Human vitronectin fragments and uses thereof

Abstract

The present invention relates to human vitronectin fragments that have improved manufacturability in E. coli, improved function in stem cell culture applications, and improved stability compared to wild-type vitronectin or previously characterized vitronectin fragments. The present invention relates to their use as substrates for cell culture and in regenerative medicine.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 239,456, filed September 1, 2021, which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via the Patent Center as an XML file entitled "0541-000017WO01" created on August 23, 2022, having a size of 5 kilobytes. With the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR §1.821(c) and the CRF required by §1.821(e). The information contained in the Sequence Listing is incorporated herein by reference. [Background technology]

[0003] Regenerative medicine is a growing field of medicine that aims to repair, regenerate, or replace damaged or diseased cells, tissues, and organs. Both mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), are currently in clinical trials for various indications in regenerative medicine. A key aspect of regenerative medicine is the ex vivo culture and expansion of stem cells and their derivatives for use as therapeutics in tissue replacement strategies (Blau and Daley, 2019, N Engl J Med;380(18):1748-1760). A critical step in generating these cells is safe and robust ex vivo culture. This involves optimization of cell culture media and substrates that are safe and reliable (from a supply perspective) while maintaining robust functionality. With regard to safety, the United States Pharmacopeia (USP) publication USP <1043> (USP43-NF38-7381, USP-NF <1043> Standards are outlined in the USP “Ancillary Materials for Cell, Gene, and Tissue Engineered Products,” Pharmacopeial Forum;43:7381), which details a tiered system for risk classification of raw materials used as ancillary materials in cell and gene therapies, including regenerative medicine (USP43-NF38). The use of animal component-containing reagents is considered high risk (tier 4), whereas non-animal derived materials produced under GMP conditions are considered low risk (tier 2). Thus, achieving low risk, non-animal derived substrates for stem cell culture is an important goal to promote more widespread use in regenerative medicine.

[0004] Conventional cell culture techniques for MSCs and PSCs use either plasma purified matrices such as fibronectin (Veevers-Lowe et al., 2011, Journal of Cell Science;124(Pt 8):1288-300; and Somaiah et al., 2015, PLoS One;10(12):e0145068), or complex tumor-derived matrices such as MATRIGEL™ or CULTREX BME™ (Xu et al., 2001, Nat Biotechnol;19(10):971-4). More recently, matrices with more homogenous composition (including laminin, fibronectin, and vitronectin) produced using recombinant manufacturing protocols have been used for stem cell culture (Miyazaki et al., 2012, Nat Commun;3:1236; Miyazaki et al., 2013, Erratum in: Nat Commun;4:1931; Braam et al., 2008, Stem Cells;26(9):2257-65; and Kalaskar et al., 2013, JR Soc Interface;10(83):20130139). These substrates can reduce variability and improve the precision of the cell culture environment. However, these proteins or protein fragments can be difficult and expensive to produce and / or are produced in animal cells.

[0005] The field of regenerative medicine requires matrix molecules that are easy to manufacture, robust and truly non-animal derived. Summary of the Invention

[0006] The present disclosure includes vitronectin polypeptide fragments, which comprise a deletion of at least 50 contiguous N-terminal amino acids compared to a full-length vitronectin polypeptide having SEQ ID NO:1; a deletion of at least C-terminal amino acids 362-478 compared to a full-length vitronectin polypeptide having SEQ ID NO:1; comprise the arginine-glycine-aspartic acid (RGD) integrin binding domain of vitronectin; do not include the N-terminal somatomedin B (SmB) domain of vitronectin; do not include the C-terminal heparin binding domain and Hp4 domain of vitronectin; and comprise about 95% sequence identity to the same fragment of a full-length vitronectin polypeptide having SEQ ID NO:1.

[0007] In some embodiments, the vitronectin polypeptide fragment comprises the Hp1, Hp2, and / or Hp3 domains of vitronectin.

[0008] In some embodiments, the vitronectin polypeptide fragment has a length of about 200 to about 350 amino acids.

[0009] In some embodiments, the vitronectin polypeptide fragment comprises the following: a deletion of 61 contiguous N-terminal amino acids compared to the full-length vitronectin polypeptide having SEQ ID NO:1; a deletion of C-terminal amino acids 293 to 478 compared to the full-length vitronectin polypeptide having SEQ ID NO:1.

[0010] In some embodiments, a vitronectin polypeptide fragment comprises about 95% sequence identity to residues 62-292 of a full-length vitronectin polypeptide having SEQ ID NO:1.

[0011] In some embodiments, the vitronectin polypeptide fragment comprises an amino acid substitution at a position corresponding to position 80 and / or position 148 of full-length human vitronectin having SEQ ID NO: 1. In some embodiments, the amino acid substitution comprises a D80Y substitution and / or a Q148E substitution.

[0012] In some embodiments, the vitronectin polypeptide fragment comprises one or more amino acid substitutions at positions corresponding to positions 63, 67, and / or 68 of full-length human vitronectin having SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise a T63G substitution and / or a V67S substitution and / or a V67N substitution and / or a F68P substitution. In some embodiments, the amino acid substitutions comprise a T63G substitution, a V67S substitution or a V67N substitution, and a F68P substitution.

[0013] The present disclosure includes a vitronectin polypeptide fragment consisting of SEQ ID NO:2.

[0014] The present disclosure includes a vitronectin polypeptide fragment consisting of SEQ ID NO: 2, which has an amino acid substitution at position D80 and / or an amino acid substitution at position Q148 compared to full-length vitronectin having SEQ ID NO: 1. In some embodiments, the amino acid substitution at position D80 comprises a D80Y substitution, and / or the amino acid substitution at position Q148 comprises a Q148E substitution.

[0015] The present disclosure includes a vitronectin polypeptide fragment consisting of SEQ ID NO:2, which has an amino acid substitution at position T63 and / or an amino acid substitution at positions V67 and / or F68, compared to full-length vitronectin having SEQ ID NO:1. In some embodiments, the amino acid substitution at position T63 comprises a T63G substitution, and / or the amino acid substitution at position V67 comprises a V67S or V67N substitution. In some embodiments, the amino acid substitution at position F68 comprises a F68P substitution.

[0016] In some embodiments, the vitronectin polypeptide fragments disclosed herein further comprise a C-terminal His tag, hi some embodiments, 1, 2, 3, 4, 5, 6, 7, 8 or more C-terminal His residues.

[0017] The present disclosure includes a vitronectin polypeptide fragment consisting of SEQ ID NO:3.

[0018] In some aspects, the vitronectin polypeptide fragments disclosed herein are of non-animal origin.

[0019] In some embodiments, the vitronectin polypeptide fragments disclosed herein are conjugated to a microcarrier. In some embodiments, the microcarrier comprises a hydrogel or a polystyrene microsphere.

[0020] The present disclosure includes compositions of vitronectin polypeptide fragments disclosed herein, or vitronectin polypeptide fragment conjugates disclosed herein. In some aspects, the compositions are of non-animal origin.

[0021] In some aspects, the vitronectin polypeptide fragment disclosed herein, the vitronectin polypeptide fragment conjugate disclosed herein, or the composition disclosed herein are for use as a cell culture substrate.

[0022] The present disclosure includes nucleotide sequences that encode the vitronectin polypeptide fragments disclosed herein.

[0023] The present disclosure includes an expression vector comprising a nucleotide sequence disclosed herein. In some aspects, the expression vector comprises an E. coli expression vector.

[0024] The present disclosure includes a host cell comprising a nucleotide sequence disclosed herein or an expression vector disclosed herein. In some aspects, the host cell comprises E. coli.

[0025] The present disclosure includes methods of producing a vitronectin polypeptide fragment, comprising expressing the vitronectin polypeptide fragment from a nucleotide sequence disclosed herein, an expression vector disclosed herein, or a host cell disclosed herein. In some aspects, the vitronectin fragment polypeptide produced is of non-animal origin.

[0026] The present disclosure includes a cell culture method, comprising culturing cells on a substrate comprising a vitronectin polypeptide fragment disclosed herein, a vitronectin polypeptide fragment conjugate disclosed herein, or a composition disclosed herein. In some embodiments, the cells comprise stem cells. In some embodiments, the cells are cultured under non-animal-derived conditions. In some embodiments, the cell culture is ex vivo.

[0027] As used herein, "isolated" refers to material that is removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus has been altered "by the hand of man" from its natural state.

[0028] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0029] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0030] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0031] "Consisting of" means inclusive and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means inclusive of any elements recited after the phrase, and limits the other elements to those elements that do not interfere with or contribute to the activity or action specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional, and that the other elements may or may not be present depending on whether they materially affect the activity or action of the recited elements.

[0032] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

[0033] As used herein, the term "or" is generally used in its ordinary sense including "and / or" unless the context clearly dictates otherwise.

[0034] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0035] The recitations herein of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0036] Here, "up to" a number (e.g., up to 50) is inclusive of the number (e.g., 50).

[0037] The terms "in the range" or "within a range" (and similar descriptions) include the endpoints of the stated range.

[0038] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed by applying ordinary rounding techniques in light of at least the number of reported significant digits.

[0039] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0040] In any method disclosed herein that includes distinct steps, the steps can be performed in any order practicable, and, where appropriate, any combination of two or more steps can be performed simultaneously.

[0041] Throughout this specification, references to "one embodiment," "an embodiment," "certain embodiment," or "some embodiments" or the like mean that a particular feature, structure, composition, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the present disclosure. Furthermore, the particular features, structures, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0042] In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. It should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0043] All headings throughout are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated. [Brief description of the drawings]

