WO2009077857A2 - Trail variants for treating cancer - Google Patents
Trail variants for treating cancer Download PDFInfo
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- WO2009077857A2 WO2009077857A2 PCT/IB2008/003720 IB2008003720W WO2009077857A2 WO 2009077857 A2 WO2009077857 A2 WO 2009077857A2 IB 2008003720 W IB2008003720 W IB 2008003720W WO 2009077857 A2 WO2009077857 A2 WO 2009077857A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Definitions
- the present invention relates to the use of a mutant TRAIL protein to treat various cancers in mammals.
- Cytokines are a family of growth factors, secreted primarily from leukocytes, and are messenger proteins that act as potent regulators, capable of controlling a wide range of cellular functions, especially the immune response and cell growth 1 . These roles include immune response regulation 2 , inflammation 3 , wound healing 4 , embryogenesis and development, and apoptosis 5 .
- TNF Tumor Necrosis Factor ligand
- TRAIL tumor necrosis factor-related apoptosis inducing ligand
- TRAIL can interact with several different receptors on the cell. Two of those, DR4 (TRAIL-Rl) and DR5 (TRAIL-R2) can induce apoptosis in the cell, while the others DcRl (TRAIL-R3) and DcR2 (TRAIL-R4), do not contain a death domain or contain a truncated death domain, respectively, and are not able to induce apoptosis.
- TRAIL in its soluble form, selectively induces apoptosis in tumor cells in vitro and in vivo. Unlike other apoptosis inducing TNF family members, TRAIL appears to be inactive against no ⁇ nal healthy tissue, therefore attracting great interest as a potential cancer therapeutic 6 . Therefore TRAIL has the potential to serve as a safe and potent therapeutic agent against tumor cells. A number of in vitro studies have shown that many tumor cell lines of divergent origins are sensitive to TRAIL induced apoptosis.
- a variant TRAIL protein which has superior selectivity for the death receptor 5, for treating a mammal diagnosed with cancer.
- TRAIL also known as TNFSFlO, TL2; APO2L; CD253; Apo-2L
- TNFSFlO also known as TNFSFlO, TL2; APO2L; CD253; Apo-2L
- TL2 TNFSFlO
- APO2L APO2L
- CD253 Apo-2L
- TRAIL is a member of the TNF ligand family 8 9 and an example of a cytokine that binds to more than one receptor, including decoy receptors, which lack or have truncated intracellular domains.
- TRAIL is a promiscuous ligand as it binds to five cognate receptors of the TNF receptor family; to the death receptor 4 (DR4, TRAIL-Rl), death receptor 5 (DR5, TRAIL-R2, KILLLER, TRICK-2) and to the decoy receptor 1, (DcRl, TRAIL-R3, TRIDD), decoy receptor 2 (DcR2, TRAIL-R4, TRUNDD) and to the soluble secreted receptor osteoprotegerin (OPG).
- DR4 death receptor 4
- DR5 death receptor 5
- DcRl, TRAIL-R3, TRIDD decoy receptor 2
- OPG soluble secreted receptor osteoprotegerin
- DR4 TRAIL-Rl
- DR5 TRAIL- R2
- DD functional death domain
- DR5 selective inducers of DR5 (TRAIL-R2) signalling
- TRAIL-R2 selective inducers of DR5
- the signalling pathway could induce the proliferative or the apoptotic pathway.
- binding of TRAIL to the decoy receptors can have an inhibitory effect on the efficiency of TRAIL to induce apoptosis.
- the inventors are therefore of the view that the use of DR5-receptor selective TRAIL variants could permit better tumor-specific therapies through escape from the decoy receptor- mediated antagonism, resulting in a higher efficacy with possibly less side effects as compared to wtTRAIL.
- receptors DR5 and/or DR4 may be up-regulated after treatment with DNA damaging chemotherapeutic drugs such as Bortezomib, 5-Flurouracil, or aspirin or irradiation. In such cells, the response to TRAIL-induced apoptosis may be significantly increased. It has been suggested in the literature, that this signalling pathway is primarily mediated through DR5. Decoy receptors can also be induced by such treatments indicating that selective targeting of DR5 would achieve more efficient tumor cell killing.
- Gynaecological cancers include but are not limited to cancers of the ovaries, cervix, uterus and vagina. Further gynaecological cancers will be known to those of skill in the art.
- the TRAIL variants of the present invention may also be useful to treat hormone dependent cancers which include but are not limited to cancers of the thyroid, prostate and breast. Other hormone-dependent cancers are known to those of skill in the art. Since the growth of such cancers is stimulated by particular hormones, it is possible to combine the treatment with the TRAIL variant, or the pharmaceutical composition comprising a TRAIL variant, with hormone therapies.
- the TRAIL variant, or the pharmaceutical composition comprising a TRAIL variant is used to treat ovarian cancer.
- the inventors have discovered that the use of the TRAIL variants, of the present invention, results in increased apoptosis of the ovarian cancer cells in vitro and in vivo, when compared to wtTRAIL.
- ovarian cancer Different types of ovarian cancer are known. There are several types of ovarian malignancies, each with its own histopathologic appearance and biologic behaviour.
- the World Health Organization (WHO) classifies ovarian tumors based on their cell type: epithelial, germ cell, and stromal whereby epithelial tumors are by far the most common type.
- WHO World Health Organization
- primary ovarian epithelial tumors are thereby particularly suitable for treatment with the TRAIL variants of the present invention since they express high levels of the DR5 receptor.
- chemotherapy and treatment with aspirin respectively can further upregulate DR5 receptor expression in these cells.
- the inventors have discovered that the D269HE195R TRAIL variant increases apoptosis by at least 2 fold in the ovarian cancer cell line A2780.
- the inventors have also discovered that the D269HE195R and D269H TRAIL variants increase apoptosis by 2.7-4.2 fold in the colon adenocarcinoma cell line Colo205.
- studies on an IP xenograft ovarian cancer mouse model have revealed that variant TRAIL protein (D269HE195R) is more effective than wtTRAIL in inducing apoptosis in vivo.
- the cancerous cells can be detected by luminescence and the treatment with the mutant TRAIL protein was found to result in higher mean signal reduction (68.3%) compared to wtTRAIL (48.8%) which indicates that the TRAIL variant demonstrated greater efficiency in decreasing the size of the ovarian tumor.
- increase apoptosis and “enhanced apoptosis induction” it is meant that the TRAIL variants of the present invention increase the number of cell that undergo apoptosis, when compared to a sample which was treated with wtTRAIL or other suitable controls, which will be evident to those skilled in the art.
- the progression of cancer is monitored by a staging process. This indicates how well developed the cancer is and if it has spread.
- Ovarian cancer is thereby usually classified according to the system established by the International Federation of Gynecology and Obstetrics (FIGO). While the example below shows the FIGO system for ovarian cancer, the classification of the various cancer stages is very similar for other gynaecological malignancies. The score runs from one to four, with the prognosis becoming progressively worse at each stage.
- FIGO International Federation of Gynecology and Obstetrics
- Stage I - malignant cells limited to one or both ovaries
- IA - involves one ovary; capsule intact; no tumor on ovarian surface; no malignant cells in ascites or peritoneal washings
- IB - involves both ovaries; capsule intact; no tumor on ovarian surface; negative washings
- IC - tumor limited to ovaries with any of the following: capsule ruptured, tumor on ovarian surface, positive washings
- Stage II - pelvic extension or implants HA - extension or implants onto uterus or fallopian tube; negative washings HB - extension or implants onto other pelvic structures; negative washings IIC - pelvic extension or implants with positive peritoneal washings
- Stage III microscopic peritoneal implants outside of the pelvis; or limited to the pelvis with extension to the small bowel or omentum IHA - microscopic peritoneal metastases beyond pelvis IHB - macroscopic peritoneal metastases beyond pelvis less than 2 cm in size HIC - peritoneal metastases beyond pelvis > 2 cm or lymph node metastases Stage IV - distant metastases
- Metastatic tumors may be derived from a primary tumor which is an ovarian tumor.
- the TRAIL variant, or the pharmaceutical composition comprising the TRAIL variant are used to treat cancers of the gastrointestinal system. These include cancers of the esophagus, stomach, liver, biliary system, pancreas, bowels, and rectum.
- the inventors have surprisingly discovered that the use of the DR5-selective TRAIL variant of the invention results in higher apoptosis in colon cancer cells, compared to cells which were treated with wtTRAIL.
- gastrointestinal cancers are not a single disease and can be classified according to the cell type which include, but are not limited to: adenocarcinoma, leiomyosarcoma, lymphoma and neuroendocrine tumors, with adenocarcinomas of the colon being the most common type.
- HNPCC heriditary non-polyposis colon cancer
- FAP familial adenomatous polyposis
- TRAIL variants of the present invention since they express high levels of the DR5 receptor.
- Another embodiment of the invention is the use of a TRAIL variant as primary prevention in a population of people at risk of hereditary colon cancers.
- the progression of colon cancers can also be monitored by a staging process.
- TNM [primary] tumor, [regional lymph] node, [remote] metastasis) staging is usually used.
- T Stage Primary Tumor
- M Remote Metastasis
- N Node
- Tumor may invade submucosa
- metastatic tumors are derived from a primary tumor, which is a colon tumor.
- Results obtained with Colo205, LoVo, SW948, ML-I, HeLa, Caski, SiHa, A2780, and BJAB cell lines and discussed herein show that the biological activity of the tested D269H, D269HE195R, and D269HT214R TRAIL variants is specifically directed towards the DR5 receptor and that these TRAIL variants have a high efficiency in inducing apoptosis in colon, ovarian and cervical cancer cell lines.
- a TRAIL variant according to the invention preferably exhibits superior selectivity for the death receptor 5 (TRAIL-R2) over the decoy receptors DcRl (TRAIL-R3) and DcR2 (TRAIL-R4).
- DcRl and DcR2 do not contain a death domain or contain a truncated death domain, respectively. Binding of TRAIL to these receptors does not induce apoptosis; on the contrary, it may actually prevent apoptosis by sequestering available TRAIL from DR4 and DR5, or by leading to NF- ⁇ B activation via DcR2. For this reason, it is preferred that the TRAIL variants of the invention are not sequestered via this route.
- the inventors have shown that the increase in association rate constant (k on ) of D269HE195R for binding to the DR5 receptor was less than 5 fold improved when compared to the k on of wtTRAIL but that kinetics of apoptosis induction in Colo205 cells surprisingly showed a 17-fold increase.
- wtTRAIL required 60.8 ⁇ 4.3 min of incubation whilst the time to reach the same value for D269HE195R was only 3.6 ⁇ 0.4 min.
- TNF- ⁇ , TNF- ⁇ , FASL, RANKL, APRIL, BAFF, Light, TLlA TNF- ⁇ , TNF- ⁇ , FASL, RANKL, APRIL, BAFF, Light, TLlA
- a variant that has to activate or block a particular receptor can be made more efficient by reducing binding to its other receptors in addition to improving binding to its target receptor.
- Such variants may be obtained by rational design, as demonstrated by the inventors in case of e.g. D269HE195R by employing a computational design method, or for example by using directed evolution methods and/or high throughput screening techniques.
- a method of enhancing therapeutic efficacy of a protein by enhancing the kinetics of activation (or blocking) of a particular target receptor by changing its receptor binding specificity.
- kinetics of activation (or blocking) of the protein is enhanced by increasing the binding and binding rate of the protein to the preferred target receptor. More preferably the kinetics of activation (or blocking) of the target receptor is increased by reducing the binding of the protein to one or more off-target receptors.
- increasing the binding and binding rate of the protein to the preferred target receptor is combined with reducing the binding of the protein to one or more of its off-target receptors.
- proteins with improved kinetic properties are produced by directed evolution or high throughput screening.
- the method of improving kinetic properties involves a combination of directed evolution and high throughput screening.
- the method of enhancing therapeutic efficacy uses a rational design approach, and in some embodiments this approach may include computational protein design. More preferably, rational design methods may be combined with directed evolution and/or high throughput screening methods.
- the protein which shows enhanced therapeutic efficacy is a cytokine. More preferably the protein is a promiscuous cytokine which binds to more than one receptor. More preferably the protein is a member of the TNF ligand family.
