Patent Application
20190177385
The present invention relates to glycoproteins with reduced acetylation rate of sialic acid residues.
Sialic acid is a generic term for the N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. After N-acetylneuraminic acid (Neu5Ac), the most frequent species are N-glycolylneuraminic acid (Neu5Gc) and O-acetylated derivatives.
Sialic acids are found widely distributed in animal tissues and to a lesser extent in other organisms, ranging from plants and fungi to yeasts and bacteria, mostly in glycoproteins where they occur at the end of glycans bound to the latter.
The covalent binding of a glycan to a protein represents an evolutionary mechanism by which the diversity of the proteome can be largely increased. The circumstance that multiple, diverse mechanisms evolved for the glycosylation of proteins argues for the evolutionary benefit and overall relevance of this type of protein modification. Such mechanisms range from non-enzymatic glycation to complex post-translational glycosylation, a multi-step and multi-cell compartment process involving several enzymatic modifications.
Acetylated sialic acids seem to be involved in many biological phenomena. Glycostructures play a role in protein-protein interactions and they can be a prerequisite for folding into a correct, functional conformation. Their predominance and variability of expression during development and malignancy, also as part of so-called “oncofetal antigens,” suggests their participation in numerous physiological and pathological processes. Not surprisingly, genetic defects impairing the synthesis or the attachment of glycan moieties to proteins cause multiple human diseases. These glycostructures can be further modified, thereby introducing additional diversification. One example of such modifications is acetylation. Its role as a potent regulator of cellular interactions classifies acetylation with other biochemical regulations of cell function such as protein phosphorylation, or modification with N-acetylglucosamine (GlcNAc) and methylation.
Acetylation of sialic acid obviously further increases diversity of glycosylation, i.e. endogenously, the attached sugars are additionally enzymatically modified. Sialic acids are prototypic examples for such modification of terminal glycans, and acetylation is a widespread type of such additional modifications. Naturally occurring sialic acids share a nine-carbon backbone and can be acetylated at all their hydroxyl groups. This means they can be acetylated at positions C4, C7, C8 and C9. Each sialic acid can be modified once, but also multiple derivatives of a single sialic acid are possible, creating several combination patterns, specifically for glycostructures carrying more than one sialic acid.
Considering the clear importance of glycosylation for the function of proteins and the obviously significant space for modifications, substantial efforts went into optimizing the therapeutic properties of proteins by targeted modification of their glycan structures. Recently the first two examples of this new generation of biopharmaceuticals have achieved marketing authorization: mogamulizumab (Poteligeo®) and obinutuzumab (Gazyvaro®/Gazyva).
However, targeted modification of glycosylation is not limited to monoclonal antibodies. Darbepoetin alfa (Aranesp®) is an example where also the characteristics of a globular protein were improved, in this case through adding two sialylated carbohydrate chains leading to a prolonged efficacy, i.e. decreased treatment frequency.
However, while some of these approaches focus on adaptation of serum half-life and other factors, it seems that modification of efficacy in a narrow sense has not yet been in the focus for potential qualitative modifications. This is in particular true for erythropoiesis-stimulating agents (ESA), which are used to increase the gas (oxygen/carbon dioxide) transport capacity of the blood.
One major risk associated with ESA therapy is increased mortality. As most prominent risk, cardiovascular events occur at a high increase of hemoglobin (Hb), which is discussed to be caused by the ESA-evoked increase in red blood cells (RBC).
It is one object of the present invention to provide a new approach for modifying efficacy of therapeutic glycoproteins.
It is another object of the present invention to provide a new approach for modifying erythropoiesis-stimulating agents to increase their efficacy.
It is still another object of the present invention to provide a new approach for modifying erythropoiesis-stimulating agents which have reduced risk of side effects and/or mortality.
These objects are achieved with methods and means according to the independent claims of the present invention. The dependent claims are related to preferred embodiments.
Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an”, and “the” include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
According to one aspect of the invention, a method or process of producing a glycoprotein that interacts with, or acts as an agonist to, the erythropoietin receptor (EpoR) is provided, which glycoprotein has modified efficacy. The method or process comprises the heterologous expression of said glycoprotein in a suitable expression system, wherein at least one step or feature is provided that results in a reduced acetylation rate of sialic acid residues in the glycoprotein.
The comparison with regard to the claimed modified efficacy can either be done with a commercially available glycoprotein of identical or similar amino acid sequence, or on a wildtype glycoprotein expressed by its natural host, or by a suitable expression system, which wildtype glycoprotein has not been modified to affect the acetylation rate of sialic acid residues.
