Preparation Method and Application of Polypeptide Self-Assembled Hydrogel Scaffold

Abstract

The present invention relates to a polypeptide hydrogel scaffold and its uses. Among them, the polypeptide hydrogel scaffold includes a self-assembling polypeptide and a functional mimic peptide, and thus can be used for synthesizing an immune microenvironment or immune tissue, rapid in vitro culture of killer immune cells, or in situ amplification of engineered killer immune cells. By using the polypeptide hydrogel scaffold of the present invention, rapid proliferation of killer immune cells can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and particularly relates to a polypeptide hydrogel scaffold containing functional sequences suitable for in-situ rapid amplification and activation of engineered immune cells (such as in-situ amplification and activation of CAR-T cells) and its uses. Background Art

[0002] T lymphocytes elicit the ultimate and most powerful anti-tumor immune response. Therefore, T cell-mediated immunotherapy is considered a key approach for cancer treatment. Chimeric antigen receptor (CAR)-T cell therapy is one of the most well-known cases in the field of cell therapy. By using genetic engineering techniques, CAR-T cells are designed to have antigen specificity and enhanced functions, and they maintain strong efficacy and assist in-situ lymphocyte attack on tumors. For CAR-T cell therapy, T cells are first removed from a patient, modified, and then cultured and stimulated in vitro. To obtain satisfactory results, a large number of functional T cells must be generated within a short time. However, current culture techniques may take 2 - 4 weeks, during which the cell functions and phenotypes for mediating effective killing and long-term memory may have been lost or altered. Therefore, there is a great need to develop more effective culture systems to activate and amplify functional T cells.

[0003] To accelerate the proliferation of CAR-T cells and improve the treatment efficiency of patients, many methods have been applied to address the above challenges, including changing cytokine mixtures, signal pathway inhibitors, and the composition of cell culture media. The rapid development of biochemical technologies and engineering methods provides an ideal platform for designing cell-based delivery systems, which are expected to be applied to disease treatment, especially immunotherapy.

[0004] There is still a need to develop a culture system that facilitates the rapid proliferation of immune cells, such as CAR-T cells. Summary of the Invention

[0005] The inventor of the present invention found through research that the application of hydrogel-synthesized immune microenvironment or immune tissue can mimic the natural extracellular matrix, maintain cell viability and achieve local immune regulation by delivering and accommodating immune cells at specific positions in the body for a long time, thereby improving the effect of immunotherapy. Specifically, the soft gel-like matrix becomes a highly hydrated and swollen three-dimensional cell microenvironment, supporting metabolic-derived mass exchange. More importantly, the soft and weakly cross-linked matrix provides an expandable space for cell development processes such as division and assembly. In the present invention, a self-assembling polypeptide hydrogel scaffold and an artificial immune cell-stimulating matrix based thereon are designed, which can be used to preserve and enhance the CAR-T cell phenotype. There has been no report on the use of such polypeptide hydrogels for the culture of immune cells, especially engineered immune cells with killing functions. The present invention provides a polypeptide hydrogel scaffold containing a functional sequence (forming a "function-mimicking peptide" with the self-assembling polypeptide). The hydrogel scaffold has adjustable composition, stiffness and biological activity, and can create a unique microenvironment suitable for the growth and activation of engineered immune cells with killing functions, so that the immune cells can proliferate and grow faster.

[0006] In one aspect, the present invention provides a polypeptide hydrogel scaffold, wherein the scaffold comprises:

[0007] A self-assembling polypeptide selected from FEFEFKFK (SEQ ID NO:1), FEFKFEFK (SEQ ID NO:2), RADARADARADARADA (SEQ ID NO:3);

[0008] A function-mimicking peptide comprising the fused self-assembling polypeptide and a functional sequence selected from KLDVGG (SEQ ID NO:4), RGDGG (SEQ ID NO:5).

[0009] In some embodiments, the function-mimicking peptide sequentially comprises the self-assembling polypeptide, a linker and the functional sequence from the N-terminus to the C-terminus. Preferably, the linkers may be the same or different and may consist of 2-4 amino acids. Further preferably, the linkers may each independently be selected from CC or CCCC (SEQ ID NO:6).

[0010] In some embodiments, the functional mimetic peptide is at least one selected from the following: 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).

[0011] In some embodiments, the self-assembling polypeptide and the functional sequence in the functional mimetic peptide self-assemble together to form a polymeric structural unit with a hydrophilic and hydrophobic surface.

[0012] In some preferred embodiments, the self-assembling polypeptide has a β-sheet structure.

