Cell attachment peptide

Abstract

The invention relates to a cell attachment peptide containing an RGD amino acid motif, characterized by a material-side motif derived from poly-L-lysine and a cell-side motif derived from vitronectin. The peptide may be used to functionalize materials for use in cell cultures, such as microcarriers, scaffolds, and substrates.

Description

[0001] DESCRIPTION

[0002] TITLE: Cell attachment peptide

[0003] TECHNICAL FIELD

[0004] The invention is in the field of peptides for attaching cells to microcarriers, substrates or similar for use in cell cultures.

[0005] BACKGROUND OF THE INVENTION

[0006] Materials for scaffolds, microcarriers, substrates, etc. for cell cultures are well known in the prior art. When performing cell cultures it is often times desired, especially for adherent cell types, that a strong attachment develops between the cells and the microcarrier or substrate material. It is well known that one risk of sudden cell detachment is cell anoikis by apoptosis. For many materials this attachment does not develop and different peptides, usually with an RGD motif, have been experimented on as attachment intermediaries between a cell and such materials. One example of such a disclosure can be found in Golunova, A. et al: Direct and Indirect Biomimetic Peptide Modification of Alginate: Efficiency, Side Reactions, and Cell Response; Int. J. Mol. Sci. 2021, 22, 5731.

[0007] In the past few years a new field of biotechnology has emerged, called cellular agriculture. One of the objectives of cellular agriculture is the replacement of entire food categories with cell-cultivated equivalents at a massive production scale - this has created a need for cheap and efficient ways to culture cells, including adherent cells that require cultivation on surfaces and good attachment to them.

[0008] Many of the cell attachment peptides known in the prior art suffer from one or several problems, namely being improper for human consumption , even in residual form, relying on specific attachment chemistry that is again incompatible with human consumption, that display poor attachment performance, that are excessively long in amino acid units and thus technically challenging to manufacture / synthetise and or prohibitively expensive for mass production needs, or that simply do not perform well, either by providing poor cell attachment or not fulfilling other expected and possibly still unknown biological functions by not correctly replicating the properties of the natural extracellular matrix and polypeptides the cells are naturally programmed to accept by their particular genome or epigenetic state. It is thus a stated objective of the inventors to solve one or more of these problems by proving a novel and inventive cell attachment peptide.

[0009] SUMMARY OF THE INVENTION

[0010] In a preferred embodiment, the invention is preferably a cell attachment peptide containing a RGD amino acid motif characterized by comprising a material-side motif before the R of RGD derived from poly-L-Lysine and a cell-side motif after the D of the RGD derived from vitronectin, preferably with the material-side motif comprising at least two K amino acids, with the first K amino acid connected to a cell culture material and the last K amino acid connected to the R of the RGD motif, preferably with the cell-side motif comprising at least a VF motif of amino acids, with the first V amino acid connected to the D of the RGD motif, preferably with the material-side motif being KK or any of Kx, where x is larger than 2 and represents the number of K amino acids, preferably with the cell attachment peptide comprising the motif KKRGDVFT, or KKKRGDVFT, or KKRGDVF, or KKKRGDVF, preferably with the cell attachment peptide being attached to a base material, preferably with the cell attachment peptide being attached to a cell.

[0011] In another preferred embodiment, the invention is a functionalized base material for a cell culture characterized by comprising a cell attachment peptide as in the previous embodiment.

[0012] In another preferred embodiment, the invention is a cell culture comprising a functionalized base material of the previous preferred embodiment with one of the peptides of the first preferred embodiment and at least a cell type that displays connection affinity to vitronectin.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention described herein aims primarily to solve the problem of replacing known cell adhesion peptides with an improved version.

[0015] These cell adhesion peptides are used during the performing of animal cell cultures. Cell cultures consist of animal cells, isolated from their original living organism (the animal), subject to artificial conditions that replicate the biological processes that allow them to proliferate and / or differentiate. These artificial conditions may comprise placing the cells in an appropriate bioreactor, spinner flask, well-plate, petri dish or any other industrial or laboratory equipment adequate for the culture of cells. The cells are mostly surrounded by, or immersed in, a cultivation medium appropriate for culturing the specific cell type. This medium can be specifically tailored to promote proliferation, differentiation, or both. Proliferation is understood to be the phase of the lifetime of a cell during which the cell divides, creating new cells, but maintaining its pluripotent or multipotent abilities, i.e. the ability to differentiate into different cell types. Differentiation is understood to be the phase of the lifetime of a cell during which it settles onto a specific phenotype, for example becoming an adipose cell or a muscle cell, and losing both of either of its pluri or multipotency (also known as “sternness”) depending on the specific phenotype acquired.

