Protein complexes comprising a mutant SRC family kinase and a probe activatable by stimuli

Abstract

The present invention relates to an immune cell comprising a protein complex, said protein complex comprising an SRC family kinase and at least one probe activatable by stimuli, for use as a medicament. The invention also relates to protein complexes.

Description

[0001] DESCRIPTION

[0002] TITLE: PROTEIN COMPRISES COMPRISING A MUTANT SRC FAMILY KINASE AND A STIMULOACTIVABLE PROBE

[0003] FIELD OF INVENTION

[0004] The present invention relates to an innovative therapy. More specifically, it relates to the field of dynamic cell therapy and that implemented in the context of tumor cells.

[0005] STATE OF THE ART

[0006] Cancer is a disease characterized by the abnormal and uncontrolled growth of cells in the body, which can form tumors. In France, cancer remains a major public health challenge. According to statistics, approximately 400,000 new cases of cancer are diagnosed each year, and more than 150,000 cancer-related deaths occur annually. The most common types of cancer include breast, lung, colon, and prostate cancer. Women and men are affected relatively equally, but the incidence increases with age.

[0007] Cancer treatments have evolved considerably over the years, incorporating multidisciplinary approaches. The main treatment modalities include surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapies. Surgery aims to remove the tumor, while radiotherapy uses radiation to destroy cancer cells. Chemotherapy involves the use of cytotoxic drugs, while immunotherapy mobilizes the immune system to target cancer cells. Aside from surgery, other cancer treatments help to shift cancers toward the concept of chronic diseases. This implies that these diseases result from dynamic and adaptive cellular behaviors over time. Finally, targeted therapies aim at specific alterations at the molecular level.The choice of treatment depends on the type of cancer, its stage, and other individual factors. Constant advances in cancer research aim to improve the effectiveness of treatments and develop more personalized approaches to combat this complex disease. Among these new approaches, cell therapies represent an innovative category of medical treatments that harness the properties of cells to fight various diseases (these therapies are applicable to cancers as well as other pathologies).

[0008] Part of these cell-based therapeutic strategies involves reactivating immune cells specialized in the antitumor response. This antitumor response is often hijacked by the tumor microenvironment's ability to suppress the targeting of cancer cells by immune cells, thus creating an immunosuppressed zone. This is the case, for example, with macrophages, which can differentiate into either antitumor cells (known as M1-like macrophages) or protumor cells (known as M2-like macrophages). Macrophages that penetrate and remain within the tumor are subjected to the tumor microenvironment, which diverts M1-like macrophages toward M2-like macrophages (also known as TAMs for "Tumor Associated Macrophages").In this pro-tumor differentiation state, these TAM macrophages secrete multiple pro-tumor and pro-angiogenic cytokines and chemokines to facilitate nutrient delivery to cancer cells, promoting tumor growth. They also remodel the matrix surrounding cancer cells, promoting matrix regeneration to allow tumor growth and invasion of other tissues, thus promoting metastasis. Finally, these pro-tumor macrophages contribute to immunosuppression by inhibiting T cells and Natural Killer (NK) cells and promoting the activation of regulatory T cells (Tregs), which are responsible for inhibiting immune functions. Thus, the tumor microenvironment is able to hijack the physiological response of macrophages arriving in damaged tissues to regulate repair processes.

[0009] Furthermore, the immunosuppressive zone created by the tumor affects several other components of the immune system, in addition to macrophages. This is the case for dendritic cells, which, under normal conditions, present antigens to activate the adaptive immune system, particularly T and B cells. In the tumor context, cancer cells prevent the maturation of dendritic cells, consequently blocking the immune response. As for T and B cells, they are inhibited by the tumor microenvironment and the inhibitory effect of other immune cells. They lose their ability to detect cancer cells. Moreover, the tumor's promotion of regulatory T and Breg cell activation also contributes to the inhibition of the immune system.Finally, the remaining immune cells, including Natural Killer (NK) cells, neutrophils, eosinophils and basophils, also suffer the inhibitory effects of this immunosuppressive zone.

