CN116179481A - Preparation method and application of polypeptide self-assembled hydrogel scaffold - Google Patents
Preparation method and application of polypeptide self-assembled hydrogel scaffold Download PDFInfo
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Abstract
The invention relates to a polypeptide hydrogel scaffold and application thereof, wherein the polypeptide hydrogel scaffold comprises self-assembled polypeptides and functional mimic peptides, so that the polypeptide hydrogel scaffold can be used for synthesizing immune microenvironments or immune tissues, in-vitro rapid culture of killer immune cells or in-situ amplification of engineered killer immune cells. The polypeptide hydrogel scaffold can be used for realizing rapid proliferation of killer immune cells.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and in particular relates to a polypeptide hydrogel bracket containing a functional sequence and suitable for in-situ rapid expansion and activation of engineered immune cells (such as in-situ expansion and activation of CAR-T cells) and application thereof.
Background
T lymphocytes elicit the final and most powerful anti-tumor immune response, and therefore T cell-mediated immunotherapy is considered a key approach to cancer treatment. Chimeric antigen receptor CAR-T cell therapy is one of the most well known cases in the field of cell therapies. By utilizing genetic engineering techniques, CAR-T cells are designed to have antigen specificity and enhanced function, they retain strong efficacy and assist in situ lymphocyte attack on tumors. For CAR-T cell therapy, T cells are first removed from the patient, engineered, cultured and stimulated in vitro. To obtain satisfactory results, a large number of functional T cells must be produced in a short time. However, current culture techniques may take 2-4 weeks, at which time the cell function and phenotype for mediating effective killing and long-term memory may have been lost or altered. Therefore, there is a great need to develop more efficient culture systems to activate and expand functional T cells.
In order to accelerate proliferation of CAR-T cells and increase the therapeutic efficiency of patients, a number of approaches have been applied to address the above challenges, including altering the cytokine mixture, signaling pathway inhibitors, and composition of the cell culture medium. The rapid development of biochemical technology and engineering methods provides an ideal platform for designing cell-based delivery systems, with promise for application in disease treatment, particularly immunotherapy.
There is still a need to develop culture systems that facilitate rapid proliferation of immune cells, such as CAR-T cells.
Disclosure of Invention
The inventor finds through research that the application of hydrogel synthesized immune microenvironment or immune tissue can simulate natural extracellular matrix, and can maintain cell viability and realize local immune regulation by delivering and accommodating immune cells at specific positions in a body for a long time, so that the immune treatment effect is improved. In particular, the soft gel-like matrix becomes a highly hydrated, expanded three-dimensional cellular microenvironment supporting metabolically derived mass exchange, and more importantly, the soft and weakly crosslinked matrix provides an expandable space for cellular development processes, such as division and assembly. In the invention, a self-assembled polypeptide hydrogel scaffold and an artificial immune cell stimulation matrix based on the self-assembled polypeptide hydrogel scaffold are designed, and can be used for preserving and enhancing the phenotype of CAR-T cells. The use of such polypeptide hydrogels for the culture of immune cells, in particular engineered immune cells with killing function, has not been reported. The present invention provides a polypeptide hydrogel scaffold containing a functional sequence (forming a "functionally mimicking peptide" with a self-assembled polypeptide) that has adjustable composition, stiffness and biological activity that creates a unique microenvironment suitable for the growth and activation of engineered immune cells with killing functions, thereby enabling faster proliferation and growth of the immune cells.
In one aspect, the invention provides a polypeptide hydrogel scaffold, wherein the scaffold comprises:
a self-assembling polypeptide selected from fefkfield (SEQ ID NO: 1), fefkfield (SEQ ID NO: 2), radar adarada (SEQ ID NO: 3);
a functional mimetic peptide comprising said self-assembled polypeptide fused and a functional sequence selected from the group consisting of KLDVGG (SEQ ID NO: 4), RGDGG (SEQ ID NO: 5).
In some embodiments, the functional mimetic peptide comprises the self-assembled polypeptide, linker, and the functional sequence in that order from the N-terminus to the C-terminus. Preferably, the linkers, which may be the same or different, may consist of 2-4 amino acids. Further preferably, the linkers may each be independently selected from CC or CCCC (SEQ ID NO: 6).
