Patent Application
20200085928
The present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding PD-L1. In particular, the present invention relates to said attenuated strain of Salmonella for use in the treatment of cancer.
An important characteristic of the immune system is its ability to discriminate between “self” and “foreign.” To this end, it uses “checkpoint”—molecules on certain immune cells that need to be activated (or inactivated) to start an immune response.
Principally, the immune system has the capacity to recognize and destroy neoplastic cells. T-cells appear to be major effectors in anti-cancer immunity. However, many tumors develop mechanisms to evade the immune system to enhance their survival. Immune regulatory proteins such as checkpoint proteins and their ligands play vital roles in immune suppression and tolerance induction of anti-cancer immune responses.
Programmed cell death 1 (PD-1) is expressed on the surface of T-cells and transmits inhibitory signals that maintain T-cell functional silence against cognate antigens. Its ligand PD-L1 is normally expressed on antigen-presenting cells, placental cells and non-hematopoietic cells in inflammatory microenvironments. PD-L1 has been reported to be expressed on immunosuppressive myeloid-derived suppressor cells (MDSC). In addition, PD-L1 is extensively expressed on the surface of various types of cancer cells, which use the PD-1/PD-L1 signaling axis to escape the host immune system. Expression of PD-L1 by cancer cells was shown to correlate with disease stage and poor patient prognosis.
The potential of targeting the PD-L1/PD-1 signaling pathway has been demonstrated in clinical trials evaluating several monoclonal anti-PD-1 and anti-PD-L1 antibodies, which function by inhibiting binding of PD-L1 to PD-1. These antibodies are designed to unleash or enhance pre-existing anti-cancer immune responses. Different agents are currently investigated in clinical trials in patients like Opdivo (nivolumab, an anti-PD-1 antibody) in various solid tumours and hematological malignancies, pembrolizumab (Keytruda, an anti-PD-1 antibody) in various solid tumours and hematological malignancies, CT-011 (anti-PD-1) in conjunction with a dendritic cell vaccine in AML following chemotherapy-induced remission, and lirilumab (anti-KIR) combined with rituximab in relapsed, refractory or high-risk untreated patients with CLL. Furthermore, anti-PD-L1 monoclonal antibodies including atezolizumab, avelumab or durvalumab are in advanced clinical development in various cancer indications.
Only recently, the existence of PD-L1-specific T-cells has been described.
WO 2013/056716 discloses a HLA (human leukocyte antigen)-A2-restricted PD-L1 peptide vaccine.
WO 2014/005683 discloses an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding a VEGF receptor protein for use in cancer immunotherapy, particularly for use in the treatment of pancreatic cancer.
WO 2016/202459 discloses a cancer therapy approach comprising the combined administration of an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding a VEGF receptor protein and a checkpoint inhibitor antibody.
To the inventor's knowledge, no bacterial DNA vaccine targeting PD-L1 with the aim to induce PD-L1-specific cytotoxic immune cells that directly target PD-L1 positive tumor cells has been reported. Furthermore, no oral cancer vaccine targeting PD-L1 has been described.
In view of the prior art, it is an object of the present invention to provide a novel safe and efficient PD-L1 targeting cancer treatment. Such a novel therapy approach would offer major advantages for improving the treatment options for cancer patients.
The present invention is based on the surprising finding that a Salmonella-based DNA delivery vehicle carrying a recombinant DNA construct encoding either full length PD-L1 or truncated PD-L1 comprising the extracellular domain of PD-L1 exhibits high antitumor efficacy in C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia.
Thus, in a first aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding PD-L1.
In particular embodiments, the attenuated strain of Salmonella is of the species Salmonella enterica. Particularly, the attenuated strain of Salmonella is Salmonella typhi Ty21a.
In particular embodiments, the expression cassette is a eukaryotic expression cassette. Particularly, the expression cassette comprises a CMV promoter.
In particular embodiments, PD-L1 is selected from the group consisting of full length PD-L1 and a truncated PD-L1 comprising the extracellular domain of PD-L1. A truncated PD-L1 may comprise an amino acid sequence of amino acids 19 to 238 of of SEQ ID NO 13, the amino acid sequence of SEQ ID NO 13, the amino acid sequence of SEQ ID NO 2 or may comprise an amino acid sequence that shares at least 80% sequence identity with amino acids 19 to 238 of SEQ ID NO 13, with SEQ ID NO 13 or with SEQ ID NO 2. In particular embodiments the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 1 and a protein that shares at least 80% sequence identity therewith. In particular other embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 2 and a protein that shares at least 80% sequence identity therewith. In particular other embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 13 and a protein that shares at least 80% sequence identity therewith. In particular other embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of amino acids 19 to 238 of SEQ ID NO 13 and a protein that shares at least 80% sequence identity therewith. Particularly, PD-L1 has the amino acid sequence as found in SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 13, preferably PD-L1 comprises the amino acid sequence of amino acids 19 to 238 of SEQ ID NO 13. In one embodiment PD-L1 comprises at least the extracellular domain with or without the signaling peptide.
In particular embodiments, PD-L1 is encoded by the nucleic acid sequence as found in SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 14.
In particular embodiments, the DNA molecule further comprises the kanamycin antibiotic resistance gene, the pMB1 ori and a CMV promoter.
Particularly, the DNA molecule further comprises the DNA sequence as found in SEQ ID NO 5.
In a second aspect, the present invention relates to the attenuated strain of Salmonella according to the present invention for use as a medicament.
In particular embodiments, the attenuated strain of Salmonella is for use in the treatment of cancer.