[0010] The field of cell therapy has been profoundly transformed by the introduction of genetically modified cells, such as CAR-T cells in the case of liquid cancers (cancers where tumor cells are primarily found in the bloodstream or lymphatic system rather than as a solid tumor mass in a specific organ). Nevertheless, there is a real need to expand the range of cell therapy-based solutions, particularly in the context of cancer prevention and treatment, in order to provide healthcare teams with more options.

[0011] Dynamic cell therapies represent a field that is still largely unexplored in oncology and other pathologies, such as autoimmunity, chronic inflammatory diseases, and neurodegeneration, despite their very promising prospects for the prevention and treatment of all these conditions. Indeed, the reactivation of certain functions independently of the immune cell differentiation program and the immunomodulatory capacities of the tumor microenvironment allows for the development of so-called "Trojan horse" strategies to modulate and counteract the growth and adaptation dynamics of tumor cells. A significant obstacle to the study and development of dynamic cell therapies is the complexity of the dynamics of cellular signaling processes combined with the pleiotropy of the cells in which these cellular processes are implemented.

[0012] The inventors focused on the roles of all immune cells, and particularly those of macrophages, given their very high functional pleiotropy. Indeed, pathophysiological transitions, such as tumor progression or tissue repair, are characterized by a large number of changes in which macrophages have often been described as having a key function comparable to that of a conductor. The ability to finely control the functional pleiotropy of macrophages, as well as all immune cells, independently of their microenvironment, is a significant limitation to the use of these cells in cell therapies, especially antitumor cell therapies. In this context, the inventors targeted the SRC family of kinases (SFK), many members of which are expressed in different immune cells and even within the same immune cell.Macrophages can express six members of this family (Src, Yes, Fyn, Fgr, Lyn, and Hck). This family comprises eight members (Src, Yes, and Fyn, which are ubiquitous, as well as Fgr, Blk, Lyn, Lck, and Hck), which are regulatory signaling proteins involved in a variety of cellular processes. Among these members is SRC, the first kinase identified and the source of the family's name, which plays a key role in cell signaling, growth, and differentiation. Fyn, a kinase similar to Src, is known for its regulation of cell growth, immune signaling, and synaptic plasticity in the nervous system. Yes, a kinase similar to Src, is involved in the regulation of cell growth. Src, Fyn, and Yes are ubiquitous kinases found in all cell types at varying levels of expression.Lyn, a kinase involved in immune signaling, regulates cell proliferation and differentiation. Hck, expressed primarily in immune system cells, participates in the regulation of the immune response. Blk, specific to B lymphocytes, regulates B cell receptor-related signaling. Fgr, similar to Fyn but expressed mainly in immune system cells, is involved in various cellular processes, including growth and differentiation. Finally, Lck, specific to T lymphocytes, plays a crucial role in T cell receptor-related signaling. However, other specific functions of each SFK in immune cells have been little studied. The activation mechanisms of each member, as well as the mechanisms by which they are activated within each immune cell type, remain poorly understood.For example, we do not yet know why macrophages express so many different members of the SCR kinase family (6 out of the 8 existing ones) compared to other immune cells.

[0013] Building on work related to chemogenetic and optogenetic approaches, which are truly revolutionary in the field of signaling biology because they allow the induction of spatial and temporal control of each of these signaling elements via the integration of genetically encoded and specifically activatable elements (for example via a small molecule or light), the inventors succeeded in creating a protein complex including the SRC kinase and a photosensitive probe, which was activated by light in these epithelial cells in order to demonstrate the effectiveness of the control of the spatio-temporal activation of this complex (Kerjouan et al., 2021, Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage).