In some embodiments, the functional mimetic peptide is at least one selected from the group consisting of: KFKFEFEFCCKLDVGG (SEQ ID NO: 7), KFEFKFEFCCCCKLDVGG (SEQ ID NO: 8), RADARADARADARADACCKLDVGG (SEQ ID NO: 9), KFKFEFEFCCRGDGG (SEQ ID NO: 10), KFEFKFEFCCCCRGDGG (SEQ ID NO: 11), RADARADARADARADACCRGDGG (SEQ ID NO: 12).
In some embodiments, the self-assembling polypeptides and functional sequences in the functional mimetic peptides self-assemble together to form a polymeric building block having a hydrophilic-hydrophobic surface.
In some preferred embodiments, the self-assembled polypeptide has a β -sheet structure.
The concentration of the hydrogel formed by the polypeptide can be adjusted to form scaffolds with different rigidities for in vitro rapid culture of killer cells. In some preferred embodiments, the self-assembled polypeptide forms a hydrogel having two distinct surfaces, one of which is a hydrophobic surface and the other of which is a hydrophilic surface, and also contains charge-complementary amino acid residues, which may further facilitate β -folding of the polypeptide.
In some preferred embodiments, the molar ratio of the functionally mimicking peptide to the self-assembled polypeptide in the scaffold is 1:1 to 1:4.
In some preferred embodiments, the scaffold may be in the form of a hydrogel, and the stiffness of the gel may be adjusted, for example, to 0.5 to 3kPa, to obtain a polypeptide hydrogel scaffold with optimal binding ligand density.
Further, the invention provides a rapid, efficient and economical method for amplifying and activating engineered killer cells by providing a polypeptide hydrogel scaffold containing functional sequences that can be designed to have optimal stiffness and adhesion ligand density to alter cellular responses through mechanical transduction signals.
In another aspect, the invention also provides the use of a polypeptide hydrogel scaffold comprising a functional sequence in: synthetic immune microenvironments or immune tissues, in vitro rapid culture of killer immune cells, or in situ expansion of engineered killer immune cells.
In this context, the polypeptide hydrogel scaffold can be applied in the field of tumor immunotherapy, and can help to improve the efficiency of immune cell therapy by rapidly culturing immune cells.
The polypeptide hydrogel scaffold promotes rapid in-situ expansion and activation of cells by adjusting rigidity and functional sequences, realizes rapid proliferation of engineered killer cells in a short time and improves the treatment efficiency of tumor patients.
The beneficial effects of the invention are as follows:
1. polypeptide-based hydrogels can be used as a promising substrate for in vitro culture of various types of immune killer cells (e.g., CAR-T, CAR-NK, CAR-KIR, TCR-T, etc.) in a three-dimensional culture environment that mimics an ecological environment.
2. The mechanical rigidity of the hydrogel can be further controlled within the elastic modulus range suitable for the growth of the cells by changing the concentration of the polypeptide hydrogel, which is more beneficial to accelerating the proliferation of the cells.
3. Screening the characteristic modulus to obtain the optimal modulus suitable for cell survival, and maintaining cell viability and proliferation.
4. Through the customized design of the polypeptide hydrogel bracket, the rapid expansion of cells in a short period is realized, and precious time is provided for further clinical treatment of patients.
5. By using different functional sequences, the performance of polypeptide hydrogels in biomedical applications is improved.
6. Functional sequences and the like may be attached to the polypeptide hydrogel by amide linkage (but not limited thereto, other covalent or non-covalent attachment means are also within the scope of the invention) via solid phase synthesis to produce a polypeptide hydrogel having functional sequences.
7. The function of lymphocytes can be activated effectively by recognizing the above functional sequences (such as LDV, etc.) by integrins α4β1, α5β1, which are specifically distributed on lymphocytes, monocytes, etc.
8. Cell network interactions alter cell morphology and function by mechanically transducing signals, thereby promoting cell elongation.
9. By adjusting the molar ratio of polypeptide hydrogels to functional sequences, the viability and proliferation of engineered killer immune cells can be further optimized.