In particular embodiments, the attenuated strain of Salmonella is for use in cancer immunotherapy.
In particular embodiments, the treatment of cancer further comprises chemotherapy, radiotherapy or biological cancer therapy. Particularly, the attenuated strain of Salmonella is administered before, during and/or after the chemotherapy or the radiotherapy treatment or the biological cancer therapy. More particularly, the attenuated strain of Salmonella is administered before and during the chemotherapy or the radiotherapy treatment or the biological cancer therapy.
In particular embodiments, the biological cancer therapy comprises administration of at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen. In particular embodiments, the at least one further DNA vaccine is selected from at least one attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a tumor antigen and/or a tumor stroma antigen. This includes at least one further DNA vaccine comprising at least one copy of a DNA molecule comprising an expression cassette encoding a tumor antigen and/or at least one further DNA vaccine comprising at least one copy of a DNA molecule comprising an expression cassette encoding a tumor stroma antigen. Particularly, said at least one further attenuated strain of Salmonella is Salmonella typhi Ty21a comprising a eukaryotic expression cassette.
In one embodiment said tumor antigen is selected from Wilms' Tumor Protein (WT1), Mesothelin (MSLN), carcinoembryonic antigen (CEA) and CMV pp65. In particular embodiments, said tumor antigen encoded by said at least one further DNA vaccine is selected from the group consisting of Wilms' Tumor Protein (WT1) having the amino acid sequence as found in SEQ ID NO 6 and a protein that shares at least about 80% sequence identity therewith, Mesothelin (MSLN) having the amino acid sequence as found in SEQ ID NO 7 and a protein that shares at least about 80% sequence identity therewith, CEA having the amino acid sequence as found in SEQ ID NO 8 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 9 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 10 and a protein that shares at least about 80% sequence identity therewith, and CMV pp65 having the amino acid sequence as found in SEQ ID NO 11 and a protein that shares at least about 80% sequence identity therewith. Particularly, Wilms' Tumor Protein (WT1) has the amino acid sequence as found in SEQ ID NO 6, Mesothelin (MSLN) has the amino acid sequence as found in SEQ ID NO 7, CEA has the amino acid sequence as found in SEQ ID NO 8, and CMV pp65 has the amino acid sequence as found in SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. In one embodiment said tumor stroma antigen is VEGFR-2 or fibroblast activation protein (FAP), preferably VEGFR-2. Particularly, said tumor stroma antigen encoded by said at least one further DNA vaccine is selected from the group consisting of VEGFR-2 having the amino acid sequence as found in SEQ ID NO 12 and a protein that shares at least about 80% sequence identity therewith and human fibroblast activation protein (FAP). Particularly, VEGFR-2 has the amino acid sequence as found in SEQ ID NO 12.
In particular embodiments, the attenuated strain of Salmonella is administered orally.
In particular embodiments, the cancer is selected from lymphoma, leukemia, myeloma, lung cancer, in particular non-small cell lung cancer (NSCLC), melanoma, renal cell cancer, ovarian cancer, glioblastoma, merkel cell carcinoma, bladder cancer, head and neck cancer, colorectal cancer, esophagial cancer, cervical cancer, gastric cancer, hepatocellular cancer, prostate cancer, breast cancer, pancreatic cancer, and thyroid cancer.
In particular embodiments, the single dose of the attenuated strain of Salmonella comprises from about 105 to about 1011, particularly from about 106 to about 1010, more particularly from about 106 to about 109, more particularly from about 106 to about 108, most particularly from about 106 to about 107 colony forming units (CFU).
In particular embodiments, the attenuated strain of Salmonella is for use in individualized cancer immunotherapy comprising the step of assessing the PD-L1 expression pattern and/or the pre-immune response against PD-L1 of a patient.
In a first aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding PD-L1.
The live attenuated Salmonella strain according to the present invention stably carries a recombinant DNA molecule encoding PD-L1. It can be used as a vehicle for the oral delivery of this recombinant DNA molecule.
In the context of the present invention, the term “attenuated” refers to a bacterial strain of reduced virulence compared to the parental bacterial strain, not harboring the attenuating mutation. Attenuated bacterial strains have preferably lost their virulence but retained their ability to induce protective immunity. Attenuation can be accomplished by deletion of various genes, including virulence, regulatory, and metabolic genes. Attenuated bacteria may be found naturally or they may be produced artificially in the laboratory, for example by adaptation to a new medium or cell culture or they may be produced by recombinant DNA technology. Administration of about 1011 CFU of the attenuated strain of Salmonella according to the present invention preferably causes Salmonellosis in less than 5%, more preferably less than 1%, most preferably less than 1‰ of subjects.
In the context of the present invention, the term “comprises” or “comprising” means “including, but not limited to”. The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. The term “comprising” thus includes the more restrictive terms “consisting of” and “essentially consisting of”. In one embodiment the term “comprising” as used throughout the application and in particular within the claims may be replaced by the term “consisting of”.
The DNA molecule comprising an expression cassette encoding PD-L1 is suitably a recombinant DNA molecule, i.e. an engineered DNA construct, preferably composed of DNA pieces of different origin. The DNA molecule can be a linear nucleic acid, or preferably, a circular DNA plasmid generated by introducing an open reading frame encoding PD-L1 into an expression vector plasmid. Suitable expression vector plasmids are for instance, without being limited thereto, pVAX1™ (Invitrogen, San Diego, Calif.) or pcDNA™3.1 (Invitrogen, San Diego, Calif.), or expression vector plasmids derived thereof. The expression vector plasmids may contain a high copy origin, such as a pUC ori, or a low copy origin, such as a pMB1 ori.
