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T cell compositions for immunotherapy

A composition and cell technology, applied in the field of T cell composition for immunotherapy, can solve problems such as inconsistency and ineffectiveness

Pending Publication Date: 2019-05-03
GENEIUS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during tumor progression, the immune response to the tumor is focused on a small number of "dominant" antigens that are ineffective at promoting tumor regression
In past attempts to use ex vivo expanded T cells for immunotherapy, tumor-associated dominant antigen-reactive T cells were inadvertently expanded, leading to inconsistent results
[0004] In the study by Kawakami et al., most melanoma patients showed cytotoxic T lymphocyte (CTL) activity against human melanocyte-specific antigen (MART-1 / Melan A), but only a few against another Tumor-associated antigen gp100

Method used

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  • T cell compositions for immunotherapy
  • T cell compositions for immunotherapy
  • T cell compositions for immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] Example 1. T cell expansion platform

[0209] Experimental procedure

[0210] Antigen selection: PepMix is ​​a pool of peptides (also referred to herein as "polypeptides") derived from peptide scans of target antigens of interest (each polypeptide is 15 amino acids with 11 amino acid overlaps) capable of stimulating CD4+ and CD8+ T cells without knowledge of HLA restriction. PepMix for LMP1, LMP2, EBNA1, CMV, NYESO-1 and survivin was purchased from JPT Peptide Technologies, Berlin. Each pepmix vial consisted of approximately 15 nmol or 25 micrograms of each peptide and was 70% pure. Pools of individual LMP2 peptides and custom epitope mapping matrices were also purchased from JPT Peptide Technologies, Berlin.

[0211] Listed are the sources of the Pepmix composition and protein sequences for the antigens used to expand T cells from normal donors and cancer patients:

[0212] - PepMix EBV (LMP1): pool of 94 peptides derived from latent membrane protein 1, Swiss-Prot ...

Embodiment 2

[0224] Example 2. T cell culture conditions

[0225] Experimental procedure

[0226] Cytokines: GMP grade cytokines for T cell expansion were purchased from Miltenyi Biotech and stock solutions were prepared at 25ug / ml in sterile dH20 and stored at -70°C. Cytokines were used at a final concentration of 100 IU / ml for human IL-2 and 10 ng / ml for IL-7 and IL-1.

[0227] Frozen PBMCs were stimulated with 1 ug / ml Pepmix during prolonged culture or 3 ug / ml during 2 hr pulses and then pooled. The DMSO concentration was 0.4% with lug / ml Pepmix cultures and no cytotoxicity was observed. PBMCs pulsed with 3ug / ml Pepmix had a 1.2% DMSO concentration during incubation, which was washed out prior to prolonged cell culture. Aliquots from days 0, 7, 14, 21 and 28 of T cell expansion were subjected to flow cytometry. Frozen day 0 PBMC samples were stained for antibodies to characterize the starting cell population: live / dead, anti-CD3 for total T cells, CD8 and CD4 subsets, CD14 / CD4 for m...

Embodiment 3

[0232] Example 3. T-directed small-scale expansion of non-Hodgkin's lymphoma clinical samples.

[0233] Experimental procedure:

[0234] Flow Cytometry: 200,000 cells were stained with the antibody panel following standard flow cytometry procedures.

[0235] PBMC group: live / dead, CD3, CD4, CD8, CD14, CD56, CD19.

[0236] Intracellular cytokine expression: live / dead, CD3, CD4, CD8, CD45RO, CD45RA, CD107a, TNFa, IFNg, IL-2.

[0237] T cell activation panel: antigen-specific pentamer, live / dead staining, CD3, CD4, CD8, CD56, CD45RA, CD45RO, CD25, CD62L, CD137, CD197, and CD279.

[0238] T cell memory panel: composed of live / dead, CD3, CD4, CD8, CD45RO, CD45RA, CD197, CD28, CD122, CD127, CD183, CD95 and CD62L.

[0239] Small-scale expansion protocol: On day 0, 2 vials of NHL-frozen PBMC (HemaCare, donor NHL 14103815) were thawed using CTL anti-agglutination solution according to the manufacturer's protocol. Cells were washed and resuspended in CellGro DC medium (CellGenix) + ...

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Abstract

The invention relates to compositions comprising a heterogeneous population of T cells with reactivity to selected antigens that are useful for adoptive immunotherapy and methods for making the T cellcompositions.

Description

technical field [0001] The present invention relates to compositions comprising a heterogeneous population of T cells reactive to selected antigens suitable for adoptive immunotherapy; and methods for preparing the T cell compositions. Background technique [0002] During the period of several days after a person's immune system first sees an antigen, a population of T cells that recognize the antigen is generated, and these T cells thereafter determine the nature of the response to the antigen. Antigen recognition and specificity of T cells are conferred by structural features of the T cell receptor (TCR) expressed on the cell surface. A single T cell possesses a TCR capable of binding to a single antigen presented in combination with a specific major histocompatibility complex molecule or MHC. Thus, antigen specificity of T cells is characterized by the presence and function of specific TCRs exhibited by the cells. Although multiple cell subtypes are involved, commonly o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/0783C12N5/0786C07K14/54C07K14/55A61K35/17
CPCC12N5/0636C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/2321A61P37/04Y02A50/30A61K39/4611A61K39/464401A61P35/00C12N2501/998C12N2501/599C12N2501/515A61K39/464838A61K2121/00A61K2300/00
Inventor A.E.斯莱恩茨T.Y.纳卡加瓦M.A.赫尔曼
Owner GENEIUS BIOTECH
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