[0014] DESCRIPTION OF THE INVENTION

[0015] Unexpectedly and surprisingly, the inventors succeeded in developing controllable protein complexes for each of the kinases in the SRC family. These complexes can be used in immune cells to specifically activate or inhibit the kinase within the complex, depending on the desired cellular response. To achieve this, the inventors deconstructed each of the SRC family kinases using synthetic biology and site-directed mutagenesis approaches. They were thus able to determine, remarkably, that each SRC family kinase does not activate the same cellular responses in immune cells, indicating that they are not entirely functionally redundant.

[0016] Building on this discovery, the inventors were able to produce immune cells in which targeted cellular processes can be activated or inhibited remotely and at the desired time. In other words, the protein complexes developed by the inventors enable dynamic intracellular control of multiple cellular processes involving one or more kinases of the SRC family, ultimately making it possible to reactivate cellular functions of interest. For example, the inventors were able to reactivate macrophage functions of interest (antitumor, anti-inflammatory, anti-neurodegenerative, pro-repair, etc.) in a highly dynamic manner via their protein complexes.Through simple light activation (and without the addition of any exogenous activating factor), the inventors successfully mimicked the effect of pro-tumorigenic M2-like macrophages (induced by IL-4) and anti-tumorigenic M1-like macrophages (induced by lipopolysaccharide LPS) in in vitro tumor models (spheroids), thereby making the microenvironment in which the macrophages were located less conducive to tumor growth. Thus, protein complexes and immune cells comprising these complexes offer very promising prospects for the prevention and treatment of pathologies, such as cancers, through dynamic cell therapies based on the immune cells and protein complexes according to the invention.One objective of the present invention is therefore to provide new solutions for healthcare teams in the context of dynamic cell therapies, particularly in the context of antitumor dynamic cell therapies.

[0017] Thus, the present invention relates to an immune cell comprising a protein complex, said protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe for use as a drug.

[0018] The present invention makes a significant contribution to the understanding of cellular processes in immune cells involving at least one of the kinases of the SRC family. Furthermore, the invention makes a significant contribution to the field of cell therapies and to the solutions available to healthcare teams for providing the most appropriate treatment for each patient, particularly cancer patients, especially those with solid tumors for whom treatments involving dynamic cell therapies are scarce.

[0019] A protein complex is defined as a molecular entity composed of two or more distinct proteins that interact in a specific and coordinated manner to perform a particular biological function. This complex is characterized by stable and reversible physical and biochemical bonds between its constituent proteins, thus creating a functional molecular structure. The proteins within the complex can play a variety of roles, such as regulating cellular processes, transmitting signals, or carrying out specific biochemical reactions.

[0020] An "immune cell" is defined as any functional unit of the immune system, a vital component of the human body responsible for defending the organism against pathogens and maintaining cellular health. These immune cells can include various types, such as lymphocytes, monocytes, macrophages, neutrophils, and others, each with specific functions in the immune response. Immune cells can recognize and eliminate pathogens such as bacteria, viruses, and even abnormal or cancerous cells. Their ability to work in a coordinated manner and regulate the immune response is essential for preventing disease and maintaining the balance of bodily homeostasis. An "SRC family kinase" is defined as a modular protein belonging to the SRC family of kinases (SFKs) and possessing multiple functional domains.These domains include a membrane-anchoring domain, an SH3 domain (Src Homology 3, facilitating protein-protein interactions by binding to proline-rich motifs, playing a role in regulating enzymatic activity and assembling protein complexes), an SH2 domain (Src Homology 2, responsible for the specific recognition of phosphotyrosine motifs, enabling the regulation of SRC kinase activity in response to cellular signals), a proline-rich region (PRR), a kinase domain (catalyzing the phosphorylation of specific tyrosine residues on substrate proteins, thus regulating various cellular processes), and an intramolecular binding and regulatory domain at the C-terminus. Together, these domains generate two intramolecular bonds whose dynamics lead to the release of the two lobes of the kinase domain, thereby controlling the rate of substrate phosphorylation.These functional domains allow SRC family kinases, particularly when aggregated together to form clusters, to participate in extramolecular protein-protein interactions crucial for the regulation of various biological processes, as well as to catalyze phosphorylation reactions involved in cell signaling.

