Co-differentiation of monocytes from allogeneic donors
Inventors
Karlsson-Parra, Alex • Andersson, Bengt
Assignees
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Publication Number
US-10626371-B2
Publication Date
2020-04-21
Expiration Date
Abstract
Disclosed is a method of producing non-exhausted immature dendritic cells (DCs) originating front at two different, allogeneic donors. In the method, a mixture of allogeneic leukocytes, which allogeneic leukocytes have been obtained from at least two different, allogeneic donors is provided. Subsequently, allogeneic monocytes are isolated from the mixture of allogeneic leukocytes. Thereafter, non-exhausted immature DCs are generated from said isolated allogeneic monocytes.
Core Innovation
The invention provides a method of producing pro-inflammatory dendritic cells (DCs) by generating non-exhausted immature DCs from monocyte-enriched allogeneic leukocytes. A mixture of allogeneic leukocytes obtained from at least two different allogeneic donors is provided, and allogeneic monocytes are isolated from the mixture to obtain monocyte-enriched allogeneic leukocytes. The non-exhausted immature DCs are generated by co-culturing the monocyte-enriched allogeneic leukocytes in aqueous cell culture medium free from non-human serum, supplemented with interleukin-4 (IL-4) and GM-CSF.
The invention addresses premature DC activation and/or exhaustion in standard co-culture of mixed allogeneic populations. It describes that serum-free aqueous culture conditions supplemented with IL-4 and GM-CSF prevent premature maturation and exhaustion even when monocytes are from mixed allogeneic populations, including monocytes enriched from buffy coats or leukocyte-depletion filters.
After generating non-exhausted immature DCs, the method activates the non-exhausted immature DCs to obtain pro-inflammatory DCs. Activation is performed by adding poly-I:C (TLR3-ligand), a TLR7/8-ligand selected from Resiquimod, Gardiquimod and Imiquimod, and interferon gamma (IFN-b3) to induce activation. The resulting DCs produce sustained pro-inflammatory chemokines/cytokines after activation factor withdrawal while maintaining non-maturation phenotypes until intentional activation.
The generated DCs and pro-inflammatory DCs, including mixtures involving PI-DCs, are connected to anti-cancer vaccine compositions. The disclosed vaccine context includes antigen loading and activation using TLR ligands plus IFN-b3, with washing to remove activation factors and reduce exhaustion risk.
Claims Coverage
The partial content identifies 1 independent claim and its dependent refinements. The independent claim is directed to producing pro-inflammatory DCs through a defined allogeneic leukocyte sourcing and a two-stage process, yielding pro-inflammatory DCs.
Allogeneic leukocyte mixture from multiple donors
providing a mixture of allogeneic leukocytes obtained from at least two different allogeneic donors
Monocyte enrichment by isolating allogeneic monocytes
isolating allogeneic monocytes from said mixture of allogeneic leukocytes to provide monocyte-enriched allogeneic leukocytes
Non-exhausted immature DC generation in serum-free IL-4/GM-CSF medium
generating non-exhausted immature DCs from said monocyte-enriched allogeneic leukocytes by co-culturing in aqueous cell culture medium free from non-human serum, supplemented with IL-4 and GM-CSF
Activation to pro-inflammatory DCs with poly-I:C, TLR7/8-ligand, and IFN-b3
activating the non-exhausted immature DCs to obtain pro-inflammatory DCs by addition of poly-I:C, a TLR7/8-ligand selected from Resiquimod, Gardiquimod and Imiquimod, and IFN-b3 to induce activation
Across the independent claim, the core inventive coverage is the combination of allogeneic leukocyte sourcing from at least two donors with monocyte enrichment, generation of non-exhausted immature DCs in aqueous non-serum medium supplemented with IL-4 and GM-CSF, and induction of pro-inflammatory DC activation with poly-I:C, a TLR7/8-ligand, and IFN-b3.
Stated Advantages
Prevents premature maturation and exhaustion of DCs even with mixed allogeneic populations.
Maintains phenotypic non-maturation (low CD83/CD86 until intentional activation).
Supports sustained pro-inflammatory chemokine/cytokine production after activation factor withdrawal.
Reduces exhaustion risk in the context of anti-cancer vaccine use, with washing to remove activation factors.
Documented Applications
Anti-cancer vaccine compositions using DCs/pro-inflammatory DCs, including antigen loading onto immature DCs and activation with TLR ligands plus IFN-b3 with washing to remove activation factors.
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