[0013] By adjusting the concentration of the hydrogel formed by the above polypeptides, scaffolds with different stiffnesses can be formed for the rapid in vitro culture of killer cells. In some preferred embodiments, the hydrogel formed by the self-assembling polypeptide has two different surfaces, one of which is a hydrophobic surface and the other is a hydrophilic surface, and also contains charge-complementary amino acid residues, which can further facilitate the β-sheet folding of the polypeptide.

[0014] In some preferred embodiments, the molar ratio of the functional mimetic peptide to the self-assembling polypeptide in the scaffold is from 1:1 to 1:4.

[0015] In some preferred embodiments, the scaffold can be in the form of a hydrogel, and for example, the stiffness of the gel can be adjusted to 0.5 - 3 kPa to obtain a polypeptide hydrogel scaffold with an optimal adhesive ligand density.

[0016] Furthermore, the present invention provides a polypeptide hydrogel scaffold containing a functional sequence that can be designed to have an optimal stiffness and adhesive ligand density, so as to change cell responses by mechanical transduction signals, and can provide a rapid, efficient and economical method for the expansion and activation of engineered killer cells.

[0017] On the other hand, the present invention also provides the use of a polypeptide hydrogel scaffold containing a functional sequence in the following aspects: synthesizing an immune microenvironment or immune tissue, rapidly culturing killer immune cells in vitro, or in situ expansion of engineered killer immune cells.

[0018] In this article, the above-mentioned polypeptide hydrogel scaffold can be applied to the field of tumor immunotherapy, and by rapidly culturing immune cells, it helps to improve the efficiency of immunotherapy.

[0019] The polypeptide hydrogel scaffold described in this article promotes the rapid in-situ amplification and activation of cells by adjusting stiffness and functional sequences, realizes the rapid proliferation of engineered killer cells in a short time, and improves the treatment efficiency of tumor patients.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The polypeptide-based hydrogel can be used as a promising matrix for in vitro culture of various types of immune killer cells (such as CAR-T, CAR-NK, CAR-KIR, TCR-T, etc.) by simulating the ecological environment in a three-dimensional culture environment.

[0022] 2. By changing the concentration of the polypeptide hydrogel, the mechanical stiffness of the hydrogel can be further controlled within the range of elastic modulus suitable for the growth of the above cells, which is more conducive to accelerating cell proliferation.

[0023] 3. Screen the characteristic modulus to obtain the optimal modulus suitable for cell survival and maintain cell viability and proliferation.

[0024] 4. Through the customized design of the polypeptide hydrogel scaffold, rapid cell expansion in a short time is achieved, providing valuable time for further clinical treatment of patients.

[0025] 5. By adopting different functional sequences, the performance of the polypeptide hydrogel in biomedical applications is improved.

[0026] 6. Functional sequences and the like can be linked and combined with the polypeptide hydrogel through amide bonds of solid-phase synthesis (but not limited to this, other covalent or non-covalent connection methods also fall within the scope of the present invention) to produce a polypeptide hydrogel containing functional sequences.

[0027] 7. By specifically recognizing the above functional sequences (such as LDV, etc.) through integrin α4β1 and α5β1 distributed on lymphocytes, monocytes, etc., lymphocyte function can be effectively activated.

[0028] 8. Cell network interactions change cell morphology and function through mechanical transduction signals, thereby promoting cell elongation.

[0029] 9. By adjusting the molar ratio of the polypeptide hydrogel to the functional sequence, the viability and proliferation of engineered killer immune cells can be further optimized.

[0030] The present invention can connect different functional sequences to a polypeptide hydrogel through, for example, amide bond bonding. By adjusting the pH, concentration, stiffness of the hydrogel, and the molar ratio of the functional sequences, a unique microenvironment suitable for the growth of engineered killer immune cells can be assembled, enabling the cells to proliferate rapidly. The biomimetic customized artificial matrix can improve cell proliferation and tumor targeting, enabling the resulting cells to be better used for the immunotherapy of solid tumors. The application of hydrogel-synthesized immune microenvironments or immune tissues can mimic the natural extracellular matrix, maintaining cell viability and achieving local immune regulation by delivering and accommodating immune cells at specific locations in the body for a long time, thereby improving the effect of immunotherapy.

[0031] The self-assembled polypeptide hydrogel scaffold has high water hydration and softness, supporting the exchange of metabolites. More importantly, the soft and weakly cross-linked matrix provides an extensible space for cell development processes such as division and assembly. Therefore, it can be designed to mimic the cell living environment for 3D culture, tissue repair, and immunotherapy.

