Process for t cell expansion

a technology process, which is applied in the field of t cell expansion, can solve the problems that the number of donor immune cells necessary to effect immune reconstitution against a specific pathogen cannot be obtained through simple mechanical selection systems, and many of these may not be functioning or may be functioning sub-optimally, so as to reduce the time and resources required, and minimise the risk of contamination

Inactive Publication Date: 2015-02-12
ALLOVIR INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]The process of the present disclosure has many advantages in that it reduces the time and resources required to expand the antigen-specific T cells, it is robust and minimises the risk of contamination and these aspects are of huge practical significance because the process can be made GMP compliant and will make the therapy accessible to a larger number of patients in need thereof.

Problems solved by technology

Immune compromised patients are susceptible to opportunistic virus infection.
This is a huge problem in bone marrow transplant patients because their immune cells are routinely depleted as part of the bone marrow transplant procedure and other times rendered non-functional due to steroid treatment for Graft versus Host Disease (GvHD) which is a common complication of bone marrow transplantation.
In some cases, the number of donor immune cells which are necessary to effect immune reconstitution against a specific pathogen cannot be obtained through simple mechanical selection systems.
That is to say there may be a large number of the desired cells present in the population but many of these may not be functioning or may be functioning sub-optimally, for example hyporesponsive in one more or more functional aspects.

Method used

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  • Process for t cell expansion
  • Process for t cell expansion
  • Process for t cell expansion

Examples

Experimental program
Comparison scheme
Effect test

example 1

Process for 226 CMV

[0214]Day −0

[0215]Buffer Preparation

[0216]IL-4

[0217]A 50 μg vial of IL-4 (USP grade, CellGenix cat 1003-050) was diluted with 2504 of WFI (USP Grade Invitrogen Cat A12873) to produce a 200 μg / ml stock solution. The stock solution was stored at −80° C. with an aliquot being left for use in the pot inoculation

[0218]IL-7

[0219]A 50 μg vial of IL-4 (GMP, Cellgenix cat 1010-050) was diluted with 200 μl of WFI (USP Grade Invitrogen Cat A12873) to produce a stock. The stock was diluted 1:25 with WFI to produce a working stock, which was then stored at −80° C. with an aliquot being left for use in the pot inoculation.

[0220]CMV PepTivator Peptide

[0221]A 60 nmol / peptide peptide (GMP PepTivator pp65) was reconstituted in 2 ml of WFI (USP Grade Invitrogen Cat A12873). 100 μl of the reconstituted peptide was removed and further diluted in 900 μl of WFI. 20 μL of the diluted peptide was retained for pot inoculation with the remaining volume aliquoted and stored at −80° C.

[0222]R...

example 2

Process for 226 ADV

[0254]Day −0

[0255]Buffer Preparation

[0256]IL-4

[0257]A 50 μg vial of IL-4 (USP grade, CellGenix cat 1003-050) was diluted with 250 μl of WFI (USP Grade Invitrogen Cat A12873) to produce a 200 μg / ml stock solution. The stock solution was stored at −80° C. with an aliquot being left for use in the pot inoculation

[0258]IL-7

[0259]A 50 μg vial of IL-4 (GMP, Cellgenix cat 1010-050) was diluted with 2004 of WFI (USP Grade Invitrogen Cat A12873) to produce a stock. The stock was diluted 1:25 with WFI to produce a working stock, which was then stored at −80° C. with an aliquot being left for use in the pot inoculation.

[0260]ADV PepTivator Peptide

[0261]A 60 nmol / peptide peptide (GMP PepTivator hexon V) was reconstituted in 2 ml of WFI (USP Grade Invitrogen Cat A12873). 100 μl of the reconstituted peptide was removed and further diluted in 900 μl of WFI. 20 μL of the diluted peptide was retained for pot inoculation with the remaining volume aliquoted and stored at −80° C.

[026...

example 3

[0296]The starting population of cells was cultured in an adapted G-Rex system as shown in the Figures employing RPMI 1640 media in the presence of 10% human serum, IL4, IL7 and an overlapping peptide pool specific for the desired antigen. For CMV specific expansion then the peptide employed was pp65. For adenovirus (ADV) overlapping peptides for the ad 5 hexon were employed. A seed density of 0.5, 1 or 2×106 / cm2 was employed. The results established that 2×106 / cm2 lead to maximal cell expansion see Table 1 Seeding Density for CMV expansion:

Day 0Day 5Day 9Day 12Donor Ino cyto20000000183000001350000014100000IL4 / 72000000029100000108000000120000000no cyto10000000780000069000008100000IL4 / 710000000135000004350000081000000no cyto5000000180000021000001900000IL4 / 75000000420000064000003900000Donor IIno cyto20000000135000001500000022800000IL4 / 7200000001470000034500000120000000no cyto10000000450000055000005000000IL4 / 710000000125000003200000075000000no cyto5000000420000021000002700000IL4 / 750000...

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Abstract

An in vitro expansion process for rapid expansion of antigen specific T cells, such as allogeneic antigen specific T cells comprising the steps culturing in a gas permeable vessel a population of PBMCs (such as allogeneic PBMCs) in the presence of antigen, for example a peptide or peptide mix relevant to a target antigen(s), in the presence of an exogenous cytokine characterized in that the expansion to provide the desired population of T cells is 14 days or less, for example 9, 10, 11 or 12 days, such as 10 days. The disclosure also extends to T cell populations generated by and obtained from the method and the use of same in therapy.

Description

[0001]The present invention relates to a novel process for expanding T cells, in particular antigen specific T cells, such as allogeneic T cells, cell populations therefrom, pharmaceutical compositions comprising the said cell populations and use of the cells and compositions for treatment, particular the treatment or prophylaxis of virus infection, especially in immune compromised patients.BACKGROUND[0002]Immune compromised patients are susceptible to opportunistic virus infection. This is a huge problem in bone marrow transplant patients because their immune cells are routinely depleted as part of the bone marrow transplant procedure and other times rendered non-functional due to steroid treatment for Graft versus Host Disease (GvHD) which is a common complication of bone marrow transplantation. Latent viruses such as Cytomegalovirus (CMV) and Adenoviruses (ADV) become re-activated and the body is unable to fight the infection. The term bone marrow transplantation as used herein d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0783A61K39/235A61K39/245C12N7/00A61K39/12
CPCC12N5/0636C12N7/00A61K39/12A61K39/245A61K39/235A61K2039/5158C12N2501/2304C12N2506/11C12N2710/16134C12N2710/10034C12N2501/2307C12M23/24A61K2039/55527A61P31/12A61P31/14A61P31/20A61P31/22A61P37/04Y02A50/30C12N2710/18034A61K35/17
Inventor KNAUS, RAINER LUDWIGNEWTON, KATY REBECCA
Owner ALLOVIR INC
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