The erythropoietin receptor (EpoR) is a protein that in humans is encoded by the EPOR gene. EpoR is a 52 kDa peptide with a single carbohydrate chain resulting in an approximately 56-57 kDa protein found on the surface of EPO responding cells. EpoR pre-exists as a dimer which upon binding of a suitable ligand changes its homodimerized state.
The cytoplasmic domains of the EpoR contain a number of phosphotyrosines which, upon the conformational changes caused by ligand binding, are phosphorylated by Jak2, and serve as docking sites for a variety of intracellular pathway activators and Stats (such as Stat5).
The primary role of EpoR is to promote proliferation of erythroid progenitor cells and rescue erythroid progenitors from cell death. Based on current evidence, it is however still unknown whether EpoR directly causes “proliferation and differentiation” of erythroid progenitors in vivo.
Another role EpoR is thought to be involved in is to promote erythroid differentiation. EpoR's PI3-K/AKT signaling pathway augments GATA-1 activity. One hypothesis is that erythroid differentiation is primarily dependent on the presence and induction of erythroid transcriptional factors such as GATA-1, FOG-1 and EKLF.
It is also known that EpoR can activate mitogenic signaling pathways and can lead to cell proliferation in erythroleukemic cell lines in vitro. EpoR expression can extend as far back as the hematopoietic stem cell compartment. It is, however, unknown whether EpoR signaling plays a permissive or an instructive role in early, multipotent progenitors in order to produce sufficient erythroblast numbers.
The term “modified efficacy” is discussed elsewhere herein.
The term “acetylation rate of sialic acid residues”, as used herein, refers to the overall percentage of sialic acids of a given glycoprotein type which carry one or more acetyl residues. Preferably, the term relates to O-acetyl residues on these sialic acids.
The term “glycoprotein”, as used herein, refers to proteins and peptides that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation.
Preferably, the reduced acetylation is reduced O-acetylation. This applies for all embodiments disclosed herein which recite the term acetylation. In all cases, O-acetylation is a preferred embodiment.
In one embodiment, the step or feature that results in a reduced acetylation rate of sialic acid residues is at least one selected from the group consisting of:
The term “expressor cell line”, as used herein, relates to a cell line that is capable of expressing the glycoprotein either homologously or heterologously.
Preferably, steps/features a), b and e) relate to in vitro processes that are carried out, e.g., by posttranslational enzymatic or chemical treatment of the glycoprotein.
Deacetylation of sialic acid residues (step or feature a) can be accomplished, e.g., by posttranslational treatment with a suitable acetylesterase. One example is sialate O-acetylesterase (SIAE), which in humans is encoded by the SIAE gene located on chromosome 11. SIAE catalyzes the removal of O-acetyl ester groups from position 9 of the parent sialic acid.
However, other organisms, and other expressor cell lines, have other types of acetylesterases. Sialate O-acetylesterases are disclosed in Expasy enzyme entry EC 3.1.1.53. These enzyme catalyze, inter alia, the following reaction:
N-acetyl-O-acetylneuraminate+H(2)O<=>N-acetylneuraminate+acetate
It is hence in the routine of the skilled person to silence, delete or mutate the respective acetyltransferase in the respective organisms, and other expressor cell lines on the basis of the teaching disclosed herein.
Reduction of overall glycosylation (step b) can be accomplished, e.g., by posttranslational treatment with suitable enzymes, like NANase II, Endoglycosidase H, O-glycosidase, and/or Peptide-N-Glycosidase F (PNGase F). Protocols are described in Kim & Leahy (2013). A chemical approach for the deglycosylation of glycoproteins, which uses trifluoromethanesulfonic acid, is disclosed in Sojar H & Bahl (1987).
Generally, de-glycosylation removes all glycans from a glycoprotein, and hence, all sialic acid residues, or their acetyl substituents, respectively. Deglycosylation thus results, de facto, in the removal of acetyl residues conjugated to sialic acids, and thus has similar effects.
Reduction of sialic acid content in glycans of a glycoprotein can be done according to methods disclosed in WO2011061275A1.
Step/feature c) preferably refers to an expressor cell line that (i) exhibits per se, a reduced acetylation (i.e., in which the acetylation rate has not been artificially manipulated), or (ii) has been modified in such way that acetylation of sialic acid residues during post translational protein modification is affected.