- the TNF ligand is a promiscuous TNF ligand, including but not limited to TNF- ⁇ , TNF- ⁇ , FASL, TRAIL, RANKL, APRIL, BAFF, Light or TLlA.
- the protein is TRAIL.
- the variant TRAIL molecules of the present invention are of great utility in inducing apoptosis in cells.
- induces apoptosis is meant that a compound according to the present invention acts to cause cell death in target cells.
- Apoptosis may be induced in vivo, ex vivo or in vitro.
- apoptosis is induced in cancerous cells, and not in healthy cells. •
- TRAIL variants of the invention do not affect the viability of healthy, non- transformed cells. Apoptosis can be measured by a number of different assays, as will be clear to those of skill in the art.
- Examples include DNA laddering assays (see, for example, EP0835305; Immunex); detection of chromatin fragmentation and condensation with Hoechst33342, staining and detection of phosphatidylserine exposure in combination with membrane permeabilisation measured by staining with Annexin V and propidium iodide. What these assays share in common is a measurement of biochemical or morphological changes occurring in dying cells upon sustained contact with a concentration of the compound whose activity is being measured. Cell death can be expressed as an increase of the percentage of dying cells in response to exposure to the compound (i.e. the percentage of dying cells in untreated, control cell population is subtracted from that of in cell populations exposed to the drug).
- Effective concentrations are typically calculated in the form of IC50 values, which is the concentration of compound at which 50% of the cells undergo apoptosis.
- IC50 values which is the concentration of compound at which 50% of the cells undergo apoptosis.
- a compound according to the invention induces apoptosis in 50% of cells at a concentration of between 1 ng/ml and 1,000 ng/ml, more preferably between 1 ng/ml and 100 ng/ml, more preferably between 1 ng/ml and 10 ng/ml, and more preferably 44 ng/ml.
- useful compounds possess IC 50 values of between 1 ng/ml and 1,000 ng/ml, more preferably between 1 ng/ml and 100 ng/ml, more preferably between 1 ng/ml and 10 ng/ml and more preferably 4 ng/ml.
- apoptosis can also be measured by caspase activation and other assays known to those of skill in the art.
- variant TRAIL protein is meant that the TRAIL protein differs in at least one amino acid position from the wtTRAIL protein (also known as TNFSFlO, TL2; APO2L; CD253; Apo-2L), Entrez GenelD: 8743; accession number NM_003810.2; UniProtKB/Swiss-Prot: P50591; UniProtKB/TrEMBL: Q6IBA9.
- selectivity is meant that the variants of the invention have substantially greater affinity for DR5 than their affinity for the decoy receptors DcRl (TRAIL-R3) and DcR2 (TRAIL-R4) in relative terms, and preferably also for DR4.
- substantially greater affinity we mean that there is a measurably higher affinity of the TRAIL variant for DR5 as compared with its affinity for DR4, DcRl and DcR2.
- the affinity is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1, 000-fold or greater for DR5 than for one or more of DR4, DcRl and DcR2. More preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1, 000- fold or greater for DR5 than for at least two, preferably all of DR4, DcRl and DcR2.
- the binding affinity of the TRAIL variants of the invention for DR5 is also higher in absolute terms than the binding affinity of the wtTRAIL molecule for DR5.
- the binding affinity of the TRAIL variants of the invention for DR4 should be lower than the binding affinity of the wtTRAIL molecule for DR4. The same is preferably true of the binding affinity demonstrated for DcRl and DcR2.
- DR5 receptor activation by D269HE195R occurs at least 2 fold faster than with wtTRAIL, preferably at least 5 fold faster than with wtTRAIL, more preferably at least 10 fold faster than with wtTRAIL, more preferably at least 15 fold faster than with wtTRAIL, more preferably at least 17 fold faster than with wtTRAIL, and more preferably at least 20 fold faster than with wtTRAIL.
- induction of apoptosis by D269HE195R occurs at least 2 fold faster than with wtTRAIL, preferably at least 5 fold faster than with wtTRAIL, more preferably at least 10 fold faster than with wtTRAIL, more preferably at least 15 fold faster than with wtTRAIL, more preferably at least 17 fold faster than with wtTRAIL, and more preferably at least 20 fold faster than with wtTRAIL.
- the TRAIL variant of the present invention contains the mutation D269H. All references to amino acid positions in the TRAIL protein sequence presented herein, and to specific TRAIL mutants are intended to refer to the amino acid sequence given in SEQ ID NO: 1. This mutant has a highly reduced binding affinity to DR4 and an increased affinity for the DR5 receptor. Further details of the binding properties of this mutant can be found in co-pending international patent application WO05/056596.
- This mutant further has increased ability to induce apoptosis in various cancer cell lines. For example, it has been shown to induce apoptosis in the colon carcinoma cell Colo205 with around 4 times more efficiency compared to wtTRAIL. Furthermore the inventors have discovered that the mutant also has 3-4 times higher efficiency in inducing apoptosis in ovarian cancer cells. It is also preferred that the D269H TRAIL variant of the present invention further contains the mutations E195R and/or T214R. These mutants show superior selectivity for DR5 and decreased binding to DR4, when compared to wtTRAIL. The binding properties of these mutants have already been discussed in WO05/056596. In one preferred embodiment of the invention the mutant is D269HT214R.
- This mutant has been shown to increase the apoptosis rate in the colon cancer cell line Colo205 by 1.5 fold compared to wtTRAIL at a lower concentration (see Figure 8C). Furthermore, it also shows 2 times improved efficiency in inducing apoptosis in the ovarian cancer cell line A2780.
- the mutant is D269HE195R. The inventors have discovered that this mutant increases apoptosis by 3-4 fold in the ovarian cancer cell line A2780. Furthermore, studies on an IP xenograft ovarian cancer mouse model have surprisingly revealed that this mutant TRAIL protein is more effective than wtTRAIL in inducing apoptosis in vivo.
- the cancerous cells can be detected by luminescence and the treatment with the mutant TRAIL protein resulted in higher mean signal reduction (68.3%) compared to wtTRAIL (48.8%) which indicates that the TRAIL variant demonstrated greater efficiency in decreasing the size of the ovarian tumor. Furthermore, the mutant was able to increase the level of apoptosis in the colon cancer cell line Colo205 by 2.7-4.2 fold, and an enhanced ability to induce apoptosis in colon carcinoma cell lines LoVo and SW948 and cervical carcinoma cell lines HeLa, Caski and SiHa.
- the above mutations may be introduced into the full length TRAIL sequence.
- the above mutations are introduced into soluble forms of the TRAIL sequence, such as forms comprising amino acids 114-281 or comprising amino acids 95-281 ; other examples will be clear to those of skill in the art.
- Preferred TRAIL variants according to.the invention are thus variants of the soluble fragments of the full length TRAIL sequence given in SEQ ID NO: 1.
- a preferred soluble fragment template comprises amino acids 1 14-281 (herein termed TRAIL) and all mutants described herein are of this length.
- TRAIL amino acids 1 14-281
- the wtTRAIL sequence of TRAIL (114-281) preceded by a methionine is presented in SEQ ID NO: 3 and a preferred coding sequence is presented in SEQ ID NO:4; variants of the invention may thus be derived from this sequence.
- variations in these soluble templates will very •likely retain the properties of this soluble form and show biological activity if additional residues C terminal and/or N terminal of these boundaries in the polypeptide sequence are included.
- an additional 1, 2, 3, 4, 5, 10, 20 or even 30 or more amino acid residues from the wild- type TRAIL sequence, or from a homologous sequence may be included at either or both the C terminal and/or N terminal of these boundaries, without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit biological activity.
- truncated variants of this template in which one or a few amino acid residues may be deleted at either or both the C terminus or the N terminus without prejudicing biological activity.
- the invention thus provides DR5-specific TRAIL variants for use as a medicament.
- the invention also provides a method for treating a subject suffering from or at risk of contracting a disease, comprising administering to the subject a pharmaceutical composition of the invention.
- the invention also provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for treating a subject.
- diseases include cancer diseases, such as leukaemia, lymphoma, melanoma, prostate, pancreatic, bladder, kidney, head and neck, liver and breast cancer, cancers of the lung, ovaries, cervix, colon, and multiple myeloma.
- the cancer is ovarian cancer or colon cancer.
- the cancer may be cervical carcinoma. These are among the most common cancers in the world and are also a leading cause of cancer-related deaths. Both ovarian and colon cancer are usually only discovered at late stages, due to the absence of obvious early warning signs. Furthermore, it is often observed that the initial response to treatment is good in advanced stage patients, but the overall survival rate is low due to the occurrence of drug resistance. It is therefore of paramount importance to improve the treatment of these diseases.
- the TRAIL variant which is administered to the mammal diagnosed with cancer, should be present at a therapeutically effective amount e.g. an amount sufficient to induce apoptosis.
- a therapeutically effective amount e.g. an amount sufficient to induce apoptosis.
- the precise effective amount for a given patient will depend upon their size and health, the nature and extent of the disease, and the composition or combination of compositions selected for administration. The effective amount can be determined by routine experimentation and is within the judgement of the clinician.
- an effective dose will generally be from about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 50mg/kg or about 0.05 mg/kg to about 10 mg/kg, preferably about 10 mg/kg.
- a suitable dose should be used so as to achieve a serum concentration in the patient of between 0.1 and 1,000 ng/ml, preferably between 1 ng/ml and around 100 ng/ml, more preferably around 10-100 ng/ml.
- TRAIL variants may be administered in the form of salts and/or esters.
- a TRAIL variant of the first aspect of the invention may form part of a fusion protein.
- the mature TRAIL variant may be fused with another compound, such as a compound to increase the half-life of the TRAIL variant (for example, polyethylene glycol).
- heterologous proteins can be obtained by cloning a polynucleotide encoding a TRAIL variant in frame to the coding sequences for a heterologous protein sequence.
- heterologous when used herein, is intended to designate any polypeptide other than a TRAIL variant according to the invention.
- heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
- heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them 11 .
- additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called “histidine tag” 12 or by the "HA” tag, an epitope derived from the influenza hemagglutinin protein 13 .
- the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
- a proteolytic cleavage for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
- the TRAIL variant may be purified by means of a hexa-histidine peptide fused at the N-terminus or C- terminus.
- the hexa-histidine peptide fused to TRAIL may be eliminated by proteolytic cleavage, as it is know that such a hexa-histidine peptide fusion renders TRAIL more toxic towards noncancerous cells.
- the fusion protein comprises an immunoglobulin region
- the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
- Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-GIy-Gly-Gln-Phe-Met introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
- the resulting fusion protein has improved properties, such as an extended residence time in body fluids (i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
- the TRAIL variant of the invention may also be fused with a marker protein, for example a fluorescent protein, like green fluorescent protein (GFP), yellow fluorescent protein (YFP) and similar proteins. Such a protein is particularly advantageous for diagnostic purposes.
- the protein is fused to the constant region of an Ig molecule.
- Ig molecules include heavy chain regions, like the CH2 and CH3 domains of human IgGl .
- Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
- the TRAIL variant may comprise at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues.
- the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
- nucleic acid molecules that encode the TRAIL variants of the invention.
- the coding sequence for wild type TRAIL is given in accession number NM_003810.
- Nucleic acid molecules coding for TRAIL variants according to the invention may be derived from this sequence by supplementing the appropriate coding sequence at the mutation point(s).
- Examples of preferred nucleic acid molecules according to the invention are variants of the sequences presented in SEQ ID NO: 2 (full length gene); or nucleotides 88-933 (846 nucleotides long) of SEQ ID NO:2 which is the coding sequence.
- a preferred coding sequence for wild type TRAIL (amino acids 114-281) is presented in SEQ ID NO:4 (TRAIL 114-281 preceded by methionine) and so the variants of the present invention are preferably encoded by variants of this sequence.
- the nucleic acid may be DNA or RNA (or hybrids thereof), or their analogues, such as those containing modified backbones (e.g. phosphorothioates) or peptide nucleic acids (PNA). It may be single stranded (e.g.