In the context of the present invention, the term “expression cassette” refers to a nucleic acid unit comprising at least one open reading frame (ORF) under the control of regulatory sequences controlling its expression. Expression cassettes can preferably mediate transcription of the included open reading frame encoding a recombinant protein, such as PD-L1, in a target cell. Expression cassettes typically comprise a promoter, at least one open reading frame and a transcription termination signal.
In particular embodiments, the attenuated strain of Salmonella is of the species Salmonella enterica. Attenuated derivatives of Salmonella enterica are attractive vehicles for the delivery of heterologous antigens to the mammalian immune system, since S. enterica strains can potentially be delivered via mucosal routes of immunization, i.e. orally or nasally, which offers advantages of simplicity and safety compared to parenteral administration. Furthermore, Salmonella strains elicit strong humoral and cellular immune responses at the level of both systemic and mucosal compartments. Batch preparation costs are low and formulations of live bacterial vaccines are highly stable. Attenuation can be accomplished by deletion of various genes, including virulence, regulatory, and metabolic genes.
Several Salmonella typhimurium strains attenuated by aro mutations have been shown to be safe and effective delivery vehicles for heterologous antigens in animal models.
In particular embodiments, the attenuated strain of Salmonella is Salmonella typhi Ty21a. The live, attenuated S. typhi Ty21a strain is the active component of Typhoral L®, also known as Vivotif® (manufactured by Berna Biotech Ltd., a Crucell Company, Switzerland). It is currently the only licensed live oral vaccine against typhoid fever. This vaccine has been extensively tested and has proved to be safe regarding patient toxicity as well as transmission to third parties (Wahdan et al., J. Infectious Diseases 1982, 145:292-295). The vaccine is licensed in more than 40 countries and has been used in millions of individuals including thousands of children for prophylactic vaccination against typhoid fever. It has an unparalleled safety track record. There is no data available indicating that S. typhi Ty21a is able to enter the bloodstream systemically. The live attenuated Salmonella typhi Ty21a vaccine strain thus allows specific targeting of the immune system in the gut, while being safe and well-tolerated. The Marketing Authorization number of Typhoral L® is PL 15747/0001 dated 16 Dec. 1996. One dose of vaccine contains at least 2×109 viable S. typhi Ty21a colony forming units and at least 5×109 non-viable S. typhi Ty21a cells.
This well-tolerated, live oral vaccine against typhoid fever was derived by chemical mutagenesis of the wild-type virulent bacterial isolate S. typhi Ty2 and harbors a loss-of-function mutation in the galE gene resulting in its inability to metabolize galactose. The attenuated bacterial strain is also not able to reduce sulfate to sulfide which differentiates it from the wild-type Salmonella typhi Ty2 strain. With regard to its serological characteristics, the Salmonella typhi Ty21a strain contains the O9-antigen which is a polysaccharide of the outer membrane of the bacteria and lacks the O5-antigen which is in turn a characteristic component of Salmonella typhimurium. This serological characteristic supports the rationale for including the respective test in a panel of identity tests for batch release.
In particular embodiments, the expression cassette is a eukaryotic expression cassette. Particularly, the expression cassette comprises a CMV promoter. In the context of the present invention, the term “eukaryotic expression cassette” refers to an expression cassette which allows for expression of the open reading frame in a eukaryotic cell. It has been shown that the amount of heterologous antigen required to induce an adequate immune response may be toxic for the bacterium and may result in cell death, over-attenuation or loss of expression of the heterologous antigen. Using a eukaryotic expression cassette that is not expressed in the bacterial vector but only in the target cell may overcome this toxicity problem and the protein expressed typically exhibits a eukaryotic glycosylation pattern.
A eukaryotic expression cassette comprises regulatory sequences that are able to control the expression of an open reading frame in a eukaryotic cell, preferably a promoter and a polyadenylation signal. Promoters and polyadenylation signals included in the recombinant DNA molecules comprised by the attenuated strain of Salmonella of the present invention are preferably selected to be functional within the cells of the subject to be immunized. Examples of suitable promoters, especially for the production of a DNA vaccine for humans, include but are not limited to promoters from Cytomegalovirus (CMV), such as the strong CMV immediate early promoter, Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV), such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, Epstein Barr Virus (EBV), and from Rous Sarcoma Virus (RSV), the synthetic CAG promoter composed of the CMV early enhancer element, the promoter, the first exon and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta globin gene, as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. In a particular embodiment, the eukaryotic expression cassette contains the CMV promoter. In the context of the present invention, the term “CMV promoter” refers to the strong immediate-early cytomegalovirus promoter.
Examples of suitable polyadenylation signals, especially for the production of a DNA vaccine for humans, include but are not limited to the bovine growth hormone (BGH) polyadenylation site, SV40 polyadenylation signals and LTR polyadenylation signals. In a particular embodiment, the eukaryotic expression cassette included in the recombinant DNA molecule comprised by the attenuated strain of Salmonella of the present invention comprises the BGH polyadenylation site.
In addition to the regulatory elements required for heterologous gene expression, like a promoter and a polyadenylation signal, other elements can also be included in the recombinant DNA molecule. Such additional elements include enhancers. The enhancer can be, for example, the enhancer of human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
Regulatory sequences and codons are generally species dependent, so in order to maximize protein production, the regulatory sequences and codons are preferably selected to be effective in the species to be immunized. The person skilled in the art can produce recombinant DNA molecules that are functional in a given subject species.