This can be accomplished, e.g., by inhibition or reduction of gene expression of a gene coding for an enzyme that catalyzes sialic acid acetylation, or expression of a dysfunctional, or inactive enzyme that catalyzes sialic acid acetylation, or an enzyme that catalyzes sialic acid acetylation with reduced activity.
Step/feature d) preferably refers to an expressor cell line that has for example been modified in such way that protein glycosylation is affected.
This can be accomplished, e.g., by modifying an expressor cell line in such way that it expresses, or overexpresses, heterologous or homologous enzymes that catalyze deglycosylation.
Examples for enzymes that catalyze deglycosylation include NANase II, Endoglycosidase H, O-glycosidase, and/or Peptide-N-Glycosidase F (PNGase F). Overexpression of any of these genes in a suitable expressor cell line, or heterologous expression in a suitable expressor cell line results in deglycosylation during post translational protein modification.
Said heterologous expression can be accomplished e.g., by genetic engineering techniques, including non-transient transfection of a cell with a vector that encodes for a respective enzyme.
Said homologous overexpression can be accomplished e.g., by genetic engineering techniques, including induced overexpression of an intrinsic gene that encodes for a respective enzyme by means of a suitable promoter that has been inserted upstream of the encoding gene.
Said deletion or mutagenesis can be accomplished by methods described elsewhere herein.
As the inventors have shown herein, a glycoprotein according to the present invention which has a reduced acetylation rate of sialic acid residues offers the opportunity to apply a lower dose while achieving the same therapeutic effect. This results in cost savings and decreases the risk of unwanted side effects, such as formation of antibodies, by applying a smaller amount of glycoprotein to the patient. The possibility to apply lower doses also reduces the volumes to be given to the patient, adding further benefit to such a therapeutic, and increasing patient compliance.
Further, the glycoprotein according to the present invention which has a reduced acetylation rate of sialic acid may reduce a major risk associated with ESA therapy, namely increased mortality. As most prominent risk, cardiovascular events occur at a high increase of Hb, which is discussed to be caused by the ESA-evoked increase in RBC. A therapy with a glycoprotein according to the present invention leads to an increase of mean corpuscular hemoglobin (MCH) above, while the RBC increase is less pronounced. Taken together, this results in a favourable safety profile, e.g. regarding cardiovascular safety and mortality in general.
In one embodiment, the glycoprotein is an erythropoiesis-stimulating agent (ESA). In a preferred embodiment, the erythropoiesis-stimulating agent (ESA) is an erythropoietin, a modified erythropoietin or an erythropoietin mimetic.
The term “erythropoietin”, as used herein, comprises the different wild type erythropoietins (Epoetin α, Epoetin β, Epoetin γ, Epoetin δ, Epoetin ε, Epoetin ζ, Epoetin θ, Epoetin κ or Epoetin ω).
The term “modified erythropoietin”, as used herein, refers to a protein that relies, structurally and/or sequence-wise, on wild type erythropoietin, but comprises structural modifications in either (i) its amino acid sequence, its (ii) glycosylation pattern or (iii) by addition of other moieties. These modifications do not affect its agonistic interaction with the erythropoietin receptor as such, but either modify said agonistic interaction, or modify other physico-chemical, pharmacological or PK/PD properties thereof, like bioavailability, serum half-life, tissue distribution, efficacy, shelf life, and the like.
The term “erythropoietin mimetic”, as used herein, refers to proteins and peptides that agonistically interact with the erythropoietin receptor. Erythropoietin mimetics are disclosed, inter alia, in Johnson, & Jolliffe (2000), and U.S. Pat. No. 8,642,545B2. Studies have shown that in the EPO receptor, which is a 484-amino acid glycoprotein with a single transmembrane segment located between extracellular and intracellular domains each of nearly equal size, Phe93 is crucial for binding EPO, as well as binding erythropoietin mimetic peptides (Middleton et al. 1999).
Quite obviously, modified erythropoietins and erythropoietin mimetics are two classes that have a large overlap. Hence, some modified erythropoietins can also be considered erythropoietin mimetics and vice versa.
In one embodiment of the invention, the erythropoietin or the modified erythropoietin is at least one selected from the group consisting of:
In another embodiment of the invention, the modified efficacy is physiological efficacy or therapeutic efficacy. Preferably, the modified therapeutic efficacy is relative increase of MCH and/or relative stimulation of hemoglobin (Hb) synthesis.