[0044] [Figure 1] The linear structure of vitronectin with its functional domains is shown. The complete vitronectin polypeptide chain (top) and the variants generated in this study are shown. The integrin-binding RGD domain is labeled at residues 64-66. The functional domains are: somatomedin B (SmB), hemoepoxin (Hp), and heparin-binding (Hb). There is a disulfide bond between the cysteines at positions 293 and 430. [Figure 2A]A comparison of human iPSC cells cultured on different VTN fragments at 1 μg / ml, 5 μg / ml, and 10 μg / ml is shown, where full-length recombinant VTN made in NSO cells is included as a control. iPSC cells grown on VTN62-120 and VTN62-155 give rise to small round colonies, whereas VTN62-292 supports the growth of large colonies. Representative brightfield images of iPSCs are shown. [Figure 2B] Comparison of human iPSC cells cultured on different VTN fragments at 1 μg / ml, 5 μg / ml, and 10 μg / ml, where full-length recombinant VTN made in NSO cells is included as a control. Quantification of the fold expansion of iPSCs after 4 days of culture is shown in a bar graph. Note that among the variants, only the hVTN62-292 fragment can support the expansion of iPSC cells at low concentrations, and in comparison, the VTN-N variant shows reduced expansion. [Figure 3A] Comparison of human MSC cells cultured on different VTN fragments at 10 μg / ml, 5 μg / ml, and 1 μg / ml. MSC morphology is similar in all VTN variants, but numbers are reduced in VTN62-120 and VTN62-155. Representative brightfield images of MSCs are shown. [Figure 3B] Figure 1 shows a comparison of human MSC cells cultured on different VTN fragments at 10 μg / ml, 5 μg / ml, and 1 μg / ml. Quantification of MSC expansion fold after two passages is shown. Note that among the bacterially expressed variants, the hVTN62-292 fragment shows superior MSC cell expansion at low concentration (1 μg / ml). [Figure 4A] Figure 1 shows long-term maintenance of iPSCs in a pluripotent state when cultured on hVTN62-292. iPSCs are maintained on VTN62-292 variants using non-animal derived iPSC medium and maintain normal morphology and healthy expansion over 5 passages. Bar graph shows average number of total viable cells per well after 4 days of expansion from a starting population of 100,000 cells. [Figure 4B]Figure 1 shows long-term maintenance of iPSCs in a pluripotent state when cultured on hVTN62-292. After long-term culture, iPSCs maintain markers of pluripotency as shown by flow cytometry. The dark grey histograms show antibody signal and the light grey histograms show isotype control staining. iPSCs maintain high levels of stemness markers Oct3, Sox2, and SSEA-4. Conversely, iPSCs have low levels of SSEA-1, a differentiation marker for human iPSCs. The percentage of positive cells is shown in the top right of the flow graph. The gate on the flow graph was set by the isotype control. [Figure 4C] FIG. 1 shows the long-term maintenance of iPSCs in a pluripotent state when cultured on hVTN62-292. iPSCs cultured on VTN62-292 maintain the ability to differentiate into ectodermal cells. Images show the transcription factor Otx2 to visualize cells differentiated into ectoderm, and DAPI to visualize total cell number. Graph shows the increase in the percentage of cells expressing Otx2 after ectodermal differentiation compared to undifferentiated iPSCs. [Figure 4D] FIG. 1 shows long-term maintenance of iPSCs in a pluripotent state when cultured on hVTN62-292. iPSCs cultured on VTN62-292 maintain the ability to differentiate into definitive endoderm. Images show staining for transcription factor Sox17 to visualize endoderm-differentiated cells, and DAPI to visualize total cell number. Graph shows increase in the percentage of cells expressing Sox17 after endoderm differentiation compared to undifferentiated iPSCs. [Figure 5A]After long-term maintenance of iPSCs on hVTN62-292, we show that iPSCs retain the ability to differentiate into neural progenitor cells (NPCs) and forebrain neurons in a completely non-animal derived workflow. Figure 1 shows the experimental workflow. iPSCs are maintained on VTN62-292 variants using non-animal derived iPSC medium. Neural induction is performed with inhibitors SB43219 and N2-GMP supplement. Neural progenitor cells emerge 7-10 days after neural induction. After 2 passages over 28 days and conversion to neural medium with non-animal derived N21 supplement, forebrain neurons emerge. [Figure 5B] After long-term maintenance of iPSCs on hVTN62-292, we show that iPSCs retain the ability to differentiate into neural progenitor cells (NPCs) and forebrain neurons in a completely non-animal derived workflow. IBJ6 iPSC neurons were cultured under conditions that maintain stemness (day 0, d0) and differentiated after 10 days (d10) into intermediate neural progenitor cells or after 32 days (d32) into terminal forebrain neurons. Images show that naïve iPSCs express high levels of Oct3 / 4, which is greatly reduced after 10 days of NPC differentiation (left panel). After NPC differentiation, there is an increase in Pax6 expression, which is not observed in naïve iPSCs (middle panel). After 32 days of neural differentiation, there are numerous neurons with long beta-3-tubulin positive neurites, which are absent in naïve iPSCs (right panel). Smaller images show DAPI staining to visualize cells through their nuclei. [Figure 5C] After long-term maintenance of iPSCs on hVTN62-292, in a completely non-animal derived workflow, we show that iPSCs retain the ability to differentiate into neural progenitor cells (NPCs) and forebrain neurons. The bar graph shows that the majority (>80%) of iPSCs are positive for Oct3 / 4, but this stemness marker is lost in differentiated neurons. Conversely, iPSCs do not express beta III tubulin, yet >60% of differentiated cells express high levels of this neural marker. [Figure 6A]Mutations thought to improve integrin binding do not result in improved iPSC cell culture function, but mutations designed based on the interspecies consensus sequence show similar (Q148E and D80Y) iPSC cell expansion. Brightfield images of iPSCs cultured on 1 μg / ml VTN variants show that iPSC colonies grow well on VTN,D80Y and VTN,Q148E, but are smaller on VTN,T63G, VTN,T63G,V67N,F68P, and VTN,TG63,V67S. The triple mutant VTN,T63G,V67N,F68P did not support adhesion or iPSC cell expansion, and the double mutant VTN,T63G,V67S,F68P showed attenuated growth compared to the VTN62-292 variant. [Figure 6B] Mutations thought to improve integrin binding do not result in improved functionality in iPSC cell culture, whereas mutations designed based on the inter-species consensus sequence show similar (Q148E and D80Y) iPSC cell expansion. Bar graphs show total cell counts for two separate iPSC cells (BYS110 and IBJ6) on VTN variants coated at two different concentrations. Note that for both BYS110 and IBJ6 cell lines, D80Y shows similar live cell counts compared to the other variants, especially at 1 μg / ml (light grey box). The triple mutant VTN,T63G,V67N,F68P did not support adhesion or iPSC cell expansion, and the double mutant VTN,T63G,V67S,F68P showed attenuated proliferation compared to the VTN62-292 variant. [Figure 7A] Figure 1 shows the binding affinity of VTN variants to alpha V beta 5 (αVβ5) integrin heterodimer in an assay. A representative graph of an ELISA titration curve is shown. The Y-axis shows the optical density (OD) reading from the HRP secondary signal. The X-axis shows the protein titration. [Figure 7B] Figure 1 shows the binding affinity of VTN variants to the alpha V beta 5 (αVβ5) integrin heterodimer in an assay. The table summarizes the ED50 (50% effective dose) values ​​for different VTN variants in one experiment. [Figure 7C] Figure 1 shows the binding affinity of VTN variants to the alpha V beta 5 (αVβ5) integrin heterodimer in assays. Bar graphs show the ED50 average of five ELISA experiments normalized to VTN-FL. In these assays, hVTN62-292 shows a similar but slightly increased binding affinity to αVβ5 as full-length VTN (VTN-FL). They both show affinity for αVβ5 similar to the VTN-N variant (VTN-CTS), which is within 7% of VTN-FL. Surprisingly, the VTN mutants assumed to improve integrin binding, namely VTN62-292 T63G,V67N,F68P, significantly reduced αVβ5 binding affinity, increasing the ED50 value by more than 7-fold. However, the D80Y mutation showed enhanced αVβ5 affinity, decreasing the average ED50 value by more than 44%. Error bars = standard error. [Figure 8] Binding of cells to different vitronectin variants as determined by cell adhesion assay. In this assay, iPSCs were allowed to adhere to low concentrations of VTN variants (0.56ng / ml) for 1 hour, washed and assessed for adhesion. There is an almost complete loss of cell adhesion with the VTN62-292T63G,V67N,F68P mutants. Compared to the VTN-N variant, there is an increase with the VTN62-292 variant alone, with no further increase in adhesion with the Q148E or D80Y mutants. [Figure 9] Figure 1 shows the thermal stability of VTN62-292. Data is based on cell expansion compared to full length vitronectin (VTN-FL). Incubation at 37 degrees Celsius for 4 days prior to plating cells significantly reduces the ability of VTN-N to support iPSC expansion. However, VTN62-292 does not show any obvious reduction in cell proliferation compared to VTN-FL. The D80Y and Q148E mutations do not appear to confer any advantage in terms of stability as would be predicted. Error bars = standard deviation. [Figure 10A]Figure 1 shows that VTN62-292 supports pluripotency of iPSCs as demonstrated by triple germ layer differentiation analyzed by flow cytometry. Figure 2 shows the results of a flow cytometry experiment demonstrating increased expression of HNF4α in the endoderm as demonstrated by a significant increase in the mean fluorescence intensity of the marker. [Figure 10B] Figure 1 shows that VTN62-292 supports pluripotency of iPSCs as demonstrated by triple germ layer differentiation analyzed by flow cytometry. Figure 2 shows the results of a flow cytometry experiment demonstrating increased expression of Pax6 in the ectoderm as demonstrated by a significant increase in the mean fluorescence intensity of the marker. [Figure 10C] Figure 1 shows that VTN62-292 supports pluripotency of iPSCs, as shown by triple germ layer differentiation analyzed by flow cytometry. Figure 2 shows the results of a flow cytometry experiment demonstrating increased expression of Brachyury in the mesoderm, as shown by a significant increase in the mean fluorescence intensity of the marker. [Figure 10D] We show that VTN62-292 supports iPSC pluripotency as demonstrated by tri-germ differentiation analyzed by flow cytometry, and the stemness marker Nanog is significantly decreased following a protocol that induces mesoderm differentiation. [Figure 11] A, VTN62-292 supports long-term culture of iPSCs with genomic stability as shown by karyotype analysis. After long-term culture of iPSCs on VTN62-292 for two months and 13 passages, the cells maintained a normal karyotype, demonstrating genomic stability, an important characteristic for stem cell expansion for regenerative medicine. B, VTN62-292 supports long-term culture of iPSCs with genomic stability as shown by karyotype analysis. After long-term culture of iPSCs on VTN62-292 for two months and 13 passages, the cells maintained a normal karyotype, demonstrating genomic stability, an important characteristic for stem cell expansion for regenerative medicine. [Figure 12A]We demonstrate the reprogramming of peripheral blood monocytes (PBMCs) into induced pluripotent stem cells using hVTN62-292 as a substrate. This is significant in that previous studies have been unable to demonstrate this with other VTN variants (Ye and Wang, 2018, Cell Physiol Biochem;50(4):1318-1331). We show the formation of iPSC colonies at different time points after transduction with the Sendai Cytotune 2.0 reprogramming kit (Thermo-Fischer). After cloning of individual colonies, nascent iPSCs induced on hVTN62-292 display morphological characteristics of iPSCs with cells tightly packed into colonies with smooth borders. [Figure 12B] We demonstrate the reprogramming of peripheral blood monocytes (PBMCs) into induced pluripotent stem cells using hVTN62-292 as a substrate. This is significant in that previous studies have been unable to demonstrate this with other VTN variants (Ye and Wang, 2018, Cell Physiol Biochem;50(4):1318-1331). After cloning of individual colonies, nascent iPSCs induced on hVTN62-292 display morphological characteristics of iPSCs with tightly packed cells in colonies with smooth borders. These iPSCs display characteristic markers of iPSCs including Nanog, Oct3 / 4 and E-cadherin expression. [Figure 12C] We demonstrate the reprogramming of peripheral blood monocytes (PBMCs) into induced pluripotent stem cells using hVTN62-292 as a substrate. This is significant in that previous studies have been unable to demonstrate this with other VTN variants (Ye and Wang, 2018, Cell Physiol Biochem;50(4):1318-1331). Analysis of Oct3 and Sox2 by flow cytometry also demonstrated expression of stemness markers, whereas the absence of SSEA1 expression indicated the undifferentiated state of the iPSCs. [Figure 12D]We demonstrate the reprogramming of peripheral blood monocytes (PBMCs) into induced pluripotent stem cells using hVTN62-292 as a substrate. This is significant in that previous studies have been unable to demonstrate this with other VTN variants (Ye and Wang, 2018, Cell Physiol Biochem;50(4):1318-1331). iPSCs derived from a single clone have the differentiation capacity to form mesodermal cells as demonstrated by increased expression of Brachyury. [Figure 13A] Figure 1 shows that VTN62-292 and various mutants exhibit altered affinity for integrin receptors. The binding affinity of VTN62-292 variants and mutations adjacent to the RGD motif for major RGD-binding integrin heterodimers including alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) and alpha5beta5 (α5β1) (alpha5Beta5(α5β1)) was assayed via enzyme-linked immunosorbent assay (ELISA). A representative graph of an ELISA titration curve is shown. The Y-axis shows the optical density (OD) reading from the HRP secondary signal. The X-axis shows the protein titration. [Figure 13B] Figure 1 shows that VTN62-292 and various mutants exhibit altered affinity for integrin receptors. The binding affinity of VTN62-292 variants and mutations adjacent to the RGD motif for the major RGD-binding integrin heterodimers, including alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) and alpha5beta5 (α5β1) (alpha5Beta5(α5β1)), was assayed via enzyme-linked immunosorbent assay (ELISA). The table summarizes the ED50 (50% effective dose) values ​​for the different VTN variants from a representative experiment. [Figure 14]Figure 1 shows that VTN62-292 and its various variants exhibit altered affinities for integrin receptors as indicated by relative ELISA binding affinities. These bar graphs show the average ED50 of ≥2 ELISA experiments normalized to fibronectin (FN) (α5β1, αVβ1, and αVβ3) or VTN-FL (αVβ5). In these assays, hVTN62-292 exhibits a similar but slightly increased binding affinity for αVβ5 as full-length VTN (VTN-FL). However, hVTN62-292 exhibits improved binding affinity for αVβ3, αVβ1, and α5β1. VTN-N variants (VTN-CTS) show similar trends but typically have lower affinity for all four integrin heterodimers. These experiments confirmed that the VTN mutants that were postulated to improve integrin binding, namely VTN62-292 T63G,V67N,F68P, significantly decreased αVβ5 binding affinity and increased ED50 values. The VTN62-292 T63G,V67S,F68P mutant also showed decreased αVβ5 binding affinity. The triple mutant showed increased affinity for the two major fibronectin receptors, namely αVβ3 and α5β1, as well as αVβ1. This is significant in that there are mutants that are similar to fibronectin in their integrin binding ability. Graphs for αVβ3, αVβ1, and α5β1 are for fibronectin binding, and graphs for αVβ5 are for full-length vitronectin. Error bars = standard deviation. [Figure 15A] Figure 1 shows the effect of VTN variants on MSC proliferation in culture. Data presented are from three experiments with three concentrations of VTN. Cell proliferation was monitored by analyzing the confluence of MSC cell cultures after 72 hours. Brightfield images of MSCs on FN and different VTN62-292 variants are shown. [Figure 15B-1]Figure 6 shows the effect of VTN mutants on MSC proliferation in culture. Data presented are from three experiments with three concentrations of VTN. Cell proliferation was monitored by analyzing the confluence of MCS cell cultures after 72 hours. All three concentrations show that the VTN62-292, T63G, V67N, F68P and VTN62-292, T63G, V67S, F68P mutants support enhanced MSC proliferation. This is in stark contrast to the proliferation of iPSCs with the attenuated (VTN62-292, T63G, V67S, F68P) or completely suppressed (VTN62-292, T63G, V67N, F68P) triple mutants (as shown in Figure 6A and Figure 6B). [Figure 15B-2] Figure 6 shows the effect of VTN mutants on MSC proliferation in culture. Data presented are from three experiments with three concentrations of VTN. Cell proliferation was monitored by analyzing the confluence of MCS cell cultures after 72 hours. All three concentrations show that the VTN62-292, T63G, V67N, F68P and VTN62-292, T63G, V67S, F68P mutants support enhanced MSC proliferation. This is in stark contrast to the proliferation of iPSCs with the attenuated (VTN62-292, T63G, V67S, F68P) or completely suppressed (VTN62-292, T63G, V67N, F68P) triple mutants (as shown in Figure 6A and Figure 6B). [Figure 16A] Figure 1 shows cell expansion of the IBJ6 iPSC cell line in an undifferentiated state on FN, VTN62-292, and VTN62-292,T63G,V67N,F68P. This experiment shows that the same cell line in different states of stemness can switch substrate preferences. In the undifferentiated state, iBJ6 iPSCs only expand significantly on VTN62-292, as seen in brightfield images and quantified by percent confluence. Note that there is essentially no growth on VTN62-292,T63G,V67N,F68P. [Figure 16B]Cell expansion of IBJ6 iPSC cell line after differentiation into MSCs on FN, VTN62-292, and VTN62-292,T63G,V67N,F68P. This experiment shows that the same cell line in different states of stemness can switch substrate preferences. After differentiation into induced MSCs (iMSCs), the same cell line shows increased proliferation on FN but best proliferation on VTN62-292,T63G,V67N,F68P. This may be due to increased expression of αVβ3 and / or increased binding via α5β1 integrin after MSC differentiation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present disclosure provides vitronectin polypeptide fragments with improved properties for production and use as substrates for cell culture, including ex vivo culture of stem cells in non-animal derived conditions.