- nucleic acids can, of course, be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by nuclease digestion of longer molecules, by ligation of shorter molecules, from genomic or cDNA libraries, by use of polymerases etc. Accordingly, the present invention also provides vectors (e.g. plasmids) comprising nucleic acids of the invention (e.g. expression vectors and cloning vectors) and host cells (prokaryotic or eukaryotic) transformed with such vectors.
- vectors e.g. plasmids
- nucleic acids of the invention e.g. expression vectors and cloning vectors
- host cells prokaryotic or eukaryotic
- the invention also provides a process for producing a TRAIL variant of the invention, comprising the step of culturing a host cell transformed with nucleic acid of the invention under conditions that induce expression of the variant.
- Suitable expression systems for use in the present invention are well known to those of skill in the art and many are described in detail in Sambrook (1989) 14 and Fernandez et al (1998) 15 .
- any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
- the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook 14 .
- the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired peptide is transcribed into RNA in the transformed host cell.
- a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired peptide is transcribed into RNA in the transformed host cell.
- suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
- Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
- Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
- Cell-free translation systems can also be employed to produce the peptides of the invention.
- cell lines that stably express the peptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
- Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
- ATCC American Type Culture Collection
- CHO Chinese hamster ovary
- BHK baby hamster kidney
- COS monkey kidney
- C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
- the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit).
- suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
- plant cell culture and whole plant genetic expression systems include those described in US Patent 5,693,506; US Patent 5,659,122; US Patent 5,608,143 and Zenk (1991) 17 .
- all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
- Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
- prokaryotic expression systems include those that use streptococci, staphylococci, E. coli, Streptomyces and Bacillus snbtilis as host cells.
- fungal expression systems include those that use yeast (for example, S. cerevisiae) and Aspergillus as host cells.
- the TRAIL variant may be used to remove cancerous cells from a patient's body fluids.
- the TRAIL variant is used to contact a patient's blood ex vivo and thereby remove cancerous cells from the blood, since they bind to the DR5-specific TRAIL variant with higher affinity than non-cancerous cells.
- the blood can then be reintroduced into the patient. It is also possible to aspirate bone marrow from a patient and contact the bone marrow with the DR5-specific TRAIL variant, whereby the cancerous cells which have a higher affinity for the TRAIL variant compared to non-cancerous cells, are removed from the bone marrow.
- the bone marrow can then be reintroduced into the patient.
- a body fluid is contacted ex vivo with the TRAIL variant of the invention it is preferred that the TRAIL variant is immobilized on a suitable matrix. Further uses will be apparent to the person skilled in the art.
- suitable cancers which can be treated with the TRAIL variant of the invention, include cells that express the DR5 receptor on the surface as measured by flow cytometry or by immunohisfochemistry (IHC) of primary tumor samples.
- IHC immunohisfochemistry
- cancer cells are readily identifiable by various means known to those of skill in the art which include, but are not limited to, immunocytochemistry with receptor specific antibodies, Fluorescent-activated cell sorting
- DR4 specific antibodies can be obtained, for example, from Abeam (ab8414).
- DR5 antibodies are available as well (Sigma-Aldrich, D3938).
- the expression of the DR5 receptor on the cell surface is higher than the expression of the DR4 receptor.
- the expression levels of the protein can thereby be assessed by various techniques, known to those of skill in the art, which include, but are not limited to, quantitative western blot analysis, FACS with fluorescently labelled DR5 and/or DR4 specific antibodies and others.
- “Higher expression” and “upregulation” mean that the protein expression is increased 1.5fold (more preferably, 2fold, 4fold, 8fold, l ⁇ fold, 32fold, lOOfold or even l,OOOfold) relative to another protein.
- lower expression means that the protein expression is reduced 1.5fold (more preferably, 2fold, 4fold, 8fold, l ⁇ fold, 32fold, lOOfold or even l,OOOfold) relative to another protein.
- the DR4 receptor is not expressed on the cell surface at any detectable level, whereas the DR5 receptor is expressed. "No expression” thereby means that the receptor is not detectable with the techniques discussed above and other suitable techniques, known to those of skill in the art.
- the DR5 receptor is expressed at similar levels as DcRl and/or DcR2. In a more preferred embodiment the DR5 receptor is expressed more highly compared to DcRl and/or DcR2.
- the cancerous cell expresses the decoy receptors DcRl and DcR2 more highly in response to a chemotherapeutic agent.
- a chemotherapeutic agent such cells are preferred because wtTRAIL shows reduced efficiency in inducing apoptosis in these cells due to binding to the decoy receptor and sequestering of wtTRAIL. Therefore, the DR5-specific TRAIL variant of the present invention would be particularly advantageous.
- the cancer cell upregulates the DR5 receptor expression in
- the cancer cell upregulates the DR5 receptor expression in response to aspirin.
- the inventors are of the opinion that receptors DR5 and/or DR4 may be up-regulated after treatment with DNA damaging chemotherapeutic drugs or aspirin. In such cells, the response to TRAlL-induced apoptosis may be significantly increased. It has been suggested in the literature, that this signalling pathway mainly goes through DR5.
- TRAIL variants of the invention may be co-administered with one or more other compounds, preferably antitumor compounds, more preferably those which are active against the cancerous cells targeted by the variants of the invention or those which increase responsiveness of the tumor to the TRAIL variants.
- compositions of the invention may thus include one or more antitumor agents, examples of which will be known to those of skill in the art and include gamma irradiation as well as chemotherapeutic drugs such as alkylating agents, anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin derivatives, taxanes, topoisomerase inhibitors, antitumor antibiotics, monoclonal antibodies, DNA damaging drugs, histone deacetylase inhibitors, hormones, proteasome inhibitors and so on.
- antitumor agents examples of which will be known to those of skill in the art and include gamma irradiation as well as chemotherapeutic drugs such as alkylating agents, anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin derivatives, taxanes, topoisomerase inhibitors, antitumor antibiotics, monoclonal antibodies, DNA damaging drugs, histone deacety
- the chemotherapeutic agent is an anti-angiogenesis antibody such as bevacizumab, a proteasome inhibitor such as Bortezomib or a DNA damaging drug such as 5-Flurouracil.
- the chemotherapeutic agent used acts to increase the surface expression of the DR5 (TRAIL-R2) receptor on the cancerous cells and/or enhance/potentiate apoptosis-induction through DR5.
- compositions of the invention may include aspirin.
- Antitumor compounds act in a variety of ways, and each disrupt the tumor at different targeted sites when used in combination. It is therefore likely that the TRAIL variants of the invention will work well in combination with agents that target multiple oncogenic pathways such as the multiple tyrosine kinase inhibitors, including but not limited to sorafenib and sunitnib, as well as other similar agents currently in development. Pre-clinical trials have shown enhanced effected of TRAIL when used in combination with such agents (data not shown).
- the chemotherapeutic agent increases the number of cells that undergo apoptosis in the presence of the chemotherapeutic agent when compared to a sample which was not exposed to the chemotherapeutic agent.
- the increase is 1.5fold (more preferably 2fold, 4fold, 8fold, lOfold, 20fold, lOOfold or even l,OOOfold).
- enhanced apoptosis induction it is meant that aspirin increases the number of cells that undergo apoptosis in the presence of aspirin when compared to a sample which was not exposed to the chemotherapeutic agent.
- the increase is 1.5fold (more preferably 2fold, 4fold, 8fold, 10fold, 20fold, lOOfold or even l,OOOfold).
- Such cancerous cells can be identified by various means known to those of skill in the art.
- a fluorescently labelled DR5 and/or DR4 antibody can be used to decorate the cell, which is to be tested.
- the expression of DR5 and DR4 can then be evaluated by fluorescent activated cell sorting (FACS) before and after exposure to a chemotherapeutic agent or aspirin.
- FACS fluorescent activated cell sorting
- Preferred cancer cells, for treatment with the DR5-s ⁇ ecific TRAIL variant of the invention are those which upregulate the DR5 receptor 1.5fold (more preferably, 2fold, 4fold, 8fold, l ⁇ fold, 32fold, lOOfold or even l,OOOfold) compared to the control.
- Other means to assess the upregulation of the DR5 receptor, in response to a chemotherapeutic agent are known to those of skill in the art.
- the chemotherapeutic agent is cisplatin (also known as cisplatinum or cis-diamminedichloroplatinum (II) (CDDP)).
- cisplatin also known as cisplatinum or cis-diamminedichloroplatinum (II) (CDDP)
- the inventors have shown that exposure of A2780 cells to cisplatin increased the expression of the DR5 and DcR2 receptor on the cell ( Figure IA). Furthermore, it is demonstrated herein that the administration of cisplatin, in combination with the TRAIL variants of the invention, results in higher apoptosis in cancerous cells in vitro ( Figures 2 and 3) and in vivo ( Figure 6) and increased survival rates in mouse xenograft models ( Figure 6C).
- an effective dose will generally be from about 0.1 ng/ml to about 1,000 ng/ml, or about 1 ng/ml to about 100 ng/ml or about 10 ng/ml to about 100 ng/ml, in the blood.
- mice suffering from colon carcinoma induced by inoculation with Colo205 cells show an increased reduction in tumor growth when treated wtTRAIL or D269HE 195R respectively ( Figure 25).
- the TRAIL variant may be used to screen for cancerous cells, which are likely to benefit from treatment with the TRAIL variant of the invention.
- the TRAIL variant can be exposed to the cells, which are to be tested. These cells can be either cell lines derived from a tumor or samples obtained after a tissue biopsy. Cells which are likely to benefit from treatment with the TRAIL variant of the invention are those to which the TRAIL variant can bind. This can be assessed, for example, by receptor binding assays.
- the recombinant TRAIL variant is either fluorescently labelled post- translationally or expressed as a fusion protein with a fluorescent protein, such as GFP etc.
- the fluorescent TRAIL variant bound to the cell can be visualised by fluorescent microscopy. It is also possible to use FACS to assess binding of TRAIL. In all cases the results have to be compared to a control, for example an identical experiment performed with iinlabelled TRAIL.
- the cell line which can be identified by the means described above upregulates the DR5 receptor in response to a chemotherapeutic agent or aspirin. Such an upregulation can be assessed by comparing the binding of the variant TRAIL protein before and after exposure to the chemotherapeutic agent or aspirin.
- Suitable cancers are those which show an increased apoptosis rate in response to the TRAIL variants of the present invention.
- primary tumor cells and cell lines which show increased apoptosis in the presence of the TRAIL variants of the present invention when compared to wtTRAIL or other suitable controls, that will be evident to those skilled in the art, are a preferred embodiment of the present invention.
- a further aspect of the invention may comprise a pharmaceutical composition comprising a mutant cytokine, nucleic acid or vector as described above, in conjunction with a pharmaceutically-acceptable carrier.
- the invention provides a pharmaceutical composition comprising (a) TRAIL variant(s), nucleic acid or vector as described above and (b) a pharmaceutical carrier.
- Component (a) is the active ingredient in the composition, and this is present at a therapeutically effective amount e.g. an amount sufficient to induce apoptosis.
- a therapeutically effective amount e.g. an amount sufficient to induce apoptosis.
- the precise effective amount for a given patient will depend upon their size and health, the nature and extent of the disease, and the composition or combination of compositions selected for administration. The effective amount can be determined by routine experimentation and is within the judgement of the clinician.
- a suitable dose should be used so as to achieve a serum concentration of 0.1 and 1,000 ng/ml, preferably between 1 ng/ml and around 100 ng/ml, more preferably around 10-lOOng/ml.
- TRAIL variants may be included in the composition in the form of salts and/or esters.
- Carrier (b) can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity.
- Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
- Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Liposomes are suitable carriers. A thorough discussion of pharmaceutical carriers is available in Gennaro 18 .
- compositions of the invention may be prepared in various forms.
- the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the composition may be lyophilised.
- the pharmaceutical composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.
- the invention also provides a delivery device containing a pha ⁇ naceutical composition of the invention.
- the device may be, for example, a syringe.
- a compound according to the invention is administered in conjunction with another agent, such as an antitumor agent.
- another agent such as an antitumor agent.
- Suitable examples of such agents for use in combination with the pharmaceutical composition of the present invention are known in the art and examples are listed above.