In one embodiment, PD-L1 is selected from the group consisting of full length PD-L1 and a truncated PD-L1 comprising the extracellular domain of PD-L1. A truncated PD-L1 may comprise an amino acid sequence of amino acids 19 to 238 of of SEQ ID NO 13, the amino acid sequence of SEQ ID NO 13, the amino acid sequence of SEQ ID NO 2 or may comprise an amino acid sequence that shares at least 80% sequence identity with amino acids 19 to 238 of SEQ ID NO 13, with SEQ ID NO 13 or with SEQ ID NO 2. In particular embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 1 and a protein that shares at least 80% sequence identity therewith. In particular other embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 2 and a protein that shares at least 80% sequence identity therewith. In yet other particular embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence as found in SEQ ID NO 13 and a protein that shares at least 80% sequence identity therewith. In particular other embodiments, PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of amino acids 19 to 238 of SEQ ID NO 13 and a protein that shares at least 80% sequence identity therewith. Particularly, PD-L1 has the amino acid sequence as found in SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 13, preferably PD-L1 comprises the amino acid sequence of amino acids 19 to 238 of SEQ ID NO 13. In one embodiment PD-L1 comprises at least the extracellular domain with or without the signaling peptide.
In this context, the term “about” or “approximately” means within 80% to 120%, alternatively within 90% to 110%, including within 95% to 105% of a given value or range.
In the context of the present invention, the term “protein that shares at least about 80% sequence identity with a given protein sequence, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2 or 13” refers to a protein that may differ in the amino acid sequence and/or the nucleic acid sequence encoding the amino acid sequence of said reference protein, e.g., PD-L1 having the amino acid sequence of SEQ ID NO 1, 2 or 13. The protein may be of natural origin, e.g. a mutant version of a wild-type protein, e.g. a mutant version of a wild type PD-L1, or a homolog of a different species, or an engineered protein, e.g., engineered PD-L1. It is known that the usage of codons is different between species. Thus, when expressing a heterologous protein in a target cell, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon usage of the target cell. Methods for designing and constructing derivatives of a given protein are well known to anyone of ordinary skill in the art.
The protein that shares at least about 80% sequence identity with a given protein sequence, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2 or 13, may contain one or more mutations comprising an addition, a deletion and/or a substitution of one or more amino acids in comparison to the reference protein sequence, e.g., PD-L1 having the amino acid sequence of SEQ ID NO 1, 2 or 13. The same applies to PD-L1 having the amino acid sequence of amino acids 19 to 238 of SEQ ID NO 13. The one or more mutations comprising an addition, a deletion and/or a substitution of one or more amino acids include less than 10 mutations, less than 9 mutations, less than 8 mutations, less than 7 mutations, less than 6 mutations, less than 5 mutations, less than 4 mutations, less than 3 mutations, less than 2 mutations or one mutation in the amino acid sequence of SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13. According to the teaching of the present invention, said deleted, added and/or substituted amino acids may be consecutive amino acids or may be interspersed over the length of the amino acid sequence of the protein that shares at least about 80% sequence identity with a reference protein, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13. According to the teaching of the present invention, any number of amino acids may be added, deleted, and/or substitutes, as long as the amino acid sequence identity with the reference protein is at least about 80%. Preferably, the mutated protein is immunogenic. Preferably, the immunogenicity of the protein which shares at least about 80% sequence identity with a given reference protein, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13, is reduced by less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% compared to said reference protein, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13. The immunogenicity may, e.g., be measured by ELISA. Methods for designing and constructing protein homologues and for testing such homologues for their immunogenic potential are well known to anyone of ordinary skill in the art. In particular embodiments, the sequence identity with the reference protein, e.g., PD-L1 having the amino acid sequence of SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13 is at least about 80%, at least about 85%, at least about 90%, or most particularly at least about 95%. Methods and algorithms for determining sequence identity including the comparison of a parental protein and its derivative having deletions, additions and/or substitutions relative to a parental sequence, are well known to the practitioner of ordinary skill in the art. On the DNA level, the nucleic acid sequences encoding the protein that shares at least about 80% sequence identity with a given reference protein, e.g., PD-L1 having the amino acid sequence as found in SEQ ID NO 1, 2, 13 or amino acids 19 to 238 of SEQ ID NO 13, may differ to a larger extent due to the degeneracy of the genetic code.
In particular embodiments, PD-L1 is encoded by the nucleic acid sequence as found in SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 14.
In particular embodiments, the DNA molecule comprises the kanamycin antibiotic resistance gene, the pMB1 ori and a CMV promoter. In particular embodiments, the recombinant DNA molecule is derived from commercially available pVAX1™ expression plasmid (Invitrogen, San Diego, Calif.). This expression vector was modified by replacing the high copy pUC origin of replication by the low copy pMB1 origin of replication of pBR322. The low copy modification was made in order to reduce the metabolic burden and to render the construct more stable. The generated expression vector backbone was designated pVAX10.
In particular embodiments, the DNA molecule further comprises the DNA sequence as found in SEQ ID NO 5 (vector backbone pVAX10).
Inserting the ORF encoding human PD-L1 having the amino acid sequence of SEQ ID NO 1 into the expression vector backbone via NheI/XhoI yielded the expression plasmid pVAX10.PD-L1h. The DNA vaccine comprising the attenuated Salmonella strain Ty21a harboring the expression plasmid pVAX10.PD-L1h is designated VXM10h. Inserting the ORF encoding human PD-L1 having the amino acid sequence of SEQ ID NO 2 into the expression vector backbone via NheI/XhoI yielded the expression plasmid pVAX10.PD-L1ha. The DNA vaccine comprising the attenuated Salmonella strain Ty21a harboring the expression plasmid pVAX10.PD-L1ha is designated VXM10ha. Inserting the ORF encoding human PD-L1 having the amino acid sequence of SEQ ID NO 13 into the expression vector backbone via NheI/XhoI yielded the expression plasmid pVAX10.PD-L1 hb. The DNA vaccine comprising the attenuated Salmonella strain Ty21a harboring the expression plasmid pVAX10.PD-L1 hb is designated VXM10hb.
