Activation of cells with self-assembled spheres

Abstract

The invention is directed to a compound for cell activation having a mean diameter between 500 nm and 10 µm and provided with one or more antigen recognizing moieties capable of activating cells characterized in comprising - first particles with a mean diameter of less than 500 nm which are provided on the surface with one or more first oligonucleotides having a length of 5 to 50 nucleotides - second particles with a mean diameter of less than 500 nm which are provided on the surface with one or more second oligonucleotides having a length of 5 to 50 nucleotides and with one or more antigen recognizing moieties wherein the first and second oligonucleotides have complementary sequences capable of hybridizing to each other, thereby binding the first and second particles to each other.

Description

ACTIVATION OF CELLS WITH SELF-ASSEMBLED SPHERES

[0001] The present invention is directed to the activation of cells with large spheres consisting of smaller particles provided with one or more antigen recognizing moieties capable to activate target cells.BACKGROUND

[0002] The automated generation of genetically modified cells, such as CAR T cells for use in cancer therapies requires a step of cell activation before they can be transfected with lentiviruses and ultimately express a CAR on the cell surface. At the moment, only the generation of CAR T cells based on T cells is established by utilizing small scale (<500 nm) particles loaded with activation moieties. Such particles are commercial available under the trade name “TransAcf ’ from Miltenyi Biotec B.V. & Co KG.

[0003] But further cell therapies based on other cells beyond T cells are desired and needed, such as TILs, Tregs, NK, iPSCs to name a few. Unfortunately, the known and available small scale particles are uncapable of activating any other cell except for T cells.

[0004] On the other hand, it has been shown that larger micron-sized Silica Beads are able to activate NK cells and Tregs. Such particles are commercial available under the trade name “MACSi Beads” from Miltenyi Biotec B.V. & Co KG.

[0005] However, the use of large beads or particles is not feasible for automated cell processing for cell therapies for several reasons:1. They cannot be sterile filtered and will not pass epidural filters2. The automated prodigy process would require a bead removal step and assay to quantify how many beads are left,3. Silica beads are not safe to be injected into a patient (safety risk).

[0006] Accordingly, there was a need to provide larger particles capable of activating other cells beyond T cells suitable for automated cell processing but without the downsides stated above.

[0007] From a different technical field, it is known to use oligonucleotides for assembling beads into larger structures. Such technology is for example disclosed in the following scientific publications.

[0008] Chou et al., Nat Nanotechnol. DNA assembly of nanoparticle superstructures for controlled biological delivery and elimination 2014, 9, 148-155. In this publication the authors describe the usage of DNA to organize gold nanoparticles into colloidalsuperstructures. The architectures and the appropriate building blocks of the colloidal superstructures can reduce the uptake and sequestration by macrophages, enhance the accumulation in tumors, as well as facilitate the elimination from the body.

[0009] Doyen et al., Polymers DNA-Promoted Auto-Assembly of Gold Nanoparticles:Effect of the DNA Sequence on the Stability of the Assemblies 2013, 5, 1041-1055. In this publication the authors modify gold nanoparticles with 11-mer oligonucleotide strands and analyze their assembly / disassembly temperature. Additionally the influence of the oligonucleotide length, a spacer and the spacer composition were analyzed. It was shown among other things that the electrostatic repulsion between the particles has a destabilizing effect and that the assembly process is highly cooperative.

[0010] Valignat et al. PNAS Reversible self-assembly and directed assembly of DNA-linked micrometer-sized colloids, 2005, 102, 4225-4229. The authors developed a system which is based on DNA mediated assembly of micrometer sized polystyrene particles. It was shown that the autoassembly of alternate microbeads is reversible by using laser tweezers.

[0011] Oh et al. Langmuir High-Density DNA Coatings on Carboxylated Colloids by DMTMMand Azide-Mediated Coupling Reactions, 2020, 36, 3583-3589. In this publication the authors introduce a highly efficient method to coat carboxylated particles with DNA homogeneously and with a high density. The DNA-coated particles can self-assemble into colloidal crystals and exhibit a sharp melting temperature. Additionally they synthesize selflimiting aggregates using DNA-coated Janus particles.