[0032] In this article, killer immune cells (such as CAR-T cells) can achieve signal activation by recognizing functional sequences (such as KLDV) on the polypeptide hydrogel scaffold through their surface integrins (such as integrin α4β1 and α5β1), activating immune cell proliferation, and thus achieving the effect of rapid in vitro culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shows the viability of CAR-T cells cultured in polypeptide hydrogel scaffolds containing different molar ratios of functional sequences. Cell viability was determined by a dead-live assay. Among them, A is a polypeptide hydrogel scaffold formed by FEFEFKFK, B is a polypeptide hydrogel scaffold formed by FEFEFKFK: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), and E is a polypeptide hydrogel scaffold formed by FEFEFKFK:KFKFEFEFCCKLDVGG = 1:1 (molar ratio).

[0034] Figure 2The amplification fold of CAR-T cells after 3 days of culture in polypeptide hydrogel scaffolds containing functional sequences with different molar ratios according to the embodiments of the present invention is shown. Among them, A is a polypeptide hydrogel scaffold formed by FEFEFKFK, B is a polypeptide hydrogel scaffold formed by FEFEFKFK: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), and E is a polypeptide hydrogel scaffold formed by FEFEFKFK:KFKFEFEFCCKLDVGG = 1:1 (molar ratio).

[0035] Figure 3 The viability of CAR-T cells cultured in polypeptide hydrogels self-assembled from different functional mimic peptides according to the comparative examples of the present invention is shown. Among them, A is a polypeptide hydrogel scaffold formed by KFKFEFEFCCKLDVGG, B is a polypeptide hydrogel scaffold formed by KFEFKFEFCCCCKLDVGG, C is a polypeptide hydrogel scaffold formed by RADARADARADARADACCKLDVGG, D is a polypeptide hydrogel scaffold formed by KFKFEFEFCCRGDGG, E is a polypeptide hydrogel scaffold formed by KFEFKFEFCCCCRGDGG, and F is a polypeptide hydrogel scaffold formed by RADARADARADARADACCRGDGG.

[0036] Figure 4 The amplification fold of CAR-T cells after 3 days of culture in polypeptide hydrogels self-assembled from different functional mimic peptides according to the comparative examples of the present invention is shown. Among them, A is a polypeptide hydrogel scaffold formed by KFKFEFEFCCKLDVGG, B is a polypeptide hydrogel scaffold formed by KFEFKFEFCCCCKLDVGG, C is a polypeptide hydrogel scaffold formed by RADARADARADARADACCKLDVGG, D is a polypeptide hydrogel scaffold formed by KFKFEFEFCCRGDGG, E is a polypeptide hydrogel scaffold formed by KFEFKFEFCCCCRGDGG, and F is a polypeptide hydrogel scaffold formed by RADARADARADARADACCRGDGG. Detailed implementation manners

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention. The experimental methods without specific conditions noted in the embodiments and the reagents without formula description are all according to the conventional conditions in the art.

[0038] Example 1 Preparation of a polypeptide hydrogel scaffold containing a functional sequence

[0039] Entrust Hangzhou Baige Medical Technology Co., Ltd. (Hangzhou, China) to synthesize the following functional mimic peptides: 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).

[0040] Dissolve 0.5% - 3% (5 - 30 mg / mL) of the above functional mimic peptides in sterile water and form a hydrogel by adding sodium chloride. Observe the structural morphology of the obtained hydrogel (which has a hydrophilic-hydrophobic surface) through a transmission electron microscope.

[0041] Change the concentration of the hydrogel formed by KFKFEFEFCCKLDVGG to about 10 mg / mL, and control the mechanical stiffness of the obtained hydrogel within the range of 0.5 - 3 kPa. At the lowest gelling concentration, the elastic modulus is about 0.5 kPa. As the concentration of the hydrogel increases, the elastic modulus shows a relatively positive correlation with the concentration of the hydrogel before reaching 3 kPa.

[0042] Mix the above-obtained 10 mg / mL KFKFEFEFCCKLDVGG (SEQ ID NO:5) hydrogel at pH 7.4 with FEFEFKFK (SEQ ID NO:1) at different molar ratios (only FEFEFKFK, or the molar ratios of the two are 1:1, 1:2, 1:3, 1:4 respectively), so as to prepare different functional hydrogel scaffolds, and thus obtain a polypeptide hydrogel scaffold containing a functional sequence with a concentration of 5 - 30 mg / mL and pH 7.4.