The invention can connect different functional sequences to the polypeptide hydrogel through, for example, amide bond bonding, and can assemble to obtain a unique microenvironment suitable for the growth of the engineering killer immune cells by adjusting the PH and the concentration, the rigidity and the mole ratio of the functional sequences of the hydrogel, thereby enabling the cells to proliferate rapidly. The bionic custom-made artificial matrix can improve proliferation and tumor targeting of cells, so that the obtained cells can be better used for immunotherapy of solid tumors. The application of hydrogel synthesized immune microenvironment or immune tissue can simulate natural extracellular matrix, maintain cell viability and achieve local immunomodulation by delivering and containing immune cells at specific locations in the body for a long time, thereby enhancing the immunotherapeutic effect.
Self-assembled polypeptide hydrogel scaffolds have a high degree of hydration and flexibility, supporting metabolically derived substance exchange, and more importantly, the soft and weakly crosslinked matrix provides stretchable space for cellular development processes, such as division and assembly, and thus can be designed to mimic the cellular living environment for 3D culture, tissue repair and immunotherapy.
Herein, the killer immune cells (e.g., CAR-T cells) can recognize the functional sequences (e.g., KLDV) on the polypeptide hydrogel scaffold through their surface integrins (e.g., integrins α4β1 and α5β1) to achieve signal activation, activate immune cell proliferation, and thus achieve the effect of rapid culture in vitro.
Drawings
Fig. 1 shows the viability of CAR-T cells cultured in polypeptide hydrogel scaffolds containing different molar ratios of functional sequences according to an embodiment of the invention, the cell viability being determined by a dead-living assay, wherein a is a polypeptide hydrogel scaffold formed by fefefefkfk, B is a polypeptide hydrogel scaffold formed by fefefefkfk: KFKFEFEFCCKLDVGG =4:1 (molar ratio), C is a polypeptide hydrogel scaffold formed by fefefkfk: KFKFEFEFCCKLDVGG =3:1 (molar ratio), D is a polypeptide hydrogel scaffold formed by fefefkfk: KFKFEFEFCCKLDVGG =2:1 (molar ratio), E is a polypeptide hydrogel scaffold formed by fefefkfk: KFKFEFEFCCKLDVGG =1:1 (molar ratio).
Fig. 2 shows the expansion of CAR-T cells after 3 days of culture in polypeptide hydrogel scaffolds containing different molar ratios of functional sequences according to an embodiment of the present invention, wherein a is a polypeptide hydrogel scaffold formed by fefefefkfk, B is a polypeptide hydrogel scaffold formed by fefefefk: KFKFEFEFCCKLDVGG =4:1 (molar ratio), C is a polypeptide hydrogel scaffold formed by FEFEFKFK: KFKFEFEFCCKLDVGG =3:1 (molar ratio), D is a polypeptide hydrogel scaffold formed by fefefk: KFKFEFEFCCKLDVGG =2:1 (molar ratio), E is a polypeptide hydrogel scaffold formed by fefefk: KFKFEFEFCCKLDVGG =1:1 (molar ratio).
FIG. 3 shows the viability of CAR-T cells cultured in the polypeptide hydrogels formed by the self-assembly of different functionally mimicking peptides of the present invention, wherein A is the polypeptide hydrogel scaffold formed by KFKFEFEFCCKLDVGG, B is the polypeptide hydrogel scaffold formed by KFEFKFEFCCCCKLDVGG, C is the polypeptide hydrogel scaffold formed by RADARADARADARADACCKLDVGG, D is the polypeptide hydrogel scaffold formed by KFKFEFEFCCRGDGG, E is the polypeptide hydrogel scaffold formed by KFEFKFEFCCCCRGDGG, and F is the polypeptide hydrogel scaffold formed by RADARADARADARADACCRGDGG.