[0012] Cui et al. Nat. Commun Self-regulated co-assembly of soft and hard nanoparticles, 2021, 12, 5682. In this publication the authors developed a controlled co-assembly of soft block copolymer micelles and hard nanoparticles through noncovalent interactions.OBJECT OF THE INVENTION

[0013] It was found that the known nanometer scale particles can combined into larger structures which could later be controllably disassembled.

[0014] To this end, nanometer-scale particles are modified with sense and antisense oligonucleotide strands as well as with the required binders for cell activation, so that they obtain a size which is sterile filter compatible. Afterwards, the sense and antisense modified particles can be mixed and self-assembled into larger structures so that they reach the required size for activating cells. After successful activation of the cells, the self-assembled structures should be enzymatically degraded, in order to prevent a bead removal step. This concept is visualized in Fig. 1.

[0015] Accordingly, object of the invention are compounds for cell activation having a mean diameter between 500 nm and 10 pm and provided with one or more antigen recognizing moieties capable of activating cells characterized in comprisingfirst particles with a mean diameter of less than 500 nm which are provided on the surface with one or more first oligonucleotides having a length of 5 to 50 nucleotides second particles with a mean diameter of less than 500 nm which are provided on the surface with one or more second oligonucleotides having a length of 5 to 50 nucleotides and with one or more antigen recognizing moieties selected from the group consisting of anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-CD137 and anti-CD335wherein the first and second oligonucleotides have complementary sequences capable of hybridizing to each other, thereby binding the first and second particles to each other.

[0016] Another object of the invention is a method for activation of cells by providing the cells with compounds having a mean diameter between 500 nm and 10 pm and provided with one or more antigen recognizing moieties capable of binding to antigens on the cells characterized in that the compounds comprisefirst particles with a mean diameter of less than 500 nm which are provided on the surface with one or more first oligonucleotides having a length of 5 to 50 nucleotides second particles with a mean diameter of less than 500 nm which are provided on the surface with one or more second oligonucleotides having a length of 5 to 50 nucleotides and with one or more antigen recognizing moietieswherein the first and second oligonucleotides have complementary sequences capable of binding the first and second particles to each other.

[0017] The term “compounds for cell activation” refers to the reaction product of the first and second particles. Since the compounds contain particles, and have a 3-dimensional structure, the term encompasses “particles” or spheres likewise. The mean diameter of the “compounds for cell activation” is preferable between 700 nm and 1500 nm.

[0018] The term first “mean diameter” refers to the mean hydrodynamic diameter.

[0019] The first and second particles with a mean diameter of less than 500 nm may have a lower limit of mean diameter of 10 nm. Because of this small size, the first and second particles are referred to as “nanomatrix”.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. 1 shows a graphical representation of the oligomer based self-assembly according to the invention

[0021] Fig. 2 shows the effect of different loading of sense and antisense oligonucleotide strands to the Nanomatrix.

[0022] Fig. 3 shows the effect of a nuclease on the size of self-assembled spheres according to the invention

[0023] Fig. 4 shows LSM images of self-assembled structures made of high (upper row) / low (lower row) oligonucleotide loaded Nanomatries and the effect of incubation with DNase. As control, the pure Nanomatrix is depicted on the right side.

[0024] Fig. 5 shows a mean fluorescence intensity (MFI) of CD69 expression on T cells measured on day 1 and day 3 after culture start.

[0025] Fig. 6 shows a mean fluorescence intensity (MFI) of CD25 expression on T cells measured on day 1 and day 3 after culture start upon treatment with naked or loaded Nanomatrix.

[0026] Fig. 7 shows the mean fluorescence intensity of CD25 expression on NK cells in percent measured two days after the treatment of cells with naked or loaded Nanomatrix.DETAILED DESCRIPTION

[0027] The first and second particles can be made from any material, like polystyrene or silica, however, preferred is iron oxide as core material coated with a dextran matrix, for example as disclosed in EP2900809B1. Such coated particles are easily accessible to known coupling chemistry to be “loaded” with oligonucleotides and antigen recognizing moieties, for example via -SH, maleimide or amino groups.