The attenuated strain of Salmonella encoding PD-L1 may be provided in a pharmaceutical composition. The pharmaceutical composition may be in the form of a solution, a suspension, an enteric coated capsule, a lyophilized powder or any other form suitable for the intended use.
The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients.
In the context of the present invention, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication. Suitable excipients include antiadherents, binders, coatings, disintegrants, flavors, colors, lubricants, glidants, sorbents, preservatives and sweeteners.
In the context of the present invention, the term “pharmaceutically acceptable” refers to molecular entities and other ingredients of pharmaceutical compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human). The term “pharmaceutically acceptable” may also mean approved by a regulatory agency of a Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and, more particularly, in humans.
In particular embodiments, the pharmaceutical composition is provided as drinking solution. This embodiment offers the advantage of improved patient compliance and allows for rapid, feasible and affordable mass vaccination programs.
In particular, suitable drinking solutions comprise means to neutralize gastric acids to at least to a certain degree, i.e. to bring the pH of the gastric juice closer to a pH of 7. In a particular embodiment, the drinking solution is a buffered suspension obtained by suspending the attenuated strain of Salmonella according to the present invention in a suitable buffer, preferably in a buffer that neutralizes gastric acids to at least a certain degree, preferably in a buffer containing 2.6 g sodium hydrogen carbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate and 100 ml of drinking water.
In a second aspect, the present invention relates to the attenuated strain of Salmonella according to the present invention for use as a medicament.
In particular embodiments, the attenuated strain of Salmonella is for use in the treatment of cancer.
In particular embodiments, the attenuated strain of Salmonella is for use as a vaccine.
In particular embodiments, the attenuated strain of Salmonella is for use in cancer immunotherapy.
Without wishing to be bound by theory, it is believed that contrary to monoclonal anti-PD-1 and anti-PD-L1 antibodies, which mediate checkpoint inhibition by preventing binding of the PD-L1 ligand to PD-1, the attenuated strain of Salmonella according to the present invention elicits a PD-L1 specific T-cell response that leads to the destruction of PD-L1-positive tumor cells. Additionally, particularly in the case of using only the extracellular domain of human PD-L1 (SEQ ID NO 13), as in the use of plasmid pVAX10.PD-L1hb, the PD-L1-based protein product being expressed may be secreted and may result in an anti-PD-L1 antibody response, which may further support the therapeutic effect of the approach according to the present invention. Without being bound by theory using truncated human PD-L1 lacking the signal peptide, e.g., truncated human PD-L1 comprising the amino acid sequence of SEQ ID NO 2, as in the use of plasmid pVAX10.PD-L1ha, and of amino acids 19 to 238 of SEQ ID NO 13 (corresponding to the extracellular domain of PD-L1), the PD-L1-based protein product being expressed may accumulate in the cytoplasm and may result in an enhanced cytotoxic T cell response against PD-L1, which may further support the therapeutic effect of the approach according to the present invention. Thus, in one embodiment the PD-L1 comprises at least the extracellular domain of PD-L1 with or without signaling peptide.
According to the invention, the attenuated Salmonella strain functions as the bacterial carrier of the recombinant DNA molecule comprising an expression cassette encoding PD-L1 for the delivery of said recombinant DNA molecule into a target cell. Such a delivery vector comprising a DNA molecule encoding a heterologous antigen, such as PD-L1, is termed DNA vaccine.
In the context of the present invention, the term “vaccine” refers to an agent which is able to induce an immune response in a subject upon administration. A vaccine can preferably prevent, ameliorate or treat a disease.
Genetic immunization might be advantageous over conventional vaccination. The target DNA can be detected for a considerable period of time thus acting as a depot of the antigen. Sequence motifs in some plasmids, like GpC islands, are immunostimulatory and can function as adjuvants furthered by the immunostimulation due to LPS and other bacterial components.
Live attenuated Salmonella vectors produce their own immunomodulatory factors such as lipopolysaccharides (LPS) in situ which may constitute an advantage over other forms of administration such as microencapsulation. Moreover, the mucosal vaccine according to the present invention has an intra-lymphatic mode of action, which proves to be of benefit. After ingestion of the attenuated vaccine according to the present invention, macrophages and other cells in Peyer's patches of the gut are invaded by the modified bacteria. The bacteria are taken up by these phagocytic cells. Due to their attenuating mutations, bacteria of the S. typhi Ty21 strain are not able to persist in these phagocytic cells but die at this time point. The recombinant DNA molecules are released and subsequently transferred into the cytosol of the phagocytic immune cells, either via a specific transport system or by endosomal leakage. Finally, the recombinant DNA molecules enter the nucleus, where they are transcribed, leading to massive PD-L1 expression in the cytosol of the phagocytic cells, and potentially, particularly in the case of using only the extracellular domain of human PD-L1 (e.g., SEQ ID NO 13), as in the use of plasmid pVAX10.PD-L1 hb, in the secretion of the PD-L1-based protein product. The infected cells undergo apoptosis, loaded with the PD-L1 antigen, and are taken up and processed by the gut's immune system. The danger signals of the bacterial infection serve as a strong adjuvant in this process, leading to a strong target antigen specific CD8+ T-cell and antibody response at the level of both systemic and mucosal compartments. The immune response peaks around ten days after vaccination. The lack of anti-carrier response allows boosting with the same vaccine over many times. Additionally, the secretion of the PD-L1-based protein product may result in an anti-PD-L1 antibody response, which may further support the therapeutic effect of the approach according to the present invention.