[0021] In the context of this invention, the term "stimulus-activatable probe" refers to a molecular entity designed to respond specifically to external stimuli, thereby triggering a measurable reaction or a predetermined biological function. "Stimuli" refers to any physical, chemical, or biological signals or factors that elicit a specific response in a system, whether an organism, a cell, or a particular mechanism. These stimuli can be varied and include elements such as light, heat, chemicals, electrical signals, pressure variations, or other forms of external stimulation. The nature of the response depends on the type of stimulus and the sensitivity of the receiving system. In the context of this invention, stimuli are signals that trigger aggregation and conformational responses within the kinase of the relevant protein complex.When a probe is stimulated by light, it is called a photosensitive probe. Such a probe has the advantage of allowing a very high activation timeframe (on the order of a second, and this can be maintained for several hours in vivo) and spatial control. It does, however, require the implantation of an illumination system. Nevertheless, a simple LED can activate such photosensitive probes, making their use inexpensive. When a probe is stimulated by the presence of one or more chemical substances, it is called a chemosensitive probe. This type of probe is advantageous because it does not require the use or implantation of an activation system, but it has a lower activation timeframe and less precise spatial control than a photosensitive probe.

[0022] The term "expression vector" means any genetic vector known for implementation in genetic engineering, including a plasmid, a cosmid, a virus, a lentivirus, a bacteriophage, and containing the elements necessary for the transcription and translation of one of the sequences encoding the protein complexes according to the invention.

[0023] The expression "a sequence showing at least 65% identity with a reference sequence" means that the sequence shows 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity with said reference sequence.

[0024] Preferably, the present invention relates to an immune cell for use as a medicinal product having the following technical characteristics, taken alone or in combination:

[0025] - the stimuloactivatable probe is a photosensitive and / or chemosensitive probe;

[0026] - said immune cell is chosen from: macrophage, T lymphocyte, NK cell, B lymphocyte, plasma cell, mast cell, dendritic cell, osteoclast, megakaryocyte, basophil, eosinophil and neutrophil;

[0027] - The SRC family kinase is chosen from:

[0028] - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a macrophage,

[0029] - Lck, Fyn, Lyn and Src, when said immune cell is a T lymphocyte.

[0030] - Fyn, Src, Lck and Lyn, when said immune cell is an NK cell,

[0031] - Blk, Lyn and Fyn, when said immune cell is a B lymphocyte or a plasma cell,

[0032] - Src and Lyn, when said immune cell is a mast cell,

[0033] - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a dendritic cell

[0034] - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is an osteoclast, - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a megakaryocyte,

[0035] - Lyn, when said immune cell is a basophil,

[0036] - Lyn, Hck, Fgr and Lck, when said immune cell is an eosinophil, or

[0037] - Fgr, Hck and Lyn, when said immune cell is a neutrophil;

[0038] - said protein complex comprises or consists of an amino acid sequence selected from sequences SEQ ID NO: 1 to 48 or comprises or consists of a sequence exhibiting at least 65% identity with an amino acid sequence selected from sequences 1 to 48;

[0039] - said immune cell comprises several protein complexes, each protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe;

[0040] - said immune cell is a macrophage comprising at least one protein complex including a Lyn kinase and a photosensitive probe and at least one protein complex including a Src kinase and a photosensitive probe;

[0041] - the use of said immune cell as a medicine in the prevention or treatment of tumor cells.

[0042] The invention also relates to a combination of at least two immune cells according to any of the variants previously described for use as a drug. Advantageously, said combination comprises a macrophage, which includes a protein complex comprising a Lyn kinase and a photosensitive probe, and a T lymphocyte, which includes a protein complex comprising an Src kinase and a photosensitive probe, for use in the prevention or treatment of tumor cells.

[0043] The present invention also relates to a method for the prevention or treatment of tumor cells in humans comprising the implantation of at least one immune cell according to any of the variants described above. Preferably, said method is a method for the prevention or treatment of tumor cells in humans. Advantageously, said implanted immune cell is a macrophage.