[0028] Particles already provided with antigen recognizing moieties are obtainable as “transact” from Miltenyi Biotec B.V. & Co KG. These particles can be provided with the necessary oligonucleotides with known chemistry.

[0029] The term “first and second oligonucleotides have complementary sequences capable of binding the first and second particles to each other” refers to any pair of oligonucleotide sequences having “sense” and “anti-sense” sequences which can easily hybridize with each other, thereby forming a permanent and stable bond. Preferable, the first and second oligonucleotides are selected such the resulting bound survives heat treatment of for example 60° C.

[0030] Preferred sequences are as followsSEQ la 5’-CTACACGACGCTCTTCCGATCTAAAAAAAAAA-3’SEQ lb 5'-AGATCGGAAGAGCGTCGTGTAGAAAAAAAAAA-3'SEQ 2a 5'-GAGGGATCGTTGTTAAAAAAAAAAA-3'SEQ 2b 5'-TAACAACGATCCCTCAAAAAAAAAA-3'SEQ 3a 5’-CGAGCCTATAAAAAAAAAASEQ 3b 5’-ATAGGCTCGAAAAAAAAAA-3’SEQ 4a 5 ’-ACGCTATTGCATCCATGGATCTGCATCCGTAAAAAAAAAA-3 ’ SEQ 4b 5 ’ - ACGGATGC AGATCC ATGGATGC AATAGCGTAAAAAAAAAA-3 ’ SEQ 5a 5’-CGTATGCCTAAAAAAAAAAA-3’SEQ 5b TAGGCATACGAAAAAAAAAA-3’

[0031] The compound according to the invention may comprises any molar ratio between first particles and second particles. However, since the molar ratio of the first particles and second particles determines to e certain extent the final size of the compounds, a molar ratio of the first particles and second particles of 2 - 1 to 1 - 2. is preferred. Most preferred is a molar ratio of 1:1.

[0032] The compounds according to the invention may be provided with any kind of antigen recognizing moieties suitable for cell activation, for example with antibodies, Fabs, Nanobodies, lipids, proteins or saccharides. More specifically the antigen recognizing moieties are selected from the group consisting of anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-CD137 and anti-CD335 and / or the antigen recognizing moieties are capable of activating NK cells, Tregs, TILs, HSC or CAR T cells.

[0033] The compounds according to the invention allows not only assembly after sterile filtration in which particles having a mean diameter greater than the nanomatrices (more than 500nm) are removed, but also after assembly after activation of cells.

[0034] In the method of the invention, the first particles and second particles can be subjected to sterile filtration before being bound to each other. Preferable, the sterile filtration excludes particles having a mean diameter of more than 500 nm.

[0035] Downstream, i.e. after activation of the cells, the compounds can be dissociated by enzymatic digestion, for example using nucleases or DNAses. With this variant, potentially larger (>500nm) particles can be removed from the activated cells, which reduces risks in clinical applications of the activated cells.

[0036] The assemble / disassembly of the compounds of the invention allows their use in automated, sterile production of cell products ready to be administered into a patient.EXAMPLES

[0037] In order to self-assemble the Nanomatrix into larger structures first the Nanomatrix needs to be modified with sense and anti-sense oligonucleotide strands. Therefore maleimidefunctionalized Nanomatrix is reacting with thiol-modified oligos, whereby the oligonucleotides are covalently attached to the Nanomatrix. The loading can be controlled by varying the amount of oligonucleotides added. The maximum loading for both sense and antisense oligonucleotide strands is reached at roughly 0.3 nmol / OD.

[0038] In order to self-assemble Nanomatrix into larger structures, the sense oligonucleotide strand loaded Nanomatrix is mixed with anti-sense oligonucleotide loaded strand Nanomatrix in a 1 : 1 ratio in PBS Pluronic (0.03%) buffer and incubated for different time points (2h, 5h and 24h) at 37°C (500rpm). Thereby either a high oligonucleotide strand loading (-0.29 nmol / OD) or a low oligonucleotide strand loading (-0.14 nmol / OD) was chosen.