[0016] In nature / in vivo, organisms possess an extracellular matrix (ECM), which is secreted by the cells, filling the intercellular space and that provides, in simplified terms, a scaffold for the cells to grow / survive on. The cells are connected to the ECM by attachment proteins, such as vitronectin, laminin, fibronectin, etc., that usually connect to specific integrin receptors present in the cell membrane. Adherent cell types are particularly adapted to survive and grow only if supported by the ECM in the presence of attachment proteins. Most vertebrate derived cells are of this type, especially cells belonging to the mesenchymal line.

[0017] When performing cell cultures, it is thus at times important to provide certain cells, especially adherent types, with a means to attach to a surface, preferably by mimicking the properties of the cells’ natural attachment proteins of the extracellular matrix (ECM) by providing them with a material that is biologically appropriate for this purpose.

[0018] The actual materials the cells attach to may or may not provide additional biological cues to the cells. In general, the cells probably receive purely biological / biochemical cues from the attachment peptide and eventually biomechanical cues from the base material, derived from its stiffness, topography or other properties. The base material may present in several shapes, for example as a microcarrier, i.e., a mostly spherical individual portion of material, as a type of scaffolding, for example in fibre like-shapes presenting individually or entangled, simply as a surface when shaped like a coating on a flask for example, and many other configurations. The cell adhesion peptides are chemically bound to the base material, preferably only at its surface to avoid wastage (i.e. excess deployment of peptide that is not physically available to be used by the cells, due to being constrained inside the volume of the base material). For ease of writing, the process of creating a base material with the attachment peptide will be called functionalization. So, a base material displaying the peptide will be a functionalized material. The term “base” will be mostly omitted since it is clear that it is implied in “functionalized material”. The reasonings behind the choice of materials for cell culture and this invention in particular are detailed further ahead in the section Materials.

[0019] An example of cells that can be cultured are cells obtained from a biopsy of an animal tissue, when the animal is living. For example, in the area of cellular agriculture, cells normally used for human consumption, such as primary cells like Fibro Adipogenic Precursors (FAPs) or Satellite Cells (SCs), also known as myosatellite cells, are obtained by biopsy from animals usually consumed by humans, for example mammals such as bovines, ovines or porcines among others. It is also usual to use man-made cell lines or other types of specialised cell lines. Most cells used in cellular agriculture are of the mesenchymal lineage.

[0020] Several cell attachment peptides are known in the prior art and usually contain the amino acids Arginine (Arg or R), Glycine (Gly or G) and Aspartate (Asp or D) in that order, at least once, and other amino acids before or after this arginylglycylaspartic acid (RGD) motif (i.e., sequence). A well-known example is GRGDSP. It is generally understood that the RGD motif is the minimal recognition sequence required for cell attachment, binding to the integrin receptors of animal cells, these receptors being the ones that usually would bind to the natural attachment proteins, providing cell-cell or cell-ECM attachment. It is also known that the RGD motif is a part of much larger cell attachment proteins, such as vitronectin, fibronectin and others, and that amino acids present in the peptide beyond the RGD motif contribute to a selective recognition of specific attachment proteins over others by a cell, with different biological results usually seen in actual cell growth in cell cultures. Different cells may present with different receptors for cell attachment proteins. Some usual integrin receptors are formed from proteins designated CD49x or as ITGAx, where x can have several values, or synonymously called alpha-x integrin. The nomenclature of the gene that encodes the protein is sometimes mixed with the protein, so for example ITGA7 encodes ITGA7, but ITGA1 encodes integrin alpha-1, also known as CD49a, but not synonymously known as ITGA1. It is a confusing nomenclature, but of note is that vitronectin is known to bind preferably to integrin alpha-V beta-3 or integrin aVp3. There are also other receptors besides integrins, such as discoidin domain receptors and syndecans, although these were not investigated for this invention. Other receptors may also bind more or less strongly to vitronectin, that is to say that integrin aVp3 is the preferred binding site but not the only possible one. Satellite cells, that mainly express ITGA7, have a strong preference for vitronectin adhesion, but Fibro- Adipogenic Progenitors (FAPs), that express mainly ITGA5, also adhere perfectly well to vitronectin. Some integrins are thus less specific than others and an attachment peptide may work to attach well within a range of integrins. Generalisations are difficult in this area, but vitronectin seems to be a polypeptide that works well with several different types of cells. We will not explain here the molecular structure of integrins and the concept of their different subunits (alfa and beta) and the different integrin types that vary from one another on the basis of the different possible combinations of these subunits. This is part of the general knowledge of the cell biology field.