- compositions of the invention may on occasion be administered in conjunction with antibodies against one or more DR4, DcRl or DcRl. In such a manner it is possible further to block signalling through the DR4 specific pathway or binding of the TRAIL variant to decoy receptors. This is advantageous since any residual binding activity to receptors other than DR5 is inhibited and specificity of the mutant cytokine of the invention is enhanced even further.
- Compositions of the invention will generally be administered directly to a subject. Direct delivery may be accomplished by parenteral injection (e.g. siibcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue; also by direct injection into the tumor).
- the TRAIL variant, or the pharmaceutical composition comprising the TRAIL variant is administered intraperitoneally.
- the rationale behind IP drug administration is to increase local drug exposure, while lowering plasma clearance.
- the inventors have discovered that injection of TRAIL by this route results in increased tumor uptake compared to intravenous injection, increased efficacy and a reduced clearance rate.
- IP injection provides reduced system toxicity compared to intravenous administration (Table 2, Figure 3).
- the inventors have also shown that specific tumor retention of 125 I-TRAIL takes place. It was demonstrated by maximal tumor activity at 60 min after IV administration, while activity in all other well perfused organs is maximal at 15 min. IP administration resulted in even higher activity in the tumor and a higher cumulative tumor to blood ratio, which shows that intraperitoneal administration of recombinant human TRAIL (herein TRAIL) results in higher tumor drug exposure compared to IV administration. Moreover, 6 h after IP injection of a low dose of TRAIL, cleaved caspase-3 was detected in the superficial layers of the tumors, suggestive of TRAIL penetration by free-surface diffusion.
- TRAIL recombinant human TRAIL
- TRAIL and TRAIL variants over monoclonal antibodies, which show limited IP tumor penetration after IP administration.
- Another advantage which TRAIL and TRAIL variants possess over monoclonal antibodies against DR4/DR5 is a reduced immunogenicity.
- Dosage treatment can be a single dose schedule or a multiple dose schedule.
- the TRAIL variant or the pharmaceutical composition, comprising the TRAIL variant is administered on a weekly basis to the patient.
- the dosing regime is within the judgement of the clinician and can be altered if the need arises or if another dosage regime is found to be more beneficial for a patient.
- Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
- B. Survival of A2780 assessed with a cytotoxicity assay after 96 h exposure to 0 - 100 ng/ml TRAIL and TRAIL-DR5. * p 0.008
- DR5-selective variant D269H/E195R show enhance apoptosis in combination with cisplatin in A2780 cells in a solely DR5-dependent manner.
- DcR2 antibody or medium after which the cells were exposed to 100 or 250 ng/ml TRAIL for 4 h.
- Anti-DcR2 antibody co-incubation did not cause significant changes in apoptosis induction.
- Data represent the mean ⁇ SD of at least three independent experiments.
- MFI mean fluorescence intensities
- A2780 cells transfected with siRNA against luciferase (Luc siRNA) or siRNA against DR5 (DR5 siRNA) were pre-incubated with medium or 30 ⁇ M of cisplatin for 4 h and then washed. After 20 h, the cells were exposed for 4 h to 100 ng/ml TRAIL or TRAIL- DR5, and apoptosis was assessed by means of acridine orange staining. Data represent the mean ⁇ SD of three independent experiments.
- Tumor-to-blood ratio versus time of 125 I-TRAIL administered intravenously or intraperitoneally Tumor to blood ratios were calculated by dividing the average tumor activity in %ID/g per time point through the average blood activity in % ID/g per time point.
- IP IP
- FV FV
- paraffin embedded tissue was obtained 15 min after 125 I-TRAILinjection and stained with cleaved caspase-3, showing no cleavage in samples obtained at this time point.
- IP IP
- IV IV
- paraffin embedded tissue was obtained 360 min after 125 I-TRAILinjection and stained with cleaved caspase-3, showing increased caspase-3 cleavage staining near the surface of the tumor and near small blood vessel with IP administration ( Figure 5A) whereas IV administration induced detectable caspase-3 activity near blood vessels but not near the tumor border ( Figure 5B).
- Figure 6 In-vivo efficacy of rhTRAIL and D269H/E195R monotherapy and in combination with cisplatin.
- Figure 7 Cell surface expression of TRAIL receptors in Colo205 and ML-I cells.
- FIG. 8 Biological activity of TRAIL and DR5-selective variants.
- A Apoptosis-inducing activity of 100 ng/ml TRAIL in the presence of 1 ⁇ g/ml DR4 (aDR4), DR5 (aDR5), or DR4 and DR5 (+aDR4+aDR5) receptor-neutralizing antibodies in Colo205 and ML-I cells.
- B Apoptosis-inducing activity in Colo205 cells of 100 ng/ml TRAIL or DR5- selective variants without the presence of neutralizing DR4 or DR5 antibodies (no AB) or in the presence of neutralizing antibody [aDR4, aDR5, or both (aDR4 aDR5)].
- TRAIL or DR5-selective variants in Colo205 (Q, ML-I (D), and A2780 (E) and of 1, 10, or 100 ng/ml TRAIL (WT) or D269HE195R (DE) relative to cycloheximide control (0.33 ⁇ g/ml) in BJAB cells responsive to both DR4- and DR5-mediated cell death (BJAB wt ), BJAB cells deficient for DR5 (BJAB DR5 DEF ), and BJAB cells deficient for DR5 stably transfected with DR5 (BJAB DR5 DEF +DR5) (F).
- BJAB wt BJAB cells responsive to both DR4- and DR5-mediated cell death
- BJAB DR5 DEF BJAB wt
- BJAB DR5 DEF BJAB DR5 DEF
- BJAB DR5 DEF +DR5 BJAB DR5 DEF +DR5
- Figure 9 DR5 knock-down reduces apoptosis of TRAIL in combination with cisplatin in A2780 cells.
- MFI mean fluorescence intensities
- Apoptosis rate in A2780 DR5 knock-down and control cells in response to TRAIL and cisplatin was assessed by means of acridine orange staining.
- Colo205 cells were treated with increasing concentrations (5-30 ng/ml) of wtTRAIL or DR5- variants and induction of apoptosis was monitored by measuring phosphatidyl serine exposure (A), caspase activation (B) and pro-caspase-8 processing (C).
- A Cell death induction by wtTRAIL and DR5-selective variants, D269H and D269HE195R. The graph shows averaged percentage apoptosis induced ⁇ SEM.
- B DEVDase activity in Colo205 cells following treatment with wtTRAIL, D269H and D269HE195R.
- DEVDase activity was measured in whole- cell lysates with a kinetic assay as described in Example 6. Enzyme activity was expressed in nmole AMC released per min by 1 mg of total cellular protein.
- C Western blot analysis of pro- caspase-8 cleavage in Colo205 cells treated with wtTRAIL and DR5 variants, showing cleavage of caspase-8 at lower concentrations of DR5-specific variants compared to wtTRAIL.
- the graphs (A, B) show averaged values of three independent experiments, while part C shows one representative picture of two independent experiments.
- FIG. 13 DR5-selective variants are more efficient in A2780 tumor cell killing than wtTRAIL.
- A Cell death induction by wtTRAIL and D269HE195R. A2780 cells were treated with increasing concentrations (10-50 ng/ml) of wtTRAIL or D269HE195R. Apoptotic cell death was measured 24 h post-treatment by Annexin V assay.
- B DEVDase activity in A2780 cells following treatment with increasing concentrations' of wtTRAIL and D269HE195R for 24 h. DEVDase activity was measured in whole-cell lysates with a kinetic assay as described in Example 6.
- FIG. 14 Inhibition of wtTRAIL and DR5-variant-induced apoptosis by decoy receptors.
- wtTRAIL, D269H and D269HE195R were pre-incubated for 30 min with increasing concentrations of soluble DcRl- and DcR2-Ig, then added to Colo205 cell cultures at a 10 ng/ml concentration for 3 h.
- the graph shows significantly less cell death in the TRAIL treated group on addition of soluble DcRl with no effect on D269H or D269HE195R induced cell death measured by Annexin V assay
- B Effect of soluble recombinant DcR2-Ig (sDcR2) on apoptosis induction by TRAIL and DR5-selective variants.
- the graph shows that higher concentration of sDcR2 is required to reduce apoptosis (measured by Annexin V binding) induced by the DR5-seIective variants.
- D Neutralizing antibodies against DcRl and DcR2 can augment wtTRAIL- but not DR5-variant induced cell death. Colo205 cells were incubated for 1 h with increasing concentrations of neutralizing antibodies to DcRl and DcR2, followed by treatment with wtTRAIL (20 ng/ml) and D269HE195R (4 ng/ml).
- the graph shows fold increase in apoptosis calculated by dividing the percentage apoptosis induced by wtTRAIL or D269HE195R in the presence of neutralizing decoy receptor antibodies with percentage apoptosis induced by the ligands in the absence of the antibodies.
- the graphs show the average ⁇ SEM of three independent experiments.
- D269HE195R activates death-inducing TRAIL receptors and apoptosis a magnitude faster than wtTRAIL.
- Colo205 cells were treated with 30 ng/ml of wtTRAIL or D269HE195R for the times indicated, after which the ligands were washed out and the cells were incubated in normal growth medium for 180 min (for TRAIL receptor activation) or for 240 min (for PS exposure) in total.
- A Kinetics of death-inducing TRAIL receptor activation by wtTRAIL and D269HE195R measured by monitoring pro-caspase-8 processing with Western blotting.
- Colo205 cell line was treated with 5 mM Aspirin for 24 h or 10 ⁇ M of 5-Flurouracil for 24 h, and then harvested for receptor expression for TRAIL Rl, R2, R3 and R4. Aspirin leads to induction TRAIL R2, R3 and R4 receptor expression. 5-Flurouracil leads to an induction of all TRAIL receptors.
- Figure 18. D269HE195R used in combination with aspirin result in enhanced synergism compared to WT TRAIL.
- Colo205 cells were treated with 5 mM aspirin for 24 h and cell surface expression of DR4, DR5, DcRl and DcR2 was measured by immunostatning and detected with flow cytometry.
- the histograms shown include isotype control (black, open peak), untreated Colo205 (grey, closed peak) and aspirin- treated Colo205 (grey, open peak) samples. The histograms are representatives of three independent experiments.
- DEVDase activity was expressed as average nmol AMC released per minute by 1 mg total cellular protein ⁇ SD of 3 independent experiments.
- FIG. 19 Comparison of 2 and 5xlO 6 Colo205 cells for inoculation. Athymic nude mice were subcutaneously injected with 200 ⁇ l of medium containing 2 or 5 *10 ⁇ 6 of Colo205 cells and tumour growth was followed for several weeks.
- FIG. 20 Aspirin as single treatment on Colo205 tumors.
- rhTRAIL proteins were tested in doses of 1 and 5 mg/kg, .given by intraperitoneal injections for 2x5 days (day 1-5 and 8-12) with a daily doses of aspirin (in the same syringe as protein).
- Figure 22 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of Colo205 cells with and without combination therapy either with Aspirin or 5-Flurouracil
- DR5 mutants (D269H, D269HE195R and Ml) lead to significantly less cell viability than TRAIL with the largest differences seen at lower concentrations of the ligand.
- B Effect of increasing concentrations of Aspirin on cell viability.
- C Effect of increasing concentrations of 5-Flurouracil on cell viability.
- D Combination therapy with 5 mM Aspirin pre-incubated for 24 h lead to further decreases in cell viability in cells treated with both TRAIL and two of the DR5 mutants.
- E 30 minute incubation of DR5 mutants, were sufficient decrease cell viability which was further augmented when cells were pre-incubated for 24 h with Aspirin.
- TRAIL stands for rhTRAIL WT
- D269H stands for rhTRAIL carrying mutation D269H
- E195R stands for rhTRAIL carrying mutations D269H and E195R
- Ml stands for rhTRAIL carrying mutations D269H, El 941 and I196S.
- Figure 23 Effect of wtTRAIL, single therapy and combination with aspirin on Colo205 tumors.
