bicyclic peptide complexes of the invention are provided herein below:
A mixture of N-(acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0 eq.), HATU (51.2 mg, 134.7 μmol, 1.2 eq.) and DIEA (29.0 mg, 224.4 μmol, 40 μL, 2.0 eq.) was dissolved in DMF (2 mL), and mixed for 5 min. Then BCY8116 (294.0 mg, 135.3 μmol, 1.2 eq.) was added. The reaction mixture was stirred at 40° C. for 16 hr. LC-MS showed a small fraction of compound 2 remained (MW: 2172.49, observed m/z: 1087.1) and one main peak with desired m/z (MW: 2778.17, observed m/z: 1389.3 ([(M/2+H+]), 926.7 ([(M/3+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). Compound 2 (194.5 mg, 66.02 μmol, 29.41% yield, 94.3% purity) was obtained as a white solid.
A mixture of Compound 2 (100.0 mg, 36.0 μmol, 1.0 eq), BCY8928 (160.0 mg, 72.0 μmol, 2.0 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 180 μL, 1.0 eq) and VcNa (28.5 mg, 143.8 μmol, 4.0 eq), THPTA (31.2 mg, 71.8 μmol, 2.0 eq) were added. Finally, 0.2 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed BCY8928 remained and desired m/z (calculated MW: 7213.32, observed m/z: 1444.0 ([M/5+H]+)) was also detected. The reaction mixture was directly purified by prep-HPLC. First purification resulted in BCY11863 (117.7 mg, 15.22 μmol, 42.29% yield, 93.29% purity) as TFA salt, while less pure fractions were purified again by prep-HPLC (TFA condition), producing BCY11863 (33.2 mg, 4.3 μmol, 11.92% yield, 95.55% purity) as TFA salt.
To a mixture of compound 1 (BP-23825, 60.0 mg, 96.2 μmol, 1.0 eq) in DMF (3 mL) was added DIEA (12.4 mg, 96.2 μmol, 16.8 μL, 1.0 eq) and HATU (38.4 mg, 101 μmol, 1.05 eq) and the mixture stirred for 5 min. Then BCY9594 (243 mg, 101 μmol, 1.05 eq) was added to the mixture and purged with N2 3 times, then stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was purified by preparative-HPLC to give (BP-23825)-BCY9594 (154 mg, 48.1 μmol, 50.0% yield, 94.0% purity) as a white solid. Calculated MW: 3006.48, observed m/z: 1002.8 [M/3+H]+, 1504.4 [M/2+H]+
A mixture of compound 1 (56.0 mg, 18.6 μmol, 1.0 eq.), BCY8928 (83.0 mg, 37.2 μmol, 2.0 eq.), and THPTA (17.0 mg, 39.1 μmol, 2.1 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 94.0 μL, 2.0 eq.) and VcNa (15.0 mg, 74.5 μmol, 4.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40° C. for 3 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY12491 (59.2 mg, 7.79 μmol, 41.81% yield, 97.9% purity) was obtained as a white solid. Calculated MW: 7441.63, observed m/z: 1861.1 ([M/4+H]+), 1489.0 ([M/5+H]+).
A mixture of (BP-23825)-BCY9594 (20.0 mg, 6.65 μmol, 1.0 eq), BCY12153 (27.8 mg, 13.3 μmol, 2.0 eq) and THPTA (5.8 mg, 13.3 μmol, 2.0 eq) was dissolved in t-BuOH (0.5 mL) and H2O (0.5 mL) (all solvents were pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 33.3 μL, 13.3 μmol, 2.0 eq), VcNa (0.4 M, 66.5 μL, 26.6 μmol, 4.0 eq) were added to the mixture under N2 atmosphere. Then NH4HCO3 was added to the mixture until pH is 8. The mixture was stirred at 25° C. for 2 hr under N2 atmosphere. LC-MS showed one main peak with desired m/z was detected. The reaction mixture was purified twice by prep-HPLC to give compound BCY12730 (7.50 mg, 0.84 μmol, 12.7% yield, 96.3% purity) as a white solid. Calculated MW: 7185.39, observed m/z: 1197.5 [M/6+H]+, 1438.4 [M/5+H]+.
A mixture of BP-23825 (12.0 mg, 19.24 μmol, 1.2 eq.), and HATU (7.32 mg, 19.24 μmol, 1.2 eq.) was dissolved in NMP (0.3 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (5.12 mg, 40.26 μmol, 7 μL, 2.4 eq.), and then the solution was activated at 40° C. for 5 min. Compound 2 (33.0 mg, 16.03 μmol, 1.0 eq.) was dissolved in NMP (0.5 mL), and then dropped to the activated solution, the pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 40° C. for 0.5 hr. LC-MS showed BCY12860 was consumed completely and one main peak with desired m/z (MW: 2667.12, observed m/z: 1334.2 ([(M/2+H+]), 889.8 ([(M/3+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). BP-23825-BCY12860 (26.5 mg, 7.88 μmol, 49.12% yield, 79.26% purity) was obtained as a white solid.
A mixture of compound 3 (26.5 mg, 9.94 μmol, 1.0 eq.), compound 4 (47.0 mg, 20.87 μmol, 2.1 eq.), and THPTA (0.4 M, 58 μL, 2.3 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 58 μL, 2.3 eq.) and VcNa (0.4 M, 115 μL, 4.6 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40° C. for 1 hr under N2 atmosphere. LC-MS showed compound 4 remained and one main peak with desired m/z (calculated MW: 7102.28, observed m/z: 1776.4 ([M/4+H]+), 1421.3 ([M/3+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY13048 (14.1 mg, 1.91 μmol, 19.00% yield, 96.2% purity) was obtained as a white solid.
A mixture of BP-23825 (10.0 mg, 16.03 μmol, 1.2 eq.), and HATU (6.10 mg, 16.03 μmol, 1.2 eq.) was dissolved in NMP (0.3 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (4.45 mg, 34.37 μmol, 6 μL, 2.6 eq.), and then the solution was stirred at 25° C. for 6 min. Compound 2 (33.0 mg, 13.36 μmol, 1.0 eq.) was dissolved in NMP (0.5 mL), and then added dropwise into the activated solution. The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 40° C. for 0.5 hr. LC-MS showed BCY12862 was consumed completely and one main peak with desired m/z (MW: 3018.49, observed m/z: 1007.0 ([(M/3+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). BP-23825-BCY12862 (20.9 mg, 6.92 μmol, 51.82% yield, 94.9% purity) was obtained as a white solid.
A mixture of compound 3 (20.9 mg, 6.92 μmol, 1.0 eq.), compound 4 (32.2 mg, 14.54 μmol, 2.1 eq.), and THPTA (7.0 mg, 15.93 μmol, 2.3 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 39 μL, 2.3 eq.) and VcNa (6.3 mg, 31.85 μmol, 4.6 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40° C. for 1 hr under N2 atmosphere. LC-MS showed compound 4 remained and one main peak with desired m/z (calculated MW: 7453.66, observed m/z: 1864.2 ([M/4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY13050 (6.0 mg, 0.77 μmol, 11.24% yield, 96.7% purity) was obtained as a white solid.
BP-23825 (14.0 mg, 22.45 μmol, 1.2 eq) and HATU (8.5 mg, 22.35 μmol, 1.2 eq) were first dissolved in 0.5 mL of NMP, then was added DIEA (7.8 μL, 44.77 μmol, 2.4 eq), the mixture was stirred at 25° C. for 6 minutes, and then BCY12865 (40.0 mg, 18.65 μmol, 1.0 eq) was added. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed one peak with desired m/z (calculated MW: 2750.21, observed m/z: 1375.5 ([M/21-H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and compound 1 (15.9 mg, 5.78 μmol, 31.0% yield, 96.69% purity) was obtained as a white solid.
Compound 1 (15.9 mg, 5.78 μmol, 1.0 eq) and BCY8928 (26.0 mg, 11.72 μmol, 2.1 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 29.0 μL, 2.0 eq), VcNa (4.6 mg, 23.2 μmol, 4.0 eq) and THPTA (5.1 mg, 11.7 μmol, 2.0 eq) were added. Finally, 0.2 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 3 times. The reaction mixture was stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired m/z (calculated MW: 7185.38, observed m/z: 1796.7 ([M/4+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and BCY13053 (21.8 mg, 3.03 μmol, 52.84% yield, 98.01% purity) was obtained as a white solid.
A mixture of compound 1 (14.0 mg, 22.45 μmol, 1.20 eq.) and HATU (8.5 mg, 22.37 μmol, 1.20 eq.) was dissolved in NMP (0.3 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (5.8 mg, 44.86 μmol, 7.8 μL, 2.40 eq.), and then the solution was activated at 25° C. for 6 min. BCY12865 (40.0 mg, 18.65 μmol, 1.00 eq.) was dissolved in NMP (0.2 mL), and then added to the activated solution dropwise. The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY12865 was consumed completely and one main peak with desired m/z (MW: 2750.21, observed m/z: 1375.5 ([(M/2+H+]) and 917.3 ([(M/3+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). Compound 2 (20.6 mg, 7.24 μmol, 38.83% yield, 95.51% purity) was obtained as a white solid.