FIG. 4 shows the expansion of CAR-T cells after 3 days of incubation in the polypeptide hydrogels formed by the self-assembly of different functionally mimicking peptides of the present invention, wherein A is the polypeptide hydrogel scaffold formed by KFKFEFEFCCKLDVGG, B is the polypeptide hydrogel scaffold formed by KFEFKFEFCCCCKLDVGG, C is the polypeptide hydrogel scaffold formed by RADARADARADARADACCKLDVGG, D is the polypeptide hydrogel scaffold formed by KFKFEFEFCCRGDGG, E is the polypeptide hydrogel scaffold formed by KFEFKFEFCCCCRGDGG, and F is the polypeptide hydrogel scaffold formed by RADARADARADARADACCRGDGG.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
EXAMPLE 1 preparation of polypeptide hydrogel scaffolds containing functional sequences
The following functional mimetic peptides were synthesized by the assigned Hangzhou Bagge medical science and technology Co., ltd (Hangzhou, china): KFKFEFEFCCKLDVGG (SEQ ID NO: 7), KFEFKFEFCCCCKLDVGG (SEQ ID NO: 8), RADARADARADARADACCKLDVGG (SEQ ID NO: 9), KFKFEFEFCCRGDGG (SEQ ID NO: 10), KFEFKFEFCCCCRGDGG (SEQ ID NO: 11), RADARADARADARADACCRGDGG (SEQ ID NO: 12).
0.5% -3% (5-30 mg/mL) of the functional mimetic peptide is dissolved in sterile water, and sodium chloride is added to form hydrogel. The structural morphology of the resulting hydrogel (which has a hydrophilic and hydrophobic surface) was observed by transmission electron microscopy.
The mechanical rigidity of the resulting hydrogel was controlled to be in the range of 0.5 to 3kPa by changing the concentration of the hydrogel formed by KFKFEFEFCCKLDVGG to about 10 mg/mL. At the lowest gel concentration, the modulus of elasticity was about 0.5kPa. As the concentration of the hydrogel increased, the elastic modulus showed a relatively positive correlation with the concentration of the hydrogel before 3kPa.
10mg/mL of KFKFEFEFCCKLDVGG (SEQ ID NO: 5) obtained above, pH7.4 hydrogel and FEFEFK (SEQ ID NO: 1) were mixed in different molar ratios (FEFEFK alone, or molar ratios of 1:1, 1:2, 1:3, 1:4, respectively) to prepare different functional hydrogel scaffolds, thereby obtaining 5-30 mg/mL, pH7.4 polypeptide hydrogel scaffolds containing functional sequences.
According to the method, FEFKFEFK and KFEFKFEFCCCCKLDVGG, RADARADARADARADA and RADARADARADARADACCKLDVGG, FEFEFKFK and KFKFEFEFCCRGDGG, FEFKFEFK and KFEFKFEFCCCCRGDGG, RADARADARADARADA and RADARADARADARADACCRGDGG are respectively adopted to prepare different functional hydrogel scaffolds according to the molar ratios of 4:1, 3:1, 2:1 and 1:1.
Example 2 in vitro evaluation of the Effect of polypeptide hydrogel scaffolds containing functional sequences on the viability of engineered killer immune cells
To examine the effect of different rigidities (elastic moduli controlled to be 0.5kPa, 1kPa, 2kPa, 3.0kPa, respectively) of the polypeptide hydrogel scaffold containing a functional sequence prepared in example 1 on the activity of human peripheral blood-derived CAR-T cells, 10 were measured 5 After 3 days of incubation of the CAR-T cells in the hydrogel scaffolds, the cells were stained with dye diluted Live/Dead Fixable Green Dead Cell Stain (thermo fisher) in a mass ratio of 1:1000 with PBS (pH 7.4) for 15 minutes, washed with PBS (pH 7.4) and read on BD FACS Calibur. From this, it was determined that in polypeptide hydrogel scaffolds containing different molar ratios of functional sequences (constructed using KFKFEFEFCCKLDVGG and FEFEFKK in molar ratios of 1:1, 1:2, 1:3, 1:4, respectively), 5% CO at 37℃was obtained 2 Viability of CAR-T cells after 3 days of culture in a culture environment. The results are shown in fig. 1, which demonstrate that the inclusion of different proportions of functionally mimicking peptides in a polypeptide hydrogel scaffold does not significantly reduce the viability of CAR-T cells cultured in the hydrogel scaffold, which are 92%, 91%, respectively.