[0039] Fig. 2 shows both variants produced from “low oligonucleotide strand loading” and “high oligonucleotide strand loading” each with a 1 : 1 ratio of the first and second particles. The “high” or “low” loading results in assembled compound having a mean diameter of 1500 and 700 nm, respectively

[0040] Since the Nanomatrix is only around 100 nm in size, it cannot be visualized by LSM. Once the Nanomatrix is self-assembled into larger structures, their size increase can be recorded using LSM. For the low (0.14 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix the LSM images were comparable to the DLS results. After 2h of incubation self-assembled structures were visible but there was no further increase in size with a longer incubation time up to 24h. The high (0.29 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix also showed an increase in size after 2h of incubation. This size was further increased over longer incubation times, which is in accordance with the DLS results. The DLS results together with the LSM images could clearly demonstrate that it is possible to self-assemble low (0.14 nmol oligonucleotide / OD) and high (0.29 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix into larger structures. It could also be demonstrated that the amount of oligonucleotides coupled to the Nanomatrix has an influence on the size. With low (0.14 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix smaller self-assembled structures can be synthesized whereas bigger self-assembled structures can be synthesized with high (0.29 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix.

[0041] In the previous experiments the sense oligonucleotide strand loaded Nanomatrix and the antisense oligonucleotide strand loaded Nanomatrix were always mixed in a 1 : 1 ratio. In order to find out if an increase in the ratio between the sense oligonucleotide strand loaded Nanomatrix and the antisense oligonucleotide strand loaded Nanomatrix has an influence on size, different ratios were tested. Therefore, high (0.29 nmol oligonucleotide / OD) or low (0.14nmol oligonucleotide / OD) sense / antisense oligonucleotide strand loaded Nanomatrix were mixed in a defined ratio and incubated for 24h at 37°C.

[0042] In order to prevent a bead removal step after the cells were activated, the selfassembled structures should disassemble into smaller beads after desired cell activation. Therefore, the self-assembled structures were either incubated with DNase or without DNase and afterwards DLS and LSM measurements were performed. Fig. 3 shows the DLS measurements of self-assembled structures made of low (light grey, 0.14 nmol oligonucleotide / OD) and high (dark grey, 0.29 nmol oligonucleotide / OD) oligonucleotide strand loaded Nanomatrix after they were incubated with (right) or without (left) DNase at 37°C for 30 minutes. After incubation without DNase, a size of 736 nm was measured for the self-assembled Nanomatrix made of high oligonucleotide loaded Nanomatrix and a size of 249 nm was measured for the self-assembled Nanomatrix made of low oligonucleotide loaded Nanomatrix. In both cases the size decreased when the samples were incubated with DNase. For the self-assembled structures made of high oligonucleotide loaded Nanomatrix the size decreased down to 91 nm and for the self-assembled structures made of low oligonucleotide loaded Nanomatrix the sized decrease to 103 nm. DLS measurements of the pure Nanomatrix without any modification revealed a size of 71 nm (black line), clearly showing that it is possible to disassemble the self-assembled structures to sizes which are comparable to the pure Nanomatrix.

[0043] Fig. 4 shows the LSM images of self-assembled structures made of low (lower row) and high (upper row) oligonucleotide loaded Nanomatrix after they were incubated with (right) or without (left) DNase at 37°C for 30 minutes. After incubation without DNase selfassembled structures were still visible whereas after incubation with DNase nearly no selfassembled structure were visible anymore.

[0044] Example 2: Activation of T cells with high oligonucleotide loaded Nanomatrix. First, the high oligonucleotide loaded Nanomatrix was modified with antisense-antibody conjugates. Therefore, Nanomatrix was incubated with 20 pmol of CD3 (clone: OKT3)-antisense conjugate and 30 pmol of CD28 (clone: 15E8)-antisense conjugate at 1 OD / mL Nanomatrix concentration in a final reaction volume of 200 pL in a ThermoShaker at 37°C and 500 rpm for 24h. The antibody modified Nanomatrix was purified using MS column via magnetic cell isolation and utilized for the T cell activation.