[0021] As such, the cell attachment peptide of the current invention contains the expected RGD motif and varies from previously known cell attachment peptides by the specific presence of other amino acids, and is able to interact with several integrins, in particular the ones that would normally display affinity to vitronectin.

[0022] It is to be understood that, as far as this invention discloses, a cell may also refer to, or be a part of, an aggregate of cells, connected between themselves for example through means of direct cell-to-cell attachment, natural ECM and natural attachment polypeptides or manmade attachment polypeptides or peptides, where at least one of those cells of the outer surface of the aggregate may connect to a material by the cell attachment peptide of the invention.

[0023] For ease of explanation, the peptide of the invention will be divided into three parts, one a sequence of amino acids appearing before the Arginine (R) of the RGD motif, followed by the RGD motif part, and another part appearing after the Aspartate (D) of the RGD motif. The sequence before the R will be the one connected to the surface of the support material, and the sequence after the D will be the one connected to the integrin receptors on the cell. These parts on sides before and after the RGD motif will respectively be called material-side and cellside.

[0024] The essays / trials described in this invention were performed in well plates and generic stir tank vessels, also known as stirred vessels. These are known respectively as 2D and 3D testing methods. It is known that trials in well plates with positive results may or may not translate to stirred vessels, but, negative results do translate negatively. So, when a test fails in a well plate, in 2D, there is no need to run it in a stirred vessel, in 3D, since it will fail there. A successful test in a well plate needs to (or should) be retested in a stirred vessel, in 3D, and it may then be successful or not, but success is not guaranteed. This is the reason why in the data presented below, some tests were not performed beyond well plates, when already unsuccessful.

[0025] The material-side sequence

[0026] A material-side sequence must, first of all, be able to attach to the material’s surface and to the R amino acid. Attachment to the R is trivially fulfilled by the simple fact that the material-side sequence is comprised of amino acids (AA), which are naturally bondable between themselves by amide or peptide bonds, so typical state-of-the-art peptide synthesis techniques are appropriate to use here and require no further explanation (and for any other AA to AA bond on the peptide chain). Another requirement for the material-side sequence is that it must be appropriate in dimensions (length) or freedom-of-movement to allow the cell to physically attach to the RGD motif and another requirement is that the attachment of the sequence to the material must be strong enough to hold the cell, or aggregate of cells, in place without breaking.

[0027] The proposed material side sequence of this invention is a double Lysine motif, i.e., Lys-Lys or KK. The inventors recognized that cell cultures with materials coated in poly-L- lysine (PLL), for example coated alginate microcarriers, plus a known cell attachment peptide such as GRGDSP display better cell growth characteristics than the same culture without the coating present. Tests were performed using bovine primary satellite cells cultures with alginate hydrogel microcarriers both coated or uncoated with PLL and functionalized with GRGDSP, and it is clear from the results shown in table 1 that cell confluence (i.e., number of cells per microcarrier area) was much higher in the PLL coated microcarriers than in the ones with no coating (see table 1) after 7 days.

[0028] Table 1- Cell growth trial, performed in well plates, of a bovine satellite cell culture starting from a population of 5000 cells / cm2and a microcarrier area of 6.67 cm2 / ml, after 7 days. This result gave rise to the notion that the lysine must have an important biological function that should be kept, and the inventors wished to replicate this by trying to connect an RGD motif to lysine, at the same time avoiding the application of a full coating, which is technically difficult, expensive, wasteful in material and time consuming, and trying to replicate the effects of the lysine coating with the minimal use of amino acids. Another issue with PLL coatings is that this shields the microcarrier underneath the coating from dissolution agents. For example, in cultivated meat or other applications it may be desirable at a certain time to separate the cells from the microcarriers, which can usually be achieved by dissolving the carriers and filtering out the cells. This lysine coating inspired insight gave rise to the realisation that at least one K amino acid should be present to allow the peptide to chemically connect to a base material’s surface and that either that same amino acid should also connect to the R in the RDG motif or that more amino acids should be present to allow for a more efficient connection of the RGD motif to the integrin receptors of a cell. Based on the properties of poly-L-lysine, which has more than one lysine amino acid, it was trialled to have another K amino acid, forming a KKRGD- motif, and this proved to be the best performing motif. A wellplate cell culture with a two-lysine motif in the material side peptide performs better than one with just a single lysine and the same as with more than two lysins, showing that two lysins is the optimal minimum quantity, which both solves the issue of replicating the properties of the poly-L-lysine coating and the waste of amino acids. In table 2 below, the different times for a population doubling to happen can be seen in a test run of motifs with different quantities of K amino acids and the same cell-side motif (VFT) in this case. A different material-side motif was included in this particular experiment for comparison purposes (GK). It is clear that the fastest growth occurs with the double Lys motif, and no large difference is observed for the triple K motif. The single K motif and the non-K only motif show more than double the time for a population doubling to occur, so, much worse performance (faster / less time is better). The presence of G and only one K (in GKRGDVFT) or only a single lonely K (KRGDVFT) proves that a large positive charge is required in the beginning of the sequence, given by the KK motif. It should be noted that the triple K version does work fairly well but shows no statistically significant improvement over the two KK version, and is not the most economical or technically easiest motif to make, hence the double K version being preferred (with the triple K version being also appropriate for use in this invention, of course). It is also expected that a four or more K version of the motif should also work similarly to the triple K version, so, technically, this could be used but is not the optimal minimum of amino acids.