[0044] Furthermore, some of the protein complexes manufactured by the inventors are novel and inventive in themselves. As such, the present invention also relates to a protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe comprising or consisting of an amino acid sequence selected from sequences 2 to 48, or comprising or consisting of a sequence exhibiting at least 65% identity with an amino acid sequence selected from sequences 2 to 48.

[0045] The present invention also relates to a nucleic acid molecule comprising or consisting of a nucleic acid sequence selected from sequences 50 to 96 or comprising or consisting of a sequence having at least 65% identity with a nucleic acid selected from sequences 50 to 96, said nucleic acid molecule encoding a protein complex according to the invention.

[0046] The present invention also relates to an expression vector comprising a nucleic acid molecule according to the invention.

[0047] The present invention also relates to a host cell comprising a protein complex according to the invention, a nucleic acid molecule according to the invention or an expression vector according to the invention.

[0048] Figure:

[0049] [Fig. 1]: Figure 1 represents A / the two pathways of evolution of the tumor microenvironment after macrophage recruitment, B / the results of the evolution of tumor microenvironments within a 3D in vitro model and C / the results of the activation of three protein complexes according to the invention within macrophages on the evolution of a tumor microenvironment;

[0050] [Fig. 2]: Figure 2 depicts macrophages and lymphocytes expressing optoSRC (OS) or optoHCK (OH), which exhibit specific functions. A. TIRF images show the light-dependent recruitment of control complexes (OSACry2, optoSRC with mutated and non-photosensitive Cry2), OS, or OH in RAW 264.7 macrophages, with colocalization to podosomes (Lifeact-GFP, 2 min). The images show that optoSRC (OS) colocalizes with podosomes, unlike optoHCK (OH). B. The proportion of podosomes over 10 minutes of photostimulation reveals that OS promotes their formation, while OH disrupts them (n = 20 cells / condition; mean ± SEM; two-way ANOVA, Tukey's post hoc test). C. After global light stimulation, OS reduces macrophage migration, while OH increases it (n < 30 cells / condition; Mann-Whitney test). D.3D invasion in Matrigel (xCELLigence) shows that only OS increases invasion capacity (N = 3; one-way ANOVA, Tukey's test). E. Western blot shows that OH specifically induces STAT3 phosphorylation after 15 min of stimulation, an effect inhibited by PP2, whereas OS does not induce it (n = 4; Kruskal-Wallis test, Dunn's test). F. In Jurkat cells (human T lymphocytes cultured in suspension), OS induces membrane clustering and actin ring formation. G. OS activation leads to Zap70 phosphorylation, marking T lymphocyte activation. Light gray squares represent light-free conditions (0 min), and dark gray squares indicate photostimulation. TIRF microscopy images and WB were acquired with a high stimulation frequency (100 MHz). Migration and invasion experiments were acquired with a slow stimulation frequency (4 mHz).Statistical significance: ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale: 5 pm.

[0051] The present invention is illustrated in a non-limiting way by the following examples.

[0052] EXAMPLES OF ACHIEVEMENTS

[0053] Obtaining corn and cells said complexes

[0054] Cloning OptoSFKs

[0055] Each expression plasmid was constructed by amplification of the coding nucleic acid sequence of each SFK (species: chicken, mouse, or human) using the high-fidelity DNA polymerase PHUSION. The primers used for PCR are listed in Table 1 and correspond to SEQ ID NO: 97 to 120. The nucleic acid sequences encoding these opto-SFK protein complexes are SEQ ID NO: 1 to 24. The amplification products (amplicons) were then inserted using the Gibson assembly (NEB) method into a pSico-CRY2-mCherry vector linearized by double enzymatic digestion with Nhel / PacL. The optogenetic control used for image analyses and Western blotting was OSACry2 (SRC chicken Amber / R175L / Y527F fused in-phase with Cryptochrome 2 D387A and mCherry at the C-terminus).