A mixture of compound 2 (20.6 mg, 7.49 μmol, 1.00 eq.), BCY12353 (31.5 mg, 15.08 μmol, 2.01 eq), and THPTA (7.0 mg, 16.11 μmol, 2.15 eq) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 37.5 μL, 2.00 eq) and VcNa (6.0 mg, 30.29 μmol, 4.04 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40° C. for 1 hr under N2 atmosphere. LC-MS showed one main peak with desired m/z (calculated MW: 6929.13, observed m/z: 1386.5 ([M/5+H]+) and 1155.8 ([M/6+H]+)). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (first run in TFA condition and second run in AcOH condition), and BCY13341 (10.3 mg, 1.49 μmol, 19.85% yield, 93.48% purity) was obtained as a white solid.
A mixture of BP-23825 (13.0 mg, 20.84 μmol, 1.2 eq.), and HATU (8.0 mg, 20.84 μmol, 1.2 eq.) was dissolved in NMP (0.3 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (5.4 mg, 41.69 μmol, 7.3 μL, 2.4 eq.), and then the solution was activated at 25° C. for 5 min. Compound 2 (35.8 mg, 17.37 μmol, 1.0 eq.) was dissolved in NMP (0.5 mL), and then dropped to the activated solution, the pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY12860 was consumed completely and one main peak with desired m/z (MW: 2667.12, observed m/z: 1334.4 ([(M/2+H+])). The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). BP-23825-BCY12860 (25.2 mg, 9.16 μmol, 52.76% yield, 97.0% purity) was obtained as a white solid.
A mixture of compound 3 (25.2 mg, 9.45 μmol, 1.0 eq.), compound 4 (40.4 mg, 19.37 μmol, 2.05 eq.), and THPTA (9.5 mg, 21.73 μmol, 2.3 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2 3 times), and then CuSO4 (0.4 M, 54.3 μL, 2.3 eq.) and VcNa (8.7 mg, 43.51 μmol, 2.5 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 1 hr under N2 atmosphere. LC-MS showed compound 3 was also consumed completely and one main peak with desired m/z (calculated MW: 6846.04, observed m/z: 1370.3 ([M/5+H+])) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY13343 (28.2 mg, 3.61 μmol, 38.23% yield, 87.7% purity) was obtained as a white solid.
TCA-PEG10-N3 (22.0 mg, 10.58 μmol, 1.0 eq) and BCY11015 (26.0 mg, 34.72 μmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 26.4 μL, 1.0 eq), VcNa (4.2 mg, 21.2 μmol, 2.0 eq) and THPTA (4.6 mg, 10.58 μmol, 1.0 eq) was added. Finally, 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30° C. for 16 hr under N2 atmosphere. LC-MS showed one main peak with desired m/z (calculated MW: 4143.75, observed m/z: 1040.50 ([(M+18]/4+H]+), and 1381.27 ([M/3+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and TCA-PEG10-BCY11015 (11.0 mg, 2.50 μmol, 23.66% yield, 94.26% purity) was obtained as a white solid.
Compound 2 (5.5 mg, 1.33 μmol, 1.0 eq) and BCY8928 (5.9 mg, 2.66 μmol, 2.0 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 10.0 μL, 3.0 eq), VcNa (1.0 mg, 5.05 μmol, 3.8 eq) and THPTA (1.0 mg, 2.30 μmol, 1.7 eq) were added. Finally, 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 35° C. for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m/z (calculated MW: 8578.91, observed m/z: 1430.6 ([M/6+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11027 (2.8 mg, 0.32 μmol, 24.5% yield, 91.71% purity) was obtained as a white solid.
Compound 1 (20.0 mg, 7.20 μmol, 1.0 eq) and BCY11607 (32.0 mg, 14.9 μmol, 2.1 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 36.0 μL, 2.0 eq), VcNa (6.0 mg, 30.3 μmol, 4.2 eq) and THPTA (6.4 mg, 14.7 μmol, 2.0 eq) were added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m/z (calculated MW: 7077.7 observed m/z: 1416.3 ([M/5+H]+), 1180.4 ([M/6+H]+), 1011.9 ([M/7+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY12967 (20.6 mg, 2.82 μmol, 39.17% yield, 96.82% purity) was obtained as a white solid.
A mixture of 1 (BP-23825, 155.5 mg, 249.40 μmol, 1.2 eq.), and HATU (95.0 mg, 249.92 μmol, 1.2 eq.) was dissolved in NMP (1.0 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg, 499.83 μmol, 87.0 μL, 2.4 eq.), and then the solution was allowed to stir at 25° C. for 5 min. Compound 2 (BCY13118, 500.0 mg, 207.83 μmol, 1.0 eq.) was dissolved in NMP (5.0 mL), and then added to the reaction solution, the pH of the resulting solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 45 min. LC-MS showed BCY13118 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by preparative-HPLC to give BP-23825-BCY13118 (1.35 g, 403.46 μmol, 64.71% yield, 90% purity) as a white solid. Calculated MW: 3011.53, observed m/z: 1506.8 ([M/2+H]+), 1005.0 ([M/3+H]+.
A mixture of BCY8928 (644.0 mg, 290.55 μmol, 2.5 eq.), THPTA (50.5 mg, 116.22 μmol, 1.0 eq.), CuSO4 (0.4 M, 145.0 μL, 0.5 eq.) and Vc (82.0 mg, 464.89 μmol, 4.0 eq.) were dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 6.0 mL), The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and then the solution was stirred at 25° C. for 3 min. BP-23825-BCY13118 (350.0 mg, 116.22 μmol, 1.0 eq.) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 11.0 mL), and then dropped into the stirred solution. All solvents here were pre-degassed and purged with N2 3 times. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 6 hr under N2 atmosphere. LC-MS showed one main product peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA condition), and BCY13272 (1.75 g, 235.01 μmol, 67.40% yield, 94% purity) was obtained as a white solid. Calculated MW: 7446.64, observed m/z: 1242.0 ([M/6+H]+), 1491.0 ([M/5+H]+.
A mixture of compound 1 (10.0 mg, 3.60 μmol, 1.0 eq.), compound 2 (6.73 mg, 2.88 μmol, 0.8 eq.), and THPTA (3.0 mg, 6.90 μmol, 2.0 eq.) was dissolved in t-BuOH/H2O (1:1, 0.5 mL, degassed and purged with N2), and then aqueous solution of CuSO4 (0.4 M, 9 μL, 1.0 eq.) and VcNa (2.0 mg, 10.10 μmol, 2.8 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 1 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and compound 3 (5.0 mg, 0.93 μmol, 25.88% yield, 95.33% purity) was obtained as a white solid. Calculated MW: 5115.80, observed m/z: 1278.95 ([M+41-1]4+).
A mixture of compound 1 (5.0 mg, 9.77 μmol, 1.0 eq.), compound 2 (2.4 mg, 1.08 μmol, 1.1 eq.), and THPTA (0.4 M, 3 μL, 1.0 eq.) was dissolved in t-BuOH/H2O (1:1, 0.5 mL, degassed and purged with N2), and then aqueous solution of CuSO4 (0.4 M, 3 μL, 1.0 eq.) and VcNa (0.4 M, 3 μL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 1 hr under N2 atmosphere. LC-MS showed compound 3 and compound 4 also remained, and desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY12733 (3.3 mg, 0.41 μmol, 42.42% yield, 94.60% purity) was obtained as a white solid. Calculated MW: 7307.33, observed m/z: 1827.1 ([M+41-1]4+), 1462.1 ([M+51-1]5+).
A mixture of compound 1 (50.0 mg, 16.6 μmol, 1.0 eq.), compound 2 (29.5 mg, 13.3 μmol, 0.8 eq.), and THPTA (36.1 mg, 83.1 μmol, 5.0 eq.) was dissolved in t-BuOH/H2O (1:1, 8 mL, degassed and purged with N2), and then aqueous solution of CuSO4 (0.4 M, 20.8 μL, 0.5 eq.) and VcNa (65.9 mg, 332.6 μmol, 20.0 eq.) were added under N2. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.5 mL 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. Then the reaction mixture was stirred at 25° C. for 24 hr under N2 atmosphere. The reaction was set up for two batches in parallel. LC-MS showed compound 1 and little amount of compound 2 remained, and desired m/z was detected. The reaction mixture was filtered to remove the insoluble residue. The crude product was purified by preparative HPLC, and compound 3 (31.5 mg, 5.44 μmol, 16.36% yield, 90.22% purity) was obtained as a white solid. Calculated MW: 5224.07, observed m/z: 1306.9 ([M+4H]4+), 871.6 ([M+6H]6+).