[0045] To test the potential of self-assembled Nanomatrix to activate and expand human T cells, pan T cells were isolated from human PBMCs using the pan T Cell Isolation Kit (Cat # : 130-096-535, Miltenyi Biotec) according to manufacturer’s instructions. The isolated 2xl05pan T cells were seeded in 200 pl TexMACS medium (Cat#: 130-097-196. Miltenyi Biotec) supplemented with 12.5 ng / ml human IL-7 (Cat #: 170-076-184, Miltenyi Biotec) and human IL- 15 (Cat #: 170-076-114. Miltenyi Biotec) in 96 well flat bottom culture plate. Cells were treated with either PBS as a negative control, 1 : 100 TransAct (Cat #: 130-111-160, Miltenyi Biotec) as positive control, the assembled high oligonucleotide loaded Nanomatrix alone, but without binders, to test the influence of the matrix alone or lastly with assembled high oligonucleotide loaded Nanomatrix modified with anti-CD3 and anti-CD28 antibodies. One and 3 days after treatment start, cells were analysed using MACSQuant 10 for viability and expression of CD69 (clone: REA824) and CD25 (clone: REA570). For staining prior to MACSQuant 10 acquisition, one fourth of the culture volume was stained according to the “Cell surface flow cytometry staining protocol” from Miltenyi Biotec.

[0046] Fig. 5 shows a mean fluorescence intensity (MFI) of CD69 expression on T cells measured on day 1 and day 3 after culture start upon treatment with naked or loaded Nanomatrix.

[0047] Experimental details were as follows: PBS: cells cultured only in TexMACS media containing 12.5ng / ml IL-7 and IL-15; “TransAct”: cells cultured in TexMACS media with IL-7 / 15 and treated with TransAct; “Matrix” naked: cells cultured in TexMACS media with IL-7 / 15 and treated with assembled nanomatrix lacking any binders; “Matrix loaded”: cells cultured in TexMACS media with IL-7 / 15 and treated with assembled Nanomatrix loaded with anti-CD3 and anti-CD28 binders. Measurements were done in triplicates.

[0048] Similar, the mean fluorescence intensity (MFI) of CD25 expression on T cells was measured as shown in Fig. 6, with the following experimental details: PBS: cells cultured only in TexMACS media containing 12.5ng / ml IL-7 and IL-15; “TransAct”: cells cultured in TexMACS media with IL-7 / 15 and treated with TransAct; “Matrix” naked: cells cultured in TexMACS media with IL-7 / 15 and treated with assembled nanomatrix lacking any binders; “Matrix loaded”: cells cultured in TexMACS media with IL-7 / 15 and treated with assembled nanomatrix loaded with anti-CD3 and anti-CD28 binders. Measurements were done in triplicates.

[0049] Compared to cells treated with Nanomatrix which was not modified with antibodies (“Matrix naked”), cells treated with CD3 and CD28 modified Nanomatrix (“Matrix loaded”) show higher CD69 expression indicating a higher degree of activation. Similar to CD69 expression, higher CD25 expression was observed for the cells treadted with CD3 and CD28 modified Nanomatrix (“Matrix loaded”) compared to cells treated with “Matrix naked”, indicating a higher degree of activation as depicted in Fig.6.

[0050] Example 3: Activation of NK cells with high oligonucleotide loaded Nanomatrix. First, the high oligonucleotide loaded Nanomatrix was modified with antisense-antibody conjugates. Therefore, Nanomatrix was incubated with 30 pmol of CD2 (clone: REA982)-antisense conjugate and 30 pmol of CD335 (clone: REA808)-antisense conjugate at 1 OD / mL Nanomatrix concentration in a final reaction volume of 200 pL in a ThermoShaker at 37°C and 500 rpm for 24h. The antibody modified Nanomatrix was purified using MS column via magnetic cell isolation and utilized for the NK cell activation.

[0051] To test the potential of self-assembled Nanomatrix to activate and expand human NK cells, primary human NK cells were isolated from peripheral blood of healthy donors by magnetic CD3 depletion and CD56 enrichment. Isolated NK cells were seeded in NK MACS medium with 1 % NK MACS supplement, 5 % AB serum, 500 U / mL IL-2 and 140 U / mL IL- 15 at 1 x 106cells / mL in flat-bottom 96 well flat bottom culture plate (100 pL / well) with or without the loaded self-assembled matrices at 1 pg / mL. Naked self-assembled matrices, which have not been loaded with antibodies, served as negative control.