In yet another embodiment of the invention, the step that results in a reduced acetylation rate of sialic acid residues also affects bioavailability, exposure, serum half-life or absolute serum concentration of the glycoprotein.
The inventors have for example shown that reduction of sialic acid acetylation may on the one hand side reduce bioavailability of an ESA, but at the same time increase MCH, compared to a non-modified ESA, as shown to a degree even overcompensating the lower exposure as measured by the Hb level. Hence, although seemingly contra-productive in terms of Hb achieved, the reduction of bioavailability surprisingly does not translate to a reduced Hb. The example shows that the decreased exposure is even overcompensated by the increased MCH resulting from the treatment with the modified ESA. This may have multiple consequences. From a manufacturing perspective, a lower dose of protein required to correct the patients hemopoiesis may translate into lower costs of goods. From a patient perspective this lower dose together with the decreased exposure due to the fast elimination would imply a further benefit. Exposure to a therapeutic protein correlates with the risk of the individual to develop anti-drug antibodies (ADA). In case of ESAs the likelihood for such ADA formation is typically low. A lower exposure would thus also decrease the immunogenicity risk. Lastly and most importantly, the increase of Hb without the otherwise rather linear increase of RBC decouples the Hb rise from an increased thrombosis risk.
In one embodiment of the invention, the modified glycoprotein has at least one selected from the group consisting of:
a) an absolute acetylation rate of ≤10%, and/or
b) an acetylation rate that is reduced by ≥50%.
As used herein, the term “absolute acetylation rate (%)” refers to the overall percentage of sialic acids of a given glycoprotein type which carry one or more O-acetylation. It is important to emphasize that in the meaning of the present disclosure, a partial or total deglycosylation would result in a reduction of the acetylation rate, too.
Preferably, the absolute acetylation rate is ≤9, 8, 7, 6, 5, 4, 3, 2 or 1%. Most preferably, the absolute acetylation rate is 0%. In this embodiment the sialic acids of all glycans of a given glycoprotein are completely de- or non-acetylated.
However, under some conditions the absolute acetylation rate should at the same not be smaller than 0.1; 0.2; 0.3; 0.4 or 0.5%.
As used herein, the term “acetylation rate that is reduced by X %” refers to relative reduction of sialic acids of a given glycoprotein type which carry one or more acetyl groups compared with (i) a commercially available glycoprotein of identical or similar amino acid sequence, or (ii) on a wildtype glycoprotein of identical or similar amino acid sequence expressed by its natural host, or by a suitable expression system, wherein further in either case, the respective commercially available glycoprotein or the wildtype glycoprotein has not been modified to affect the acetylation rate of sialic acid residues.
Preferably, the acetylation rate is reduced by ≥51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95%.
However, under some conditions the acetylation rate should not be reduced by more than 99; 98; 97 or 96%.
For example, the absolute acetylation rate of an ESA (darbepoietin alpha) according to the invention is <1%, while the commercially available ESA of identical or similar amino acid sequence (Aranesp®) has an absolute acetylation rate of 29.5%. Hence, the acetylation rate is reduced to about 3% of that of commercially available ESA of identical or similar amino acid sequence.
Preferably, the acetylation rate is determined with at least one method selected from the group consisting of:
For an overview of methods suitable for acetylation rate determination, see e.g. Reuter and Schauer, 1994). In case the reduction of acetylation rate has been accomplished indirectly, i.e., by partial or total glycosylation, the acetylation rate can as well be determined with, inter alia, chemical deglycosylation with trifluoromethane sulfonic acid (disclosed in Sojar & Bahl (1987)).
According to another aspect of the invention, a glycoprotein that interacts with, or acts as an agonist to, the erythropoietin receptor (EpoR) is provided, which glycoprotein has modified efficacy. The glycoprotein is produced with a method or process according to the above description.
According to another aspect of the invention, the use of such glycoprotein for the manufacture of a medicament for the treatment of a human or animal patient or subject is provided. Alternatively, the use of such glycoprotein for the treatment of a human or animal patient or subject is provided.
According to another aspect of the invention, a method of treatment of a human or animal patient or subject is provided, which method encompasses the administration of a glycoprotein according to the above description in a pharmaceutically effective amount.
According to another aspect of the invention, a pharmaceutical preparation comprising a glycoprotein according to the above description in a pharmaceutically acceptable carrier is provided.
Because of the increased efficacy, such preparation could be administered in smaller doses or volumes, which in turn may help to decrease drug aggregation risk, increase shelf life, reduce storage needs, reduce administration related discomfort and increase patient compliance.