[0056] Lentivirus production, cell infection, and line sorting

[0057] Lentiviruses were produced by co-transfection of the pC57GPBEB GagPol MLV and pSUSVSVG plasmids and each plasmid of interest using Lipofectamin2000® on HEK293 FT cells (generous donation from Dr. Nègre, ANIRA platform) plated in 6-well plates at 50% confluence. The medium was changed after 24 hours. Viral particles present in the culture supernatant were collected 72 hours later and filtered with a 0.45 µm filter. RAW 264.7 cells, a murine macrophage cell line, were plated in 6-well plates at 60% confluence on the day of infection, and the filtered supernatant was used directly to infect these cells of interest. The culture medium was then changed 24 hours after infection. After 10 days of decontamination, the cells were sorted by FACS (Aria cell sorter 2000, BD) based on the expression level of the fluorescent optoSFK mCherry using a 561nm laser.For conditions with lifeact-GFP where there is double fluorescent labeling, the cells were sorted by FACS based on the expression level of optoSFK-mCherry with the 561 nm laser at the same time as with the expression of lifeact-GFP with the 488 nm laser.

[0058] [Table 1]

[0059] Primers (SEQ ID sequences NO: 97 to 120) for PCR amplification of various OptoSFKs. Cell culture and experimental conditions: Cells were cultured at 37°C and 5% CO2 in RPMI 1640 glutamax medium (Gibco™, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (GE Healthcare) and 1% (v / v) penicillin-streptomycin (Dutscher, P06-07100) to obtain a complete RPMI medium. The BALB / c mouse colon carcinoma cell line, CT26 (from ATCC™), was generously donated by Dr. Carole Fournier (HAINAUT team, IAB laboratory, Grenoble). These cancer cells were also cultured in a complete RPMI medium. Mouse embryonic fibroblasts (MEFs) were donated by Prof.Reinhard Fàssler (Martinsried, Germany) and were cultured in a complete DMEM medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin before being co-cultured with CT26 and RAW cells in a complete RPMI medium.

[0060] 3D Tumor Models

[0061] 3D tumor models (spheroids and organoids) were generated in 96-well ultra-low attachment (ULA) culture plates (174925, Nunclon™ Sphera™ 96-Well, ThermoFisher Scientific). Three cell lines were used: 800 cells per CT26 condition, 400 cells per MEF condition, and 400 cells per RAW (or optoSFK-RAW) condition. The cells were placed in the 96-well plate with complete RMPI. After centrifugation at 1200 rpm for 5 minutes, the cells were incubated for 7 days. Phase-contrast images were acquired on days 3, 5, and 7. LPS (LPS-EB Ultrapure, InvivoGen, tlrl-3pelps) and IL-4 (recombinant murine IL-4, Peprotech, 214-14) were used at concentrations of 1.5 pg / mL and 15 ng / mL, respectively. The medium remained unchanged throughout the experiment.Cells expressing the OptoSFK-RAW construct were illuminated for 3 hours at 0.005 Hz light stimulation and were kept in darkness for 1 hour for rest.

[0062] TIRF microscopy and podosome quantification (Fig. 2A-B)

[0063] Fluorescence images were obtained by TIRF microscopy, allowing specific observation of the basement membrane of macrophages expressing OptoSFK-mCherry and Lifeact-GFP. Cells (RAW 264.7 cell line) were seeded onto microscopy chambers the day before in complete RPMI medium. Rapid light stimulation (pulses every 10 seconds for 10 minutes) was applied to observe short-term effects. Podosome dynamics were analyzed by quantifying the number of Lifeact-GFP dots over time. Acquisition was performed using a Zeiss AxioVert 200M motorized inverted microscope equipped with a 100x oil immersion objective (NA 1.46), a Coolsnap HQ2 camera (Photometries), and the TIRF2 Slider module (ZEISS) with 488 nm and 561 nm lasers. The cells were maintained at 37 °C with CO2 control (XL incubator, PeCon). MetaMorph software was used for data acquisition and experimental control.The brightness and contrast of the images were adjusted after acquisition using ImageJ, uniformly between conditions to facilitate comparison.