[0046] Vitronectin (VTN or VN) is a glycoprotein of the hemopexin family found abundantly in serum and the extracellular matrix where it is involved in cell adhesion, migration, cell spreading, and extracellular fixation. The full-length vitronectin polypeptide is 478 amino acid residues. When the signal sequence is removed, the mature vitronectin protein is a 54 kDa glycoprotein consisting of 459 amino acid residues.

[0047] The amino acid sequence of full-length human vitronectin (NCBI accession number NM_000638.4) is shown below:

[0048] TIFF2024533113000002.tif71170

[0049] The secondary structure of full-length vitronectin is illustrated in FIG. 1, showing the following domains: - A signal peptide from amino acid residues 1 to 19. - N-terminal somatomedin B (SMB) domain, amino acid residues 20 to 63. The SMB domain is a compact disulfide knot, with four disulfide bonds within approximately 35 residues. - an arginine-glycine-aspartic acid (RGD) integrin-binding motif at amino acid residues 64–66; - three central domains with hemopexin homology: these hemopexin-like domains are Hp1, from amino acid residues 158-202, Hp2, from amino acid residues 203-250, and Hp3, from amino acid residues 251-305. - a heparin-binding domain (Hb) comprising amino acid residues 365-395. - a C-terminal domain with hemopexin homology (Hp4) at amino acid residues 419–472.

[0050] The vitronectin polypeptide fragments of the present disclosure include vitronectin polypeptide fragments lacking one or more of the signal peptide, the N-terminal somatomedin B (SMB) domain, the heparin binding domain (Hb), and / or the C-terminal Hp4 domain. In some embodiments, the vitronectin polypeptide fragments of the present disclosure include vitronectin polypeptide fragments lacking the signal peptide, the N-terminal somatomedin B (SMB) domain, the heparin binding domain (Hb), and the C-terminal Hp4 domain.

[0051] In some embodiments, a vitronectin polypeptide fragment of the present disclosure comprises the arginine-glycine-aspartic acid (RGD) integrin binding domain of vitronectin at amino acid residues 64-66.

[0052] In some aspects, a vitronectin polypeptide fragment of the present disclosure may comprise any one, any two, or all three of the Hp1, Hp2, and Hp3 domains of vitronectin.

[0053] In some aspects, the vitronectin polypeptide fragments of the present disclosure include vitronectin polypeptide fragments that contain the arginine-glycine-aspartic acid (RGD) integrin binding domain of vitronectin at amino acid residues 64-66, and any one, any two, or all three of the Hp1, Hp2, and Hp3 domains of vitronectin, and that are missing the signal peptide from amino acids, the N-terminal somatomedin B (SMB) domain, the heparin binding domain (Hb), and the C-terminal Hp4 domain.

[0054] The vitronectin polypeptide fragment of the present disclosure has a length shorter than full-length vitronectin (478 amino acids) or mature vitronectin (459 amino acids). For example, the vitronectin polypeptide fragment of the present disclosure may have a length of about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 350 amino acids, or about 300 amino acids to about 350 amino acids. The vitronectin polypeptide fragment of the present disclosure may have a length of about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, or about 350 amino acids, or any range thereof. Vitronectin polypeptide fragments of the present disclosure can have a length of about 230, about 231, about 232, about 232, about 234, about 235, about 236, about 237, about 239, about 240 amino acids, or any range thereof.

[0055] A vitronectin polypeptide fragment of the present disclosure may comprise a deletion of 61 contiguous N-terminal amino acids and a deletion of C-terminal amino acids 293-478 as compared to a full-length vitronectin polypeptide having SEQ ID NO: 1. In some embodiments, such a vitronectin polypeptide fragment is a fragment of amino acids 62-292 of human vitronectin (SEQ ID NO: 1), also referred to herein as hVTN(62-292), VTN62-292, or hvitronectin:62-292, and has the following amino acid sequence: MVTRGDVFTM PEDEYTVYDD GEEKNNATVH EQVGGPSLTS DLQAQSKGNP EQTPVLKPEE EAPAPEVGAS KPEGIDSRPE TLHPGRPQPP AEEELCSGKP FDAFTDLKNG SLFAFRGQYC YELDEKAVRP GYPKLIRDVW GIEGPIDAAF TRINCQGKTY LFKGSQYWRF EDGVLDPDYP RNISDGFDGI PDNVDAALAL PAHSYSGRER VYFFKGKQYW EYQFQHQPSQ EE (SEQ ID NO:2) has.

[0056] Vitronectin polypeptide fragments of the present disclosure include vitronectin polypeptide fragments that are at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the vitronectin polypeptide fragment of SEQ ID NO: 2. As used herein, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. As discussed herein, whether any particular polypeptide is at least 40 percent (%), at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Advances in Applied Mathematics 2:482-489 to find the best segment of homology between two sequences. When using BESTFIT or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, parameters are set such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and allows gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0057] Vitronectin polypeptide fragments of the present disclosure include vitronectin polypeptide fragments having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions relative to SEQ ID NO: 2. Such amino acid substitutions include, but are not limited to, any of those shown in Table 1.

[0058] Such amino acid substitutions may be at positions adjacent to the RGD integrin binding site within the region corresponding to amino acids 62-70 of SEQ ID NO: 1. For example, such substitutions may be at the following positions: position 63 of full-length human vitronectin having SEQ ID NO:1 (position 3 of SEQ ID NO:2), including but not limited to the T63G amino acid substitution; position 67 of full-length human vitronectin having SEQ ID NO:1 (position 7 of SEQ ID NO:2), including but not limited to the V67S amino acid substitution; Position 68 of full-length human vitronectin having SEQ ID NO:1 (position 8 of SEQ ID NO:2), including but not limited to the F68P amino acid substitution; at positions 63 and 67 of full-length human vitronectin having SEQ ID NO:1, including but not limited to T63G or V67S amino acid substitutions; and / or Positions 63, 67, and 68 of full-length human vitronectin having SEQ ID NO:1, including but not limited to T63G, V67N, and F68P amino acid substitutions.

[0059] Such an amino acid substitution can include a substitution at position 80 of full-length human vitronectin having SEQ ID NO:1 (position 20 of SEQ ID NO:2), including, but not limited to, a D80Y amino acid substitution.

[0060] Such an amino acid substitution can include a substitution at position 143 of full-length human vitronectin having SEQ ID NO:1 (position 83 of SEQ ID NO:2), including, but not limited to, the H143D amino acid substitution.

[0061] Such an amino acid substitution can include a substitution at position 148 of full-length human vitronectin having SEQ ID NO:1 (position 88 of SEQ ID NO:2), including, but not limited to, a Q148E amino acid substitution.

[0062] Such an amino acid substitution can include a substitution at position 149 of full-length human vitronectin having SEQ ID NO:1 (position 89 of SEQ ID NO:2), including, but not limited to, the P149S amino acid substitution.

[0063] Such amino acid substitutions can include substituting a cysteine ​​at C293 of full-length human vitronectin having SEQ ID NO:1.

[0064] In some embodiments, vitronectin polypeptide fragments of the present disclosure include a fragment corresponding to amino acids 62-292 of human vitronectin (SEQ ID NO:1) in which aspartic acid (Asp, D) at position 80 is replaced by tyrosine (Tyr, Y) (D at position 20 of SEQ ID NO:2 is replaced by Y), referred to herein as VTN62-292,D80Y (also referred to herein as VTN62-292(D80Y), VTN62-292 D80Y, and VTN62-292D80Y).

[0065] In some embodiments, vitronectin polypeptide fragments of the present disclosure include a fragment corresponding to amino acids 62-292 of human vitronectin (SEQ ID NO:1) in which glutamine (Gln, Q) at position 148 has been replaced with glutamic acid (Glu, E) (Q at position 20 of SEQ ID NO:2 has been replaced with E), also referred to herein as VTN62-292(Q148E).

[0066] In some embodiments, the vitronectin polypeptide fragments of the present disclosure may include the mutations VTN62-292,T63G,V67N,F68P and VTN62-292,T63G,V67S,F68P. VTN fragments with these mutations may confer alterations in binding to RGD-binding integrins, namely alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) and alpha5beta5 (α5β1) (alpha5Beta5(α5β1)), in a manner similar to fibronectin binding to these integrins. Thus, given the difficulty in expressing fibronectin (FN) in non-animal derived systems such as E. coli, the vitronectin polypeptide fragments and variants thereof disclosed herein provide FN-like molecules that can be produced on a large scale in non-animal derived systems. This is beneficial for additional applications in cell therapy and regenerative medicine that are improved via signaling through the FN receptors αVβ3 and α5β1. Examples of such benefits include cardiomyocyte differentiation and osteoblast differentiation.

[0067] Also included in the present disclosure are vitronectin polypeptide fragments having additional heterologous amino acid residues at the N-terminus and / or C-terminus of the vitronectin polypeptide fragment that are not of vitronectin origin, such heterologous amino acid residues may, for example, code for an enzymatic activity or other additional components, such as a linker, a detectable marker, or a fusion protein.

[0068] In some embodiments, the C-terminus of the vitronectin polypeptide fragment of the present disclosure can be conjugated to a detectable marker, such as a polyhistidine tag or a streptavidin tag. Vitronectin can be indirectly detected using a fluorescently conjugated antibody against the polyhistidine tag or labeled biotin. These tags can provide quantification related to the amount of protein and its interaction with the corresponding integrin receptor. In addition, these tags can be utilized for conjugation to hydrogel polymers (alginate, polyethylene glycol, etc.) via antibody conjugates. Alternatively, vitronectin peptides can be directly conjugated to hydrogels via reactive groups such as free amine groups, carboxylic acid groups, or sulfones for the formation of vitronectin-functionalized hydrogels. In some embodiments, the C-terminus can be modified by adding lysines to increase side chain reactive amino groups or cysteines to increase reactive thiols for conjugation reactions.