According to one embodiment of the invention, the human or animal patient or subject suffers from, is at risk of developing, and/or is diagnosed for, at least one disease or symptom selected from the group consisting of:
Said anemia can have different origins, e.g., kidney diseases deficiency, radiotherapy, cancer such as myelodysplastic syndrome with or without bone marrow suppressive chemotherapy, iron or iron metabolism deficiency, symptomatic anemia in predialysis, or bone marrow deficiency or disease.
According to one embodiment of the invention, the human or animal patient or subject is subject of a condition or undergoes a treatment selected from the group consisting of:
According to another aspect of the invention, a cell for heterologous expression of a glycoprotein according to the above description is provided, which cell
In one embodiment, the expression of glycoproteins that have de- or non-acetylated sialic acid residues, or a reduced acetylation rate of sialic acid residues, is accomplished by at least one of:
Preferably, said enzyme of option a)-c) catalyzes sialic acid O-acetylation. Examples for such enzymes are shown in the following list, which is however not to be construed as limiting:
These enzymes catalyse reactions of e.g., the following types:
Acetyl-CoA+N-acetylneuraminate->CoA+N-acetyl-7-O (or 9-O)-acetylneuraminate
Acetyl-CoA+an alpha-2,8-linked polymer of sialic acid->CoA+polysialic acid acetylated at O-7 or O-9
Silencing, deletion or mutagenesis of said gene in a suitable human expressor cell line results in a lacking or dysfunctional enzyme, which in turn leads to a reduced or lacking acetylation of sialic acid residues during post-translational protein modification.
However, other organisms, and other expressor cell lines, have other types of sialic acid acetyltransferases. It is hence in the routine of the skilled person to silence, delete or mutate the respective acetyltransferase in the respective organism or expressor cell line on the basis of the teaching disclosed herein.
Preferably, said enzyme of option d) catalyzes de-o-acetylation of sialic acid. Examples for such enzymes are shown in the following list, which is however not to be construed as limiting:
These enzymes catalyze, inter alia, the following reaction:
N-acetyl-O-acetylneuraminate+H2O->N-acetylneuraminate+acetate
Human sialate O-acetylesterase (SIAE) is for example encoded by the SIAE gene located on chromosome 11. SIAE catalyzes the removal of O-acetyl ester groups from position 9 of the parent sialic acid.
Overexpression of a gene from the above list in a suitable human expressor cell line, or heterologous expression in a non-human expressor cell line, results in post-translational deacetylation of sialic acid residues during post translational protein modification.
However, other organisms, and other expressor cell lines, have other types of acetylesterases. It is hence in the routine of the skilled person to silence, delete or mutate the respective acetyltransferase in the respective organism or expressor cell line on the basis of the teaching disclosed herein.
In one embodiment, the expression of glycoproteins that are deglycosylated, or have reduced glycosylation, is accomplished by heterologous expression or homologous overexpression of a gene coding for an enzyme that catalyzes deglycosylation.
In one embodiment, the inhibition or reduction of gene expression has been achieved by at least one genetical engineering step selected from the group consisting of:
The term “gene expression”, as used herein, is meant to encompass at least one step selected from the group consisting of: DNA transcription into mRNA, mRNA processing, non-coding mRNA maturation, mRNA export, translation, protein folding and/or protein transport.
The inhibition or reduction of gene expression of a gene refers to methods which directly interfere with gene expression, encompassing, but not restricted to, inhibition or reduction of DNA transcription, e.g., by use of specific promoter-related repressors, by site specific mutagenesis of a given promoter, by promoter exchange, or inhibition or reduction of translation, e.g., by RNAi induced post-transcriptional gene silencing. The expression of a dysfunctional, or inactive enzyme, or an enzyme with reduced activity, can, for example, be achieved by site specific or random mutagenesis, insertions or deletions within the coding gene.
The inhibition or reduction of the activity of an enzyme can, for example, be achieved by administration of, or incubation with, an inhibitor to the respective enzyme, prior to or simultaneously with protein expression. Examples for such inhibitors include, but are not limited to, an inhibitory peptide, an antibody, an aptamer, a fusion protein or an antibody mimetic against said enzyme, or a ligand or receptor thereof, or an inhibitory peptide or nucleic acid, or a small molecule with similar binding activity. Some inhibitors described in literature for N-acetylneuraminate 7-O (or 9-O)-acetyltransferase activity are iodoacetate, CoA, diethyl carbonate, N-bromosuccinimide, Triton X-100 or p-chloromercuribenzoate.