Example 3 in vitro evaluation of the Effect of polypeptide hydrogel scaffolds containing functional sequences on proliferation of engineered killer immune cells
To examine the effect of different rigidities (elastic modulus controlled to 0.5kPa, 1kPa, 2kPa, 3.0kPa, respectively) of the polypeptide hydrogel scaffolds containing functional sequences prepared in example 1 on CAR-T cell proliferation, CFSE dyes were used to quantify CAR-T cell expansion. Briefly, 10 will be 6 The CAR-T cells were resuspended in 1mL PBS (pH 7.4), mixed with 100. Mu.M CFSE for 5 min at room temperature, and washed twice with PBS (pH 7.4, available from ThermoFisher) to remove the undyed dye. Then, 10 is to 5 The CAR-T cells were cultured in different polypeptide hydrogel scaffolds prepared in example 1 (formed by FEFEFKFK self-assembly alone, or with molar ratios of functional mimetic peptide to self-assembled polypeptide of 1:1, 1:2, 1:3, 1:4, respectively) at 37 ℃, 5% CO 2 Cells were collected and analyzed after 3 days of culture. CFSE fluorescence intensity was measured using a flow cytometer. The cell proliferation was analyzed using FlowJo, the diluted CFSE fluorescence peak represents the population after each round of cell division, and the results are shown in fig. 2, which indicate that the proliferation efficiency of stimulated CAR-T cells can be improved when CAR-T cells were cultured with a polypeptide hydrogel scaffold containing a functional mimetic peptide (constructed using different molar ratios of KFKFEFEFCCKLDVGG to FEFEFKFK) compared to fefefefk alone; after 3 days of culture, the proliferation multiple of the CAR-T cells can reach more than 4 times; particularly, the functional mimetic peptide KFKFEFEFCCKLDVGG and the self-assembled polypeptide KFKEFE have a significantly higher capacity of promoting the proliferation of CAR-T cells when the molar ratio is 1:2 to 1:4.
The ability of the polypeptide hydrogel scaffolds prepared in example 1 using fefkferfk and KFEFKFEFCCCCKLDVGG, RADARADARADARADA and RADARADARADARADACCKLDVGG, FEFEFKFK and KFKFEFEFCCRGDGG, FEFKFEFK and KFEFKFEFCCCCRGDGG, RADARADARADARADA and RADARADARADARADACCRGDGG, respectively, to promote CAR-T cell proliferation was evaluated according to the methods described above, and these hydrogel scaffolds all exhibited better ability to promote CAR-T cell proliferation. Wherein, at a molar ratio of the functional mimetic peptide KFEFKFEFCCCCKLDVGG to the self-assembled polypeptide fefkferffk of 1:1 to 1:4, a significantly higher ability to promote proliferation of CAR-T cells is exhibited (cell proliferation fold up to about 4.6 fold, 4.8 fold, 4.6 fold, 4.0 fold, respectively). Likewise, significantly higher capacity to promote CAR-T cell proliferation was demonstrated at molar ratios of functional mimetic peptide RADARADARADARADACCKLDVGG to self-assembled polypeptide radar adaradarada of 1:2 to 1:4 (cell proliferation fold up to about 3.8 fold, 4.1 fold and 3.9 fold, respectively).
Comparative example 1 Effect of functionally mimicking peptide self-Assembly formed polypeptide hydrogel scaffolds on cell viability
To examine the effect of polypeptide hydrogel scaffolds formed from separate self-assembly of different functional mimetic peptides on CAR-T cell viability, the following functional mimetic peptides were each dissolved in sterile water at 10mg/mL and self-assembled by addition of sodium chloride to form control hydrogel scaffolds: KFKFEFEFCCKLDVGG, KFEFKFEFCCCCKLDVGG, RADARADARADARADACCKLDVGG, KFKFEFEFCCRGDGG, KFEFKFEFCCCCRGDGG, RADARADARADARADACCRGDGG.