[0069] In some embodiments, vitronectin polypeptide fragments of the present disclosure include a fragment corresponding to amino acids 62-292 of human vitronectin (SEQ ID NO:1) with a C-terminal polyhistidine tag, which can be 2, 3, 4, 5, 6, 7, 8, or more His residues, including, for example, a fragment corresponding to amino acids 62-292 of human vitronectin (SEQ ID NO:1) with a C-terminal polyhistidine tag of six histidine residues, which fragment is also referred to herein as human vitronectin fragment 62-292 C-His or VTN62-292his, hvitronectin:62-292 / His6, and has the following sequence: MVTRGDVFTM PEDEYTVYDD GEEKNNATVH EQVGGPSLTS DLQAQSKGNP EQTPVLKPEE EAPAPEVGAS KPEGIDSRPE TLHPGRPQPP AEEELCSGKP FDAFTDLKNG SLFAFRGQYC YELDEKAVRP GYPKLIRDVW GIEGPIDAAF TRINCQGKTY LFKGSQYWRF EDGVLDPDYP RNISDGFDGI PDNVDAALAL PAHSYSGRER VYFFKGKQYW EYQFQHQPSQ EEHHHHHH (SEQ ID NO:3) has.

[0070] The histidine tag serves many purposes. It provides affinity purification on nickel columns, thereby isolating the vitronectin polypeptide fragment from other cellular material in cell lysates. It can serve as an antigen for anti-His antibodies, which are used to quantitate the vitronectin polypeptide fragment in immunoassays. The His tag can also be used to bind the vitronectin polypeptide fragment to a cell culture substrate.

[0071] The vitronectin polypeptide fragments of the present disclosure may have one or more of the properties described herein. Characterization of the vitronectin polypeptide fragments of the present disclosure may be by any of a variety of available methods, including, but not limited to, any of the methods described in the Examples section contained herein.

[0072] For example, vitronectin polypeptide fragments of the present disclosure may exhibit improved yields when expressed in a host cell, such as, for example, E. coli, as compared to full-length vitronectin or other fragments.

[0073] Vitronectin polypeptide fragments of the present disclosure may exhibit improved solubility when expressed in a host cell, such as, for example, E. coli, as compared to full-length vitronectin or other fragments.

[0074] Vitronectin polypeptide fragments of the present disclosure may exhibit increased thermal stability as compared to full-length vitronectin or other fragments.

[0075] Vitronectin polypeptide fragments of the present disclosure, including but not limited to the mutations VTN62-292,T63G,V67N,F68P and VTN62-292,T63G,V67S,F68P, may exhibit reduced binding affinity to RGD-binding integrins, such as alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) or alpha5beta5 (α5β1) (alpha5Beta5(α5β1)). Reduced binding can be determined in comparison to full-length vitronectin (SEQ ID NO: 1) or full-length fibronectin.

[0076] Vitronectin polypeptide fragments of the present disclosure, including but not limited to the mutations VTN62-292,T63G,V67N,F68P and VTN62-292,T63G,V67S,F68P, may exhibit increased binding affinity to RGD-binding integrins, such as alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) or alpha5beta5 (α5β1) (alpha5Beta5(α5β1)). Increased binding may be determined relative to full-length vitronectin (SEQ ID NO: 1) or full-length fibronectin.

[0077] The vitronectin polypeptide fragments of the present disclosure can function as suitable cell-binding substrates for the adhesion / attachment of various cell types, including, but not limited to, human pluripotent stem cells (e.g., embryonic stem cells and induced pluripotent stem cells) and mesenchymal stem cells, to cell culture surfaces.

[0078] The vitronectin polypeptide fragments of the present disclosure can function as cell culture matrices for two-dimensional cell culture (e.g., coating tissue culture plastic), for conjugation to microcarriers (e.g., hydrogel-based or polystyrene microspheres), or for culture in three-dimensional matrices (e.g., as components of synthetic hydrogels).

[0079] The vitronectin polypeptide fragments of the present disclosure can support the undifferentiated proliferation and expansion of various cell types in culture, including, but not limited to, human pluripotent stem cells (e.g., embryonic stem cells and induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs), where the cells maintain an undifferentiated phenotype.

[0080] The vitronectin polypeptide fragments of the present disclosure may have an enhanced ability to support pluripotent stem cell viability, proliferation, pluripotency, maintenance of cloned cells, differentiation, and / or derivation of induced pluripotent cells compared to full-length vitronectin or full-length fibronectin.

[0081] The vitronectin polypeptide fragments of the present disclosure can support the differentiation of stem cells in a differentiation protocol, for example, supporting the differentiation of human pluripotent stem cells (hPSCs) into mesendoderm lineage cells and hepatocyte lineage cells, or supporting the osteogenic differentiation of human mesenchymal stem cells (hMSCs).

[0082] The vitronectin polypeptide fragments of the present disclosure can be produced in non-animal derived conditions.

[0083] The vitronectin polypeptide fragments of the present disclosure can function as substrates for cell culture, including ex vivo culture of stem cells in non-animal derived conditions.

[0084] nucleic acid In another aspect, the present disclosure describes an isolated polynucleotide molecule encoding a vitronectin polypeptide fragment described herein.In some embodiments, the isolated polynucleotide molecule comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence encoding the vitronectin polypeptide fragment described herein.In some embodiments, the isolated polynucleotide molecule comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence encoding the vitronectin polypeptide fragment of human vitronectin having SEQ ID NO:1.

[0085] In another aspect, the present disclosure describes a recombinant vector comprising an isolated polynucleotide of the present disclosure. The vector may be, for example, in the form of a plasmid, a viral particle, or a phage. The appropriate DNA sequence can be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) in the vector by procedures known in the art. Such procedures are considered to be within the scope of one of ordinary skill in the art. Many suitable vectors and promoters are known to those of ordinary skill in the art and are commercially available. The following vectors are provided as examples. Bacterial vectors include, for example, pQE70, pQE60, pQE-9, pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryotic vectors include, for example, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, and pSVL.

[0086] In some embodiments, suitable vectors are expression vectors for the expression of vitronectin polypeptide fragments in prokaryotic host cells, non-eukaryotic host cells, or non-animal host cells (host cells not belonging to the animal kingdom).Non-limiting examples include expression vectors for the expression in prokaryotic host cells, such as E. coli, fungal host cells, such as Pichia pastoris, or plant host cells.However, any other plasmid or vector may be used.

[0087] In a further aspect, the present disclosure also includes a host cell containing at least one of the above nucleotide sequences or vectors. The host cell may be a higher eukaryotic cell, such as a mammalian cell or an insect cell, or a lower eukaryotic cell, such as a yeast cell. Alternatively, the host cell may be a prokaryotic cell, for example, a bacterial cell, a fungal cell, or a plant cell. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is Pichia pastoris. The introduction of the vector construct into the host cell may be by any suitable technique, such as, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, or nucleofection. The vitronectin polypeptide fragment of the present disclosure can be expressed in mammalian cells, plant cells, yeast, bacteria, or other cells under the control of a suitable promoter. Examples of eukaryotic cells for such recombinant expression include HEK293, CHO, NSO, Vero, COS, HeLa, SF21 cells, Pichia pastoris, Saccharomyces cerevisiae, and Oryza sativa cells. Prokaryotic expression systems include E. coli variants such as DH5 alpha, DE3, and ROSETTA® (Novagen) cell strains.

[0088] Cell-free translation systems can be used to produce vitronectin polypeptide fragments of the present disclosure using RNA derived from the DNA constructs of the present disclosure. Alternatively, vitronectin polypeptide fragments of the present disclosure can be chemically synthesized by procedures known in the art.

[0089] As shown in the examples contained herein, expression of the vitronectin polypeptide fragments of the present disclosure in a host cell such as E. coli has demonstrated improved yields and improved solubility compared to those of full-length vitronectin or previously known fragments such as VTN62-398 and VTN62-478. The full-length native version of human vitronectin is difficult, if not impossible, to express in large quantities in E. coli, likely due to post-translational processing of the protein. The same is true when compared to larger fragments that contain amino acids C293 and C430 that form disulfide bonds. When expressed in E. coli, the fragment VTN62-398 shows poor expression and oxidation, while the commercially available VTN fragment VTN62-478 (Cell Therapy Systems) accumulates in inclusion bodies, requiring an additional solubilization step and complicating large-scale production. In contrast, the vitronectin polypeptide fragments of the present disclosure, including, for example, the vitronectin polypeptide fragments of SEQ ID NO: 2 and SEQ ID NO: 3 and variants thereof, result in soluble protein products when expressed in a host cell such as E. coli.

[0090] The vitronectin polypeptide fragments of the present disclosure can be expressed or synthesized as described herein to obtain preparations of vitronectin polypeptide fragments that are non-animal derived and free of animal contamination. Non-animal derived products can be expressed and isolated from non-animal host cells, such as, for example, E. coli. Non-animal derived products can be made without the use of any animal component-containing raw materials in the primary, secondary, or tertiary processes. Ex vivo culture of cells for therapeutic applications, including but not limited to immune cells and stem cells, requires safe and robust culture conditions. Safety standards for cell culture media and substrates used in such cultures are set forth in the United States Pharmacopeia (USP) publication USP <1043> In USP43-NF38, which details a tiered system for risk classification of raw materials used as auxiliary materials in cell and gene therapy, including regenerative medicine. The use of animal component-containing reagents is considered high risk (tier 4), while non-animal derived materials produced under GMP conditions are considered low risk (tier 2). The vitronectin polypeptide fragments of the present disclosure provide such low risk, non-animal derived products for ex vivo culture of cells for therapeutic applications.

[0091] The present disclosure also includes compositions comprising one or more of the vitronectin polypeptide fragments described herein. Such compositions can be used in cell cultures that contain such compositions as substrates for cell attachment. Such compositions may be of non-animal origin. Such compositions can function as defined culture media, i.e., culture media in which all components of the medium are fully disclosed and characterized.

[0092] The vitronectin polypeptide fragments of the present disclosure can be associated or conjugated to various substrates, including two-dimensional or three-dimensional cell culture substrates. The vitronectin polypeptide fragments of the present disclosure can be conjugated to microcarriers, including, for example, hydrogels or polystyrene microspheres. The hydrogels can be dissolvable hydrogels. The hydrogels can include compositions of multiple alginate molecules and multiple branched polymer molecules, where multiple alginate molecules are conjugated or blended with the branched polymer molecules to form a hydrogel, as described in more detail in U.S. Patent No. 9,927,334, U.S. Patent No. 9,790,467, and U.S. Patent No. 10,739,338, each of which is incorporated herein in its entirety.

[0093] The vitronectin polypeptide fragments, conjugates thereof, and compositions thereof described herein can be used for the culture of a variety of cell types for use in applications ranging from basic cell biology research to cell therapy and regenerative medicine workflows. Such vitronectin polypeptide fragments can function as cell attachment and / or growth substrates for a variety of cell types.

[0094] Such culturing may include ex vivo culturing of cells in addition to in vitro culturing. As used herein, ex vivo means "outside of living organism". In ex vivo culturing, cells or tissues can be taken directly from a living organism and cultured under defined culture conditions. Such cells can then be returned to the organism after ex vivo culturing.

[0095] The vitronectin polypeptide fragments, conjugates thereof, and compositions thereof described herein can be used to culture stem cells and stem cell-derived cells, such as adult stem cells, fetal stem cells, progenitor cells, peripheral hematopoietic stem cells, endothelial progenitor cells, amniotic stem cells, mesenchymal stem cells, adipose-derived stem cells, intestinal stem cells, skin stem cells, neural stem cells, or cancer stem cells. Stem cells can include pluripotent stem cells and multipotent stem cells (Salzig et al., 2016, Stem Cells Int;2016:5246584;Braam et al., 2008, Stem Cells;26(9):2257-65). Stem cell-derived cells include, but are not limited to, mesoderm and cells derived therefrom, such as cardiomyocytes, osteoblasts, chondrocytes, definitive endoderm and cells derived therefrom, and ectoderm and cells derived therefrom.

[0096] As used herein, the term "pluripotent stem cell" refers to a cell that can differentiate into cells of all three germ layers. Examples of pluripotent cells include embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. As used herein, "iPS cells" refers to pluripotent cells derived from somatic cells that exhibit similar characteristics to cells with higher potential, such as ES cells. Examples of multipotent stem cells include mesenchymal stem cells.

[0097] The vitronectin polypeptide fragments, compositions thereof, and conjugates thereof described herein can be used in the culture of cells for engineering and / or expansion of cells for use in adaptive T cell therapy systems, such as chimeric antigen receptor (CAR) T cell immunotherapy (reviewed, for example, in Feins et al., 2019, Am J Hematol;94(S1):S3-S9; Mohanty et al., 2019, Oncol Rep;42(6):2183-2195; and Huang et al., 2020, J Hematol Oncol, 13(1):86).