[0029] Table 2- Time (in hours) for a bovine satellite cell culture in well plates population doubling to occur (Doubling time DT)

[0030] The result for KKRGDVFT was confirmed in a stirred tank (see table 4 below and its discussion).

[0031] The lysine may be connected to the material by different chemistries, as explained in the section Connection chemistries further ahead.

[0032] As a further explanation, and not wishing to be bound by theory, it is known that cells have a negative net electrical charge. When cells are considered as particles these particles are negative. Lysine has a positive net electrical charge. By including positive charges in the peptide the cells are attracted towards the substrate by means of electrostatic interactions, 'forcing' the cells to come into contact with the peptide, promoting the subsequent attachment to the peptide via the integrin. A double K motif could be seen to provide the minimum required electrical charge for this phenomenon to occur, assuming this theory is correct. Considering motifs with more than two KK’s, it is envisioned that excess of positive charges might at some point become cytotoxic and lead to cell membrane instability. The point at which this occurs has not been directly investigated at this point but from simulations is expected to happen from high numbers of K repetitions.

[0033] The use of this motif and avoiding the use of a PLL coating allows the microcarrier to be exposed to dissolving agents if required.

[0034] The cell-side sequence

[0035] For the cell-side sequence, the inventors took inspiration from the natural molecule vitronectin. Vitronectin (VTN) is a glycoprotein synthesised and secreted by the liver and abundantly found in serum, the extracellular matrix and bone. VTN has 478 amino acid residues and, as expected, includes a RGD motif (positions 45-47) as a specific binding site for integrin receptors, specifically the vitronectin receptor (vitronectin binds to integrin alpha-V beta-3, or ITGA5) or other receptors with some affinity to vitronectin, and is thus involved in cell adhesion biology. Vitronectin, when used as a substrate, is known to improve cell growth for certain types in cultures, and its motifs are thus candidates for research into cell attachment peptide motifs. The inventors ran tests with peptides with no RGD and none would attach to the cells. RGD seems to be essential for this application.

[0036] Several shorter peptide sequences based on VTN are available in the market, such as GGGGKGGPQVTRGDVFTMP or GKGGPGVTRGDYFTFP. These vitronectin derived peptides display very good properties when used as attachment motifs in cell culture and were used as the standard to be achieved during the creation of this invention. Through experimentation, the inventors surprisingly realised that there is a minimum motif that will communicate the necessary biological cues to the cells to favour good growth, meaning that a much shorter VTN based sequence may be used, and also unexpectedly realised that there is only a need to replicate the cell-side sequence of any VTN derived peptide as long as there is already a well performing replacement to the material side available, such as the previously identified lysine based motif, and as such that performance is basically independent of the material side sequence for this case. As such, using the KK motif while excising all the amino acids appearing before the RGD sequence in the VTN derived peptides provides similar results when compared to the original sequences and the identified shorter sequence also does not show much worse performance. A particularly well performing motif was identified in VFT. This can be seen in tables 3 and 4 below, where the GGGGKGGPQVTRGDVFTMP peptide (designated as VN1) was compared to the peptide KKRGDVFT, with similar results for well plates (2D), but with the peptide of the invention displaying much better results in spinners (3D).

[0037] Table 3- Cell culture of bovine satellite cells in well plates at 7 days, starting from 5.000 cells / cm2and 6.67 microcarrier cm2 / ml.

[0038] Table 4- Cell culture of bovine satellite cells in spinners at 9 days, starting from 3.000 cells / cm2and with a microcarrier area of 40 cm2 / ml.

[0039] This shows, as explained before, that a good result in well plates could translate in a bad result in spinners, as seen in the VNI behaviour, that was much worse in spinners when compared to KKRGDVFT.