- the rhTRAIL protein was tested in doses of 1 and 5 mg/kg, given by intraperitoneal injections for 2x5 days (day 1-5 and 8-12) with or without the doses of aspirin (in the same syringe as protein).
- ASA indicates combination with aspirin.
- Figure 24 Effect of D269HE195R TRAIL, single therapy and combination with aspirin on Colo205 tumors.
- the rhTRAIL protein were tested in doses of 1 and 5 mg/kg, given by intraperitoneal injections for 2x5 days (day 1-5 and 8-12) with or without the doses of aspirin (in the same syringe as protein).
- ASA indicates combination with aspirin.
- Figure 25 Apoptosis assays showing enhanced effect of TRAIL and DR5 mutants with Aspirin, with the mutants showing more apoptosis compared to TRAIL.
- Apoptosis was measured using AnnexinV/PI staining at 3 h following the addition of 5 and 10 ng/ml of TRAIL or the DR5 mutant (D269HE195R), in cells which were pre-incubated with or without 5 Mm Aspirin or 10 ⁇ M 5-Flurouracil.
- the DR5 mutant treated cells display more apoptosis which can be enhanced by Aspirin and not 5-Flurourail.
- TRAIL treated cells show lesser apoptosis which can be enhanced by Aspirin and to a lesser extent by 5-Flurouracil.
- Colo205 cells were harvested at various time points shows a greater caspase activation following treatment with 10 ng/ml of the DR5 mutant (D269HE195R).
- the caspase activity is further enhanced in cells pre-incubated for 24 h with 5 mM of Aspirin but not 5-Flurouracil.
- Minimal caspase activation is seen for the given time points in cells treated with 10 ng/ml of TRAIL.
- Figure 27 Figure 27.
- TRAIL and the DR5 mutants (D269H, D269HE195R and Ml) lead to decreased cell viability with TRAIL leading to greater loss in cell viability compared to that of the DR5 mutant at higher concentrations.
- B Effect of increasing concentration of Aspirin on SW948 cell.
- C ' Combination therapy with 2.5 nM Aspirin pre-incubated for 24 h lead to further decreases in cell viability in cells treated with both TRAIL and two of the DR5 mutants.
- D Combination therapy with 10 ⁇ M 5-Flurouracil pre-incubated for 24 h lead to significant enhancement only in TRAIL treated cells.
- Figure 29 Apoptosis assays showing enhanced effect of the TRAIL and DR5 mutants with Aspirin, with 5-Flurouracil enhancing only TRAIL treated cells.
- Apoptosis was measured using AnnexinV/PI staining at 3 h following the addition of 5 and 10 ng/ml of TRAIL or the DR5 mutant (D269HE195R), in cells which were pre-incubated with or without 5 Mm Aspirin or 10 ⁇ M 5-Flurouracil.
- the DR5 mutant treated cells display more apoptosis which can be enhanced by Aspirin and not 5-Flurourail.
- TRAIL treated cells show lesser apoptosis which can be enhanced by Aspirin and to a lesser extent by 5-Flurouracil.
- FIG 31 Receptor expression in LOVO cell line following treatment with Aspirin and 5- Flurouracil Lovo cell line was treated with 2.5 mM Aspirin for 24 h or 10 ⁇ M of 5-Flurouracil for 24 h, and then harvested for receptor expression for TRAIL Rl, R2, R3 and R4. Aspirin leads to an induction TRAIL R4 and to a lesser extent TRAIL R2 receptor expression. 5-Flurouracil leads to an induction of TRAIL R2, R3 and R4 receptors.
- Figure 32 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of Lovo cells with and without combination therapy either with Aspirin or 5-Flurouracil
- TRAIL and the DR5 mutants lead to an equal decrease in cell viability
- B Effect of increasing concentration of Aspirin on Lovo cell.
- C Effect of increasing concentration of 5-Flurouracil on Lovo cells.
- D Pre-incubation with 2.5 mM Aspirin for 24 h lead to significantly decreased cell survival in both TRAIL and DR5 mutant treated cells.
- E Pre-incubation with 10 ⁇ M 5-Flurouracil for 24 h lead to significantly decreased cell survival in both TRAIL and DR5 mutant treated cells.
- Figure 33 Apoptosis assays showing enhanced effect of the TRAIL and DR5 mutants with Aspirin and 5-Flurouracil
- Apoptosis was measured using AnnexinV/PI staining at (A) 7 and (B) 15 h following the addition of 5 and 50 ng/ml of TRAIL or the DR5 mutant (D269HE195R), in cells which were pre-incubated with or without 5 Mm Aspirin or 10 ⁇ M 5-Flurouracil. Enhanced apoptosis in cells pretreated with Aspirin and 5-Flurouracil following TRAIL and DR5 mutant (D269HE195R) treatment. Enhanced apoptosis with Aspirin is best seen at 15 h.
- Lovo cells were harvested at various time points, shows an earlier caspase activation following treatment with 50 ng/ml of DR5 mutant (D269HE195R) compared to TRAIL 50 ng/ml.
- the caspase activity is further enhanced in cells pre-incubated for 24 h with 5 mM of Aspirin and 10 ⁇ M 5-Flurouracil.
- FIG. 35 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of HCT15, HT29 and RKO cells with and without combination therapy either with 5- Flurouracil TRAIL and DR5 mutant (D269HE195R) show equal decrease in cell viability following treatment with or without combination therapy with 5-Flurouracil. 5-Flurouracil does not further decrease cell viability compared to ligand alone in all cell lines tested (A) & (B) HCT 15, (C) RKO and (D)HT29.
- Figure 36 Top (left) and side (right) view of the trimer-trimer complex of the extracellular moiety of wtTRAIL (grey) and of DR5 (black).
- FIG. 37 SPR analysis of the binding of wtTRAIL and D269HE195R to immobilized receptor.
- Figure 38 Dose-response curve of the apoptotic activity of wtTRAIL and D269HE195R in colon carcinoma cell line Colo205.
- TRAIL-induced cell death as a function of concentration was measured with the MTS assay.
- Figure 39 Induction of p53 following treatment with Bortezomib in cervical cancer cell lines.
- All three cervical cancer cell lines were treated with increasing concentration of Bortezomib to assess expression of p53 protein.
- A Induction of p53 protein can be seen at concentrations of 1 nM with largest induction seen at 5 and 10 nM in Caski cell line
- B Induction of p53 in SIHA cells can be seen at 5 and 10 nM.
- C In HELA cells induction of p53 is seen also at 5 and 10 nM, though relatively smaller to that of SIHA and Caski cell lines.
- Caski cervical cell line was treated with (A) 5 nM Bortezomib for 24 h or (B) incubated for 24 h following 10Gy radiotherapy, and then harvested for receptor expression for TRAIL Rl, R2, R3 and R4.
- Figure 41 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of Caski cells with and without combination therapy either with Bortezomib of Radiotherapy
- TRAIL and DR5 mutants appear comparable as a single agent in affecting cell viability.
- B Effect of increasing concentrations of Bortezomib on cell viability.
- C Combination therapy with 5 nM Bortezomib incubated for 24 h lead to further decreases in cell viability in cells treated with both wtTRAIL and two of the DR5 mutants
- D Radiation had no enhancing effect in combination with wtTRAIL or the DR5 mutants.
- Figure 42 Apoptosis assays showing enhanced effect of TRAIL and DR5 mutant with Bortezomib
- FIG 44 Receptor expression in SIHA cell line following treatment with Bortezomib and Radiotherapy
- SIHA cervical cell line was treated with (A) 10 nM Bortezomib for 24 h or (B) incubated for 24 h following 10Gy radiotherapy, and then harvested for receptor expression for TRAIL Rl, R2, R3 and R4.
- (A) Bortezomib treatment increases receptor expression of all TRAIL receptors.
- Figure 45 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of SIHA cells with and without combination therapy either with Bortezomib of Radiotherapy
- DR5 mutant shows a greater apoptosis compared to TRAIL in SIHA cells following incubation with Bortezomib. Apoptosis was measured using Acridine Orange staining and cells were measured 7 h following treatment with TRAIL and DR5 mutant D269HE195R, in SIHA cells with or without preincubation for 24 h with 10 nM Bortezomib. The DR5 mutant show greater apoptosis compared to TRAIL at the given time point. Radiotherapy had no enhancing effect compared to cells treated with TRAIL or DR5 mutant alone (data not shown).
- Figure 48 Receptor expression in HELA cell line following treatment with Bortezomib and Radiotherapy
- HeIa cervical cell line was treated with (A)IO nM Bortezomib for 24 h or (B) incubated for 24 h following 10Gy radiotherapy, and then harvested for receptor expression for TRAIL Rl, R2, R3 and R4.
- A Bortezomib treatment increases receptor expression of all TRAIL receptors.
- B Radiotherapy also leads to an increase of all TRAIL receptors.
- Figure 49 Cell survival assays looking at the effect of TRAIL/DR5 mutants on viability of HELA cells with and without combination therapy with either Bortezomib of Radiotherapy
- TRAIL and DR5 mutants appear comparable as a single agent in affecting cell viability.
- B Effect of increasing of Bortezomib on cell viability. HeIa cells appear to tolerate higher concentration of Bortezomib compared to Caski and SIHA cells
- C Combination therapy with 10 nM Bortezomib incubated for 24 h lead to further decreases in cell viability in cells treated with both TRAIL and two of the DR5 mutants
- D Radiation therapy in combination with TRAIL or the DR5 mutants further decrease cell viability in HELA cells. The DR5 mutant show decrease cell viability at lower concentration of ligand compared to TRAIL.
- Figure 50 Apoptosis assay showing enhanced effect of TRAIL and DR5 mutant with Bortezomib and 10Gy Radiotherapy
- Apoptosis was measured using Acridine Orange staining and cells were measured 7 h following treatment with TRAIL and DR5 mutant D269HE195R, in HeIa cells with or without pre- incubation for 24 h with 10 nM Bortezomib or 10Gy Radiotherapy. Both TRAIL and the DR5 mutant show almost equal enhancement of apoptosis with either combination therapy. Combination of Bortezomib, Radiotherapy and TRAIL or DR5 mutant had no further additive effect. Figure 51. Both TRAIL and DR5 mutant show enhance caspase activity to Bortezomib and Radiotherapy.
- Figure 52 Human colon adenoma cells are sensitive to TRAIL and D269H/E195R.
- the colon adenoma cell lines VACO-235 and VACO330 were treated with TRAIL or D269H/E195R for 96 hours.
- the adenoma cell lines were more sensitive to D269H/E195R. Sensitivity could be further enhanced using TRAIL or D269H/E195R in combination with the cdk inhibitor roscovitine.
- Figure 53 P-Akt inhibition for 17 h selectively sensitizes Colo205 colon carcinoma to TRAIL-DR5 (D269H/E195R)
- TRAIL and D269H/E195R induced apoptosis by PDK inhibition in Colo205, as assessed by Annexin V assay.
- Cells were pre-incubated for 15-18 hours with 20 ⁇ M LY294002, then either left untreated or exposed to rhTRAIL (0.1 ⁇ g/ml) or D269H/E195R (0.1 ⁇ g/ml) for 3 additional hours before harvest. While sensitivity to TRAIL slightly decreased, sensitivity to D269H/E195R was enhanced.
- FIG. 54 HeLa xenografts treated with wtTRAIL or TRAIL-DR5(D269H/E195R).
- Xenografts of the HeLa cervical cancer cell line were established with matrigel in athymic nude mice. Treatment was started when tumor volume was +/-150 mm3. No effect was observed in HeLa cervical cancer xenografts following treatment of TRAIL or (D269H/E195R).
- Figure 55 Mathematical modeling of TRAIL receptor complex formation triggered by wtTRAIL and D269HE195R over time.
- Figure 56 Mathematical modeling of TRAIL receptor complex formation triggered by wtTRAIL and D269HE195R over time.
- the wild-type TRAIL and TRAIL mutant constructs were transformed to Escherichia CoIi BL21 (DE3) (Invitrogen). Wild-type TRAIL and Ml were grown at a 5 1 batch scale in a 7.5 1 fermentor (Applicon) using 4 x LB medium, 1% (w/v) glucose, 100 ⁇ g/ml ampicillin and additional trace elements. The culture was grown to mid-log phase at 37 0 C, 30% oxygen saturation and subsequently induced with 1 mM IPTG. ZnS ⁇ 4 was added at a concentration of 100 ⁇ M to promote trimer formation. Temperature was lowered to 28 0 C and the culture was grown until stationary phase.