[0043] According to the above method, different functional hydrogel scaffolds were prepared by mixing FEFKFEFK and KFEFKFEFCCCCKLDVGG, RADARADARADARADA and RADARADARADARADACCKLDVGG, FEFEFKFK and KFKFEFEFCCRGDGG, FEFKFEFK and KFEFKFEFCCCCRGDGG, RADARADARADARADA and RADARADARADARADACCRGDGG in molar ratios of 4:1, 3:1, 2:1, and 1:1 respectively.

[0044] Example 2: In vitro evaluation of the effect of a polypeptide hydrogel scaffold containing a functional sequence on the viability of engineered killer immune cells

[0045] To detect the effect of the different stiffnesses (elastic moduli were controlled to be 0.5 kPa, 0.5 kPa, 1 kPa, 2 kPa, 3.0 kPa respectively) of the polypeptide hydrogel scaffolds containing functional sequences prepared in Example 1 on the viability of CAR-T cells derived from human peripheral blood, 10 5 of the CAR-T cells were cultured in the hydrogel scaffold for 3 days. After that, the cells were stained with a dye obtained by diluting Live / Dead Fixable Green Dead Cell Stain (ThermoFisher) with PBS (pH 7.4) at a mass ratio of 1:1000 for 15 minutes, washed with PBS (pH 7.4), and read on a BD FACS Calibur. Thus, the viability of CAR-T cells cultured in polypeptide hydrogel scaffolds containing different molar ratios of functional sequences (constructed using KFKFEFEFCCKLDVGG and FEFEFKFK with molar ratios of 1:1, 1:2, 1:3, and 1:4 respectively) was determined after culturing for 3 days in a culture environment of 37 °C and 5% CO2. The results are as Figure 1 shown, indicating that including different proportions of functional mimic peptides in the polypeptide hydrogel scaffold does not significantly reduce the viability of CAR-T cells cultured in the hydrogel scaffold, and the cell viabilities are 92%, 92%, 92%, 91%, and 91% respectively.

[0046] Example 3: In vitro evaluation of the effect of a polypeptide hydrogel scaffold containing a functional sequence on the proliferation of engineered killer immune cells

[0047] To detect the effect of the different stiffnesses (elastic moduli were controlled to be 0.5 kPa, 0.5 kPa, 1 kPa, 2 kPa, 3.0 kPa respectively) of the polypeptide hydrogel scaffolds containing functional sequences prepared in Example 1 on the proliferation of CAR-T cells, CFSE dye was used to quantify CAR-T cell expansion. Briefly, 106 The CAR-T cells were resuspended in 1 mL of PBS (pH 7.4), mixed with 100 μM CFSE at room temperature for 5 minutes, and washed twice with PBS (pH 7.4, purchased from ThermoFisher) to remove the unstained dye. Then, 10 5 of the CAR-T cells were cultured in different polypeptide hydrogel scaffolds prepared in Example 1 (formed only by self-assembly of FEFEFKFK, or the molar ratios of the functional mimic peptide to the self-assembled polypeptide were 1:1, 1:2, 1:3, 1:4 respectively), collected and analyzed after culturing in 37 °C and 5% CO2 for 3 days. The CFSE fluorescence intensity was measured using a flow cytometer. The cell proliferation was analyzed using FlowJo. The diluted CFSE fluorescence peaks represent the populations after each round of cell division. The results are as Figure 2 shown, indicating that when culturing CAR-T cells using polypeptide hydrogel scaffolds containing functional mimic peptides (constructed using different molar ratios of KFKFEFEFCCKLDVGG to FEFEFKFK) compared to only FEFEFKFK, the proliferation efficiency of the stimulated CAR-T cells can be improved; and after 3 days of culture, the proliferation multiple of CAR-T cells can reach more than 4 times; especially when the molar ratio of the functional mimic peptide KFKFEFEFCCKLDVGG to the self-assembled polypeptide KFKFEFE is from 1:2 to 1:4, it shows a significantly higher ability to promote the proliferation of CAR-T cells.

[0048] According to the method described above, the ability of the polypeptide hydrogel scaffolds self-assembled from the functional mimic peptides prepared in Example 1 using FEFKFEFK and KFEFKFEFCCCCKLDVGG, RADARADARADARADA and RADARADARADARADACCKLDVGG, FEFEFKFK and KFKFEFEFCCRGDGG, FEFKFEFK and KFEFKFEFCCCCRGDGG, RADARADARADARADA and RADARADARADARADACCRGDGG to promote the proliferation of CAR-T cells was evaluated, and these hydrogel scaffolds all showed good ability to promote the proliferation of CAR-T cells. Among them, when the molar ratio of the functional mimic peptide KFEFKFEFCCCCKLDVGG to the self-assembled polypeptide FEFKFEFK was from 1:1 to 1:4, a significantly higher ability to promote the proliferation of CAR-T cells was shown (the cell proliferation multiples could reach about 4.6-fold, 4.8-fold, 4.6-fold, and 4.0-fold respectively). Similarly, when the molar ratio of the functional mimic peptide RADARADARADARADACCKLDVGG to the self-assembled polypeptide RADARADARADARADA was from 1:2 to 1:4, a significantly higher ability to promote the proliferation of CAR-T cells was shown (the cell proliferation multiples could reach about 3.8-fold, 4.1-fold, and 3.9-fold respectively).