A mixture of compound 1 (31.5 mg, 6.03 μmol, 1.0 eq.), compound 2 (14.4 mg, 6.33 μmol, 1.05 eq.), and THPTA (2.62 mg, 6.03 μmol, 1.0 eq.) was dissolved in t-BuOH/H2O (1:1, 1.0 mL, degassed and purged with N2), and then aqueous solution of CuSO4 (0.4 M, 15.07 μL, 1.0 eq.) and VcNa (4.78 mg, 24.12 μmol, 4.0 eq.) were added under N2. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.5 mL 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. Then the reaction mixture was stirred at 25° C. for 3 hrs under N2 atmosphere. LC-MS showed little amount of compound 2 remained, compound 1 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered to remove the insoluble residue. The crude product was purified by preparative HPLC, and BCY14413 (22.5 mg, 3.00 μmol, 43.10% yield, 86.63% purity) was obtained as a white solid. Calculated MW: 7498.75, observed m/z: 938.3 ([M+8H]8+), 1072.2 ([M+7H]7+), 1250.9 ([M+6H]6+), 1500.8 ([M+5H]5+).
A mixture of BCY14413 (10.0 mg, 1.33 μmol, 1.0 eq.) and biotin-Peg12-NHS (2.6 mg, 2.80 μmol, 2.6 eq.) was dissolved in DMF (0.3 mL). The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY14413 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY14415 (10 mg, 1.07 μmol, 80.49% yield, 90.2% purity) was obtained as a white solid. Calculated MW: 8324.73, observed m/z: 1388.4 ([M+6H]6+), 1190.2 ([M+7H]7+), 1041.5 ([M+8H]8+), 926.0 ([M+9H]9+)
A mixture of compound BCY14413 (5.1 mg, 0.68 μmol, 1.0 eq.) and Alexa Fluor 488 NHS ester (0.5 mg, 8.16e-1 μmol, 1.2 eq.) was dissolved in DMF (0.3 mL). The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed that some BCY14413 remained and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and the main peak was collected as two fractions with different purity, and BCY14416 (0.7 mg, 0.065 μmol, 9.84% yield, 96.4% purity) and (0.5 mg, 0.047 μmol, 7.03% yield, 91.2% purity) were obtained as red solid. Calculated MW: 8015, observed m/z: 1336.5 ([M+7H]7+).
A mixture of BCY14964 (55.0 mg, 18.26 μmol, 1.0 eq), BCY8928 (32.4 mg, 14.61 μmol, 0.8 eq), and THPTA (39.8 mg, 91.32 μmol, 5.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 23.0 μL, 0.5 eq) and sodium ascorbate (72.0 mg, 365.27 μmol, 20.0 eq) were added under N2. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 1.5 h under N2 atmosphere. LC-MS showed BCY14964 remained, compound BCY8928 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY14798 (51 mg, 9.17 μmol, 33.37% yield, 94% purity) was obtained as a white solid. Calculated MW: 5229.07, observed m/z: 1308.3 ([M+4H]4+), 1046.7 ([M+5H]5+).
A mixture of BCY14798 (21.0 mg, 4.02 μmol, 1.0 eq), BCY13389 (10.0 mg, 4.42 μmol, 1.1 eq), and THPTA (1.8 mg, 4.02 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 5.0 μL, 0.5 eq) and sodium ascorbate (2.8 mg, 16.06 μmol, 4.0 eq) were added under N2. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3) and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N2 atmosphere. LC-MS showed BCY14798 was consumed completely, some BCY13389 remained and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative and BCY14414 (20 mg, 2.40 μmol, 59.73% yield, 90.9% purity) was obtained as a white solid. Calculated MW: 7503.74, observed m/z: 1251.5 ([M+5H]5+), 1072.9 ([M+7H]7+).
A mixture of BCY14414 (13.0 mg, 1.73 μmol, 1.0 eq) and biotin-PEG12-NHS ester (CAS 365441-71-0, 4.2 mg, 4.50 μmol, 2.6 eq) was dissolved in DMF (0.5 mL). The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY14414 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC and BCY14417 (9.0 mg, 1.07 μmol, 80.49% yield, 90.8% purity) was obtained as a white solid. Calculated MW: 8329.74, observed m/z: 1389.6 ([M+6H]6+), 1191.9 ([M+7H]7+).
A mixture of BCY14414 (5.6 mg, 0.75 μmol, 1.0 eq) and Alexa Fluor® 488 (0.9 mg, 1.49 μmol, 2.0 eq) was dissolved in DMF (0.3 mL). Then pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 1.0 hr. LC-MS showed BCY14414 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY14418 (2.3 mg, 0.25 μmol, 32.89% yield, 85.6% purity) was obtained as a red solid. Calculated MW: 8020.19, observed m/z: 1337.2 ([M+6H]6+).
A mixture of BCY14964 (20.0 mg, 6.64 μmol, 1.0 eq), BCY14601 (30.5 mg, 13.95 μmol, 2.1 eq), and THPTA (2.9 mg, 6.64 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 16.6 μL, 1.0 eq) and sodium ascorbate (4.7 mg, 26.56 μmol, 4.0 eq) were added under N2. The pH of this solution was adjusted to 8, and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N2 atmosphere. LC-MS showed BCY14964 remained, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY15217 (19.7 mg, 2.41 μmol, 36.26% yield, 96.2% purity) was obtained as a white solid. Calculated MW: 7362.5, observed m/z: 1473.5 ([M+5H]5+), 1228.2 ([M+6H]6+), 1052.8 ([M+7H]7+)
A mixture of BCY14798 (30.0 mg, 5.74 μmol, 1.0 eq), BCY14601 (15.0 mg, 6.88 μmol, 1.2 eq), and THPTA (2.5 mg, 5.74 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 14.0 μL, 1.0 eq) and sodium ascorbate (4.0 mg, 22.95 μmol, 4.0 eq) were added under N2. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 2 h under N2 atmosphere. LC-MS showed BCY14798 was consumed completely, BCY14601 remained, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY15218 (22 mg, 2.67 μmol, 46.61% yield, 95.0% purity) was obtained as a white solid. Calculated MW: 7404.6, observed m/z: 1234.8 ([M+6H]6+).
Compound 1 (N,N-Bis[3-(Fmoc-amino)propyl]glycine potassium sulfate, 10.0 mg, 15.78 μmol, 1.0 eq) and HATU (7.2 mg, 18.94 μmol, 1.2 eq) were first dissolved in 1 mL of DMF, then added DIEA (11.0 μL, 63.15 μmol, 4.0 eq). The mixture was stirred at 30° C. for 30 minutes, and then BCY9594 (80.0 mg, 30.09 μmol, 1.0 eq) was added. The reaction mixture was stirred at 25° C. for 2 hr. LC-MS showed one main peak with desired m/z (calculated MW: 3016.51, observed m/z: 1006.4 ([M+31-1]3+). The reaction mixture was used in the next step without purification.
Compound 2 (47.6 mg, 15.78 μmol, 1.0 eq) was first dissolved in 1 mL of DMF, then was added piperidine (0.2 mL, 2.03 mmol, 128.0 eq). The mixture was stirred at 30° C. for 30 minutes. LC-MS showed one main peak with desired m/z (calculated MW: 2572.04, observed m/z: 1286.8 ([M+2H]2+), 858.1 ([M+3H]3+). The reaction mixture was purified by preparative HPLC and compound 3 (24.4 mg, 9.06 μmol, 57% yield, 95% purity) was obtained as a white solid.
Compound 3 (24.4 mg, 9.06 μmol, 1.0 eq) and compound 4 (10.0 mg, 23.13 μmol, 2.4 eq), were dissolved in 2 mL of MeCN/H2O (1:1), 1 M NaHCO3 was added to adjust pH to 8. The mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired m/z (calculated MW: 3206.71, observed m/z: 1069.7 ([M+31-1]3+) was detected. The reaction mixture was purified by preparative HPLC and compound 5 (12.8 mg, 3.99 μmol, 42.08% yield, 88.62% purity) was obtained as a white solid.
Compound 5 (12.8 mg, 3.99 μmol, 1.0 eq) and BCY8928 (18.0 mg, 8.12 μmol, 2.0 eq) were first dissolved in 2 mL of t-BuOH/H2O (1:1), and then CuSO4 (0.4 M, 20.0 μL, 2.0 eq), VcNa (3.2 mg, 16.1 μmol, 4.0 eq) and THPTA (3.5 mg, 8.0 μmol, 2.0 eq) was added. Finally, 1M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2. The reaction mixture was stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed compound 5 was consumed completely and one main peak with desired m/z. The reaction mixture was purified by preparative HPLC and BCY12979 (16.0 mg, 2.02 μmol, 51% yield, 96.6% purity) was obtained as a white solid. Calculated MW: 7641.87, observed m/z: 1911.2 ([M+4H]4+), 1528.3 ([M+5H]5+), 1247.5 ([M+6H]6+), 1092.2 ([M+7H]7+).