- mutants were grown in shake flasks at a 1 1 scale at 250 rpm, using a similar protocol. Protein expression was induced when the culture reached OD 60O 0.5 and induction was continued for 5 h. In this case, the medium used was 2 x LB without additional trace elements.
- the isolated pellet was resuspended in 3 volumes extraction buffer (PBS pH 8, 10% (v/v) glycerol, 7 mM ⁇ -mercapto-ethanol). Cells were disrupted using sonication and extracts were clarified by centrifugation at 40,000 g. Subsequently, the supernatant was loaded on a nickel charged IMAC Sepharose fast-flow column and wild-type TRAIL and TRAIL mutants were purified as described by Hymowitz 24 with the following modifications: 10 % (v/v) glycerol and a minimal concentration of 100 mM NaCl were used in all buffers. This prevented aggregation during purification.
- Example 2 In vitro activity of TRAIL-DR5, TRAIL and cisplatin on A2780
- A2780 is a human ovarian cancer cell line, which expresses DR5 and low levels of DcR2 at its cell surface, whereas DR4 and DcRl are undetectable ( Figure IA).
- Treatment with cisplatin resulted in a dose dependent upregulation of DR5 and DcR2 expression ( Figure IA).
- Long term exposure (96 h) of A2780 cells to low concentrations of TRAIL and TRAIL-DR5 induced a dose dependent loss of viability (Figure IB).
- DR5 The involvement of the DR5 receptor in inducing apoptosis in these cells was further assessed in cells in which the DR5 receptor had been knocked down by RNA interference.
- A2780 cells were transfected with siRNA against DR5 (5'GACCCUUGUGCUCGUUGUC- dTdT3' (sense) and 5'GACAACGAGCACAAGGGUC-dTdT3' (anti-sense)) or luciferase, as a control or remained untransfected. 24 h after transfection the cells were exposed for 4 h to 2.5 ⁇ M, 10 ⁇ M and 30 ⁇ M cisplatin, after which all cells including those unexposed to cisplatin, were washed.
- DR5 expression is represented as mean fluorescence intensity (MFI).
- MFI mean fluorescence intensity
- TRAIL TRAIL-induced apoptosis
- a small fraction of the siRNA treated cell suspension was plated in a 96-wells plate and apoptosis levels were determined with acridine orange apoptosis assays counting at least 200 cells.
- Data represent the mean ⁇ SD of at least three independent experiments.
- the results show that apoptosis in the control cells was increased, when compared to the untreated cells.
- the DR5 siRNA cells did not show increased apoptosis in response to TRAIL ( Figure 9B), even when it was administered together with cisplatin. Therefore, DR5 siRNA resulted in a complete inhibition of TRAIL-induced apoptosis and blocked sensitization by cisplatin of TRAIL-induced apoptosis in A2780.
- the A2780-Luc cell line was generated as follows.
- the luciferase gene was excised from pGL3-basic (Promega, Madison, WI) with HindIII and Xbal restriction enzymes (Roche Applied Science, Almere, The Netherlands) and ligated into a pcDNA3 vector under the control of the cytomegalovirus promotor.
- A2780 cells were cultured to 70% confluency. and transfected by incubation with 2.5 ⁇ g plasmid DNA and 5 ⁇ l Fugene ⁇ (Roche) in 250 ⁇ l OptiMem (Invitrogen, Breda, the Netherlands).
- transfectants Two days after transfection, transfectants were selected by adding geneticin (1 mg/ml) (Roche Applied Science, Almere, The Netherlands). Stable transfectants were obtained with a clonogenic assay followed by subcloning of positive clones by limiting dilution.
- the cell lines were cultured in RPMI 1640 (Life Technologies Breda, the Netherlands), supplemented with 10% heat inactivated fetal calf serum (FCS) (Bodinco BV 3 Alkmaar, the Netherlands) and 0.1 M L-glutamine in a humidified atmosphere with 5% CO 2 at 37°C. Geneticin was added once a month to the A2780-Luc culture.
- FCS heat inactivated fetal calf serum
- Luciferase expression was regularly tested with the luciferase assay (# El 500, Promega, Leiden, The Netherlands) and the BioRad ChemiDoc XRS system (BioRad, Veenendaal, The Netherlands).
- the microculture tetrazolium assay performed as described earlier 36 , was used to measure cytotoxicity.
- the cells were cultured in HAM/F12 and DMEM medium, supplemented with 20% FCS and 0.1 M L-glutamine.
- wtTRAlL and TRAIL-DR5 were produced as we have described earlier 11 ' 20 . Binding capacity to DR4 and DcRl is virtually absent for TRAIL-DR5, whereas affinity for DcR2 is reduced 11 .
- Treatment consisted of continuous incubation with 0 - 100 ng/ml TRAIL-DR5 or wtTRAIL.
- the cells were pre-incubated for 4 h (h) with 2.5 ⁇ M cisplatin (inhibitory concentration 20% - IC20), before addition of 0 - 25 ng/ml TRAIL or TRAIL-DR5.
- TRAIL-DR5 or TRAIL were added for various times. Thereafter the cells were harvested with trypsin and washed twice with ice- cold PBS. Before performing the caspase-3 activity assay according to the manufacturer's protocol, protein content of the lysates was determined with Bradford analysis 21 . The acridine orange staining served as a marker for end/stage apoptosis. For the apoptosis assay 10,000 cells were incubated in 96-well tissue-culture plates. The cells were exposed to 2.5, 10 or 30 ⁇ M of cisplatin for 4 h, after which they were washed with PBS twice and incubated in regular culture medium.
- Staining intensity was determined by fluorescence microscopy. Apoptosis was defined by the appearance of apoptotic bodies and/or chromatin condensation and expressed as the percentage of apoptotic cells counted in three fields containing minimally 300 cells.
- enhancement ratio % induced by combination therapy / (% induced by cisplatin alone + % induced by ligand).
- TRAIL-receptor membrane expression was performed by FACS analysis as described previously 35 .
- For death receptor expression after cisplatin exposure cells were exposed for 4 h, washed with PBS and incubated for 20 h in regular culture medium, after which FACS analysis was performed. Cells were subsequently washed twice with cold PBS containing 2% FCS and 0.1% sodium azide and incubated with phycoerythrin (PE)-conjugated mouse monoclonal antibodies against DR4, DR5, DcRl and DcR2.
- PE phycoerythrin
- Mouse PE-labeled IgGl and IgG2B were used as isotype controls. All PE-labeled antibodies were purchased from R&D systems (Oxon, UK).
- Membrane receptor expression was analyzed with Winlist and Winlist 32 software (Verity Software House, Inc., Topsham, ME) and is shown as mean fluorescent intensity (MFI) of all analyzed cells.
- DR5 siRNA small interfering RNA
- SiRNAs specific for human DR5 were designed and synthesized by Eurogentec (Seraing, Belgium).
- the double-stranded siRNA specific for human DR5 was 5'GACCCUUGUGCUCGUUGUC-dTdT3' (sense) and 5'GACAACGAGCACAAGGGUC- dTdT3' (anti-sense).
- Double-stranded luciferase siRNA sequence was 5' CUUACGCUGAGUACUUCGA-dTdT 3' (sense) and 5'UCGAAGUACUCAGCGUAAG- dTdT 3' (anti-sense).
- A2780 cells were transfected in 6-wells plates (at 30-50% confluency) with siRNA duplexes ( 133 nM) using Oligofectamine transfection reagent according to the manufacturer's instructions (Invitrogen-Life Technologies). After 24 h, media was aspired and cells were harvested and plated. Then, cells were exposed to various cisplatin concentrations for 4 h, washed with PBS and incubated for 20 h in regular culture medium. Finally, cells were harvested and used for flow cytometry or Western blotting.
- apoptosis assay cells were incubated in regular culture medium without or with TRAIL-DR5 or TRAIL (100 ng/ml) for an additional 4 h and apoptosis was dete ⁇ nined with the acridine orange assay.
- the following antibodies were applied: rabbit anti-DR5 antibody from Cell Signaling Technology (Leusden, the Netherlands) and mouse-anti-actin as the control for equal protein loading from ICN Biomedicals (Zoetermeer, the Netherlands). Secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from DAKO Cytomation (Glostrup, Denmark). Chemiluminescence was detected using BM Chemiluminescence Blotting Substrate (POD) or Lumi-LightPLUS Western blotting substrate Roche Diagnostics (Almere, the Netherlands).
- HRP horseradish peroxidase
- Radioiodination of TRAIL was performed with a TRAIL solution of 1 mg/ml in a pH 7.4 TRIS buffer, containing 100 ⁇ M zinc sulphate and 10% glycerol. 45 ⁇ g TRAIL and 50 ⁇ g chloramine
- Non-bound ' 25 I was removed by gel filtration chromatography.
- the PD-10 column (SephadexTM G-25M, Amersham Biosciences AB, Uppsala, Sweden) was eluted with TRIS buffer, containing
- A2780-Luc xenografts were prepared, as described above.
- mice The in vivo biodistribution study was conducted in 50 mice after establishment of A2780-Luc IP xenografts.
- 125 I-TRAIL (0.15 ml; 150 kBq, 0.5 ⁇ g) was administered IV through retro-orbital injection in 25 mice, and IP in 25 mice.
- t 15, 30, 60, 90 and 360 min
- groups of 5 mice were sacrificed and organs and tissues were excised, rinsed for residual blood and weighed. Tumor tissue was additionally fixed in 10% buffered formalin for histological assessment. Samples were counted for radioactivity in a calibrated well-type LKB- 1282-CompuGamma counter. Tissue activity was expressed as percentage of the injected dose/g tissue (% ID/g).
- Tumor-to- blood and tumor-to-muscle ratios were also calculated. All data were corrected for physical decay and compared with a known standard sample. Pharmacokinetic parameters were . derived using the KINFIT module of the MW/PHARM computer program package (version 3.50, MediWare, Groningen, The Netherlands). Clearance rates of 125 I-TRAIL from the circulation were calculated using non-linear regression analysis. The administration route influenced the disposition of 125 I-TRAIL. Blood activity (% ID/g) was higher at 15 (43.29 ⁇ 11.04 vs. 25.30 ⁇ 5.04) and 30 min (30.51 ⁇ 12.40 vs. 15.33 ⁇ 3.78) after IV vs.
- IP administration resulted in high tumor activity at 15 min (11.31 ⁇ 1.51) and 60 min (12.91 ⁇ 3.29) with a gradual decrease to 360 min, whereas after IV administration initial low tumor activity increased to a maximum at 60 min (6.85 ⁇ 1.29 IV).
- the tumor to blood ratios were higher after IP administration at 15 (0.13 ⁇ 0.02 vs 0.48 ⁇ 0.03) and 60 min (0.38 ⁇ 0.04 vs. 0.55 ⁇ 0.06). Tumor to blood ratios remained constant over time after IP injection and increased over time after IV administration (Figure 3B).
- TRAIL injection was stained for cleaved caspase-3. While almost no cleaved caspase-3 was detected in samples obtained at 15 min ( Figure 4A, B), tissue obtained at 360 min showed increased cleaved caspase-3 staining.
- IP administration resulted in caspase-3 activation near the surface of the tumor and near small blood vessels ( Figure 5A), whereas IV administration induced detectable caspase-3 activity near blood vessels but not near the tumor border ( Figure
- IP drug administration is to increase local drug exposure, while lowering plasma clearance.
- IP TRAIL-administration resulted in a higher area under the curve and a reduced clearance.
- the high kidney activity confirms the function of the kidney as main site of TRAIL-clearance, which is not influenced by IP administration. Activity in most organs followed that of blood pool activity, suggesting that distribution to normal tissues was limited.