[0049] Effect of the polypeptide hydrogel scaffold self-assembled from the functional mimic peptide in Comparative Example 1 on cell viability

[0050] To detect the effect of the polypeptide hydrogel scaffolds self-assembled from different functional mimic peptides alone on the viability of CAR-T cells, the following functional mimic peptides were respectively dissolved in sterile water at 10 mg / mL and self-assembled by adding sodium chloride to form control hydrogel scaffolds: KFKFEFEFCCKLDVGG, KFEFKFEFCCCCKLDVGG, RADARADARADARADACCKLDVGG, KFKFEFEFCCRGDGG, KFEFKFEFCCCCRGDGG, RADARADARADARADACCRGDGG.

[0051] Take 10 5After culturing the CAR-T cells in the hydrogel scaffolds formed above (the elastic moduli of which were measured to be 1.07 kPa, 1.19 kPa, 2.16 kPa, 1.87 kPa, 1.73 kPa, and 2.52 kPa respectively; these scaffolds had a high stiffness, which was not conducive to maintaining the viability of the CAR-T cells) at 37 °C and 5% CO2 for 3 days, the cells were stained with a dye obtained by diluting Live / Dead Fixable Green Dead Cell Stain (ThermoFisher) at a mass ratio of 1:1000 with PBS (pH 7.4) for 15 minutes, and then washed with PBS (pH 7.4) and read on a BD FACS Calibur.

[0052] The results were as Figure 3 shown. The viability of the CAR-T cells maintained by the control polypeptide hydrogel scaffolds prepared above was 90%, 89%, 86%, 89%, 88%, and 86% respectively.

[0053] Example 2 In vitro evaluation of the effect of polypeptide hydrogel scaffolds formed by self-assembly of different functional mimic peptides on the proliferation of CAR-T

[0054] The CAR-T cells were inoculated into the control hydrogel scaffolds prepared in Comparative Example 1 at a density of 10 5 cells / mL, and after culturing at 37 °C and 5% CO2 for 3 days, they were stained with CFSE dye according to the operation described in Example 2 for quantifying the results of CAR-T cell expansion. The cells were collected and analyzed 3 days after culturing. The CFSE fluorescence intensity was measured using a flow cytometer, and cell proliferation was analyzed using FlowJo.

[0055] The results were as Figure 4 shown. Although the control hydrogel scaffolds could improve the proliferation of CAR-T, the CAR-T proliferation rates in each group were 3.5-fold, 3.3-fold, 3.2-fold, 3.3-fold, 3.1-fold, and 2.8-fold respectively.

[0056] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements made should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide hydrogel scaffold, wherein, The scaffold includes: Self-assembling polypeptides selected from FEFEFKFK, FEFKFEFK, RADARADARADARADA; Function-mimicking peptides selected from KFKFEFEFCCKLDVGG, KFEFKFEFCCCCKLDVGG or RADARADARADARADACCKLDVGG, The molar ratio of the function-mimicking peptide to the self-assembling polypeptide in the scaffold is 1:2 to 1:

4.

2. The polypeptide hydrogel scaffold according to claim 1, wherein, The self-assembling polypeptide has a β-sheet structure.

3. The polypeptide hydrogel scaffold according to claim 1 or 2, wherein The scaffold is a hydrogel, and the stiffness of the gel is adjusted to 0.5~3 kPa.

4. Use of the polypeptide hydrogel scaffold according to any one of claims 1-3 for the following aspects: synthesizing an immune microenvironment or immune tissue, or rapid in vitro culture of cytotoxic immune cells.

5. The use according to claim 4, wherein, The cytotoxic immune cells are selected from CAR-T, CAR-NK, CAR-KIR, TCR-T, CD4 + T cells, CD8 + T cells, monocytes, NK cells.

6. Use according to claim 4 or 5, wherein, The polypeptide hydrogel scaffold is used to promote the in vitro proliferation of CAR-T cells.

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