Other ways to inhibit the enzyme comprise the reduction of specific cofactors of the enzyme in the medium.
Gene silencing, gene knock-down and gene knock-out refer to techniques by which the expression of a gene is reduced, either through genetic modification or by treatment with an oligonucleotide with a sequence complementary to either an mRNA transcript or a gene. If genetic modification of DNA is done, the result is a knock-down or knock-out organism. If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this results in a temporary change in gene expression without modification of the chromosomal DNA and is referred to as a transient knock-down.
In a transient knock-down, which is also encompassed by the above term, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through blocking of transcription (in the case of gene-binding), degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) or RNase-H dependent antisense) or blocking either mRNA translation, pre-mRNA splicing sites or nuclease cleavage sites used for maturation of other functional RNAs such as miRNA (e.g., by morpholino oligos or other RNase-H independent antisense). Other approaches involve the use of shRNA (small hairpin RNA, which is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference), esiRNA (endoribonuclease-prepared siRNAs, which are a mixture of siRNA oligos resulting from cleavage of long double-stranded RNA (dsRNA) with an endoribonuclease), or the activation of the RNA-induced silencing complex (RISC).
Another approach for genetic modifications that can be used in the present context comprises the use of CRISPR Cas (Baumann et al., 2015), TALEN or ZFN (Gaj et al., 2013).
Other approaches to carry out gene silencing, knock-down or knock-out are known to the skilled person from the respective literature, and their application in the context of the present invention is considered as routine.
Gene knock-out refers to techniques by which the expression of a gene is fully blocked, i.e. the respective gene is inoperative, or even removed. Methodological approaches to achieve this goal are manifold and known to the skilled person. Examples are the production of a mutant which is dominantly negative for the given gene. Such mutant can be produced by site directed mutagenesis (e.g., deletion, partial deletion, insertion or nucleic acid substitution), by use of suitable transposons, or by other approaches which are known to the skilled person from the respective literature, the application of which in the context of the present invention is thus considered as routine. One example for a newly developed technique which the skilled person would consider as useful in the context of the present invention is knock-out by use of targeted zinc finger nucleases. A respective kit is provided by Sigma Aldrich as “CompoZR knockout ZFN”. Another approach encompasses the use of Transcription Activator-Like Effector Nucleases (TALENs).
The delivery of a dominant negative construct involves the introduction of a sequence coding for a dysfunctional enzyme, e.g., by transfection. Said coding sequence is functionally coupled to a strong promoter, in such way that the gene expression of the dysfunctional enzyme overrules the natural expression of the wild type enzyme, which, in turn, leads to an effective physiological defect of the respective enzyme activity.
A conditional gene knock-out allows to block gene expression in a tissue- or time-specific manner. This is done, for example, by introducing short sequences called loxP sites around the gene of interest. Again, other approaches are known to the skilled person from the respective literature, and their application in the context of the present invention is considered as routine. One other approach is gene alteration which may lead to a dysfunctional gene product or to a gene product with reduced activity. This approach involves the introduction of frame shift mutations, nonsense mutations (i.e., introduction of a premature stop codon) or mutations which lead to an amino acid substitution which renders the whole gene product dysfunctional, or causing a reduced activity. Such gene alteration can for example be produced by mutagenesis (e.g., deletion, partial deletion, insertion or nucleic acid substitution), either unspecific (random) mutagenesis or site directed mutagenesis.
Protocols describing the practical application of gene silencing, gene knock-down, gene knock-out, delivery of a dominant negative construct, conditional gene knock-out, and/or gene alteration are commonly available to the skilled artisan, and are within his routine. The technical teaching provided herein is thus entirely enabled with respect to all conceivable methods leading to an inhibition or reduction of gene expression of a gene coding for an enzyme, or to the expression of a dysfunctional, or inactive enzyme, or an enzyme with reduced activity.
The teaching disclosed herein comprises
Hence, the technical teaching of this application enables the skilled person to develop an expressor cell line that has either impaired or defect O-acetylation or has increased de-O-acetylation, in order to produce an EpoR agonist with a given modified efficacy.
In one embodiment of the invention, the cell is a eukaryotic cell. The term “eukaryotic cell” encompasses, but is not restricted to, animal cells, like, e.g., insect cells, plant cells and fungal cells. Preferably, the cell is an animal cell and/or a plant cell. More preferably, the cell is a mammalian cell.