[0064] 2D Migration (Fig. 2C)

[0065] For 2D migration assays under continuous stimulation, cells were seeded the day before and fasted for at least 1 hour prior to imaging. Transmission images were acquired every 4 minutes for 3–4 hours with a 20x objective. OptoSFK-positive cells were identified as Rhodamine channel-positive. Optogenetic activation was achieved via FITC illumination with slow light stimulation (pulses every 4 minutes), and time-lapse imaging continued for up to 16 hours.

[0066] 3D Invasion - xCELLigence (Fig.2D)

[0067] Three-dimensional invasion assays were performed using the xCELLigence RTCA DP system (Agilent) with 16-well CIM plates. Matrigel (3.3% in serum-free RPMI) was polymerized overnight. The following day, cells in serum-free medium were loaded into the upper chamber; the lower chamber contained RPMI + 10% FBS as a chemoattractant. Invasion was monitored in real time by impedance for 36 hours. For the activated conditions, cells were stimulated every 4 minutes (4 MHz) with a LITOS LED device.

[0068] Western blot (Fig. 2E)

[0069] Western blot analysis was used to assess protein phosphorylation in response to light activation of OptoSFK constructs. RAW macrophages expressing OSACry2, OS, or OH were stimulated with blue light pulses every 10 seconds for 15 minutes. Some conditions were pretreated with the inhibitor PP2 (20 pM) for 30 minutes to inhibit SFK-dependent phosphorylation. Cells were lysed in a modified RIPA buffer containing protease and phosphatase inhibitors. Proteins were detected using anti-STAT3, pSTAT3 (Tyr705), and GAPDH antibodies. Membranes were visualized using the Vilber Newton 7.0 system, and densitometric analysis was performed with ImageJ, with signals normalized to GAPDH.

[0070] Obtaining corn and cells said corn

[0071] Cloning, viral production and infection of Jurkat cells

[0072] The optogenetic constructs used in Jurkat cells are identical to those previously described for macrophages, in particular the optoSRC. Cloning was performed using the same strategy (amplification with the PHUSION® polymerase, Gibson assembly in the pSico-CRY2-mCherry vector linearized by Nhel / Pacl).

[0073] Lentiviruses were produced as described above for macrophages from the corresponding constructs and then used to infect Jurkat cells, a human T-lymphocyte cell line. Cells were sorted by FACS based on mCherry expression (561 nm laser), and in cases of double labeling with lifeact-GFP, a double sort was performed with the 561 nm (mCherry) and 488 nm (GFP) lasers, as with RAW 264.7 cells.

[0074] Cell culture and experimentation conditions

[0075] Jurkat cells are human T lymphocytes cultured in suspension. They are maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% of

[0076] T1 RF microscopy (Fig. 2 F)

[0077] Prior to imaging, Jurkat cells are deposited onto microscopy chambers pretreated with a poly-L-lysine (PLL) coating followed by fibronectin to promote T cell adhesion to the surface. Live-cell imaging is performed using TIRF microscopy, enabling visualization of the basement membrane and actin dynamics (Lifeact-GFP) in the presence of the OptoSRC-mCherry optogenetic probe. Images are acquired every 10 seconds for 15 minutes using a Zeiss AxioVert 200M inverted microscope equipped with a 100x oil immersion objective (NA 1.46), a Coolsnap HQ2 camera, and the TIRF2 Slider module (ZEISS). Illumination is provided by 488 nm and 561 nm lasers. The cells are maintained at 37 °C with CO2 control. Acquisition and experimental control are performed using MetaMorph software. Image analysis is carried out using ImageJ, with standardized intensity parameters for all conditions.