Will 10 5 The hydrogel scaffolds formed above (measured as elastic moduli of 1.07kPa, 1.19kPa, 2.16kPa, 1.87kPa, 1.73kPa, 2.52kPa; these scaffolds have higher rigidity, which is disadvantageous for maintaining the viability of the CAR-T cells) were each 1.07kPa, 1.19kPa, 2.16kPa, 1.87kPa, 2.52kPa, 5% CO 2 After 3 days of culture, the cells were stained with PBS (pH 7.4) at a mass ratio of 1:1000 with dye diluted Live/Dead Fixable Green Dead Cell Stain (ThermoFisher) for 15 minutes, washed with PBS (pH 7.4) and read on BD FACS Calibur.
The results are shown in fig. 3, and the polypeptide hydrogel scaffolds prepared above for the control maintained CAR-T cell viability at 90%, 89%, 86%, 89%, 88%, 86%, respectively.
Comparative example 2 in vitro evaluation of the Effect of polypeptide hydrogel scaffolds formed from self-assembly of different functionally mimicked peptides on CAR-T proliferation
At 10 5 Density of individual/mL CAR-T cells were seeded in control hydrogel scaffolds prepared in comparative example 1 at 37℃with 5% CO 2 After 3 days of culture, staining was performed as described in example 2 using CFSE dye for quantification of CAR-T cell expansion results. Cells were collected and analyzed 3 days after culture. CFSE fluorescence intensity was measured using a flow cytometer, and cell proliferation was analyzed using FlowJo.
As shown in FIG. 4, although the hydrogel scaffolds for the respective controls could increase the proliferation of CAR-T, the proliferation rates of CAR-T in the respective groups were 3.5-fold, 3.3-fold, 3.2-fold, 3.3-fold, 3.1-fold and 2.8-fold, respectively.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art should and are intended to be comprehended within the scope of the present invention without departing from the principles of the present invention.
Claims (10)
1. A polypeptide hydrogel scaffold, wherein the scaffold comprises:
a self-assembling polypeptide selected from FEFEFKFK, FEFKFEFK, RADARADARADARADA; a functional mimetic peptide comprising said self-assembled polypeptide fused and a functional sequence selected from the group consisting of KLDVGG, RGDGG.
2. The polypeptide hydrogel scaffold of claim 1, wherein the functional mimetic peptide comprises the self-assembled polypeptide, linker, and the functional sequence in that order from N-terminus to C-terminus; preferably, the linker is composed of 2-4 amino acids; more preferably, the linkers may each be independently selected from CC or CCCC.
3. The polypeptide hydrogel scaffold of claim 1 or 2, wherein the functional mimetic peptide is at least one selected from the group consisting of: KFKFEFEFCCKLDVGG, KFEFKFEFCCCCKLDVGG, RADARADARADARADACCKLDVGG, KFKFEFEFCCRGDGG, KFEFKFEFCCCCRGDGG, RADARADARADARADACCRGDGG.
4. The polypeptide hydrogel scaffold of any one of claims 1-3, wherein the self-assembled polypeptide and functional sequence in the functional mimetic peptide self-assemble together to form a polymeric structural unit having a hydrophilic-hydrophobic surface.
5. The polypeptide hydrogel scaffold of any one of claims 1-4, wherein the self-assembled polypeptide has a β -sheet structure.
6. The polypeptide hydrogel scaffold of any one of claims 1-5, wherein the molar ratio of the functionally mimicking peptide to the self-assembled polypeptide in the scaffold is 1:1 to 1:4.
7. The polypeptide hydrogel scaffold of any one of claims 1-6, wherein the scaffold is a hydrogel, the stiffness of the gel being adjusted to 0.5-3 kPa.
8. Use of the polypeptide hydrogel scaffold of any one of claims 1-7 for: synthetic immune microenvironments or immune tissues, in vitro rapid culture of killer immune cells, or in situ expansion of engineered killer immune cells.
9. The use of claim 8, wherein the killer immune cell is selected from CAR-T, CAR-NK, CAR-KIR, TCR-T, CD4 + T cells, CD8 + T cells, monocytes, NK cells.
10. Use according to claim 8 or 9, wherein the polypeptide hydrogel scaffold is for promoting CAR-T cell proliferation.
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