Preferably, the cell is at least one selected from the group consisting of:
In one preferred embodiment, the cell is a recombinant cell. As used herein, the term “recombinant cell” is used to refer to a cell with exogenous and/or heterologous nucleic acid incorporated within, either incorporated stably so as to remain incorporated in clonal expansion of the cells, or introduced transiently into a cell (or a population of cells). Such exogenous and/or heterologous nucleic acid can either code for a heterologous protein to be expressed, or it can affect the inhibition or reduction of gene expression of a gene coding for an enzyme, or the expression of a dysfunctional or inactive enzyme, or an enzyme with reduced activity.
To provide a comprehensive disclosure without unduly lengthening the specification, the applicant hereby incorporates by reference each of the patents and patent applications referenced above.
The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.
Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, and the following description of the respective figures and examples, which, in an exemplary fashion, show preferred embodiments of the present invention. However, these drawings should by no means be understood as to limit the scope of the invention.
In a first study, the effect of complete removal of glyco-structures on the pharmacokinetics of a glycoprotein biologic was investigated. For this experiment, a glycosylated IgG1-type monoclonal antibody was used. While there is some evidence that galactosylation of the Fc domain of antibodies plays a role for the efficient recruitment of effector cells, the role of glycosylation as such, and in particular sialylation and O-acetylation of sialic acids, for the remaining characteristics such as pharmacokinetics, is less understood.
For this purpose, a human IgG1 mAb was enzymatically deglycosylated according to standard protocols (see, e.g., Kim & Leahy 2013), and the pharmacokinetics were assessed following a single i. v. infusion, as summarized in Table 1.
Dense serum samples were taken to closely monitor the entire time course up to 14 days following treatment, stored frozen and quantified for the human IgG concentrations using conventional ELISA, which was validated for this purpose.
It was tested whether two batches of the CTLA-FC fusion protein abatacept (Orencia®) which have different levels of O-acetylation would differ in their exposure/bioavailability upon single, s.c. administration. The respective di-O-acetylation rates were 11.4% for batch No 1 and 6.5% for batch No 2. Tri-O-acetylated sialic acids were not observed.
Dense serum samples were taken up to 14 days following treatment, to allow a close monitoring of serum levels, stored frozen and quantified for abatacept concentrations using conventional ELISA.
In this example the causal relationship between O-acetylation rates and exposure/bioavailability of a selected glycoprotein biologic are investigated.
To this end, a sufficient amount of a single batch of abatacept (Orencia®) was purchased, reconstituted and desalted into a 10 mM Sodium Phosphate/1 mM MgCl2 buffer at pH 7. The batch was split into two halves. The first half was incubated with sialate-9-O-acetylesterase (Applied BioTech, Angewandte Biotechnologie GmbH) for two hours at 37° C. The second half was treated the same way, yet no enzyme was added. Subsequently the esterase was removed by affinity chromatography. The characteristics of the two resulting materials is summarized in Table 3 and
Dense serum sampling (at high frequency of sampling allowing a close and reliable monitoring of serum levels over time) was employed up to 14 days following treatment, with samples stored frozen and quantified for Orencia® concentrations using conventional ELISA.
Doses of the two batches were then administered s.c. to rabbits. Results are shown in
As typical example of erythropoiesis stimulating agents, Aranesp® (darbepoetin alfa) was selected. Aranesp® is highly sialylated and carries O-acetylated sialic acids as well. Rats were chosen as model due to the excellent predictivity of the results obtained for humans. As injection route, subcutaneous injection was chosen again, representing a typical route for clinical praxis. The dose range was selected to follow clinical praxis, as well.
For the preparation of de-O-acetylated Aranesp® to be tested in vivo Aranesp® was used. In short, several syringes were pooled to provide about 1 mg darpepoetin starting material for which the buffer was exchanged by dialysis into 50 mM Na-phosphate buffer pH 7.6 containing 140 mM NaCl, before incubation with 1 ml=which was treated with 1 U of sialate 9-O-acetylesterase for 20 h at 37° C.
After incubation the enzyme was removed by affinity chromatography on an anti-Epo antibody column. The column eluate was again buffer exchanged against 20 mM Na-phosphate buffer pH 6.2, containing 140 mM NaCl, filter-sterilized, aliquoted and stored below −60° C. until used for the treatment of the animals as shown in this example. Content determination was done by RP-HPLC. The concentration measured by RP-HPLC was 0.136 mg/ml, which was used as basis to calculate the dosing.