[0098] The present invention is defined in the claims. However, the following provides a non-exhaustive list of non-limiting exemplary aspects. Any one or more of the characteristics of these aspects can be combined with any one or more characteristics of another example, embodiment, or aspect described herein. Exemplary embodiments of the present invention include, but are not limited to:

[0099] Embodiment 1. A vitronectin polypeptide fragment comprising: comprising a deletion of at least 50 consecutive N-terminal amino acids compared to the full-length vitronectin polypeptide having SEQ ID NO:1; comprising a deletion of at least the C-terminal amino acids 362 to 478 compared to the full-length vitronectin polypeptide having SEQ ID NO:1; Contains the arginine-glycine-aspartic acid (RGD) integrin-binding domain of vitronectin; does not contain the N-terminal somatomedin B (SmB) domain of vitronectin; does not contain the C-terminal heparin-binding domain and Hp4 domain of vitronectin; and A vitronectin polypeptide fragment comprising about 95% sequence identity to the same fragment of a full-length vitronectin polypeptide having SEQ ID NO:1.

[0100] Embodiment 2. A vitronectin polypeptide fragment according to embodiment 1, comprising the Hp1, Hp2 and / or Hp3 domains of vitronectin.

[0101] Embodiment 3. The vitronectin polypeptide fragment according to embodiment 1 or 2, which has a length of about 200 to about 350 amino acids.

[0102] Embodiment 4. A vitronectin polypeptide fragment according to any one of embodiments 1 to 3, comprising: A deletion of 61 consecutive N-terminal amino acids compared to the full-length vitronectin polypeptide having SEQ ID NO:1; and The vitronectin polypeptide fragment, which comprises a deletion of C-terminal amino acids 293 to 478 compared with the full-length vitronectin polypeptide having SEQ ID NO:1.

[0103] Embodiment 5. A vitronectin polypeptide fragment according to any one of embodiments 1 to 4, comprising about 95% sequence identity to residues 62 to 292 of a full-length vitronectin polypeptide having SEQ ID NO:1.

[0104] Embodiment 6. A vitronectin polypeptide fragment according to any one of embodiments 1 to 5, comprising an amino acid substitution at a position corresponding to positions 80 and / or 148 of full-length human vitronectin having SEQ ID NO:1.

[0105] Embodiment 7. The vitronectin polypeptide fragment of embodiment 6, wherein the amino acid substitution comprises a D80Y substitution and / or a Q148E substitution.

[0106] Embodiment 8. A vitronectin polypeptide fragment consisting of SEQ ID NO:2.

[0107] Embodiment 9. A vitronectin polypeptide fragment consisting of SEQ ID NO:2, which has an amino acid substitution at position D80 and / or an amino acid substitution at position Q148 compared to the full-length vitronectin having SEQ ID NO:1.

[0108] Embodiment 10. A vitronectin polypeptide fragment according to embodiment 9, wherein the amino acid substitution at position D80 comprises a D80Y substitution and / or the amino acid substitution at position Q148 comprises a Q148E substitution.

[0109] Embodiment 11. A vitronectin polypeptide fragment according to any one of embodiments 1 to 10, comprising amino acid substitutions at positions corresponding to positions 63, 67, and / or 68 of full-length human vitronectin having SEQ ID NO:1.

[0110] Embodiment 12. The vitronectin polypeptide fragment of embodiment 11, wherein the amino acid substitution at position 63 comprises a T63G substitution, the amino acid substitution at position 67 comprises a V67S substitution or a V67N substitution, and / or the amino acid substitution at position 68 comprises a F68P substitution.

[0111] Embodiment 13. A vitronectin polypeptide fragment according to any one of embodiments 1 to 12, further comprising a C-terminal His-tag.

[0112] Embodiment 14. The vitronectin polypeptide fragment of embodiment 13, comprising 1, 2, 3, 4, 5, 6, 7, 8 or more C-terminal His residues.

[0113] Embodiment 15. A vitronectin polypeptide fragment consisting of SEQ ID NO:3.

[0114] Embodiment 16. The vitronectin polypeptide fragment according to any one of embodiments 1 to 15, which is of non-animal origin.

[0115] Embodiment 17. A vitronectin polypeptide fragment according to any one of embodiments 1 to 16, which is conjugated to a microcarrier.

[0116] Embodiment 18. The vitronectin polypeptide fragment conjugate of embodiment 17, wherein the microcarrier comprises a hydrogel or a polystyrene microsphere.

[0117] Embodiment 19. A composition comprising a vitronectin polypeptide fragment according to any one of embodiments 1 to 16, or a vitronectin polypeptide fragment conjugate according to embodiment 17 or 18.

[0118] Embodiment 20. The composition of embodiment 19, wherein the composition is of non-animal origin.

[0119] Embodiment 21. A vitronectin polypeptide fragment according to any one of embodiments 1 to 16, a vitronectin polypeptide fragment conjugate according to embodiment 17 or 18, or a composition according to embodiment 19 or 20 for use as a cell culture substrate.

[0120] Embodiment 22. A nucleotide sequence encoding a vitronectin polypeptide fragment according to any one of embodiments 1 to 16.

[0121] Embodiment 23. An expression vector comprising the nucleotide sequence of embodiment 22.

[0122] Embodiment 24. The expression vector of embodiment 23, comprising an E. coli expression vector.

[0123] Embodiment 25. A host cell comprising a nucleotide sequence according to embodiment 22 or an expression vector according to any one of embodiments 23 or 24.

[0124] Embodiment 26 The host cell of embodiment 25, comprising E. coli.

[0125] Embodiment 27. A method for producing a vitronectin polypeptide fragment, comprising expressing the vitronectin polypeptide fragment from a nucleotide sequence described in embodiment 22, an expression vector described in embodiment 23 or 24, or a host cell described in embodiment 25 or 26.

[0126] Embodiment 28 The method of embodiment 27, wherein the vitronectin fragment polypeptide produced is of non-animal origin.

[0127] Embodiment 29. A method for culturing cells, comprising culturing cells on a substrate comprising a vitronectin polypeptide fragment described in any one of embodiments 1 to 16, a vitronectin polypeptide fragment conjugate described in embodiment 17 or 18, or a composition described in embodiment 19 or 20.

[0128] Embodiment 30. The cell culture method of embodiment 29, wherein the cells comprise stem cells.

[0129] Embodiment 31. The cell culture method of embodiment 30, wherein the stem cells comprise human induced pluripotent stem cells (iPSCs).

[0130] Embodiment 32. The cell culture method of embodiment 30, wherein the stem cells comprise human mesenchymal stem cells (MSCs).

[0131] Embodiment 33. A cell culture method according to any one of embodiments 29 to 32, wherein the cells are cultured under non-animal-derived conditions.

[0132] Embodiment 34. A cell culture method according to any one of embodiments 29 to 33, wherein the cell culture is ex vivo.

[0133] The invention is illustrated in the following examples, it being understood that the particular examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0134] Example 1 Vitronectin 62-292 and VTN62-292,D80Y The production of full-length recombinant vitronectin (VTN) under non-animal-derived conditions is problematic, and expression in the E. coli protein expression system results in insoluble protein and low yields (Table 1). In this example, various vitronectin variants with improved solubility and expression were produced. The secondary structure of the vitronectin polypeptide and the variants produced and tested in this example are shown in Figure 1. [Table 1]

[0135] Previous studies have suggested that removal of the N-terminal SMB domain alone (VTN-N, or VTN62-478) or in combination with deletion of the C-terminal V10 domain (VTN-NC, or VTN62-398) results in recombinant VTN variants made in E. coli that support pluripotent stem cells in culture. These variants retain or even improve adhesion and survival bioactivity in culture, but produce insoluble proteins when made in E. coli, necessitating additional solubilization steps and complicating large-scale production. The poor solubility and resulting low yields of these two VTN variants (VTN62-478 and VTN62-398) are shown in Table 1.

[0136] The vitronectin variants produced and tested in this example retain the integrin-binding RGD site at amino acids 64-66, but lack the compact N-terminal somatomedin B (SmB) domain (amino acids 20-63). The heparin-binding (Hb) region (amino acids 362-395) was present in some of the variants produced and tested in this example. See Figure 1.

[0137] The VTN62-292 variant was constructed to remove the disulfide bond between cysteine ​​residues 293 and 430 (see FIG. 1 and Table 1).

[0138] In the two VTN truncation variants produced and tested in this example (VTN62-155 and VTN62-120), the glycosylation sites at amino acids 169 and 242 were removed (see FIG. 1 and Table 1).

[0139] The protein yields of these various constructs were tested in standard E. coli cultures for heterologous expression of recombinant proteins. Expression of longer fragments up to amino acid 398 resulted in low solubility and low yields in E. coli, whereas the VTN62-120, VTN62-155, and VTN62-292 variants were soluble and resulted in high expression (see FIG. 1 and Table 1). Due to low yields and / or low solubility, variants 1-3 in Table 1 were not compatible with non-animal-derived production in E. coli and were excluded from further analysis.

[0140] Next, in this example, we identified high-yield soluble variants that are suitable substrates for cell culture. To this end, we tested two cell types important for potential cell therapy applications: human induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs). One important goal of the stem cell workflow is to eliminate animal component-containing raw materials. Therefore, the VTN variants were tested in a unique media formulation that contains all growth factors and whose components are of non-animal origin.

[0141] iPSC line 12-10a was grown as single cells at 20,000 cells / cm 2 VTN62-120 and VTN62-155 variants were plated at 100 μg / ml and colonies were allowed to grow for 5 days with daily medium changes. To identify more subtle performance differences, three different concentrations of a given VTN variant were tested, including the more typical concentrations of 10 μg / ml and 5 μg / ml and the lower concentration of 1 μg / ml. Indeed, iPSCs cultured with the VTN62-292 variant showed typical morphology and high expansion even at lower concentrations compared to animal-derived full-length VTN and the commercially available VTN62-498 variant (VTN-N, commercially available from ThermoFisher) (Figures 2A and 2B). However, the VTN62-120 and VTN62-155 variants did not support iPSC colony growth, especially at lower concentrations.

[0142] Human MSC cultures should yield similar, though less dramatic, results (Figures 3A and 3B). Although MSCs attached and had normal morphology in VTN62-120 and VTN62-155, the yield of viable cells was significantly lower than in the VTN62-292 variant. These results indicate that the VTN62-292 variant confers similar biological properties to full-length wild-type VTN in its ability to support stem cell culture.

[0143] An important property of pluripotent stem cells is their ability to spread over long periods of time and after multiple passages while maintaining potency. Therefore, we tested the ability of VTN62-292 variants to support iPSC expansion and stemness over time. After 5 passages (3 weeks in culture), iPSCs maintained typical morphology with high expansion rates (Figure 4A). Using flow cytometry, we showed that pluripotency markers Oct3, Sox2, and SSEA-4 remained highly expressed, while the differentiation marker SSEA-1 was expressed at low levels in cells grown on VTN62-292 over long periods in cell culture (Figure 4B).

[0144] Next, it was demonstrated that iPSCs maintained on VTN62-292 could differentiate into different germ layers and diverse lineages. Ectoderm is patterned by inhibition of TGFβ signaling via Noggin. Under these conditions, iPSCs lose expression of Oct3 / 4, while ectodermal transcription factor Otx-1 is significantly increased (Figure 4C). Definitive endoderm develops in vivo by a combination of Activin A and Wnt signaling. Emulating these signals ex vivo resulted in increased expression of Sox17 and decreased Oct3 / 4 in iPSCs maintained on VTN62-292, thus indicating the potential of these cells to differentiate into endodermal cell lineages (Figure 4D). In other experiments, iPSCs maintained on VTN62-292 could differentiate into cardiomyocytes, a mesodermal derivative. Thus, iPSCs maintained in cell culture for long periods on VTN62-292 maintain pluripotency.

[0145] The results shown in Figures 4A-4D were confirmed in separate experiments using flow cytometry, which showed increased expression of brachyury, HNF4α, and Pax6 for mesoderm, endoderm, and ectoderm, respectively, as indicated by significant increases in the mean fluorescence intensity of these markers (Figures 10A-C). In addition, the stemness marker Nanog is significantly decreased following a protocol inducing mesoderm differentiation (Figure 10D).