- the inventors have also shown that specific tumor retention of 125 I-TRAIL takes place. It was demonstrated by maximal tumor activity at 60 min after IV administration, while activity in all other well perfused organs is maximal at 15 min. IP administration resulted in even higher activity in the tumor and a higher cumulative tumor .to blood ratio, which shows that IP TRAIL administration results in higher tumor drug exposure compared to FV administration in this model. Moreover, 6 h after IP injection of a low dose of TRAIL, cleaved caspase-3 was detected in the superficial layers of the tumors, suggestive of TRAIL penetration by free-surface diffusion. This is a possible advantage of TRAIL and -variants over monoclonal antibodies, which show limited IP tumor penetration after IP administration.
- Example 4 Cell killing of ovarian carcinoma cell line A2780 by wtTRAIL and DR5- selective TRAIL
- the ovarian carcinoma cell line A2780 expresses very low levels of DR4, and high levels of the other three membrane-bound TRAIL receptors. Sensitivity of these cells to wild type TRAIL and the DR5-specific variant was compared initially as a single agent. The sensitivity pattern is illustrated in the Table 6, with + indicating minimally sensitive, ++ moderately sensitive and +++ very sensitive.
- A2780 cells showed increased sensitivity to the DR5-selective variant compared to wild type TRAIL. These effects were enhanced when the ligands were used in combination with Cisplatin chemotherapy, which is currently the active agent used in the treatment of ovarian carcinoma. Again, the DR5-selective variant showed higher efficacy than wtTRAIL.
- Example 5 In vivo efficacy of TRAIL, TRAIL-DR5 and cisplatin on IP xenografts determined by bioluminescence imaging
- the A2780-Luc cell line was generated with Hin ⁇ Wl and Xbal restriction enzymes (Roche Applied Science, Almere, The Netherlands).
- the luciferase gene was excised from pGL3-basic (Promega, Madison, WI) and ligated into a pcDNA3 vector under the control of the cytomegalovirus promoter.
- A2780 cells were cultured to 70% confluency and transfected by incubation with 2.5 ⁇ g plasmid DNA and 5 ⁇ l Fugene ⁇ (Roche) in 250 ⁇ l OptiMem (Invitrogen, Breda, the Netherlands). Two days after transfection, transfectants were selected by adding geneticin (1 mg/ml) (Roche Applied Science, Almere, The Netherlands). Stable transfectants were obtained with a clonogenic assay followed by subcloning of positive clones by limiting dilution.
- the cell lines were cultured in RPMI 1640 (Life Technologies Breda, the Netherlands), supplemented with 10% heat inactivated fetal calf serum (FCS) (Bodinco BV, Alkmaar, the Netherlands) and 0.1 M L-glutamine in a humidified atmosphere with 5% CO 2 at 37 0 C. Geneticin was added once a month to the A2780-Luc culture. Luciferase expression was regularly tested with the luciferase assay (# E1500, Promega, Leiden, The Netherlands) and the BioRad ChemiDoc XRS system (BioRad, Veenendaal, The Netherlands).
- mice Female nude mice (HsdrAthymic Nude-r ⁇ ) were obtained from Harlan Nederland (Horst, The Netherlands) at 6-8 weeks of age (- 21 g). Inoculation was performed after 10 days of acclimatization. All animal studies were conducted in accordance with the Law on Animal Experimentation and local guidelines, and were approved by the local ethical committee.
- Imaging was conducted with the IVIS 100 series (Xenogen Corporation Alameda, CA) 5 composed of a cooled charge-coupled device camera connected to a light tight black chamber. Before in vivo imaging animals were anesthetized with 4% isoflurane and injected IP with D- luciferin (150 mg/kg, Xenogen) reconstituted in sterile PBS. Mice were placed in prone position on a warmed stage (37 0 C) in the imaging chamber, and grayscale reference images were obtained under dim illumination. Pseudocolor images representing bioluminescent intensity were acquired with Livinglmage software (version 2.50, Xenogen) 10 and 15 min after D-luciferin injection in complete darkness.
- IP A2780-Luc xenografts to treatment with TRAIL, TRAIL-DR5 and cisplatin or a combination of either TRAIL or TRAIL-DR5 with cisplatin was assessed.
- Tumor regression was not visible within the first 48 h after treatment initiation at day 5, but was clearly evident at the end of the first treatment period (day 9), with the largest signal reduction seen after combination of TRAIL or TRAIL-DR5 with cisplatin (Figure 6A). Signals rose in the days between both treatments. All treatment groups except the TRAIL-treated arm had significantly smaller tumors at day 16 than the vehicle treated group.
- Colo205 colon carcinoma cells and ML-I chronic myeloid leukemia cells express all four TRAIL receptors on the cell surface, as shown by using FACS analysis (Fig. 7), and are sensitive to
- TRAIL-induced apoptosis To test the involvement of DR4 versus DR5 in TRAIL-induced cell death, Colo205 cells were treated with neutralizing anti-DR4 or anti-DR5 antibody for 1 h before the addition of TRAIL. Both antibodies reduced TRAIL-mediated cell death and had an additive effect when used in combination (Fig. 8A). However, the DR5-neutralizing antibody was 3x more effective than the DR4-neutralizing antibody, demonstrating that TRAIL-induced apoptosis in Colo205 cells is primarily mediated by DR5. In contrast, the DR4 pathway is the major mediator of TRAIL-induced apoptosis in ML-I cells (Fig.
- Colo205 and ML-I cells were then treated with increasing concentrations of TRAIL or the DR5- spectfic variants D269H, D269HE195R, and D269H/T214R, and their cytotoxic potential was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
- MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- TRAIL was able to induce cell death in ML- I cells (Fig. 8D). Similar results were obtained by using EM-2 chronic myeloid leukemia cells expressing only the DR4 receptor and lacking the DR5 receptor and by using the ovarian cancer cell line A2780, which expresses DR5 but lacks DR4 on its surface and is relatively insensitive toward TRAIL- induced cell death. Although EM-2 cells were sensitive to TRAIL-induced cell death (50 ng/ml TRAIL initiating >80% cell death), treatment with any of the DR5 mutants failed to induce significant cell death (data not shown).
- D269H, D269HE195R, and D269HT214R are significantly increased, showing both an increased maximum response and drastically decreased EC50 values when compared with wild-type TRAIL (Fig. 8E and Table 3).
- An additional experiment using D269HE195R in wild-type BJAB cells responsive to both DR4- and DR5-mediated cell death (BJABwt), BJAB cells deficient in DR5 (BJABDR5 DEF), and BJAB cells deficient in DR5 and stably transfected with DR5 (BJABDR5 DEF+DR5) confirm our findings.
- D269HE195R was able to induce cell death in BJABwt cells but was unable to induce significant cell death in BJABDR5 DEF cells when compared with wild-type TRAIL.
- the cytotoxic potential was restored (Fig. 8F).
- the cytotoxic effects of these TRAIL variants on noncancerous human umbilical vein endothelial cells was assessed by incubating these cells in the presence of 100 ng/ml TRAIL or TRAIL variants. However, no cytotoxic effects were observed for TRAIL and the receptor- selective TRAIL variants (data not shown).
- Example 7 DR5-slelective TRAIL variants are more effective in tumor cell killing Colo205 cells express all four TRAIL receptors, and are sensitive to TRAIL-induced apoptosis, which is mediated predominantly via DR5 in these cells 10 .
- Colo205 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 niM L-glutamine, 50 U/ml penicillin and 5 mg/ml streptomycin and 10 mM sodium pyruvate and seeded at 2 x 10 5 cells/ml concentration 24 h before treatment (all reagents were from
- Annexin V assay wherein externalization of phosphatidyl serine (PS) on the plasma membrane of apoptotic cells was detected using Annexin V-FITC (IQ Corporation, Groningen, The Netherlands). Briefly, cells were trypsinized and were then collected by centrifugation at 400 g, washed in ice-cold calcium buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ) and incubated with Annexin V-FITC for 15 min in the dark prior to acquisition on a FACSCalibur flow cytometer (Becton Dickinson).
- the two DR5-selective TRAIL-variants displayed similar cytotoxic potential, which was 2.7-4.2 fold higher than that of wtTRAIL based on the IC50 values (determined from the linear range of the dose response curve).
- Caspase activation in response to WT TRAIL and DR5-selective variants was also measured and showed that 2.9-4.1 fold lower concentrations of the DR5-selective variants were sufficient to induce the same level of caspase activity (Figure 10B).
- Protein samples were denatured in Laemmli sample buffer and boiled for 5 min. Proteins were separated by 10% SDS- PAGE and transferred onto nitrocellulose membrane. Membranes were blocked for Ih in PBS containing 0.05% Tween20 and 5% (w/v) non-fat dried milk. The membranes were then incubated for Ih at room temperature with antibodies to actin (1:500; Sigma) or overnight at 4 0 C with antibodies to caspases-3 and -8 (1 : 1,000; Cell Signaling Technologies). This was followed by 2h incubation at room temperature with appropriate secondary antibodies (1 :5,000 for all antibodies). Protein bands were visualized using Supersignal Ultra Chemiluminescent Substrate (Pierce) on X-ray film (Agfa). The result of the Western blot is shown in Figure 1OC.
- A2780 ovarian carcinoma cells were cultured in DMEM with the same supplements as for Colo205 cells and seeded at 3.5 x 10 5 cells/ml concentration 24h before treatment.
- A2780 carcinoma cells were found to express high levels of DR5, DcRl DcR2 and no detectable DR4 on their surface and to be partially resistant to wtTRAIL-induced apoptosis ( Figures 1 1 & 13A). Similar to Colo205 cells, D269HE195R appeared to be a more effective inducer of apoptosis than wtTRAIL in A2780 cells. While wtTRAIL could not induce more than 24.3 ⁇ 4.5% cell death even at the highest concentration tested (250 ng/ml), 10 ng/ml D269HE195R was sufficient to induce 30.6 ⁇ 2.5% apoptosis measured by Annexin V assay as described above ( Figure 13A).
- a surface plasmon resonance (SPR) based receptor binding assay was carried out to compare the binding affinity of wtTRAIL to D269H and D269HE195R towards immobilized DcRl- and DcR2-Ig fusion proteins ( Figure 12). Binding experiments were performed at 37°C using a surface plasmon resonance-based biosensor (Biacore 3000, Biacore AB, Uppsala, Sweden) as previously described 10 . In short, DcRl-Ig and DcR2-Ig receptor chimeras (R&D Systems) were captured at a 35 ⁇ l/min flow rate using a protein A (Sigma) modified CM5 sensor chip (Biacore).
- Receptor chimeras were captured at a level of -500-800 response units (RU).
- Purified wtTRAIL and TRAIL variants were injected in three-fold at concentrations ranging from 250 nM to 2 nM at 70 ⁇ l/min flow rate using PBS (pH 7.4) (Invitrogen) supplemented with 0.005% vol/vol P20 (Biacore) as running and sample buffer. Binding of ligands to the receptors was monitored in real-time. Between injections the protein A sensor surface was regenerated using a 30s pulse of 0.1 M glycine 0.5 M NaCl pH 3. Due to the very slow dissociation of the TRAIL- receptor complex, only pre-steady state binding data could be obtained.
- Affinity of the ligands was assessed by measuring receptor binding in a concentration range of 0.1 - 250 nM.
- the binding of D269H and D269HE195R towards DcRl was approximately 20 fold reduced when compared to wtTRAIL ( Figure 12A). Binding of D269H and D269HE195R toward DcR2 was also reduced, however the effect was much less pronounced compared to the reduction in binding observed with the DcRl ( Figure 12B).
- wtTRAIL, D269H and D269HE195R were incubated for 30 min with increasing concentrations of soluble, recombinant DcRl-Ig (sDcRl) and DcR2-Ig (sDcR2), prior to addition to Colo205 cell cultures at 10 ng/ml final ligand concentration.
- Induction of cell death was assessed by Annexin V assay 3 h after treatment ( Figure 14), as described above.
- Pre-incubation of wtTRAIL with 0.5 ⁇ g/ml sDcRl completely blocked its pro-apoptotic activity.
