Method for polyclonal stimulation of t cells by mobile nanomatrices

Abstract

The present invention provides a method for polyclonal stimulation of T cells, the method comprising contacting a population of T cells with a nanomatrix, the nanomatrix comprising a) a matrix of mobile polymer chains, and b) attached to said matrix of mobile polymer chains one or more stimulatory agents which provide activation signals to the T cells; thereby activating and inducing the T cells to proliferate; wherein the nanomatrix is 1 to 500 nm in size. At least one first and one second stimulatory agents are attached to the same or to separate mobile matrices. If the stimulatory agents are attached to separate nanomatrices, fine-tuning of nanomatrices for the stimulation of the T cells is possible. Closed cell culture systems also benefit from this method.

Description

FIELD OF INVENTION[0001]The present invention relates generally to the field of immunology, in particular to processes for polyclonal stimulation of T cells by nanomatrices.BACKGROUND OF THE INVENTION[0002]Antibodies against CD3 are a central element in many T cell proliferation protocols. Immobilized on a surface, anti-CD3 delivers an activating and proliferation-inducing signal by crosslinking of the T cell receptor complex on the surface of T cells. By immobilizing anti-CD3 and anti-CD28 to simultaneously deliver a signal and a co-stimulatory signal, proliferation can be increased (Baroja et al (1989), Cellular Immunology, 120: 205-217). In W009429436A1 solid phase surfaces such as culture dishes and beads are used to immobilize the anti-CD3 and anti-CD28 antibodies. Regularly, the immobilization on beads is performed on DynaBeads®M-450 having a size of 4.5 μm in diameter.[0003]EP01257632B1 describes a method for stimulating a population of T-cells by simultaneous T-cell concentr...

AI Technical Summary

Benefits of technology

[0019]Surprisingly, it was found that polyclonal T cell stimulatory agents such as antibodies, e.g. against CD3 and CD28, attached to nanomatrices, which are characterised by a mobile polymeric matrix backbone (non-solid surface), which may have embedded within the matrix additional functional compounds, such as magnetic nanocrystals, can be used to stimulate naïve and memory T cells in vitro, although their diameter is smaller than 1 μm, preferentially smaller than 500 nm, more preferentially smaller than 200 nm. Contrary thereto, it was found that beads with solid surfaces of the same size as the nanomatrices used herein are not able to stimulate T cells at all or to a similar level like the nanomatrices which is in accordance with the well established opinion of the person skilled in the art. Due to their small size the nanomatrices per se, without antibodies attached thereto, do not alter structure, function, activity status or viability of cells, i.e. they do not cause perturbance in the cells and do not interfere with subsequent analyses, experiments and therapeutic applications of the stimulated cells. In addition, preferentially, the nanomatrix is biodegradable and non-toxic to living cells, i.e. the nanomatrix is a biologically inert entity with regard to alterations of the cell function. Therefore the nanomatrix used in the method of the present invention improves the in-vitro stimulation of T-cells by saving the viability of the cells. In addition sterile filtration of the small nanomatrices is possible which is an important feature for long term T cell in vitro expansion under conditions which are compliant with rigorous GMP standards and is a valuable option for clinical application of the in vitro expanded T cells. Moreover these nanomatrices are well suited for use in sterile and closed cell culture system such as described e.g. in WO2009 / 072003 (CliniMACS®Prodigy, Miltenyi Biotec GmbH, Germany).

[0020]In addition surprisingly, it was found that the T cell stimulatory agents such as antibodies, e.g. against CD3 and CD28, attached to nanomatrices may be conjugated to separate nanomatrices (instead of conjugating to the same nanomatrix), which can be mixed hereafter for optimised use. In general, the ratio of nanomatrices to cells is larger than 100:1, preferentially larger than 500:1, most preferentially larger than 1000:1. This results in the possibility of fine-tuning of the nanomatrices used for stimulation of the target T cells, e.g. it facilitates the production process and quality control of the single nanomatrices and improves the flexibility of the reagent, e.g. facilitating the optimisation of the activation conditions for specialised T cell subsets by titrating various CD3 and CD28 concentrations and ratios.

[0024]Alternatively, due to being soluble or colloidal the nanomatrices can easily be diluted by repeated washing steps to effective concentrations below the T cell activation threshold after the T-cell stimulation process.

Problems solved by technology

Below 4 microns, responses decreased rapidly with decreasing particle size, and large numbers of small particles could not compensate for suboptimal size.

According to U.S. Pat. No. 8,012,750B2 nanospheres do not provide enough cross-linking to activate naive T-cells and thus can only be used with previously activated T-cells.

In addition, US2010 / 284965A1 does not disclose polyclonal stimulation with the nanoparticles.

Beads of this size have several disadvantages with regard to their potential to interact with T cells as well as their production, handling and safety in clinical T cell therapy procedures.1. Due to the solid surface of the bead the size of interaction area between the bead and cells is limited.2. Their preparation is complex and costly as compared to soluble antibodies and it is especially inconvenient to generate them in cGMP quality, e.g. due to their size no sterile filtration is possible, sedimentation complicates handling, i.e. constant particle number / volume during filling and antibody loading.3. They are inconvenient to use for in vitro processes to generate T cell therapeutics for in vivo use,since they have to be added to cells in defined cell / bead ratios at defined density cell / beads per surface area,adaption of stimulation strength is only possible to some extent, since the T cell stimulation strength is mostly determined by the density of antibodies on the cell surface and not by the number of beads / cellaliquoting is inaccurate due to sedimentation,sterile filtration is not possibledue to their size they might affect cell viability and function and they cannot simply be removed from cells by centrifugation.

However both methods suffer from inaccuracies with regard to the actual number of residual beads after the removal process, leaving behind a certain risk for toxic effects if T cell stimulatory beads are injected into patients.

This problem is particularly relevant because of the size of the particles, since each single particle on its own might still have retained the capacity to activate T cells in vivo.

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