[0146] In a different cell line, the BXS0114 line (available from ATCC) also showed pluripotency after multiple passages on VTN62-292 in three experiments. Definitive endoderm differentiation was achieved by Wnt3a and activin signaling and assessed by flow cytometry using the marker HNF4α (Figure 10A). There was a significant increase in HNF4α, indicating successful differentiation to endoderm. Differentiation to embryonic ectoderm was performed by dual SMAD inhibition with Noggin and the small molecule SB413452. Successful differentiation was observed by staining with the neuroectoderm marker Pax6, which increased dramatically after the differentiation protocol (Figure 10B). Differentiation to mesoderm lineages was achieved by the small molecule CHIR 99021, a GSK 3 inhibitor. Flow cytometry of brachyury showed a significant increase in mesoderm differentiation (Figure 10C) and a concomitant decrease in the stemness marker Nanog (Figure 10D). Taken together, these data indicate that the VTN62-292 variant supports the culture of pluripotent stem cells under naïve conditions and maintains the ability to differentiate into all three major germ layers.

[0147] Given the potential concerns regarding the culture and expansion of rapidly dividing stem cells, attention should be paid to genomic stability. Therefore, after 2 months of cell culture and 13 passages, karyotype analysis was performed (WiCell, Madison, Wi). Analysis of 1210a cells showed a normal karyotype / banding pattern of chromosomes, providing evidence of genomic stability (Figures 11A and 11B). After long-term culture of iPSCs on VTN62-292 for 2 months and through 13 passages, the cells maintained a normal karyotype, indicating genomic stability, a property important for the expansion of stem cells for regenerative medicine.

[0148] It has been reported that VTN does not support the reprogramming of somatic cells into induced pluripotent stem cells, whereas other undefined matrices such as basement membrane extract (BME, also known as Matrigel) and laminin can support reprogramming. Since these matrices are not non-animal derived, VTN62-292 was used as a substrate to support a non-animal derived workflow after reprogramming with Yamanaka factors (Oct3 / 4, Klf4, Sox2, c-Myc), and peripheral blood monocytes (PBMCs) were reprogrammed into induced pluripotent stem cells using the Sendai reprogramming kit (CytoTune2.0) with hVTN62-292 as a substrate following a modification of the manufacturer's guidelines. After day 7 of transduction, the cultures were fully transitioned to completely non-animal derived conditions.

[0149] Figures 12A-12D show the reprogramming of peripheral blood monocytes (PBMCs) into induced pluripotent stem cells using hVTN62-292 as a substrate. This is significant in that previous studies have not been able to show this with other VTN variants. Figure 12A shows the formation of iPSC colonies at different time points after transduction with the Sendai Cytotune2.0 reprogramming kit (Thermo-Fischer). After cloning of individual colonies, nascent iPSCs induced on hVTN62-292 show morphological characteristics of iPSCs with tightly packed cells in colonies with smooth borders. These iPSCs show characteristic markers of iPSCs including Nanog, Oct3 / 4 and E-cadherin expression (Figure 12B). Analysis of Oct3 and Sox2 by flow cytometry also showed expression of stemness markers, whereas the absence of SSEA1 expression indicated the undifferentiated state of the iPSCs (Figure 12C). In addition, iPSCs derived from a single clone have the differentiation potential to form mesodermal cells, as indicated by increased expression of brachyury (FIG. 12D).

[0150] Figure 12A shows the formation of iPSC colonies at different time points after transduction with Sendai virus. The colony formation efficiency was similar to that on BME matrix. After cloning of individual colonies, nascent iPSCs derived on hVTN62-292 show morphological characteristics of iPSCs with tightly packed cells in colonies with smooth borders. These iPSCs show characteristic markers of iPSCs including Nanog, Oct3 / 4 and E-cadherin expression (Figure 12B). Analysis of Oct3 and Sox2 by flow cytometry also showed expression of stemness markers, whereas the absence of SSEA1 expression indicated the undifferentiated state of iPSCs (Figure 12C). In addition, iPSCs derived from a single clone have the differentiation capacity to form mesodermal cells, as indicated by increased expression of Brachyury (Figure 12D). The reprogramming experiment was successfully repeated in two independent experiments with two different donors.

[0151] The primary goal of this example was to develop a VTN variant for use in an entirely non-animal derived workflow to generate differentiated cells from pluripotent stem cells. Such variants have broad applications for improving the safety and efficacy of workflows related to regenerative medicine. Therefore, VTN62-292 was tested as a cell matrix in a protocol to generate forebrain neurons from iPSCs ex vivo using (Figure 5A). All other components, such as culture medium, supplements, growth factors, and small molecules, were completely devoid of animal component-containing materials. After expanding iPSCs for 4 passages on VTN62-292, cells were plated at high density in non-animal derived N2 medium containing the TGFβ inhibitor SB43152-GMP (Tocris biosciences) based on previous studies (Chambers et al., 2009, Nat Biotechnol;27(3):275-80;and Chambers et al., 2009, Erratum in: Nat Biotechnol;27(5):485). After 7–12 days, this results in neural progenitor cells (NPCs) with high expression of neural stem cell transcription factors Pax6 and Sox1 (Figure 5B). These NPCs can then be further differentiated into forebrain neurons after transferring the cells to neuronal medium containing non-animal derived N21 neural supplement. Thirty-two days after the start of differentiation, more than 60% of the cells had long neurite outgrowths and were positive for the neural marker beta III tubulin (Figure 5C). In addition, expression of the stemness marker Oct3, which was prominent in iPSCs, was absent after the differentiation protocol (Figure 5C). Taken together, these data indicate that VTN62-292 can support iPSCs in a naive pluripotent state, as well as the differentiation of iPSCs into downstream lineages, indicating that VTN62-292 is a promising agent for stem cell culture.

[0152] To improve the function of VTN as an adhesion protein, we introduced targeted mutations into the VTN62-292 variant.

[0153] First, amino acids adjacent to the RGD integrin binding site were modified. Six mutants of the VTN62-292 variant were produced and tested to determine whether these amino acid changes could promote improved biological function in the context of the larger VTN polypeptide in cell culture. These are variants 9-15, listed in Table 1. Vitronectin binds to integrin heterodimers via the RGD motif. αVβ5 and αVβ3 are the major VTN receptors that bind with high affinity, but α5β1 integrin can also interact weakly with VTN. The RGD motif is conserved among other integrin-binding proteins such as fibronectin, laminin, and osteopontin, but these proteins show different affinities for integrin heterodimers. The RGD motif itself is important for optimal interaction with integrins in any of these proteins; point mutations here reduce or eliminate integrin binding (Cherny et al., 1993, J Biol Chem;268(13):9725-9). There is growing evidence that the amino acid sequence flanking the RGD motif can at least partially explain the differential binding of various proteins that bind to integrins via this tripeptide. For example, biochemical studies using small peptides have investigated their binding affinity to various integrin heterodimers (Kapp et al., 2017, Sci Rep;7:39805). Taking advantage of this information, we attempted to modulate the function of VTN62-292 as an adhesion protein for stem cell culture by modifying amino acids in the region surrounding the RGD site. For example, the sequence GRGDSP was shown to increase the binding affinity to αVβ5, αVβ3, and α5β1, a significant improvement over the RGD peptide in isolation (Kapp et al., 2017, Sci Rep;7:39805).

[0154] Second, we used interspecies sequence alignment to identify consensus sequences of VTN polypeptides that differ in the human sequence (Jones et al., 2020, Methods Enzymol;643:129-148). Consensus sequence alignments have been used to study the molecular evolution of proteins and, more recently, to identify mutations that may alter function. In particular, consensus sequences may indicate differences in the stability of the molecular structure, which would be beneficial for recombinant protein expression. Using publicly available online sequence alignment tools, we identified various single amino acid variations in the human sequence compared to the majority of other vitronectin sequences (Jones et al., 2020, Methods Enzymol;643:129-148). The two most prevalent amino acid changes, namely, an aspartic acid to tyrosine change at position 20 (D80Y) and a glutamine to glutamic acid change at position 148 (Q148E), were further tested. These point mutations were made and tested to see if they conferred any advantage to the function and stability of the recombinant vitronectin protein in the 62-292 variant, which are variants 7 to 8 listed in Table 1.

[0155] The screen for these mutations on VTN62-292 focused on its ability to support pluripotent stem cell culture. Two different iPSC cell lines (BYS110 and iBJ6) as well as two concentrations were tested. Surprisingly, the amino acid substitutions predicted to improve integrin binding actually reduced iPSC cell proliferation (Figures 6A and 6B). For example, the triple mutation VTN62-292,T63G,V67N,F68P was predicted to improve cell adhesion because peptide studies showed a significant improvement in binding of various integrin heterodimers, including αVβ5, αVβ3, and α5β1 (Kapp et al., 2017, Sci Rep;7:39805). However, in these studies, this mutation was detrimental to the function of vitronectin as a substrate for iPSCs, especially at low concentrations (Figures 6A and 6B). Interestingly, two mutations tested using consensus sequence alignment showed promise as matrices for iPSCs (VTN62-292, Q148E and VTN62-292, D80Y). Notably, the D80Y mutation supported improved cell expansion of both iPSC lines at both concentrations, but more pronounced at the 1 μg / ml concentration. In the case of the iBJ6 cell line, this was a more than 50% increase in total viable cells (Figure 6B).

[0156] Given the improved cell expansion observed with the VTN62-292 variant and the D80Y mutation, the ability of VTN variants to bind to the αVβ5 heterodimer was tested in an ELISA assay.

[0157] VTN62-292 variants and mutations therein were compared to full-length vitronectin made in NSO cells (a mouse cell line) and the commercially available version VTN62-498 (so-called VTN-N, or VTN-CTS from Thermo-Fisher). These assays demonstrated that VTN62-292 has a similar binding affinity for αVβ5 as full-length VTN and VTN-CTS (Figure 7A), and that the VTN62-292 triple mutant had significantly reduced binding to αVβ5, with an ED50 of 523 ng / ml (indicating poor binding), an almost notable increase compared to VTN-FL or VTN62-292. This correlated well with previous experiments in which VTN62-292,T63G,V67N,F68P failed to support iPSCs in cell culture (Figures 7A, 7B, and 7C).

[0158] In contrast, the VTN62-292, D80Y mutant showed a nearly two-fold increase in αVβ5 binding affinity, which correlated with the improved function of this variant in expanding iPSC cells. This is shown by a nearly 50% decrease in the ED50 value for αVβ5 binding (Figure 7B). The Q148E mutant did not show a significant increase in αVβ5 binding. This data indicates that the improved function of the D80Y mutant in supporting stem cell expansion in culture may be related to increased affinity for integrins and integrin-mediated adhesion or cell signaling.

[0159] Mutants flanking the RGD domain did not show improved αVβ5 binding, but it is possible that these changes may affect binding to other integrin heterodimers. Additional ELISA experiments were performed to examine the binding affinity of VTN62-292 variants and mutants generated from it to the major RGD receptors: alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3) and alpha5beta5 (α5β1) (alpha5Beta5(α5β1)). αVβ3 and α5β1 are the major fibronectin (FN) receptors. αVβ3 and αVβ5 are VTN receptors. FN and full-length VTN were used for comparison. Figures 13A and 13B and Figure 14 show the binding affinity of VTN62-292 variants and mutations adjacent to the RGD motif for the major RGD-binding integrin heterodimers, including alphaVbeta5 (αVβ5), alphaVbeta1 (αVβ1), alphaVbeta3 (αVβ3), and alpha5beta5 (α5β1) (alpha5Beta5(α5β1)). In these assays, hVTN62-292 shows similar but slightly increased binding affinity for αVβ5 as full-length VTN (VTN-FL). However, hVTN62-292 shows improved binding affinity for αVβ3, αVβ1, and α5β1. This may explain why hVTN62-292 outperforms FL-VTN in some cell proliferation assays. VTN-N variants (VTN-CTS) show a similar trend, but typically have lower affinity for all four integrin heterodimers.

[0160] These experiments confirmed that the VTN mutants postulated to improve integrin binding, namely VTN62-292 T63G,V67N,F68P, significantly reduced αVβ5 binding affinity and increased ED50 values. The VTN62-292 T63G,V67S,F68P mutant also showed reduced αVβ5 binding affinity, albeit to a lesser extent. Interestingly, the triple mutant showed increased affinity for the two major fibronectin receptors, namely αVβ3 and α5β1, as well as αVβ1. This is significant in that there are mutants that are similar to fibronectin in their integrin binding ability. Because the VTN62-292 T63G,V67S,F68P and VTN62-292 T63G,V67N,F68P mutants can be efficiently produced in non-animal derived conditions, whereas full length fibronectin cannot, these mutants may offer the advantage of achieving non-animal derived cell culture conditions with cell types that utilize FN and / or these integrins for optimal growth.