- D269HE195R Although high concentration of soluble DcR2 was able to limit the activity of D269HE195R, cell surface expressed DcR2 was not sufficient to reduce apoptosis-induction by D269HE195R. This may be due to a different ratio or of DR5:DcR2 or difference in structure of full length, surface expressed DR5 and DcR2 compared to the soluble, recombinant, Fc-fusion receptors.
- Example 9 DR5-seIective variants are not toxic towards non transformed cells The importance of decoy receptors in protecting non-transformed cells against TRAIL has been suggested by many reports. 37 Thus, the toxic effect of the DR5-selective variants on noncancerous cells was assessed.
- Human dermal fibroblasts were grown in low glucose DMEM (Sigma) supplemented with 10% fetal calf serum, 50 U/ml penicillin and 5 mg/ml streptomycin; human umbilical vein endothelial cells (HUVEC, PromoCell) were cultured in Endothelial Cell Growth Medium with SupplementMix (PromoCell).
- Fibroblasts and HUVEC cells were seeded at 1.3 x 10 5 cells/ml 24 h before treatment with increasing concentrations of TRAIL and DR5- selective variants, D269H and D269HE195R (50-500 ng/ml) for 24 h and cell viability was measured using MTT assay, wherein 200 ⁇ g/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide) was added to control and treated cells and incubated for 3 h at 37°C.
- MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide
- Example 10 DBS-selective variants activate DR5 17-fold faster than wtTRAIL
- Colo205 cells were incubated with 30 ng/ml concentration of the ligands (at which concentration both wtTRAIL and D269HE195R induce maximal cell death) for varying times (5 - 180 min), after which the ligands were washed out and the incubation was continued in normal growth medium to 180 min in total, when the cells were harvested and as a marker of receptor activation, pro- caspase-8 processing was measured.
- Treatment with D269HE195R resulted in significant pro- caspase-8 processing already after 5 min of incubation, while 60 min was required for a comparable level of pro-caspase-8 processing by wtTRAIL (Figure 16A).
- Colo205 cells were treated with 30 ng/ml wtTRAIL or D269HE195R for the times indicated, after which the ligand was washed out and the incubation continued up to 240 min in total in normal growth medium.
- the cells were harvested and cell death was quantified with Annexin V labelling ( Figure 16B), as described above.
- the data was fitted with an interpolating spline using the software MatlabR. From the fitted curve the incubation time required for 50% efficacy was determined to be 60.81 min for wtTRAIL.
- the same value for D269HE195R was 3.55 min, showing that receptor activation by D269HE195R occurs 17 fold faster than with wtTRAIL.
- Colo205 cells were pre-incubated with wtTRAIL for 5 or 10 min, washed and then D269HE195R was added to the cells and incubated for another 5 or 10 min (enough to induce cell death) and removed. The cells were then incubated to a total of 180 min in normal growth medium and then the level of cell death was determined (Figure 16C). The results show that pre-incubation with the wtTRAIL did not affect apoptosis induction by the double mutant.
- Example 11 TRAIL and DR5-selective variants exhibit higher synergism with aspirin in Colo205 cells and tumors induced by inoculation with Colo205 cells.
- Example 12 Cell killing of three colon carcinoma cell lines by wtTRAIL and DR5-selective TRAIL
- Colo205 and LoVo showed higher sensitivity to the DR5-selective variant compared to wtTRAIL.
- SW948 cells showed higher sensitivity to wtTRAIL compared. Further studies have shown that in SW948 cells selectively transmits the death inducing signal predominantly through the DR4 receptor (data not shown).
- Example 13 TRAIL and DR5-selective variants exhibit higher synergism with aspirin in Colo205 cells and tumors induced by inoculation with Colo205 cells.
- Example 14 Cell killing of three colon carcinoma cell lines by wtTRAIL and DR5-seIective TRAIL
- Colo205 and LoVo showed higher sensitivity to the DR5-selective variant compared to wtTRAIL.
- SW948 cells showed higher sensitivity to wtTRAIL compared. Further studies have shown that in SW948 cells selectively transmits the death inducing signal predominantly through the DR4 receptor (data not shown).
- chemotherapeutics namely 5-FIurouracil and Aspirin have been tested in combination with wtTRAIL and DR5-selective variant.
- Colo205 and LoVo cells both 5-FU and Aspirin enhanced apoptosis induced by wtTRAIL and DR5-selective variants ( Figures 17, 22, 25, 26 & 3 1-34).
- SW948 cells 5-FU enhanced sensitivity to wtTRAIL but not to the DR5- selective variant ( Figures 27-30).
- Aspirin enhanced the effect the DR5-selective variant in SW948 cells, suggesting that chemotherapeutics can modulate DR5 sensitivity in cells.
- Example 15 Cell killing of three cervical carcinoma cell lines by wtTRAIL and DR5- selective TRAIL
- DR5-specific/selective variant D269HE195R was compared initially as a single agent. The sensitivity pattern is illustrated in the Table 5, with — indicating resistant, +: minimally sensitive, ++: moderately sensitive, +++: very sensitive.
- TRAIL variants were made by site-directed mutagenesis of the extra-cellular domain of human TRAIL, comprising amino acids 114-281.
- TRAIL proteins were all produced from pET15b as recombinant, soluble proteins in Escherichia coli and purified in three chromatographic steps 10 .
- the purified proteins were characterised for binding to the various receptors in ELISA assays and in a pre-steady state approach using Surface Plasmon Resonance (SPR) assays, and furthermore by their ability to induce cell death in cell lines with different TRAIL receptor expression patterns.
- SPR Surface Plasmon Resonance
- variant D269HE195R 10 Several DR5-specific variants have been designed and characterised, amongst which is variant D269HE195R 10 .
- the two amino acid changes in this variant occur at the interface of TRAIL and receptor, as depicted in Figure 35.
- the positions of residues E 195 and D269 are indicated in a top and side view of the trimer-trimer complex of TRAIL and DR5.
- Figure 35A shows a typical sensorgram when TRAIL binds to immobilised TRAIL receptor. Especially at low concentrations, the curves do not show significant dissociation. In other experiments dissociation was continued for periods of up to 2,000 sec, with similar results. As a consequence it is essentially impossible to obtain a dissociation rate constant and thus to calculate the affinity from the association and dissociation rate constants. We therefore chose to use the pre-steady state approach to analyse the data 10 . SPR analysis of the ability of this mutant in comparison to that of wtTRAIL to bind to immobilised receptors is depicted in Figure 35B.
- D269HE195R The DR5-selectivity of D269HE195R was also demonstrated in cancer cell lines.
- Figure 40 the apoptotic activity of D269HE195R is shown in comparison to wtTRAIL in colon carcinoma cancer cell line Colo205, where living cells were stained with a soluble tetrazolium-based MTS proliferation reagent (Promega). Colo205 cells are sensitive to TRAIL induced apoptosis mainly via the DR5 receptor.
- our variant D269HE195R is efficient at a several fold lower concentration than needed for wtTRAIL. The increase in efficiency is not only demonstrated by a dose-response curve, but can also be observed in time.
- the model simulates the interactions occurring on the surface of Colo205 cells upon exposure of the cells to wtTRAIL or DR5-variant.
- the model describes the binding of the ligand (wtTRAIL or D269H/E195R) to the receptors in a stepwise fashion and allows for the formation of both homomeric (e.g. TRAIL-3DR5) and heteromeric receptor complexes (e.g. TRAIL-2DR5-DcR2).
- Receptor binding of TRAIL and D269HE195R on the cell surface was simulated using a mathematical model describing all possible binding events. Both the formation of homomeric (e.g. TRAIL-3DR5) and heteromeric ligand-receptor complexes (e.g. TRAIL-2DR5-DcR2) were allowed and binding was simulated in a stepwise fashion. On rates and off rates measured for TRAIL binding to monomeric DR4, DR5, DcRl 1 and DcR2 receptors were taken from the report of Lee and colleagues 40 . In contrast to the more commonly used dimeric TRAIL-receptor-IgG Fc chimeric constructs, Lee et al., employed monomeric TRAIL-receptor constructs.
- Stepwise constants for going from a single ligand-bound receptor, via two ligand-bound receptors, to the complex consisting of 3 receptors bound by a trimeric ligand were estimated in the following way: the k on reported by Lee et al., was assigned to the first binding event.
- the first association step is entropically the most unfavourable one due to loss of rotational degrees of freedom when going from an unbound state to a bound state.
- the k Qn for the second and third step was increased by 2 kcal/mol.
- the reported k ot ⁇ was assigned to the third step.
- the k ot r for the second and the first step was increased by a factor ten and hundred, respectively.
- the effect of avidity on the apparent off-rate will be larger upon binding two receptor subunits instead of one than for binding three subunits instead of two, hence the difference in factors used.
- an extra step to an "activated" receptor complex was introduced in order to mathematically describe the assembly of the intracellular DISC. Formation of the DISC reduces the likelihood of complex disassembly and participation in rebinding events with other free death or decoy receptors.
- DcRl does not contain an intracellular domain and DcR2 only contains a truncated Death domain, no "activation" step was added for the decoy receptors (Table Ia).
- the number of DR5 receptors on the surface of Colo205 was estimated by comparing the receptor expression levels on the surface of Colo205 to MD MBA-231 cells and subsequently relating this ratio to the number of surface expressed receptors determined for MD MBA-231 cells by Kim and co-workers 40 .
- Apparent affinities for D269HE195R were taken from van der Sloot et al., 10 .
- the increase in affinity of D269HE195R for DR5 was completely attributed to the k on , as changes in off-rate in the SPR sensorgram between wtTRAIL and D269HE195R could not be calculated due to minimal and very slow rate of ligand dissociation.
- the model was simulated using the stochastic next subvolume method (Elf and Ehrenberg) as implemented in SmartCell version 4.2 (2, 8) (http://smartcell.crg.es/). Receptor binding was simulated with the ligands continuously present, or by ligand removal during the course of the simulation in order to simulate the washout experiment. In the later case the system was simulated for the indicated time and then the free ligand concentration was set to zero.
- the "ED 50 " of the wtTRAIL-3DR5 complex was reached within -500 sec, while the ED 50 for DR4 was reached after -1,200 sec ( Figure 55A).
- the number of wtTRAIL-3DR5 complexes formed after 1 h was highest, while the number of TRAIL-3DR4, TRAIL-3DcRl and TRAIL-3DcR2 were 70%, 6% and 0% of the number of TRAIL-3DR5 complexes, respectively.
- the mathematical simulations showed that the reduced affinity of D269HE195R for DR4, DcRl and DcR2 in combination with an increased k on for DR5, all contributed to produce a 17-fold increase in the rate of homotrimeric DR5 receptor complex formation.
- the model explains other experimental data: it shows that D269HE195R is already bound to the majority of the available DR5 receptors within 5 min; washing out of unbound D269HE195R consequently would hardly influence apoptosis induction.
- the model also reveals that blocking the decoy receptors or improving the binding of wtTRAIL towards DR5 separately is not sufficient to enhance its activity to a level comparable to that of D269HE195R.
- wtTRAIL can still form inactive DR4/DR5 heterotrimers, therefore only an improvement of its binding to DR5 will be sufficient to prevent the formation of inactive heteromeric complexes.
- This phenomenon has implications for the design of other faster working ligands; engineering faster kinetics for other promiscuous ligands cannot be achieved by only increasing the k o ⁇ for the target receptor but also needs to decrease the affinity for the other receptors. Given the short in vivo half-life reported for wtTRAIL 46, faster receptor binding kinetics (and faster induction of apoptosis) can generate a considerable therapeutic advantage.
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JP2010538945A JP2011506592A (en) | 2007-12-17 | 2008-12-17 | TRAIL variants for the treatment of cancer |
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CN2008801269253A CN102388062A (en) | 2007-12-17 | 2008-12-17 | Trail variants for treating cancer |
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US20110165265A1 (en) | 2011-07-07 |
WO2009077857A3 (en) | 2009-12-10 |
JP2011506592A (en) | 2011-03-03 |
CA2709463A1 (en) | 2009-06-25 |
AU2008337231A1 (en) | 2009-06-25 |
EP2238161A2 (en) | 2010-10-13 |
CN102388062A (en) | 2012-03-21 |
GB0724532D0 (en) | 2008-01-30 |
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