[0040] Further experiments were done to try to further reduce the amount of cell-side amino acids.

[0041] KKRGDVF proved to be statistically similar to KKRGDVFT, with doubling times (see table 5) of 53 hours vs. 57 of KKRGDVFT. KKRGDV was not tested at this time.

[0042] Table 5- Time (in hours) for a bovine satellite cell culture in well plates population doubling (Doubling time DT).

[0043] Note

[0044] As an extra advantage, it can be seen from the positive results obtained with proliferation of cells attached with the peptides of the invention that the mechanical connection strength is at least sufficient to provide secure attachment for the duration of the culture. So, there does not seem to be any significant breakage of either the connection of the peptide to the base material or of the peptide to the cell, and the peptide provides the chemical and / or biological and / or biomechanical cues the cells required to expand.

[0045] The cell cultures may also be co-cultures, meaning that more than one cell type could be present. For example, a co-culture of bovine SCs and FAPs could be performed. Materials

[0046] The main purpose of a material used for cell culture, as described before, is basically to provide something for adherent cells to adhere to. These materials can be naturally functionalized to attach to cells, for example when using materials that already contain the RGD motif, such as vitronectin or laminin, or may need to be functionalized to achieve that attachment objective. The current invention is mostly material agnostic, as in, the attachment peptide motif described herein could in theory be used to functionalize a broad array of materials. Actual laboratory testing was conducted on alginate hydrogel as a scaffold, both in a microcarrier and a fibre-like shape, functionalized with the peptides of the invention (including the ones used for comparative testing). Alginate hydrogel and its fabrication is quite well known in the prior art, and as such its manufacture is considered to be outside the scope of the invention and well within the normal capabilities of a person skilled in the art.

[0047] Connection chemistries

[0048] To functionalize a scaffold material with the peptide(s) of the invention, several well- known state of the art reactions can be used, typically of the click-chemistry sort, such as DMTMM or sulphate click chemistry. In general, any chemical reaction that forms a C- terminal (carboxylic) N-terminal (amine) bond between the substrate and the peptide is appropriate.

[0049] Concept of Equivalence

[0050] In all the trials described above, the guiding principle was that the number of peptides available for connecting the base material to the cells was the same. This is known as equivalence. In other words, the amount of, for example, taking table 3, KKRGDVFT or VN1 is not the same in weight, but is the same in total individual units, i.e. the number of peptide chains available for the cells to connect to are the same.

[0051] Exemplary Embodiments of the Invention

[0052] As described, the invention is base material and cell type agnostic, as long as one of the attachment peptides of the invention can be attached to the base material and the cell attaches successfully and with good proliferation performance to the peptide. Nonetheless, the invention did start as a solution for cultured meat cell types, so an exemplary embodiment of the invention is of a base material consisting of alginate hydrogel, functionalized with one of the peptides of the invention, preferably KKRGDVF or KKRGDVFT, the alginate hydrogel being shaped as a microcarrier or as a fibre, and the cell type being of a mesenchymal lineage, preferably a muscle or fat cell or precursor cell.

Claims

Claims1. A peptide for use in a cell culture, containing a RGD amino acid motif characterized by consisting of three parts, the RGD motif, a material-side motif before the R of RGD comprising at least two K amino acids, and only K amino acids, with the first K amino acid being available to connect to a cell culture base material such as a scaffold, microcarrier or substrate and the last K amino acid being connected to the R of the RGD motif and a cell-side motif after the D of the RGD derived from vitronectin comprising at least a VF motif of amino acids, with the first V amino acid of the VF motif connected to the D of the RGD motif.

2. A peptide according to claim 1 characterized by the material-side motif being KK or any of Kx, where x is larger than 2 and represents the number of K amino acids.

3. A peptide according to any of claims 1 to 2 characterized by consisting of the motif KKRGDVFT, or KKKRGDVFT, or KKRGDVF, or KKKRGDVF.

4. A peptide according to any of the preceding claims characterized by comprising being attached to a base material, by the N-terminus on the material side.

5. A peptide according to any of the preceding claims characterized by comprising being attached to a cell, by the C-terminus on the cell side.

6. A functionalized base material for a cell culture characterized by comprising a cell attachment peptide as claimed in claims 1 to 5, the peptide being bonded to the base material via a chemical reaction between the N-terminus of the peptide and a C terminus of the base material7. A cell culture characterized by comprising a functionalized base material with one of the peptides claimed in claims 1 to 5 and at least a cell type that displays connection affinity to vitronectin.

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