[0161] To further investigate the role of VTN variants in supporting iPSCs, a modified adhesion assay was performed whereby cells were allowed to adhere to VTN-coated tissue culture plates for 1 hour, then washed and tested for cell adhesion to the substrate (Kueng et al., 1989, Anal Biochem;182(1):16-9; and Taooka et al., 1999, J Cell Biol;145(2):413-20). This assay is thought to measure how tightly cells are attached to the underlying matrix. Using this approach, no increase in iPSC cell attachment to VTN62-292 compared to full-length VTN (Figure 7B) was detected. The VTN62-292 variant showed higher adhesion than the VTN-N(CTS) variant. This is significant because VTN-N is the only other functional non-animal derived VTN available. However, the D80Y mutation surprisingly did not result in improved adhesion given the improved integrin binding (see Figure 8). Because this assay probes nascent adhesions formed during initial cell attachment, but not more mature focal adhesions, these results may not fully reflect the cell adhesive properties of VTNs.

[0162] The VTN62-292 and VTN62-292,D80Y variants supported the expansion of iPSCs in cell culture, whereas the triple mutants VTN62-292 T63G,V67S,F68P and VTN62-292 T63G,V67N,F68P did not. This is likely a reflection of the reduced ability of these mutants to bind to αVβ5, the major VTN receptor expressed in iPSCs. Although data are not shown, in this example it was confirmed that iPSCs lack expression of the typical VTN receptor β3, whereas mesenchymal stem cells (MSCs) express all the major subunits of the RGD receptor, including β3. MSCs generally grow on FN, and MSC differentiation is driven by α5β1 integrin. Therefore, given the ELISA data showing increased affinity for αVβ3 and α5β1, the VTN62-292 T63G,V67S,F68P mutant and / or the VTN62-292 T63G,V67N,F68P mutant may promote proliferation of MSCs.

[0163] To test this, human bone-derived MSCs were cultured on FN, VTN62-292, VTN62-292 T63G,V67S,F68P, and VTN62-292 T63G,V67N,F68P (Figures 15A and 15B). At various concentrations, VTN62-292 T63G,V67S,F68P, and VTN62-292 T63G,V67N,F68P showed improved cell proliferation compared to FN and VTN62-292 (Figure 15B). All of these matrices improved MSC proliferation compared to BME.

[0164] Figures 15A and 15B show the effect of VTN variants on MSC proliferation in culture. The data presented are from three experiments with three concentrations of VTN. Cell proliferation was monitored by analyzing the confluence of MCS cell cultures after 72 hours. Figure 15A shows bright field images of MSCs on FN and different VTN62-292 variants. All three concentrations show that the VTN62-292, T63G, V67N, F68P and VTN62-292, T63G, V67S, F68P variants support enhanced MSC proliferation (Figure 15B). This is in stark contrast to the proliferation of iPSCs on the attenuated (VTN62-292, T63G, V67S, F68P) or completely suppressed (VTN62-292, T63G, V67N, F68P) triple mutants (Figures 6A and 6B). Figures 16A and 16B show cell expansion of the IBJ6 iPSC cell line in an undifferentiated state on FN, VTN62-292, and VTN62-292,T63G,V67N,F68P, and after differentiation into MSCs. This experiment shows that the same cell line in different states of stemness can switch substrate preferences. In the undifferentiated state, iBJ6 iPSCs expand significantly only on VTN62-292, as seen in brightfield images and quantified by percent confluence (Figure 16A). Note that there is essentially no proliferation on VTN62-292,T63G,V67N,F68P. After differentiation into induced MSCs (iMSCs), the same cell line shows increased proliferation on FN, but best proliferation on VTN62-292,T63G,V67N,F68P (Figure 16B). This may be due to increased expression of αVβ3 and / or increased binding via α5β1 integrin after MSC differentiation.

[0165] MSCs derived from iPSCs (iMSCs) offer many advantages for clinical applications, including homogeneity and expandability. Therefore, VTN62-292 and VTN62-292 T63G,V67N,F68P were tested for their suitability for iMSCs. First, iBJ6 iPSCs were grown on FN, VTN62-292, and VTN62-292 T63G,V67S,F68P. Brightfield images and cell confluence measurements confirmed that VTN62-292 supported robust expansion of iPSCs, whereas FN and VTN62-292 T63G,V67S,F68P failed to support iPSC proliferation (Figure 16A). However, after differentiation of iPSCs to iMSCs, FN, VTN62-292, and VTN62-292 T63G,V67S,F68P supported cell expansion (Figure 16B). The VTN62-292 T63G,V67S,F68P mutant showed the highest cell expansion as indicated by cell confluence measurements. This likely reflects increased β3 expression and / or increased signaling through α5β1 after MSC differentiation.

[0166] This improved functionality of the VTN62-292 and D80Y variants may be related to the thermal stability of the proteins, which is predicted based on previous studies of consensus alignments in other proteins (Porebski and Buckle, 2016, Protein Eng Des Sel;29(7):245-51). Therefore, a simple test related to the goal of improving VTN as a cell culture substrate was performed. In this test, VTN variants were incubated at 37°C for 4 days before plating iPSCs. After 4 days of cell expansion, it was unexpectedly observed that the D80Y mutation did not confer additional stability over the VTN62-292 variant (Figure 9). Although moderate, the VTN62-292 variant conferred a stability advantage compared to the VTN-N variant. However, none of the E. coli-derived fragments had a stability advantage over the VTN-FL protein. Nonetheless, these results suggest that VTN62-292 may have increased stability over other E. coli -derived fragments, which may be an added advantage for cell culture.

[0167] Taken together, the results of this example show that the VTN62-292 and VTN62-292,D80Y variants confer significant advantages over full-length VTN and previously published VTN variants. First, the enhanced manufacturability of VTN62-292 in prokaryotic expression systems is a major advantage, providing large-scale production of biologically active adhesion molecules for a non-animal derived workflow for stem cell culture and differentiation. Although the smaller variants also showed high expression, these variants are inadequate for stem cell applications due to their insufficient ability to support iPSC or MSC culture. The second advantage is the improved ability of these variants to support iPSC and MSC expansion compared to full-length VTN or VTN-N variants. With respect to iPSCs, this is especially evident at low concentrations where VTN62-292 and VTN62-292,D80Y show significant improvements in cell expansion in multiple iPSC cell lines. Furthermore, the utility of these variants extends to more robust stem cell workflows, as they also support differentiation of iPSCs into definitive endoderm, neural progenitor cells, and fully differentiated neurons. A third advantage is the application to completely non-animal derived ex vivo workflows relevant to regenerative medicine.

[0168] In addition, the results here suggest that RGD integrin binding specificity can also be modulated in the context of VTN variants to tailor cell type-specific cell culture and modify the behavior of these cells. In the context of regenerative medicine, these VTN variants provide a non-animal derived substrate for enhancing MSC expansion. These variants, including VTN62-292 T63G,V67S,F68P and VTN62-292 T63G,V67N,F68P, may also prove beneficial for cellular processes enhanced by α5β1 and / or αVβ3, such as mesoderm lineage differentiation, cardiac differentiation, or osteogenesis.

[0169] Example 2 Recombinant vitronectin variants with differentially tuned integrin specificity optimize proliferation of mesenchymal and pluripotent stem cells

[0170] Vitronectin (VTN) is an important defined substrate for the ex vivo expansion of human induced pluripotent stem cells (iPSCs), but is less frequently used for mesenchymal stem cells (MSCs). The latter cell type is more frequently expanded using fibronectin (FN), but this is difficult and expensive to produce using recombinant techniques.

[0171] The targeted mutations described in more detail in Example 1 improve the ability of VTN to support MSCs through altered binding of different integrin heterodimers. These novel recombinant human vitronectin (hVTN) variants show different binding activities to the major RGD integrin heterodimers, including the canonical FN receptor alpha5-beta1. Through this different integrin binding activity, these hVTN variants differentially support the adhesion, survival, spreading, and differentiation of MSCs and iPSCs. One of these modified VTN variants (VTN62-292 T63G, V67N, F68P; also referred to herein as VN-3) was unable to support undifferentiated iPSCs in cell culture, but showed improved MSC adhesion, survival, and expansion. Furthermore, MSC osteogenic differentiation was also enhanced with this VTN variant. The VTN62-292 T63G,V67N,F68P variant showed similar binding affinity for alphaV-beta3, but a large decrease in affinity for alphaV-beta5, and an increase in affinity for alpha5-beta1. Using integrin expression profiling, it was shown that while MSCs express all major RGD-binding integrin heterodimer subunits, iPSCs lack expression of beta3 integrin, limiting the repertoire of heterodimers that iPSCs can use to bind RGD-containing ECM proteins. Blocking integrin alphaVbeta5 heterodimer binding impairs the ability of iPSCs to bind either full-length VTN or VTN variants, but does not affect binding of MSCs to VTN. Blocking only alphaVbeta3, alphaVbeta5, and alpha5beta1 significantly reduces binding of MSCs to VTN. This strongly suggests that undifferentiated iPSCs primarily use alphaV-beta5 to bind to the VTN, whereas MSCs are more versatile and can bind to the VTN using a variety of integrin heterodimers.Interestingly, when iPSCs differentiate into MSCs, they start to express beta3 integrin and are subsequently able to bind effectively to the VTN62-292 T63G,V67N,F68P variant, confirming how changes in integrin expression patterns confer sensitivity to different vitronectin variants.

[0172] Integrin receptor binding influences a variety of short-term and long-term cellular responses that drive diverse and essential cellular behaviors. Through direct binding to the actin cytoskeleton and signaling through Rho GTPases, integrins regulate actin dynamics and focal adhesion turnover, thereby influencing cell-substrate binding, cell polarity, and migration. In addition, integrins signal through diverse, interconnected transduction pathways that influence cell survival, spreading, and differentiation. Integrins are transmembrane heterodimers composed of α and β subunits, which are arranged in different combinations to form 24 unique integrin receptors. These different heterodimers can be classified into subgroups according to the binding of different extracellular matrix (ECM) proteins, such as collagen, laminin, fibronectin (FN), and vitronectin (VTN).

[0173] Fibronectin and vitronectin share a common arginine-glycine-aspartic acid (RGD) motif that is recognized by five αV integrins, two β1 integrins, and αIIbβ3 integrins (Humphries et al., 2006, J Cell Sci;119(Pt 19):3901-3). Additional ECM and adhesion molecules, including laminin, osteopontin, bone sialoprotein, and platelet endothelial cell adhesion molecule, also have RGD sites that are recognized by these integrins. Although the mechanics of the RGD binding motif are generally conserved among the various α / β dimers, different ligand affinities exist, likely due to differences in the specific conformation of the binding site (Wang et al., 2013, Int J Mol Sci;14(7):13447-62).

[0174] It is well known that the presentation of different conformations of the RGD motif can alter the binding of synthetic peptides, with cyclic RGD peptides exhibiting higher affinity than linear and different variations of cyclic peptides and different binding affinities for various integrins (Kapp et al., 2017, Sci Rep;7:39805). Indeed, integrin specificity can be achieved for synthetic peptides by altering the adjacent amino acid sequence and binding mode of the RGD peptide at the dimer interface (Kapp et al., 2017, Sci Rep;7:39805). These studies suggest that the relative affinity that a given RGD-containing ECM molecule has for various integrin dimers depends on the presentation of the RGD motif, which can be influenced by the adjacent amino acid sequence.