In particular embodiments, the treatment of cancer further comprises chemotherapy, radiotherapy or biological cancer therapy. Particularly, the attenuated strain of Salmonella is administered before, during and/or after the chemotherapy or the radiotherapy treatment or the biological cancer therapy. More particularly, the attenuated strain of Salmonella is administered before and during the chemotherapy or the radiotherapy treatment or the biological cancer therapy. For cure of cancer, complete eradication of cancer stem cells may be essential. For maximal efficacy, a combination of different therapy approaches may be beneficial.
In the context of the present invention, the term “biological cancer therapy” refers to cancer therapy involving the use of living organisms including bacteria and viruses, substances derived from living organisms or laboratory-produced versions of such substances. Some biological therapies for cancer aim at stimulating the body's immune system to act against cancer cells (so called biological cancer immunotherapy). Biological cancer therapy approaches include the delivery of tumor antigens and tumor stroma antigens, e.g. by Salmonella based DNA vaccines, particularly S. typhi Ty21a based DNA vaccines, delivery of therapeutic antibodies as drugs, administration of immunostimulatory cytokines and administration of immune cells, including engineered T-cells. Therapeutic antibodies include antibodies targeting tumor antigens or tumor stroma antigens.
In particular embodiments, the biological cancer therapy comprises administration of at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen. In particular embodiments, the at least one further DNA vaccine is selected from at least one further attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a tumor antigen and/or a tumor stroma antigen. Particularly, said at least one further attenuated strain of Salmonella is Salmonella typhi Ty21a comprising a eukaryotic expression cassette.
In one embodiment said tumor antigen is selected from Wilms' Tumor Protein (WT1), Mesothelin (MSLN), CEA and CMV pp65. In particular embodiments, said tumor antigen encoded by said at least one further DNA vaccine is selected from the group consisting of Wilms' Tumor Protein (WT1) having the amino acid sequence as found in SEQ ID NO 6 and a protein that shares at least about 80% sequence identity therewith, Mesothelin (MSLN) having the amino acid sequence as found in SEQ ID NO 7 and a protein that shares at least about 80% sequence identity therewith, CEA having the amino acid sequence as found in SEQ ID NO 8 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 9 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 10 and a protein that shares at least about 80% sequence identity therewith, and CMV pp65 having the amino acid sequence as found in SEQ ID NO 11 and a protein that shares at least about 80% sequence identity therewith. Particularly, Wilms' Tumor Protein (WT1) has the amino acid sequence as found in SEQ ID NO 6, Mesothelin (MSLN) has the amino acid sequence as found in SEQ ID NO 7, CEA has the amino acid sequence as found in SEQ ID NO 8, and CMV pp65 has the amino acid sequence as found in SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. In one embodiment said tumor stroma antigen is VEGFR-2 or FAP, preferably VEGFR-2. Particularly, said tumor stroma antigen encoded by said at least one further DNA vaccine is selected from the group consisting of VEGFR-2 having the amino acid sequence as found in SEQ ID NO 12 and a protein that shares at least about 80% sequence identity therewith and human fibroblast activation protein (FAP). Particularly, VEGFR-2 has the amino acid sequence as found in SEQ ID NO 12.
In particular embodiments, the attenuated strain of Salmonella encoding PD-L1 is administered prior to, simultaneously with and/or after the at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen.
In the context of the present invention, the term “simultaneously with” means administration of the attenuated strain of Salmonella encoding PD-L1 and the at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen on the same day, more particularly within 12 hours, more particularly within 2 hours.
In particular embodiments, administration of the attenuated Salmonella strain encoding PD-L1 and the at least further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen occurs within twelve consecutive weeks, more particularly within eight consecutive weeks, more particularly within three to six consecutive weeks. The attenuated Salmonella strain encoding PD-L1 and the at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen may be administered via the same route or via different routes. For example, in particular if the at least one further DNA vaccine is a further attenuated strain of Salmonella, it may be administered orally.
The single dose of the further attenuated strain of Salmonella may comprise from about 105 to about 1011, particularly from about 106 to about 1010, more particularly from about 106 to about 109, more particularly from about 106 to about 108, most particularly from about 106 to about 107 colony forming units (CFU).
Chemotherapeutic agents that may be used in combination with the attenuated mutant strain of Salmonella of the present invention may be, for example gemcitabine, amifostine (ethyol), cabazitaxel, carboplatin, oxaliplatin, cisplatin, capecitabine, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, nimustine (ACNU), carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), folinic acid, gemcitabine (gemzar), daunorubicin, daunorubicin lipo (daunoxome), epirubicin, procarbazine, ketokonazole, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), permetrexed, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, temozolomide and combinations thereof.
Most preferred chemotherapeutic agents according to the invention are cabazitaxel, carboplatin, oxaliplatin, cisplatin, cyclophosphamide, docetaxel, etoposide, gemcitabine, doxorubicin, lomustine, paclitaxel (taxol), irinotecan, vincristine, vinblastine, vinorelbin, folinic acid, 5-fluorouracil, bleomycin and temozolomide, especially gemcitabine.