[0052] Two days afterwards, activation of NK cells was measured by flow cytometry. To do so, NK cells were transferred to a U-bottom 96 well plate, centrifuged for 5 minutes at 300x g, washed once with PBS and then stained with CD45-VioBlue and CD56-APC to identify NK cells and with CD25-PE-Vio770 to quantify NK cell activation. Two donors were included in the experiments. Fig. 7 shows the NK cell activation in percent, indicating a higher activation degree for the cells which were treated with CD2 and CD335 modified Nanomatrix.

[0053] Experimental details were as follows: Medium: cells cultured only in NK MACS medium with 1 % NK MACS supplement, 5 % AB serum, 500 U / mL IL-2 and 140 U / mL IL- 15 at 1 x 106 cells / mL in flat-bottom 96 well plate (100 pL / well); Naked: cells cultured in NK MACS medium with 1 % NK MACS supplement, 5 % AB serum, 500 U / mL IL-2 and 140 U / mL IL-15 at 1 x 106 cells / mL in flat-bottom 96 well plate (100 pL / well) without not loaded Nanomatrix; Loaded: cells cultured in NK MACS medium with 1 % NK MACS supplement, 5 % AB serum, 500 U / mL IL-2 and 140 U / mL IL- 15 at 1 x 106 cells / mL in flat-bottom 96 well plate (100 pL / well) with CD2 and CD335 loaded Nanomatrix at 1 pg / mL.

Claims

CLAIMS1) Compound for cell activation having a mean diameter between 500 nm and 10 pm and provided with one or more antigen recognizing moieties capable of activating cells characterized in comprisingfirst particles with a mean diameter of less than 500 nm which are provided on the surface with one or more first oligonucleotides having a length of 5 to 50 nucleotidessecond particles with a mean diameter of less than 500 nm which are provided on the surface with one or more second oligonucleotides having a length of 5 to 50 nucleotides and with one or more antigen recognizing moieties selected from the group consisting of anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-CD137 and anti-CD335wherein the first and second oligonucleotides have complementary sequences capable of hybridizing to each other, thereby binding the first and second particles to each other.2) Compound according to claim 1 characterized in that the antigen recognizing moieties are antibodies, Fabs, Nanobodies, lipids, proteins or saccharides3) Compound according to claim 1 or 2 characterized in that the antigen recognizing moieties are capable of activating NK cells, Tregs, TILs, HSC or CAR T cells.4) Compound according to any of claims 1 to 3 characterized in that the compounds comprise first particles and second particles in a molar ratio of 2 - 1 to 1 - 2.5) Method for activation of cells by providing the cells with compounds having a mean diameter between 500 nm and 10 pm and provided with one or more antigen recognizing moieties capable of binding to antigens on the cells characterized in that the compounds comprisefirst particles with a mean diameter of less than 500 nm which are provided on the surface with one or more first oligonucleotides having a length of 5 to 50 nucleotidessecond particles with a mean diameter of less than 500 nm which are provided on the surface with one or more second oligonucleotides having a length of 5 to 50 nucleotides and with one or more antigen recognizing moietieswherein the first and second oligonucleotides have complementary sequences capable of binding the first and second particles to each other.6) Method according to claim 5 characterized in that the antigen recognizing moieties are antibodies, Fabs, Nanobodies, lipids, proteins or saccharides7) Method according to claim 5 or 6 characterized in that the antigen recognizing moieties are capable of activating NK cells, Tregs, TILs, HSC or CAR T cells.8) Method according to any of claims 5 to 7 characterized in that the antigen recognizing moieties are selected from the group consisting of anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-CD137 and anti-CD335.9) Method according to any of claims 5 to 8 characterized in that the compounds comprise first particles and second particles in a molar ratio of 2 - 1 to 1 - 2.10) Method according to any of claims 5 to 9 characterized in that the first particles and second particles are subjected to sterile filtration before being bound to each other.11) Method according to claim 10 characterized in that the sterile filtration excludes particles having a mean diameter of more than 500 nm.12) Method according to any of claims 5 to 11 characterized in that after activation of the cells, the compounds are dissociated by enzymatic digestion.

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