A mixture of COM00000329 (102 mg, 58.76 μmol, 1.0 eq BCY11015 (92.6 mg, 41.13 μmol, 0.7 eq) and THPTA (0.4 M, 146.9 μL, 1.0 eq) was dissolved in t-BuOH/H2O (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 146.9 μL, 1.0 eq) and VcNa (0.4 M, 293.8 μL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 25-30° C. for 12 hr under N2 atmosphere. LC-MS showed COM00000329 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was directly purified by preparative HPLC. Compound 1 (60 mg, 13.61 μmol, 23.16% yield, 90.45% purity) was obtained as a white solid. Calculated MW: 3988.52, observed m/z: 1329.97 ([M+31-1]3+), 990.56 ([M+41-1]4+).
A mixture of Compound 1 (60 mg, 15.04 μmol, 1.0 eq), BCY8928 (72.0 mg, 32.47 μmol, 2.2 eq) and THPTA (0.4 M, 37.6 μL, 1.0 eq) was dissolved in t-BuOH/H2O (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 37.6 μL, 1.0 eq) and VcNa (0.4 M, 75.2 μL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 25-30° C. for 12 hr under N2 atmosphere. LC-MS showed Compound 1 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was directly purified by preparative HPLC. BCY10918 (48 mg, 5.47 μmol, 36% yield, 96% purity) was obtained as a white solid. Calculated MW: 8423.67, observed m/z: 1404.27 ([M+6H]6+), 1203.73 ([M+7H]7+).
A mixture of Compound 1 (75 mg, 18.8 μmol, 1.0 eq), BCY11014 (93.75 mg, 43.1 μmol, 2.3 eq) and THPTA (0.4 M, 47.0 μL, 1.0 eq) was dissolved in t-BuOH/H2O (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 47.0 μL, 1.0 eq) and VcNa (0.4 M, 94.0 μL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 25-30° C. for 12 hr under N2 atmosphere. LC-MS showed Compound 1 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was directly purified by preparative HPLC. BCY10919 (96 mg, 11.39 μmol, 60.59% yield, 96.12% purity) was obtained as a white solid. Calculated MW: 8339.54, observed m/z: 1391.3 ([M+6H]6+), 1192.5 ([M+7H]7+).
A mixture of compound 1 (15.0 mg, 6.10 μmol, 1.0 eq.), BCY11016 (18.4 mg, 7.93 μmol, 1.3 eq.), and THPTA (2.65 mg, 6.10 μmol, 1.0 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (30.0 μL, 0.4M, 2.0 eq.) and VcNa (0.4 M, 30.0 μL, 2.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 4 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by preparative HPLC. Compound 3 (2.89 mg, 0.514 μmol, 8.42% yield, 83.4% purity) was obtained as a white solid. Calculated MW: 4782.46, observed m/z: 963.9 ([M+3H+2H2O]5+).
A mixture of compound 3 (2.89 mg, 0.60 μmol, 1.0 eq.), BCY7744 (4.38 mg, 1.87 μmol, 3.1 eq.), and THPTA (0.9 mg, 2.1 μmol, 3.5 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 3.0 μL, 2.0 eq.) and VcNa (0.4 M, 6.0 μL, 4.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY11021 (2.8 mg, 0.229 μmol, 37% yield, 96.4% purity) was obtained as a white solid. Calculated MW: 11795.38, observed m/z: 1310.6 ([M+9H]9+), 786.6 ([M+15H]15+).
A mixture of compound 3 (2.7 mg, 0.6 μmol, 1.0 eq.), BCY8928 (5.3 mg, 2.38 μmol, 4.0 eq.), and THPTA (0.9 mg, 2.1 μmol, 3.5 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 6.0 μL, 4.0 eq.) and VcNa (0.4 M, 6.0 μL, 4.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY11022 (1.9 mg, 1.0 μmol, 23.2% yield, 94.6% purity) was obtained as a white solid. Calculated MW: 11435.19, observed m/z: 1143.2 ([M+10H]10+).
A mixture of compound 2 (5 mg, 1.80 μmol, 1.0 eq), BCY7744 (9 mg, 3.85 μmol, 2.1 eq), THPTA (0.4 M, 9 μL, 1.0 eq) was dissolved in t-BuOH/H2O (1:1, 2 mL, pre-degassed and purged with N2), then CuSO4 (0.4 M, 9 μL, 2.0 eq) and VcNa (0.4 M, 18 μL, 4.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 16 hr under N2 atmosphere. LC-MS showed BCY7744 remained and desired m/z was detected. The reaction mixture was directly purified by preparative HPLC. BCY11864 (5.2 mg, 0.62 μmol, 34% yield, 89% purity) was obtained as a white solid. Calculated MW: 7453.44, observed m/z: 1490.70 ([M+5H]5+).
A mixture of compound 1 (40.0 mg, 21.15 μmol, 1.0 eq.), compound 2 (43.0 mg, 15.86 μmol, 0.75 eq.), and THPTA (10.0 mg, 21.20 μmol, 1.0 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (53.0 μL, 0.4M, 1.0 eq.) and VcNa (0.4 M, 53.0 μL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 4 hr, LC-MS showed compound 2 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and a residue was produced. This was purified by preparative HPLC. Compound 3 (11.7 mg, 2.44 μmol, 11% yield, 96.2% purity) was obtained as a white solid. Calculated MW: 4607.33, observed m/z: 1152.36 ([M+4H]4+).
A mixture of compound 3 (11.7 mg, 2.54 μmol, 1.0 eq.), BCY8928 (11.8 mg, 5.33 μmol, 2.1 eq.), and THPTA (2.3 mg, 5.3 μmol, 2.0 eq.) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 12.7 μL, 2.0 eq.) and VcNa (0.4 M, 25.4 μL, 4.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 40° C. for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY11780 (5.0 mg, 0.509 μmol, 20.03% yield, 92.0% purity) was obtained as a white solid. Calculated MW: 9042.48, observed m/z: 1292.8 ([M+7H]7+), 1130.96 ([M+8H]8+).
A mixture of N-(acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0 eq), HATU (51.2 mg, 134.7 μmol, 1.2 eq) and DIEA (29.0 mg, 224.4 μmol, 40 μL, 2.0 eq) was dissolved in DMF (2 mL), and mixed for 5 min. Then BCY8116 (294.0 mg, 135.3 μmol, 1.2 eq) was added. The reaction mixture was stirred at 40° C. for 16 hr. LC-MS showed one main peak with desired m/z. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by preparative HPLC. BCY12476 (194.5 mg, 66.02 μmol, 29% yield, 94% purity) was obtained as a white solid. Calculated MW: 2778.17, observed m/z: 1389.3 ([M+21-1]2+), 926.7 ([M+31-1]3+).
A mixture of BCY12476 (47.0 mg, 16.91 μmol, 1.0 eq), BCY8928 (30.0 mg, 13.53 μmol, 0.8 eq), and THPTA (36.7 mg, 84.55 μmol, 5.0 eq) was dissolved in t-BuOH/H2O (1:1, 8 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 21.0 μL, 0.5 eq) and VcNa (67.0 mg, 338.21 μmol, 20.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 1.5 h under N2 atmosphere. LC-MS showed that some BCY12476 remained, BCY8928 was consumed completely, and a peak with the desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13689 (25.3 mg, 4.56 μmol, 27% yield, 90% purity) was obtained as a white solid. Calculated MW: 4995.74, observed m/z: 1249.4 ([M+4H]4+), 999.9 ([M+5H]5+).
A mixture of BCY13689 (43.6 mg, 8.73 μmol, 1.0 eq), BCY13389 (20.8 mg, 9.16 μmol, 1.05 eq), and THPTA (3.8 mg, 8.73 μmol, 1.0 eq) was dissolved in t-BuOH/H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 22.0 μL, 1.0 eq) and VcNa (3.5 mg, 17.45 μmol, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH/H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N2 atmosphere. LC-MS showed a significant peak corresponding to the desired m/z. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13390 (33.8 mg, 4.21 μmol, 48% yield, 90% purity) was obtained as a white solid. Calculated MW: 7270.41, observed m/z: 1454.9 ([M+5H]5+), 1213.2 ([M+6H]6+).
A mixture of BCY13390 (5.0 mg, 0.6 μmol, 1.0 eq), biotin-PEG12-NHS ester (CAS 365441-71-0, 0.7 mg, 0.72 μmol, 1.1 eq) was dissolved in MeCN/H2O (1:1, 2 mL). The pH of this solution was adjusted to 8 by dropwise addition of 1.0 M NaHCO3. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY13390 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13582 (2.5 mg, 0.30 μmol, 43% yield, 96% purity) was obtained as a white solid. Calculated MW: 8096.43, observed m/z: 1351.1 ([M+6H]6+), 1158.5 ([M+7H]7+).