In particular embodiments, the attenuated strain of Salmonella is administered orally. Oral administration is simpler, safer and more comfortable than parenteral administration. However, it has to be noted that the attenuated strain of Salmonella encoding PD-L1 may also be administered by any other suitable route. Preferably, a therapeutically effective dose is administered to the subject, and this dose depends on the particular application, the type of malignancy, the subject's weight, age, sex and state of health, the manner of administration and the formulation, etc. Administration may be single or multiple, as required.
In particular embodiments, the cancer is selected from lymphoma, leukemia, myeloma, lung cancer, in particular non-small cell lung cancer (NSCLC), melanoma, renal cell cancer, ovarian cancer, glioblastoma, merkel cell carcinoma, bladder cancer, head and neck cancer, colorectal cancer, esophagial cancer, cervical cancer, gastric cancer, hepatocellular cancer, prostate cancer, breast cancer, pancreatic cancer, and thyroid cancer.
The attenuated strain of Salmonella encoding PD-L1 is surprisingly effective at relatively low doses. Administration of low doses of live bacterial vaccines minimizes the risk of excretion and thus of transmission to third parties.
In particular embodiments, the single dose of the attenuated strain of Salmonella comprises from about 10s to about 1011, particularly from about 106 to about 1010, more particularly from about 106 to about 109, more particularly from about 106 to about 108, most particularly from about 106 to about 107 colony forming units (CFU).
In this context, the term “about” or “approximately” means within a factor of 3, alternatively within a factor of 2, including within a factor of 1.5 of a given value or range.
In particular embodiments, the attenuated strain of Salmonella is for use in individualized cancer immunotherapy comprising the step of assessing the PD-L1 expression pattern and/or the pre-immune response against PD-L1 of a patient. The patient's PD-L1 expression and/or the patient's pre-immune responses against PD-L1 may be assessed in a first step for example by companion diagnostics. Methods for assessing the expression of a target gene, such as PD-L1, either on mRNA or on protein level are well known to any one of ordinary skill in the art. For instance, immunohistochemistry staining, flow cytometry methods or RNA sequencing, or alternative methods using labelling can be used to identify the level of target expression in the tumor. Similarly, methods for assessing a patient's pre-immune response against a given protein, such as PD-L1, are well known to any one of ordinary skill in the art. A patient's pre-existing PD-L1 specific T-cell pool can be detected by e.g. ELISpot or multimer FACS analysis. High tumor-specific PD-L1 expression and/or the occurrence of pre-immune responses against PD-L1 are prognostic indicators for the predisposition of a patient to respond especially favorably to the treatment with the attenuated strain of Salmonella encoding PD-L1.
The attenuated strain of Salmonella encoding PD-L1 may be provided in the form of a solution, a suspension, a lyophilisate, an enteric coated capsule, or any other suitable form. Typically, the attenuated strain of Salmonella is formulated as drinking solution. This embodiment offers the advantage of improved patient compliance. Preferably, the drinking solution comprises means to neutralize gastric acids at least to a certain degree, i.e. to bring the pH of the gastric juice closer to a pH of 7. Preferably, the drinking solution is a buffered suspension comprising the attenuated strain of Salmonella encoding PD-L1. In a particular embodiment, the buffered suspension is obtained by suspending the attenuated strain of Salmonella in a suitable buffer, preferably containing 2.6 g sodium hydrogen carbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate and 100 ml of drinking water.
It may be favorable dependent on the occurrence of possible side effects, to include treatment with antibiotics or anti-inflammatory agents.
Should adverse events occur that resemble hypersensitivity reactions mediated by histamine, leukotrienes, or cytokines, treatment options for fever, anaphylaxis, blood pressure instability, bronchospasm, and dyspnoea are available. Treatment options in case of unwanted T-cell derived auto-aggression are derived from standard treatment schemes in acute and chronic graft vs. host disease applied after stem cell transplantation. Cyclosporin and glucocorticoids are proposed as treatment options.
In the unlikely case of systemic Salmonella typhi Ty21a type infection, appropriate antibiotic therapy is recommended, for example with fluoroquinolones including ciprofloxacin or ofloxacin. Bacterial infections of the gastrointestinal tract are to be treated with respective agents, such as rifaximin.
In particular embodiments, cancer immunotherapy comprises a single or multiple administrations of the attenuated strain of Salmonella PD-L1 or a pharmaceutical composition comprising the same. The single dose of the administrations may be the same or different. In particular, cancer immunotherapy comprises 1, 2, 3, 4, 5 or 6 administrations of the attenuated strain of Salmonella encoding PD-L1, preferably wherein the multiple administrations occur within three to six consecutive months.
The aim of this study was to investigate the antitumor efficacy of two Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. VXM10m is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding murine full-length native PD-L1 (with the nucleic acid sequence of SEQ ID NO 17). VXM10ma is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding a truncated form of murine PD-L1 (with the nucleic acid sequence of SEQ ID NO 18), more specifically the N-terminus truncated by 17 amino acid residues.
The treatment started the day of randomization that was considered as day 1 (D1). Thirty healthy male C57BL/6 mice, 4-6 weeks old, were randomized according to their body weight into 5 groups of 6 animals each. Animal allocation to treatment groups is summarized in Table 1. A statistical test (Student t test) was performed to test for homogeneity between the groups (data not shown).
Group 1 was treated with the empty vector control (VXM0m_empty; S. typhimurium bacterial vector control harboring no exogenous expression plasmid). Groups 2 to 5 were treated with VXM10m or VXM10ma, at two different single doses.
VXM0m-empty, VXM10m and VXM10ma were administered by oral gavage (per os, po) in 100 μl in final volumes per application. Regardless of animal groups, each animal received pre-dose application buffer po to neutralize acid in the stomach prior dosing (100 μl/animal/application). This buffer was produced by dissolution of 2.6 g sodium hydrogen carbonate, 1.7 g L-Ascorbic acid, 0.2 g lactose monohydrate in 100 ml of drinking water and was applied within 30 min prior application of VXM0m-empty, VXM10m and VXM10ma.
