US-20260124276-A1 - BIOENGINEERING TERITIARY LYMPHOID STRUCTURES USING CHITOSAN-BASED HYDROGELS

US20260124276A1US 20260124276 A1US20260124276 A1US 20260124276A1US-20260124276-A1

Abstract

As disclosed herein, stromal cells are important for the recruitment and organization of lymphocytes in TLSs. Therefore, disclosed herein are composition and methods for bioengineering TLSs in animals, such as mice. These compositions involve chemokine-releasing microparticles or cells that can be implanted into the animals. Therefore, disclosed herein are chitosan hydrogels with chemokine-releasing microparticles or cells.

Inventors

James Mulé
Rana Falahat
Genyuan Zhu

Assignees

H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.

Dates

Publication Date: 20260507
Application Date: 20250711

Claims (13)

1 . A composition comprising chemokine-releasing microparticles or cells embedded in a chitosan hydrogel.
2 . The composition of claim 1 , wherein the microparticles or stromal cells release CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL13, or any combination thereof.
3 . The composition of claim 2 , wherein the microparticles or stromal cells release CCL-19, CCL-21, CXCL-13, or any combination thereof.
4 . The composition of claim 1 , wherein the microparticles or stromal cells further release lymphotoxin-a2/b1.
5 . The composition of claim 1 , wherein the microparticle comprises a lipid-enveloped mesoporous microparticle.
6 . The composition of claim 1 , wherein the cells are lymph node-derived stromal cells.
7 . A method for bioengineering tertiary lymphoid structures (TLSs) in animals, comprising administering to the animal the composition of claim 1 .
8 . A method for treating a solid tumor in a subject, comprising administering an effective amount of the composition of claim 1 to the solid tumor.
9 . The method of claim 8 , wherein the composition is implanted into the solid tumor.
10 . A method for enhancing immunotherapy in a subject with a solid tumor, comprising administering to the subject an effective amount of the composition of claim 1 to the solid tumor.
11 . The method of claim 10 , wherein the composition is implanted into the solid tumor.
12 . The method of claim 10 , wherein the immunotherapy comprises adoptive transfer of a therapeutic lymphocyte.
13 . The method of claim 10 , wherein the immunotherapy comprises a checkpoint inhibitor selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of copending application Ser. No. 17/052,973, filed Nov. 4, 2020, which is a National Stage of International Application No. PCT/US2019/031306, filed May 8, 2019, which claims benefit of U.S. Provisional Application No. 62/668,609, filed May 8, 2019, which is hereby incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST This invention was made with Government Support under Grant Nos. CA148995, CA184845, CA076292, and CA168536 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION Tertiary lymphoid structures (TLSs) have been identified in various tumor types including breast, lung, melanoma and colorectal cancers, and are generally predictor of a favorable clinical outcome. However, their development and therapeutic function have not been extensively investigated, partially due to the lack of appropriate mouse models. To create such models, we developed injectable chitosan-based hydrogels embedded with chemokine-releasing microparticles or stromal cells and examined their potential for inducing TLSs in mice. SUMMARY OF THE INVENTION As disclosed herein, stromal cells are important for the recruitment and organization of lymphocytes in TLSs. Therefore, disclosed herein are composition and methods for bioengineering TLSs in animals, such as mice. These compositions involve chemokine-releasing microparticles or cells that can be implanted into the subject. Therefore, disclosed herein are chitosan hydrogels with chemokine-releasing microparticles or cells. In some embodiments, the microparticles or cells release CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL13, or any combination thereof. For example, in some cases, the microparticles or cells can release CCL-19, CCL-21, CXCL-13, or any combination thereof. In some embodiments, the microparticles or stromal cells further release lymphotoxin-a2/b1. In some embodiments, the microparticle is any biocompatible particle capable of sustained release of chemokines. In some embodiments, the microparticle is a lipid-enveloped mesoporous microparticle. In some embodiments, the microparticle is an alginate/chitosan microsphere, liposomal polymeric gel, PLGA nanoparticle, avidin-coated PLGA nanoparticle, PLGA microsphere, lipid-coated PLGA microparticle, medgel microgel, or liposome PLGA particle. In some embodiments, the cells are stromal cells. In some embodiments, the cells are lymph node-derived stromal cells. In some embodiments, the cells are follicular helper T cells. In some embodiments, the cells are antigen pulsed dendritic cells. Also disclosed herein is a method for bioengineering tertiary lymphoid structures (TLSs) in animals that involves administering to the animal the TLS composition disclosed herein. Also disclosed herein is a method for treating a solid tumor in a subject that involves administering an effective amount of the TLS composition disclosed herein. For example, in some embodiments, the TLS composition is implanted into the solid tumor. Also disclosed herein is a method for enhancing immunotherapy in a subject with a solid tumor, comprising administering to the subject an effective amount of the TLS composition to the solid tumor. For example, in some embodiments, the composition is implanted into the solid tumor. In some embodiments, the immunotherapy comprises adoptive transfer of a therapeutic lymphocyte. For example, in some embodiments, the immunotherapy comprises a checkpoint inhibitor selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES FIGS. 1A to 1C show cell viability assessment in chitosan hydrogel using LIVE/DEAD fluorescence assay. FIG. 1A is a schematic diagram of cell encapsulation in a thermosensitive chitosan hydrogel. FIG. 1B shows mouse splenocytes encapsulated in chitosan hydrogel and the control group that cultured in complete medium displayed similar viability. FIG. 1C shows human non-small cell lung cancer TILs encapsulated in chitosan hydrogel maintained their viability and continued to proliferate and formed colonies during a 21-day culture period. FIGS. 2A to 2D show characterization of lipid-enveloped microparticles (MPs) encapsulated with chemokines. FIG. 2A is a schematic illustrating preparation of lipid-enveloped MPs containing chemokine. FIG. 2B contains fluorescence microscopy images of MPs containing Alexa Fluor 488-labeled CCL-19. FIG. 2C shows kinetics of CCL-19 release from MPs measured over 7 days using ELISA and the percentage of CCL-19 released from MPs compared to t