A mixture of BCY13390 (15.0 mg, 2.06 μmol, 1.0 eq) and Alexa Fluor® 488 NHS ester (2.5 mg, 4.12 μmol, 2.0 eq) was dissolved in DMF (0.5 mL). DIEA (2.6 mg, 20.63 μmol, 3.6 μL, 10 eq) was then added dropwise. The reaction mixture was stirred at 25° C. for 1 hr. LC-MS showed BCY13390 remained, and one main peak with desired m/z was detected. Additional Alexa Fluor® 488 NHS ester (2.0 mg, 3.09 μmol, 1.5 eq) was added to the reaction mixture, and the reaction mixture was stirred at 25° C. for one additional hour. HPLC showed BCY13390 was consumed completely. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13583 (5 mg, 0.61 μmol, 29% yield, 95% purity) was obtained as a red solid. Calculated MW: 7787.9, observed m/z: 1948.8 ([M+4H+H2O]4+), 1558.6 ([M+5H+H2O]5+), 1299.1 ([M+7H+H2O]7+).
A mixture of BCY13390 (5.6 mg, 0.77 μmol, 1.0 eq) and cyanine 5 NHS ester (0.5 mg, 0.85 μmol, 1.1 eq) was dissolved in MeCN/H2O (1:1, 2 mL). The pH of this solution was adjusted to 8 by dropwise addition of 1.0 M NaHCO3. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY13390 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13628 (2.9 mg, 0.36 μmol, 46% yield, 95% purity) was obtained as a blue solid. Calculated MW: 7736.06, observed m/z: 1289.9 ([M+6H]6+), 1105.5 ([M+7H]7+).
A mixture of BCY13689 (25.0 mg, 5.00 μmol, 1.0 eq), BCY14601 (13.0 mg, 6.01 μmol, 1.2 eq), and THPTA (2.0 mg, 5.00 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 12.5 μL, 1.0 eq) and Vc (3.5 mg, 20.02 μmol, 4.0 eq) were added under N2. The pH of this solution was adjusted to 8, and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N2 atmosphere. LC-MS showed BCY13689 was consumed completely, some BCY14601 remained and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY15155 (19.7 mg, 2.41 μmol, 36% yield, 97% purity) was obtained as a white solid. Calculated MW: 7171.3, observed m/z: 1434.7 ([M+5H]5+), 1196.2 ([M+6H]6+)
The following heterotandem bicyclic peptide complexes of the invention were analysed using mass spectrometry and HPLC. HPLC setup was as follows for analytical method A-C below:
HPLC setup was as follows for analytical method D below:
Gradients used are described in the table below:
and the data was generated as follows:
Further analytical data was generated as follows:
1. CD137 Reporter Assay Co-Culture with Tumor Cells
Culture medium, referred to as R1 media, is prepared by adding 1% FBS to RPMI-1640 (component of Promega kit CS196005). Serial dilutions of test articles in R1 are prepared in a sterile 96 well-plate. Add 25 μL per well of test articles or R1 (as a background control) to designated wells in a white cell culture plate. Tumor cells* are harvested and resuspended at a concentration of 400,000 cells/mL in R1 media. Twenty five (25) μL/well of tumor cells are added to the white cell culture plate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in the water bath and then added to 5 ml pre-warmed R1 media. Twenty five (25) μL/well of Jurkat cells are then added to the white cell culture plate. Incubate the cells and test articles for 6 h at 37° C., 5% CO2. At the end of 6 h, add 75 μL/well Bio-Glo™ reagent (Promega) and incubate for 10 min before reading luminescence in a plate reader (Clariostar, BMG). The fold change relative to cells alone (Jurkat cells+Cell line used in co-culture) is calculated and plotted in GraphPad Prism as log(agonist) vs response to determine EC50 (nM) and Fold Induction over background (Max).
The tumor cell type used in co-culture is NCI-H292 and HT1376 which has been shown to express Nectin-4. The tumor cell types used in co-culture for EphA2 are A549, PC3 and HT29. The tumor cell type used in co-culture for PD-L1 is RKO.
Data presented in
A summary of the EC50 (nM) and Fold Induction induced by heterotandem bicyclic peptide complexes in a CD137 reporter assay in co-culture with a Nectin-4-expressing tumor cell line is reported in Table 1A below:
A summary of the EC50 (nM) induced by heterotandem bicyclic peptide complexes BCY11863 and close analogues in a CD137 reporter assay in co-culture with a Nectin-4-expressing tumor cell line is reported in Table 1B below and visualized in
A summary of fold induction induced by Nectin-4/CD137 heterotandem peptides in a CD137 reporter coculture assay with NCI-H292 cells is shown in Table 2 below. All compounds are compared to plate control BCY10000 which has an average EC50 of 1.1±0.5 nM and Emax of 28±11 fold over background.
A summary of fold induction induced by Nectin-4/CD137 heterotandem peptides in a CD137 reporter coculture assay with HT1376 tumor cells is shown in Table 2A below with EC50 (nM) and Emax (fold induction over background) being reported. Most Nectin-4/CD137 heterotandems have EC50 below 1 nM.
Data presented in
A summary of fold induction induced by EphA2/CD137 heterotandem peptides in a CD137 reporter coculture assay with PC3 cells is shown in Table 3A below. All compounds are compared to plate control BCY9173 which has an average EC50 of 0.54 nM and Emax of 42 fold over background.
A summary of the EC50 (nM) and Fold Induction induced by BCY13272 in a CD137 reporter assay in co-culture with an EphA2 expressing tumor cell line is reported in Table 3B below:
A summary of fold induction induced by EphA2/CD137 heterotandem peptides in a CD137 reporter coculture assay with PC3 tumor cells is shown in Table 3C below with EC50 (nM) and Emax (fold induction over background) being reported. Most EphA2/CD137 heterotandems have EC50 below 1 nM.
A summary of fold induction induced by PD-L1/CD137 heterotandem peptides in CD137 reporter coculture assay with RKO cells is shown in Table 4 below.
Tumor cell lines were cultured according to suppliers recommended protocol. Frozen PBMCs from healthy human donors were thawed and washed one time in room temperature PBS, and then resuspended in R10 medium. 100 μl of PBMCs (1,000,000 PBMCs/ml) and 100 μl of tumor cells (100,000 tumor cells/ml) (Effector:Target cell ratio (E:T) 10:1) were plated in each well of a 96 well flat bottom plate for the co-culture assay. 100 ng/ml of soluble anti-CD3 mAb (clone OKT3) was added to the culture on day 0 to stimulate human PBMCs. Test, control compounds, or vehicle controls were diluted in R10 media and 50 μL was added to respective wells to bring the final volume per well to 250 μL. Plates were covered with a breathable film and incubated in a humidified chamber at 37° C. with 5% CO2 for three days. Supernatants were collected 48 hours after stimulation, and human IL-2 and IFN-γ were detected by Luminex. Briefly, the standards and samples were added to black 96 well plate. Microparticle cocktail (provided in Luminex kit, R&D Systems) was added and shaken for 2 hours at room temperature. The plate was washed 3 times using magnetic holder. Biotin cocktail was then added to the plate and shaken for 1 hour at RT. The plate was washed 3 times using magnetic holder. Streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plates were washed 3 times using magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT, and read using the Luminex 2000. Raw data were analyzed using built-in Luminex software to generate standard curves and interpolate protein concentrations, all other data analyses and graphing were performed using Excel and Prism software. Data represents one study with three independent donor PBMCs tested in experimental duplicates.
Data presented in
A summary of the EC50 (nM) and maximum IFN-γ cytokine secretion (pg/ml) induced by selected Nectin-4/CD137 heterotandem bicyclic peptide complexes in Human PBMC co-culture (cytokine release) assay is reported in Table 4A below and visualized in
Male SD Rats were dosed with each heterotandem Bicycle peptide complex formulated in 25 mM Histidine HCl, 10% sucrose pH 7 by IV bolus or IV infusion (15 minutes). Serial bleeding (about 80 μL blood/time point) was performed via submandibular or saphenous vein at each time point. All blood samples were immediately transferred into prechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4° C., 3000 g. The precipitant including internal standard was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10 minutes. The supernatant was transferred into pre-labeled polypropylene microcentrifuge tubes, and then quick-frozen over dry ice. The samples were stored at 70° C. or below as needed until analysis. 7.5 μL of the supernatant samples were directly injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported. The pharmacokinetic parameters from the experiment are as shown in Table 6A:
The pharmacokinetic parameters specifically for BCY11863 are as shown in Table 6B:
Data in Table 6B above and
Data in Table 6C and
Non-naïve Cynomolgus Monkeys were dosed via intravenous infusion (15 or 30 min) into the cephalic vein with 1 mg/kg of each Heterotandem Bicycle Peptide Complex formulated in 25 mM Histidine HCl, 10% sucrose pH 7. Serial bleeding (about 1.2 ml blood/time point) was performed from a peripheral vessel from restrained, non-sedated animals at each time point into a commercially available tube containing Potassium (K2) EDTA*2H2O (0.85-1.15 mg) on wet ice and processed for plasma. Samples were centrifuged (3,000×g for 10 minutes at 2 to 8° C.) immediately after collection. 0.1 mL plasma was transferred into labelled polypropylene micro-centrifuge tubes. 5-fold of the precipitant including internal standard 100 ng/mL Labetalol & 100 ng/mL dexamethasone & 100 ng/mL tolbutamide & 100 ng/mL Verapamil & 100 ng/mL Glyburide & 100 ng/mL Celecoxib in MeOH was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm for 10 minutes at 2 to 8° C. Samples of supernatant were transferred into the pre-labeled polypropylene microcentrifuge tubes, and frozen over dry ice. The samples were stored at −60° C. or below until LC-MS/MS analysis. An aliquot of 40 μL calibration standard, quality control, single blank and double blank samples were added to the 1.5 mL tube. Each sample (except the double blank) was quenched with 200 μL IS1 respectively (double blank sample was quenched with 200 μL MeOH with 0.5% tritonX-100), and then the mixture was vortex-mixed well (at least 15 s) with vortexer and centrifuged for 15 min at 12000 g, 4° C. A 10 μL supernatant was injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported. The pharmacokinetic parameters for three bispecific compounds are as shown in Table 7.