Prime vaccination started at day 1 and consisted of 4 administrations every second day (d1, 3, 5, 7). Prime vaccination was followed by two boost vaccinations at days 14 and 21.
Tumors were induced in all animals by I.P. injection of 5.0×106 FBL-3 cells in 500 μl RPMI 1640 on day 20. FBL-3 is a Friend leukemia virus-induced erythroleukemia cell line originated from C57BL/6 mice. This cell line expresses unique tumor specific transplantation antigens that can be recognized by the immune system. Priming syngeneic mice with FBL-3 tumor cells leads to the subsequent rejection of future live tumor challenges. Although FBL-3 is immunogenic, injection of live FBL-3 tumor cells into naïve syngeneic mice results in tumor growth, suggesting that the FBL-3 tumor cells possess mechanisms of escaping immune recognition and destruction. Of note, PD-L1 was shown to be highly expressed on the FBL-3 cell line.
Animal body weight, viability and animal behavior were monitored throughout the study.
Survival of test animals is displayed in a Kaplan-Meier plot in
The aim of this study was to investigate the long-term effect of antitumor efficacy of two Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia following re-challenge.
The Study of Example 1 was continued and mice were receiving a second tumor induction dose on day 100. Further unvaccinated control animals (n=10) that were present in the study from the start of the experiment, but received no first tumor induction, were included as a control for the tumor re-challenge. Re-challenge was performed by tumor induction in all animal of Groups 2-5 (see Table 1) and the unvaccinated control animals by I.P. injection of 5.0×106 FBL-3 cells in 500 μl RPMI 1640 on day 100.
Animal body weight, viability and animal behavior were continuously monitored. Over the treatment phase, when compared with control group, administration of VXM10m and VXM10ma generated a potent memory T cell response against the leukemia, with 100% of long-term surviving mice also after re-challenge with FBL-3 cells. No vaccination-related toxicity or body weight loss was observed throughout the study. Survival of test animals is displayed in a Kaplan-Meier plot in
The aim of this study was to investigate the antitumor efficacy of VXM10m and VXM10ma administered therapeutically in C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia.
Tumors were induced in all animals by intraperitoneal (i.p.) injection of 5.0×106 FBL-3 cells in 500 μl RPMI 1640 on day 0.
The treatment started the day of randomization that was considered as day 1 (D1, after tumor injection). For the treatment groups sixteen healthy male C57BL/6 mice, 4-6 weeks old, were randomized according to their body weight into 2 groups of 8 animals each. A statistical test (Student t test) was performed to test for homogeneity between the groups (data not shown).
VXM0m-empty, VXM10m and VXM10ma were administered by oral gavage (per os, po) at 1.0×109 CFU in 100 μl as described in Example 1.
One group of 8 mice was treated with the empty vector control (VXM0m_empty; S. typhimurium bacterial vector control harboring no exogenous expression plasmid). The other group of 8 mice were treated with VXM10m or VXM10ma, with a prime treatment 4 times on Days 1, 3, 5, 7 and 2 weekly boosts on Days 14 and 21 (
Animal body weight, viability and animal behavior were monitored 3 times weekly during the prime-boost period and upon the peak immune response, i.e., from study day 0 to 28, and then twice weekly until the end of the study.
Over the treatment phase, when compared with control group, oral administration of VXM10m and VXM10ma resulted a strong anti-tumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge (
The aim of this study was to investigate the anti-PD-L1 response to VXM10 or VXM10a administered at 108 CFU and 1010 CFU at days 1, 3, 5 and 7 with a boost at days 14 and 21 in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. Tumors were induced in all animals by I.P. injection of 5.0×106 FBL-3 cells on day 20. Vaccination and tumor induction was performed basically as described in Example 1. Soluble recombinant murine PD-L1 was used for immunization with CFA/IFA in the positive control group.
The systemic antibody response was evaluated by ELISA in the serum of animals vaccinated with either VXM10 or VXM10a, 79 days after the final vaccination on day 21 (
The aim of this study was to investigate the T-cell response induced against PD-L1 epitopes in healthy C57 BL/6 mice (n=5 per group) immunized four times every other day (days 1, 3, 5 and 7) via oral route with 1010 CFU of either VXM10, VXM10a or the empty vector control (
Ex vivo restimulation of the spenocytes was performed 10 days after the last immunization (day 17), using a pool of 5 immunogenic peptides derived from murine PD-L1. The content of the culture supernatant was tested for the presence of IFNγ and TNFα by ELISA after 6 days of in vitro stimulation.
The level of TNFα, and to a lesser extend IFNγ, was significantly increased in the supernatant of spenocytes derived from animals vaccinated with VXM10a and stimulated with PD-L1 peptides (
The aim of this study is to investigate the long-term effect of antitumor efficacy of a third Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. VXM10mb is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding a truncated form of murine PD-L1, more specifically the extracellular domain including the N-terminal signaling peptide (amino acid sequence SEQ ID NO 15; nucleic acid sequence SEQ ID NO 16). The Experiment is essentially performed as described in Examples 1 and 2.
The aim of this study is to investigate the antitumor efficacy of VXM10mb administered therapeutically in C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. The Experiment is essentially performed as described in Example 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17161666.7 | Mar 2017 | EP | regional |
| 17188941.3 | Sep 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/056721 | 3/16/2018 | WO | 00 |