[0175] The difference in the binding affinity of integrins for various RGD-containing molecules likely regulates the diverse cell behaviors on ECM matrices in cell culture. Under serum-free cell culture conditions, the choice of ECM substrate becomes especially important, since the normally high levels of fibronectin, vitronectin, and other adhesion proteins in serum are necessary for cell attachment, and numerous other growth factors, proteins, and other molecules affect cell behavior. Pluripotent stem cells (PSCs) and mesenchymal stem cells (MSCs) are two important cell types that require ex vivo expansion on ECM matrices for applications in regenerative medicine. To employ these cell types and their derivatives, it is necessary to optimize defined cell culture conditions, excluding animal- and even human-derived materials. Under xeno-free conditions, MSC culture protocols commonly use fibronectin for both expansion and differentiation (Cimino et al., 2017, Stem Cells Int;2017:6597815; and Basoli et al., 2021, Sci Rep;11(1):13089). Although less commonly used, MSCs also grow and differentiate on vitronectin, basement membrane extracts, and laminin (Lam and Longaker, 2012, J Tissue Eng Regen Med;6 Suppl 3(0 3):s80-6). The ability of MSCs to use a variety of substrates is likely due to their expression of diverse integrin subunits (Frith et al., 2012, Stem Cells Dev;21(13):2442-56). MSCs can bind and signal through a variety of integrins, and their expansion rate and differentiation potential vary depending on the substrate / binding motif.Many studies have used FN under xenogeneic conditions to promote the expansion, osteogenic and chondrogenic differentiation of MSCs, which appears to be mediated through both αV and β1 integrins (Singh and Schwarzbauer, 2012, J Cell Sci;125(Pt 16):3703-12; Di Benedetto et al., 2015, Stem Cell Res;15(3):618-628; and Basoli et al., 2021, Sci Rep;11(1):13089), with evidence of enhanced binding of α5β1, which plays a role in osteogenic differentiation (Martino et al., 2008, Biomaterials;30(6):1089-97; Hamidouche et al., 2009, Proc Natl Acad Sci USA;106(44):18587-91; Frith et al., 2012, Stem Cells Dev;21(13):2442-56). Although not commonly used as a substrate for MSCs, vitronectin also supports the attachment, expansion, and differentiation of MSCs expressing the canonical αVβ3 and alternative αVβ5 VTN receptors (Ode et al., 2010, J Biomed Mater Res A;95(4):1114-24). The move towards culturing pluripotent stem cells in xeno-free culture began with the adoption of basement membrane extract (BME) as a substrate and medium containing key growth factors, replacing the mouse fibroblast feeder system (Ludwig et al., 2006, Nat Biotechnol;24(2):185-7). BME is an undefined mixture of proteins that contains high levels of laminin, type IV collagen, perlecan, and entactin, as well as low levels of many other adhesion-related proteins, such as fibronectin. In the naive state, PSCs can be expanded and cultured long-term on purified or recombinant ECM proteins such as laminin, vitronectin, and fibronectin.However, reports of the use of fibronectin to maintain PSCs in a pluripotent state have been inconsistent, with some reporting a decreased proliferation rate and increased spontaneous differentiation (Hayashi et al., 2016, Stem Cells Int;2016:5380560).

[0176] Specific binding of different RGD integrin receptor dimers has different consequences for intracellular signaling and cell behavior. For example, the major FN receptors, αVβ3 and α5β1, synergize for proper mechanosensing of the microenvironment and motility (Schiller et al., 2013, Nat Cell Biol;15(6):625-36), but they signal intracellularly through different binding partners and pathways. This may result in subtle differences in cell behavior in the short term and more pronounced changes in cell physiology in the long term. In the short term, FN binding of α5β1 results in larger, more dynamic cells with smaller focal adhesions, whereas FN signaling through αVβ3 results in smaller, less motile cells with larger focal adhesions and stress fibers (Schiller et al., 2013, Nat Cell Biol;15(6):625-36). Long term, there is evidence that α5β1-mediated signaling is important for osteogenic differentiation of MSCs (Martino et al., 2008, Biomaterials;30(6):1089-97; Frith et al., 2012, Stem Cells Dev;21(13):2442-56) and cardiac specification of iPSCs (Neiman et al., 2019, Sci Rep;9(1):18077). α5β1 integrin is the main receptor for fibronectin, binding mainly through the RGD site in the 10th FNIII repeat domain of the central binding region, with some additive effect of the PHSRN motif in FNIII repeat domain 9 (Pankov and Yamada, 2002, J Cell Sci;115(Pt 20):3861-3). Fibronectin is essential for embryonic development; its deficiency results in severe mesodermal defects, including vascular and cardiac malformations (George et al., 1993, Development;119(4):1079-91; and George et al., 1997, Blood;90(8):3073-81).Genetic ablation of α5 integrin has mesodermal defects that overlap with vascular and cardiac abnormalities, suggesting that FN signaling through α5β1 is at least partially involved in these developmental processes (Bouvard et al., 2001, Circ Res;89(3):211-23; and Liang et al., 2014, Dev Biol;395(2):232-44).

[0177] Fibronectin molecules exist in multiple alternatively spliced ​​isoforms from the same gene. In general, FN is a dimer of two large, approximately 250 kDa glycoprotein subunits linked via disulfide bonds (Pankov and Yamada, 2002, J Cell Sci;115(Pt 20):3861-3). As such, the production of recombinant fibronectin is a laborious process, resulting in low yields in mammalian systems and essentially impossible in E. coli-based expression systems (Staunton et al., 2009, Methods Mol Biol;522:73-99). Although FN has many integrin binding partners, many of the important effects of FN are mediated through α5β1. Other RGD proteins, such as VTN, are unable to bind to and signal through α5β1 despite fundamental similarities in the RGD binding pocket (Kapp et al., 2017, Sci Rep;7:39805).

[0178] As discussed in more detail in Example 1, a VTN variant consisting of amino acids 62-292 can be effectively produced in a non-animal derived system with functional activity equivalent to or improving upon full-length VTN or other published fragments. Based on analysis of adjacent amino acid sequences from various RGD-containing ECM molecules and modifications to RGD-containing peptides (Kapp et al., 2017, Sci Rep; 7:39805), in this Example, modifications of the integrin binding site of this VTN62-292 variant provided a platform for specific binding to different integrin isoforms.

[0179] In this example, vitronectin variants were generated that altered binding to specific integrin heterodimers. Single, double, and triple mutations designed around the RGD domain of the VTN62-292 variant background show different affinities for αVβ3, αVβ5, αVβ1, and α5β1 heterodimers in ELISA assays. Surprisingly, it was observed that while single and double mutations suppress binding of αVβ3, αVβ1, and α5β1 integrins, triple mutants constructed based on these single and multiple / tiple mutants improve binding to these same integrins. In cellular assays, improved proliferation was observed for both bone-derived MSCs and iPSC-derived MSCs on these same triple mutants, whereas iPSC proliferation was attenuated or completely abolished. The latter result is likely due to the reduced ability of these triple mutants to bind to αVβ5, the major VTN receptor in iPSCs. This was confirmed in antibody blocking experiments with iPSCs cultured on VTN, showing that blocking αVβ5 alone significantly reduced iPSC cell proliferation, while blocking β1 together with αVβ5 completely inhibited iPSC proliferation. Blocking αVβ3 does not affect iPSC proliferation on VTN. iPSC-derived MSC (iMSC) proliferation on VN is not dramatically attenuated when αVβ5, αVβ3, and β1 are all blocked simultaneously, which is consistent with the integrin expression profile of these cells. Furthermore, enhanced osteogenic differentiation was observed in triple mutants of VTN, suggesting that altered integrin binding has additional effects on cell physiology. Overall, this example shows that RGD integrin binding specificity can also be modulated in the context of VTN variants to tailor cell type-specific cell cultures and modify the behavior of these cells. In the context of regenerative medicine, these VTN variants provide a non-animal derived substrate for enhanced MSC expansion. These variants may also prove beneficial to cellular processes enhanced by α5β1 and / or αVβ3, such as mesodermal lineage differentiation or osteogenesis.

[0180] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence entries in GenBank and RefSeq, amino acid sequence entries in, for example, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall prevail. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art are included within the invention as defined by the claims.

[0181] Sequence Listing Free Text SEQ ID NO: 1 Full length human vitronectin SEQ ID NO: 2 Vitronectin polypeptide fragment amino acids 62-292 (hVTN(62-292)) SEQ ID NO: 3 His-tagged vitronectin polypeptide fragment amino acids 62-292

Claims

1. 1. A vitronectin polypeptide fragment comprising: having a length of about 200 to about 350 amino acids; comprising a deletion of at least 50 consecutive N-terminal amino acids compared to the full-length vitronectin polypeptide having SEQ ID NO:1; comprising a deletion of at least the C-terminal amino acids 362 to 478 compared to the full-length vitronectin polypeptide having SEQ ID NO: 1; Contains the arginine-glycine-aspartic acid (RGD) integrin-binding domain of vitronectin; comprising the Hp1, Hp2, and / or Hp3 domains of vitronectin; does not contain the N-terminal somatomedin B (SmB) domain of vitronectin; does not contain the C-terminal heparin-binding domain and Hp4 domain of vitronectin; and A vitronectin polypeptide fragment having at least 95% sequence identity with SEQ ID NO:

2.

2. 2. The vitronectin polypeptide fragment of claim 1, comprising: of full-length human vitronectin having SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 63; an amino acid substitution at a position corresponding to position 67; an amino acid substitution at a position corresponding to position 68; an amino acid substitution at a position corresponding to position 80; and / or containing an amino acid substitution at a position corresponding to position 148, the amino acid substitutions include a T63G substitution, a V67S substitution, a V67N substitution, a F68P substitution, a D80Y substitution, and / or a Q148E substitution; The vitronectin polypeptide fragment.

3. (a) a vitronectin polypeptide fragment consisting of SEQ ID NO: 2 (b) a vitronectin polypeptide fragment consisting of SEQ ID NO: 2, which has an amino acid substitution at position D80 and / or an amino acid substitution at position Q148 compared to the full-length vitronectin having SEQ ID NO: 1, optionally, the amino acid substitution at position D80 comprises a D80Y substitution, and / or the amino acid substitution at position Q148 comprises a Q148E substitution; (c) a vitronectin polypeptide fragment consisting of SEQ ID NO: 2, which has an amino acid substitution at a position corresponding to position 63, an amino acid substitution at a position corresponding to position 67, and / or an amino acid substitution at a position corresponding to position 68, compared to a full-length vitronectin having SEQ ID NO: 1, Optionally, the amino acid substitution at position T63 comprises a T63G substitution, the amino acid substitution at position V67 comprises a V67S or V67N substitution, and / or the amino acid substitution at position F68 comprises a F68P substitution; or (d) further comprising a C-terminal His-tag; optionally containing 1, 2, 3, 4, 5, 6, 7, 8 or more C-terminal His residues; The vitronectin polypeptide fragment of claim 1.

4. (a) consisting of SEQ ID NO: 3, and / or (b) are of non-animal origin, and / or (c) conjugated to a microcarrier, optionally wherein the microcarrier comprises a hydrogel or a polystyrene microsphere; The vitronectin polypeptide fragment conjugate of claim 1.

5. A vitronectin polypeptide fragment comprising amino acids 62 to 155 (hVTN(62-155)) of human vitronectin (SEQ ID NO: 1), or A vitronectin polypeptide fragment comprising amino acids 62 to 120 (hVTN(62-120)) of human vitronectin (SEQ ID NO: 1).

6. A composition comprising a vitronectin polypeptide fragment of any one of claims 1 to 4(b), a vitronectin polypeptide fragment conjugate of claim 4(c), or a vitronectin polypeptide fragment of claim 5.

7. The composition described in claim 6, which is of non-animal origin.

8. Use of the vitronectin polypeptide fragment of any one of claims 1 to 4(b) or the vitronectin polypeptide fragment conjugate of claim 4(c) as a cell culture substrate.

9. A nucleotide sequence encoding the vitronectin polypeptide fragment according to any one of claims 1 to 4(b).

10. 10. An expression vector comprising the nucleotide sequence of claim 9, optionally comprising an E. coli expression vector.

11. 10. A host cell comprising the nucleotide sequence of claim 9, said host cell optionally comprising E. coli.

12. A method for producing a vitronectin polypeptide fragment, comprising expressing the vitronectin polypeptide fragment from the nucleotide sequence described in claim 9, wherein the produced vitronectin fragment polypeptide is optionally of non-animal origin.

13. A cell culture method, comprising culturing cells on a substrate comprising the vitronectin polypeptide fragment of any one of claims 1 to 4(b), the vitronectin polypeptide fragment conjugate of claim 4(c), or the vitronectin polypeptide fragment of claim 5. (a) the cells comprise stem cells; and / or (b) culturing the cells under non-animal-derived conditions, and / or (c) the cell culture is ex vivo, and / or (d) the cells comprise stem cells; optionally (i) the stem cells comprise human induced pluripotent stem cells (iPSCs); or (ii) the stem cells comprise human mesenchymal stem cells (MSCs); and / or (e) the vitronectin polypeptide fragment consists of SEQ ID NO: 2 or SEQ ID NO: 3; The cell culture method according to claim 13.

15. 1. A cell culture method, the method comprising culturing mesenchymal stem cells (MSCs) on a substrate comprising a vitronectin polypeptide fragment; The vitronectin polypeptide fragment consists of SEQ ID NO:2, provided that from SEQ ID NO:2: a T63G amino acid substitution at the position corresponding to position 63 relative to full-length vitronectin having SEQ ID NO: 1; A V67S or V67N amino acid substitution at the position corresponding to position 67 relative to full-length vitronectin having SEQ ID NO: 1; and modified by an F68P amino acid substitution at the position corresponding to position 68 relative to full-length vitronectin having SEQ ID NO: 1; The method.

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