6 Male CD-1 mice were dosed with 15 mg/kg of each Heterotandem Bicycle Peptide Complex formulated in 25 mM Histidine HCl, 10% sucrose pH 7 via intraperitoneal or intravenous administration. Serial bleeding (about 80 μL blood/time point) was performed via submandibular or saphenous vein at each time point. All blood samples were immediately transferred into prechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4° C., 3000 g. The precipitant including internal standard was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10 minutes. The supernatant was transferred into pre-labeled polypropylene microcentrifuge tubes, and then quick-frozen over dry ice. The samples were stored at 70° C. or below as needed until analysis. 7.5 μL of the supernatant samples were directly injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported.
Data in
6-8 weeks old C57BL/6J-hCD137 female mice were inoculated in the flank with 1×106 syngeneic Nectin-4 overexpressing MC38 cells (MC38 #13). When tumors reached 72 mm3 size on average, mice were randomized to receive vehicle or BCY11863 (intraperitoneal administration). BCY11863 was administered (n=6 mice/treatment cohort) at either 1 mg/kg or 10 mg/kg either daily (QD) or every three days (Q3D). QD dosed mice received 16 doses of BCY11863 and Q3D dosed mice received 10 doses of BCY11863. Tumor growth was monitored by caliper measurements until day 69 after treatment initiation. The results of this experiment may be seen in
Based on the circulating plasma half-life of BCY11863 in mice after IP injection (2.5 h), plasma trough levels will be close to 0 after both BCY11863 doses (1 and 10 mg/kg) and dosing intervals (QD and Q3D) thus demonstrating that less than continuous plasma exposure of BCY11863 from intermittent dosing is sufficient to lead to significant anti-tumor activity leading to durable complete responses.
On day 69, 5 animals that had responded completely to BCY11863 treatment were re-inoculated with 1×106 MC38 #13-cells. A cohort of 5 naïve C57BL/6J-hCD137 female mice were inoculated with 1×106 MC38 #13-cells as a control. The results of this experiment may be seen in
6-8 weeks old BALB/c-hCD137 female mice were inoculated in the flank with 3×105 syngeneic Nectin-4 overexpressing CT26 cells (CT26 #7). When tumors reached around 70 mm3 size on average, mice were randomized to receive vehicle or 5 mg/kg BCY11863 intraperitoneally every three days (6 doses total). Tumor growth was monitored by caliper measurements until day 14 after treatment initiation. The results of this experiment may be seen in
Based on the circulating plasma half-life of BCY11863 in mice at IP injection (2.5 h), plasma exposure will not be continuous throughout the dosing period demonstrating that less than continuous plasma exposure of BCY11863 is sufficient to lead to significant anti-tumor activity.
9. Total T Cells and CD8+ T Cells Increase in CT26 #7 Tumor Tissue 1 h after the Last (6th) Q3D Dose of BCY11863
1 hour after the last vehicle or BCY11863 dose the CD26 #7 bearing mice were sacrificed and tumors were harvested, processed for single cell suspensions and stained for flow cytometry analysis for total T cells (CD45+CD3+), CD8+ T cells (CD45+CD3+CD8+), CD4+ T cells (CD45+CD3+CD4+) and regulatory T cells (Tregs; CD45+CD3+CD4+Foxp3+). The results of this experiment may be seen in
This demonstrates that treatment with BCY11863 can lead to an increased level of T-cells locally in the tumor tissue after intermittent dosing.
10. Pharmacokinetic Profiles of BCY11863 in Plasma and Tumor Tissue of CT26 #7 Syngeneic Tumor Bearing Animals after a Single Intravenous (iv) Administration of 5 mg/kg of BCY11863
6-8 weeks old BALB/c female mice were inoculated in the flank with 3×105 syngeneic Nectin-4 overexpressing CT26 cells (CT26 #7). When tumors reached around 400 mm3 size on average, mice were randomized to receive a single intravenous dose of vehicle or 5 mg/kg BCY11863. A cohort of mice (n=3/timepoint) were sacrificed at 0.25, 0.5, 1, 2, 4, 8 and 24 h timepoints and harvested plasma and tumor tissue were analyzed for BCY11863. For tumor BCY11863 content analysis, tumor homogenate was prepared by homogenizing tumor tissue with 10 volumes (w:v) of homogenizing solution (MeOH/15 mM PBS (1:2, v:v)). 40 μL of sample was quenched with 200 μL IS1 and the mixture was mixed by vortexing for 10 min at 800 rpm and centrifuged for 15 min at 3220 g at 4° C. The supernatant was transfer to another clean 96-well plate and centrifuged for 5 min at 3220 g at 4° C., and 10.0 μL of supernatant was then injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. For plasma BCY11863 content analysis, blood samples were collected in K2-EDTA tubes and immediately processed to plasma by centrifugation at approximately 4° C., 3000 g. 40 μL of plasma sample was quenched with 200 μL IS1 and the mixture was mixed by vortexing for 10 min at 800 rpm and centrifuged for 15 min at 3220 g at 4° C. The supernatant was transfer to another clean 96-well plate and centrifuged for 5 min at 3220 g at 4° C., and 10.0 μL of supernatant was then injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte.
The results of this experiment may be seen in
6-8 weeks old C57BL/6J-hCD137 female mice were inoculated in the flank with 1×106 syngeneic MC38 cells. When tumors reached 76 mm3 size on average, mice were randomized to receive vehicle or BCY12491 (intraperitoneal administration). BCY12491 was administered (n=6 mice/treatment cohort) at either 5 mg/kg or 15 mg/kg either daily (QD) or every three days (Q3D). QD dosed mice received 22 doses of BCY12491 and Q3D dosed mice received 8 doses of BCY12491. Tumor growth was monitored by caliper measurements until day 73 after treatment initiation. The results of this experiment may be seen in
Based on the circulating plasma half-life BCY12491 in mice after IP injection (2.5 h), plasma trough levels will be close to 0 after both BCY12491 doses (5 and 15 mg/kg) and dosing intervals (QD and Q3D) thus demonstrates that less than continuous plasma exposure of BCY12491 from intermittent dosing is sufficient to lead to significant anti-tumor activity and durable complete responses.
Mouse mammary gland tumor cell line MC38 were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 1× Penicillin/Streptomycin, 10 mM HEPES, and 2 mM L-Glutamine (referred to as R10 medium). Frozen PBMCs from healthy human donors were thawed and washed once in room temperature PBS with benzonase, and then resuspended in RPMI supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 1× Penicillin/Streptomycin, 10 mM HEPES, and 2 mM L-Glutamine (herein referred to as R10 medium). 100 μl of PBMCs (1,000,000 PBMCs/ml) and 100 μl of tumor cells (100,000 tumor cells/ml) (Effector:Target cell ratio (E:T) 10:1) were plated in each well of a 96 well flat bottom plate for the co-culture assay. 100 ng/ml of soluble anti-CD3 mAb (clone OKT3) was added to the culture on day 0 to stimulate human PBMCs. Test, control compounds, or vehicle controls were diluted in R10 media and 50 μL was added to respective wells to bring the final volume per well to 250 μL. Plates were covered with a breathable film and incubated in a humidified chamber at 37° C. with 5% CO2 for two days. Supernatants were collected 24 and 48 hours after stimulation, and human IFN-γ was detected by Luminex. Briefly, the standards and samples were added to a black 96 well plate. Microparticle cocktail (provided in Luminex kit, R&D Systems) was added and shaken for 2 hours at room temperature. The plate was washed 3 times using a magnetic holder. Biotin cocktail was then added to the plate and shaken for 1 hour at RT. The plate was washed 3 times using a magnetic holder. Streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plates were washed 3 times using a magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT, and read using the Luminex 2000. Raw data were analyzed using built-in Luminex software to generate standard curves and interpolate protein concentrations, all other data analyses and graphing were performed using Excel and Prism software. Data represents one study with three independent donor PBMCs tested in experimental duplicates.
Data presented in
Similarly, PBMCs from healthy donors were co-cultured with EphA2 expressing cancer cells (MC38 and HT-1080) at a ratio of 5:1 in presence of anti-CD3 and BCY13272. Supernatants were analyzed after 48 h by Luminex for cytokines (IL-2 and IFNγ), data is shown in Table 8 and is representative of PBMCs from one donor (from a total of n=4 or 5 individual experiments).