The de-O-acetylated Aranesp® was analyzed by LC-MS to determine the efficiency of the enzymatic treatment and control for the intact protein structure. Species with O-acetylated sialic acids were not detected, indicating efficient de-O-acetylation. Otherwise the spectrum show the expected glycosylated species with high abundance of molecules carrying tetra-antennary, tetra-sialo glycan structures at all five N-linked sites and one disialylated O-linked glycan (species with 22 sialic acids) and in general a high degree of sialylation. The distribution of glycoforms is also in qualitative agreement with the results of the sialo glycan maps.
The analysis of sialic acids was conducted by DMB-labeling and RP-HPLC. The resulting chromatogram is shown in
Rats were chosen as species for the comparison due to the excellent predictivity of the results obtained for humans. As injection route, subcutaneous injection was chosen, representing a typical route for clinical praxis. The dose range was selected to follow clinical praxis, as well. Due to the sensitivity of PD read-outs, the study design was further refined in this example. In addition to the highly specific enzymatic modification, the dosing considered the change of the molecular weight by the modification. The molecular weight of native Aranesp® is about 37,100 Da. In case of a complete de-O-acetylation, the MW decreases to about 36,366 Da. While the dose was weight based (mg/kg) in the previous examples, the dose was the same in this study based on the number of molecules administered, i.e., equimolar dosing, considering the 1-2% change in molecular weight (Table 6).
The animals were housed under standard conditions and treated with a single injection as detailed in Table 6. The assignment to the treatment groups was performed randomly, prior to dosing. Blood was sampled from the tail vein and analyzed using standard hematological equipment.
Table 7 summarizes the minimal level of O-acetylation observed for a substantial number of Aranesp® lots, illustrating that O-acetylation of Neu5Ac consistently plays a major role with regard to the overall mean level of O-acetylation.
In order to compensate for minimal baseline differences of Hb between groups, i.e., prior to treatment,
To gain more insight into the details of Hb increase, reticulocyte as well as erythrocyte count was examined. Unexpectedly, the level of RBC increase in groups treated with modified Aranesp® was slightly less pronounced than observed in the Aranesp® treated groups.
Consequently, the MCH was analyzed as illustrated in
As a result, the ESA with de- or non-acetylated sialic acids showed less pronounced stimulation of RBC proliferation, but an increased MCH in the resulting RBCs, compared to unmodified ESA.
Because RBCs participate in hemostasis through exposure of procoagulant phospholipids (Peyrou et al., 1999), an ESA-mediated increase of RBCs results in an increased cardiovascular risk. However, the therapeutic goal of ESA treatment is the increase of oxygen capacity of the blood, not necessarily the increase of RBCs. Conventional ESA treatment accomplishes this by stimulation of RBC proliferation, hence resulting in a higher oxygen capacity.
The inventors have shown that ESA with de- or non-acetylated sialic acids still increase oxygen capacity of the blood, however, without increasing the RBC to the same extent as conventional ESA, but by increasing the MCH, i.e., the average load of Hb per RBC. This approach may help to reduce side effects that coincide with ESA treatment, like an increased cardiovascular risk.
Without being bound to theory, one explanation may be that the amount of Hb loaded into RBC during maturation can differ. While some conditions characterized by hypochromic anemias demonstrate normal RBC counts but low Hb, because there is a disproportionate reduction of Hb relative to the volume of the RBC, it appears that ESA with de- or non-acetylated sialic cause the opposite phenomenon appears, in that the packing of Hb/RBC is increased. Generally, sialic acids are examples for glycostructures that are known to play an important role in numerous biological processes. Genetic modification of therapeutic proteins to increase the level of sialylation is a successful and frequently used approach to maximize exposure resulting from an administered dose. It is surprising that a decreased level of O-acetylation of sialic acids in an ESA consistently leads, on one hand, to a lower level of exposure/bioavailability, but at the same time to an increased efficacy in terms of MCH. This is unexpected, because publications on erythropoetin show that O-acetylation of sialic acids actually increases the half-life (Shahrokh et al., 2011).
Accordingly, one would have expected that ESA with lower level of O-acetylation of sialic acids would have a lower efficacy in terms of oxygen capacity, but the observed effect was the opposite.
Reuter & Schauer (1994), Methods in enzymology vol 230, p. 168-199
| Number | Date | Country | Kind |
|---|---|---|---|
| 16179065.4 | Jul 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/067615 | 7/12/2017 | WO | 00 |