Data presented in
Primary patient derived tumor cells from Discovery Life Sciences (DLS) were thawed gently in 10 mL pre-warmed wash medium spiked fresh with Benzonase. The 3D spheroid kit from Greiner (cat #655840) is used to maintain cells in culture for 2 days. Briefly, tumor cells were counted with trypan blue using a haemocytometer. The cells were centrifuged at 1500 rpm for 5 min to wash, and the pellet is resuspended in 100 μL per 1×106 cells N3D nanoshuttle. To make them magnetic, cells were spun down at 1500 rpm for 5 min and resuspended; this process is repeated for a total of 4 times. After the final spin, cells were resuspended in the appropriate amount of fresh Lung DTC medium (DLS) to give 50,000-100,000 cells per well in 100 μL/well. Greiner cell-repellent, 96-well plates (cat #655976) were used for this experiment. If there were cell clumps or debris visible, sample is applied to a 70-100 μm filter before plating. At least 50,000 cells per sample were reserved for a Day 0 flow cytometry panel, these cells were stained, fixed, and stored at 4° C. for later flow analysis. Control/test compound dilutions were prepared in a separate plate at 2× in Lung DTC medium, and 100 μL/well of these 2× drug solutions were added to the wells as described by the plate map. The assay plate was then placed onto the 96-well magnetic spheroid drive in a humidified chamber at 37° C., 5% CO2. At 24 h, the magnetic spheroid drive was removed. At 48 h, medium was collected for cytokine analysis and cells were collected for a Day 2 flow cytometry panel. Cytokines were quantified using a custom-built cytokine/chemokine panel (IP-10, Granzyme B, IFNγ, IL-2, IL-6, TNFα, IL-8, MIP-1a, MIP-1b, MCP-1, IL-10, MIG) from R&D systems on a Luminex reader. Flow panels: Day 0=Live/Dead, CD45, EpCAM, Nectin4, CD3, CD4, CD8, CD137; Day 2=Live/Dead, CD45, EpCAM, Nectin4, CD3, CD8, Ki67, and counting beads. Flow data is analysed with Flowjo software.
Data shown in
14. Promega OX40 Cell-Activity Assay in Co-Culture with Tumor Cells
Promega have developed an OX40 cell-activity assay that uses NF-κB luciferase luminescence as a read-out of OX40 activation in Jurkat cells (Promega CS197704). On the day of the experiment, prepare medium by thawing FBS and adding 5% FBS to RPMI-1640. Thaw OX40 Jurkat cells in the water-bath and then add 500 μl cells to 11.5 ml pre-warmed 5% FBS RPMI-1640 medium. Add 55 μl cells/well to white cell culture plates. Harvest tumor cells from culture. 4T1 is a Nectin-4 negative murine mammary gland epithelial cancer cell and it was genetically modified to express murine Nectin-4 on the cell surface (4T1 Nectin-4 positive; clone 411-D02). Tumor cells were cultured to 80% confluency in vitro in RPM11640 medium supplemented with 10% heat-inactivated FBS, 1× Penicillin/Streptomycin, 1× L-Glutamine, 20 mM HEPES and 1×NEAA (RPMI working medium). Tumor cells were trypsinized and washed two times at 1500 rpm for 5 minutes in RPM11640 working medium prewarmed to 37° C. Count cells and resuspend at 2,000,000 cells/mL in R5 media (for 10,000 cells/well). Add 5 μL of tumor cells per well.
Proceed to dilute agonists at concentration giving the maximum fold induction and then titrate down the amount in a sterile 96 well-plate. Prepare enough reagent for duplicate samples and then perform 1/3 dilution series or 1/10 dilution series. Include positive control OX40L trimer (AcroBiosystems, R&D systems) and negative control monomeric or non-binding peptides. Add 20 μl of agonist as duplicate samples or 5% FBS RPMI-1640 alone as background control.
Co-incubate cells together with agonists for 6 hours at 37° C., 5% CO2. After 6 hours, thaw Bio-Glo™ and develop the assay at room-temperature. Add 80 μl Bio-Glo™ per well and incubate 5-10 min. Read luciferase signal on CLAIROStar plate-reader using the MARS program and normalize the fold induction relative to background (medium alone). Analyse data by transforming the data to x=log(X), then plot log (agonist) vs. response variable slope (4 parameters) to calculate EC50 values.
The results of this assay are shown in Table 9 and
6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 cells. Mice were randomized into treatment groups when average tumor volumes reached around 240 mm3 and were treated (n=6/treatment cohort) with vehicle (25 mM histidine, 10% sucrose, pH7) intravenously (IV), 15 mg/kg BCY12491 (EphA2: CD137 1:2 Heterotandem Complex) IV, 15 mg/kg BCY13626 (non-binding control for EphA2) IV or 2 mg/kg Anti-CD137 (urelumab analogue) intraperitoneally. All treatments were given Q3D for three doses and tumor tissues were harvested 1 hour after the last dose. Part of the tumor tissue was used for RNA isolation for transcriptional analysis and a part of the tumor tissue was used for formalin fixed paraffin embedded (FFPE) sample preparation for immunohistochemical (INC) analysis. RNA was isolated from tumor tissues using RNAeasy kit [Qiagen] and transcriptional analysis was performed using nCounter Mouse PanCancer IO 360 panel (Nanostring) from 100 ng RNA/tumor. Data was analysed using the nSolver Analysis Software (Nanostring). CD8+ tumor infiltrating cells were stained in FFPE tissue sections using anti-mouse CD8 antibody (Abcam, #ab217344) and Ventana Discovery OmniMap anti Rabbit-HRP Kit (Ventana #760 4310).
The results of this study are shown in
6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 80 mm3 and were treated with vehicle (25 mM histidine, 10% sucrose, pH7) intravenously (IV), 8 mg/kg BCY13272, 0.9 mg/kg BCY13272 and 0.1 mg/kg BCY13272 IV. All treatments were given twice a week (BIW) for 6 doses in total. Tumor growth was monitored until Day 28 from treatment initiation. Complete responder animals (n=7) were followed until day 62 after treatment initiation and re-challenged with an implantation of 2×106 MC38 tumor cells and tumor growth was monitored for 28 days. In parallel, naïve age-matched control huCD137 C57Bl/6 mice (n=5) were implanted with 2×106 MC38 tumor cells monitored for 28 days. The results of this experiment may be seen in
Biacore experiments were performed to determine ka (M−1s−1), kd (s−1), KD (nM) values of heterotandem peptides binding to human CD137 protein. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer's suggested protocol. The protein was desalted to remove uncoupled biotin using spin columns into PBS.
For analysis of peptide binding, a Biacore T200 or a Biacore 3000 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl of onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) and biotinylated CD137 captured to a level of 270-1500 RU. Buffer was changed to PBS/0.05% Tween 20 and a dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The top peptide concentration was 500 nM with 6 further 2-fold or 3-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/min with 60 seconds association and 900 seconds dissociation. After each cycle a regeneration step (10 μl of 10 mM glycine pH 2) was employed. Data were corrected for DMSO excluded volume effects as needed. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate.
Biacore experiments were performed to determine ka (M−1s−1), kd (s−1), KD (nM) values of BCY13272 binding to human EphA2 protein.
EphA2 were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 with a 3× molar excess of biotin over protein. The degree of labelling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture into PBS. For analysis of peptide binding, a Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N (1:1). Buffer was changed to PBS/0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500-1500 RU using a dilution of protein to 0.2 μM in buffer. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5% with a top peptide concentration was 50 or 100 nM and 6 further 2-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/min with 60 seconds association and 900-1200 seconds dissociation. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate.
The binding of BCY11863 to its primary target Nectin-4 and CD137 was characterized using surface plasmon resonance (SPR).
BCY11863 binds to cyno, rat, mouse and human Nectin-4 with KD between 5-27 nM as measured by direct binding to the extracellular domain that has been biotinylated and captured on a streptavidin sensor chip surface.
To understand whether the binding of BCY11863 to Nectin-4 was altered in the context of the ternary complex, i.e. when also bound to CD137, a multicomponent SPR binding assay was developed. BCY11863 was first captured to human CD137 immobilized on the SPR chip surface and then Nectin-4 from different species were passed over the chip to determine their affinities to the captured BCY11863 (see
Direct binding of BCY11863 to surface bound CD137 cannot be measured accurately by SPR because of avidity resulting from two CD137 binding bicycles in BCY11863 which leads to extremely slow koff (See
To understand whether the binding of BCY11863 to CD137 was altered in the context of the ternary complex, i.e. when also bound to Nectin-4, a dual binding SPR binding assay was developed. BCY11863 was first captured to human Nectin-4 immobilized on the SPR chip surface and then soluble CD137 from different species were passed over the chip to determine their affinities to the captured BCY11863 (see
Nectin-4 Paralogue screening: Binding of BCY11863 was assessed using SPR against Nectin-1 (2880-N1, R&D Systems), Nectin-2 (2229-N2, R&D Systems), Nectin-3 (3064-N3, R&D Systems), Nectin-like-1 (3678-S4-050, R&D Systems), Nectin-like-2 (3519-S4-050, R&D Systems), Nectin-like-3 (4290-S4-050, R&D Systems), Nectin-like-4 (4164-S4, R&D Systems) and Nectin-like-5 (2530-CD-050, R&D Systems) by labelling them with biotin and immobilizing them on a streptavidin surface. BCY11863 did not show any binding to these targets up to a concentration of 5000 nM.
CD137 Paralogue screening: Binding of streptavidin captured BCY13582 (biotinylated-BCY11863) was assessed using SPR against soluble TNF family receptors OX40 and CD40. BCY13582 did not bind to these targets up to a concentration of 100 nM.
Retrogenix microarray screening: Retrogenix's cell microarray technology was used to screen for specific off-target binding interactions of a biotinylated BCY11863 known as BCY13582.
Investigation of the levels of binding of the test peptide to fixed, untransfected HEK293 cells, and to cells over-expressing Nectin-4 and CD137 (TNFRSF9), showed 1 μM of the test peptide to be a suitable screening concentration. Under these conditions, the test peptide was screened for binding against human HEK293 cells, individually expressing 5484 full-length human plasma membrane proteins and secreted proteins. This revealed 9 primary hits, including Nectin-4 and CD137.
Each primary hit was re-expressed, along with two control receptors (TGFBR2 and EGFR), and re-tested with 1 μM BCY13582 test peptide, 1 μM BCY13582 test peptide in the presence of 100 μM BCY11863, and other positive and negative control treatments (
No specific off-target interactions were identified for BCY13582, indicating high specificity for its primary targets.
20. Anti-Tumor Activity of BCY11863 in a Syngeneic Nectin-4 Overexpressing MC38 Tumor Model (MC38 #13) on Dosing on Twice a Week at 5 mg/kg at 0, 24 h and 10 mg/kg at 0 h
6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 #13 (MC38 cells engineered to overexpress murine Nectin-4) cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 95 mm3 and were treated with a weekly dose of vehicle (25 mM histidine, 10% sucrose, pH7) or 10 mg/kg BCY11863 with two different dosing schedules for two dosing cycles (5 mg/kg BCY11863 at 0 h and 24 h on DO and D7, or 10 mg/kg at 0 h on DO and D7). All treatments were administered intravenously (IV). Tumor growth was monitored until Day 15 from treatment initiation.
BCY11863 leads to significant anti-tumor activity with both dosing schedules, but the dose schedule with 5 mg/kg dosing at 0 h and 24 h was superior to 10 mg/kg dosing at 0 h when complete responses were analyzed on day 15 after treatment initiation (
At 3 weekly doses of 3, 10 and 30 mg/kg with dose fractionated weekly, biweekly and daily 6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 #13 (MC38 cells engineered to overexpress murine Nectin-4) cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 107 mm3 and were treated with 21 daily doses of vehicle (25 mM histidine, 10% sucrose, pH7). BCY11863 treatment was done at three different total dose levels (3, 10 and 30 mg/kg total weekly dose) fractionated in three different schedules (QD: daily; BIW: twice a week or QW: weekly). Different BCY11863 treatment cohorts received either 21 daily doses (0.43, 1.4 or 4.3 mg/kg), 6 twice weekly doses (1.5, 5 or 15 mg/kg) or 3 weekly doses (3, 10 or 30 mg/kg). All treatments were administered intravenously (IV). Tumor growth was monitored until tumor reached volumes over 2000 mm3 or until 31 days after treatment initiation. Complete responders (animals with no palpable tumors) were followed until D52.
BCY11863 leads to significant anti-tumor activity with many of the dosing schedules the BIW dosing schedule being the most efficacious schedule, the 5 mg/kg BIW dose in particular. This is demonstrated by the number of complete responder animals on day 52. On day 52 after treatment initiation, 15/18 mice treated BIW with BCY11863 were complete responders, 12/18 mice treated QD with BCY11863 were complete responders and 6/18 mice treated QW with BCY11863 were complete responders. 5 mg/kg BIW dosing lead to 100% complete response rate with 6/6 CRs (
6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 76 mm3 and were treated with daily doses of vehicle (25 mM histidine, 10% sucrose, pH7). BCY12491 treatment was conducted at two different dose levels (5 and 15 mg/kg) and two different dosing schedules (QD: daily; Q3D: every three days). Animals received either 22 QD doses or 8 Q3D doses intraperitoneally (ip). Tumor growth was monitored until tumor reached volumes over 2000 mm3 or until 73 days after treatment initiation. After Day 73, 5 complete responder animals were re-challenged with MC38 tumor cell implantation alongside with 5 naïve C57BL/6J-hCD137 mice. Tumor growth was monitored for 20 days.
BCY12491 led to significant anti-tumor activity with all the doses and dose schedules used in the study. By day 41 after treatment initiation, 2 out of 6 BCY12491 5 mg/kg Q3D treated animals had become complete responders (CRs; no palpable tumor left), 3 out of 6 BCY12491 5 mg/kg QD treated animals became CRs, 4 out of 6 BCY12491 15 mg/kg Q3D treated animals became CRs and all (6/6) BCY12491 15 mg/kg QD treated animals became CRs. These data together with the BCY12491 mouse plasma PK-data indicate that continuous BCY12491 plasma exposure is not needed for maximal anti-tumor response to BCY12491 in the MC38 tumor model. Furthermore, complete responder animals rejected the re-challenge with MC38 tumor cell implantation and did not show any tumor growth whereas naïve mice implanted simultaneously with the same tumor cells established tumor growth at 100% take rate by day 22 after implantation of tumor cells. This indicates development of immunogenic memory upon BCY12491-treatment leading to complete tumor response (
Dependency of BCY12491 activity of different immune cell populations was determined in treating MC38 tumor bearing C57BL/6J-hCD137 mice that had been depleted of CD8+ T cells or NK 1.1+NK cells with BCY12491. 6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 #13 cells (clone of MC38 that has been engineered to overexpress Nectin-4). Three days after cell implantation mice received an ip injection of vehicle (PBS), 100 μg of depleting anti-CD8 (Rat IgG2b, clone 2.42) or anti-NK (Mouse IgG2a, clone PK136) antibodies (or their combination) or the corresponding isotype control antibodies (Rat IgG2b isotype control or Mouse IgG2a isotype control). Mice received additional doses of depletion antibodies (or isotype controls) 5 and 10 days after the first dose of antibodies. Cell depletion was verified by flow cytometry 4 and 12 days after the first dose of depletion antibody. When tumor volumes reached around 111 mm3 (5 days after the first dose of depletion antibodies), mice started receiving vehicle or BCY12491 intravenously (iv) at 15 mg/kg twice weekly (BIW). Mice received a total of 4 doses of BCY12491. Tumor growth was monitored until Day 28 or until tumor volume exceeded 2000 mm3.
BCY12491 treatment led to significantly decreased tumor growth rate and increased survival in MC38 #13 tumor bearing mice that had been treated with vehicle or isotype control antibodies. The benefit of BCY12491 treatment on decreasing tumor growth rate and survival was lost in CD8-depleted mice. Depletion of NK1.1+ cells did not affect the anti-tumor activity of BCY12491 treatment and subsequent survival benefit. This data demonstrates that the activity of BCY12491 in MC38 #13 tumor model is dependent on CD8+ T cells, but not on NK1.1+NK cells (
Anti-tumor activity of BCY12730 and BCY12723 was demonstrated alongside with BCY12491 activity. 6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 92 mm3 and were treated intravenously with Q3D doses of vehicle (25 mM histidine, 10% sucrose, pH7), 15 mg/kg of BCY12730, BCY12723 or BCY12491 (7 Q3D doses). Tumor growth was monitored for 28 days or until tumors exceeded 2000 mm3. BCY12491, BCY12730 and BCY12723 demonstrated significant anti-tumor activity leading to complete responses in 4 out of 6 BCY12491 treated animals, 3 out of 6 BCY12730 treated animals and 2 out of 6 BCY12723 treated animals (
Anti-tumor activity of BCY13048 and BCY13050 was demonstrated alongside with BCY12491 activity. 6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×106 MC38 cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 76 mm3 and were treated intravenously with twice weekly (BIW) doses of vehicle (25 mM histidine, 10% sucrose, pH7), 5 mg/kg of BCY13048, BCY13050, or BCY12491 (6 BIW doses). Tumor growth was monitored for 28 days or until tumors exceeded 2000 mm3. BCY12491, BCY13048 and BCY13050 demonstrated significant anti-tumor activity leading to complete responses in 2 out of 6 BCY12491 treated animals, 5 out of 6 BCY13048 treated animals and 3 out of 6 BCY13050 treated animals (
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2020/051831 | 7/30/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63024715 | May 2020 | US | |
| 63022667 | May 2020 | US | |
| 62931442 | Nov 2019 | US | |
| 62910088 | Oct 2019 | US | |
| 62880191 | Jul 2019 | US |