[0078] Western blot (Fig. 2G)

[0079] To assess intracellular activation in response to optogenetic activation, Jurkat OptoSFK cells were exposed, or not, to rapid light stimulation (flashes every 10 seconds) for 10 minutes using the LITOS LED device. After stimulation, the cells were lysed in a modified RIPA buffer containing protease and phosphatase inhibitors. Protein extracts were analyzed by Western blot to detect phosphorylation of Zap70, a marker of T cell activation, in comparison with unstimulated conditions.

[0080] Example 3: Results and conclusions

[0081] As previously mentioned, macrophages (MOs) infiltrate strongly into solid tumors (called tumor-associated macrophages -TAMs) and can be reprogrammed by the tumor microenvironment (TME) into M2-like MOs, thereby supporting tumor growth (Fig. IA). Other studies have shown that M1-like macrophages can reduce tumor size (Fig. IA). Based on current knowledge, it is extremely difficult to bypass the TME, since it very efficiently reprograms M2-like macrophages. Based on the described 3D in vitro models, a heterotypic tumor spheroid model was developed to test the ability of the protein complexes according to the invention to directly reprogram macrophages into M1-like MOs within a TME model.To achieve this, a mixed spheroid of tumor cells (CT26), fibroblasts (MEF) and macrophages (RAW), simply but effectively mimicking many features of a solid 3D tumor, was constructed (Fig. IB).

[0082] The first step was to test the model to confirm its ability to reproduce certain characteristics of pro- or antitumor macrophage activity. To do this, these mixed spheroids were cultured in the presence of IL-4 (inducing macrophage differentiation into M2-type MOs) or LPS (inducing macrophage differentiation into M1-type MOs). After 7 days of tumor growth, IL-4 treatment specifically increased tumor growth compared to the control, demonstrating the M2-type protumor characteristics of RAW macrophages (Fig. 1B). Conversely, LPS treatment reduced the size and homogeneity of the spheroids, thus demonstrating the ability of LPS-treated RAW macrophages to induce M1-type antitumor characteristics (Fig. 1B).These highly reproducible results (n=6) confirmed the possibility of modulating tumor growth through different activations of the RAW macrophage cell line.

[0083] Using modified macrophages comprising protein complexes according to the invention (specifically, the three optogenetic optoSFK complexes: OptoSrc, OptoHck, and OptoLyn), the inventors determined that RAW macrophages comprising the photo-activated optoSFK protein complexes reproduced pro- or antitumor activities in vitro. Using the model described above, in the presence of such RAW macrophages genetically modified to express the OptoSFK complexes, photostimulated for 7 days with a cyclic stimulation frequency, the experiments conducted demonstrated that:

[0084] - Activation of RAW OptoSrc macrophages induces a reproducible increase in spheroid size (Fig. IC), mimicking the effect of IL-4 dependent M2 type RAW macrophages;

[0085] - Activation of RAW OptoLyn macrophages reduces or blocks the growth of heterotypic spheroids (Fig. IC), mimicking the effect of LPS;

[0086] - Activation of RAW OptoHck macrophages has no significant effect on spheroid growth, suggesting that this construct has no pro- or antitumor activity (Fig. IC).

[0087] The second experiment, also conducted on macrophages but activated only using the OptoSrc and OptoHck complexes (Fig. 2A-E) (Src and Hck being the two most redundant kinases of the SRC family in myeloid tissues), allowed the inventors to demonstrate that the optogenetic activation mode of these kinases controls the functional specificity between the different members of the SRC kinase family. To do this, they compared the effects of optogenetic activation of the two aforementioned members within macrophages and demonstrated the ability of the OptoSrc complexes, once activated, to colocalize with podosomes (adhesion structures of macrophages allowing them to probe the rigidity of the extracellular environment and degrade it) (Fig. 2A), thus promoting the formation of said podosomes (Fig. 2B), reducing the migratory capacity of macrophages (Fig. 2B).2C), all resulting in an increased invasion capacity of said macrophages (Fig. 2D). Conversely, the inventors demonstrated that the activation of optoHCK complexes within macrophages disrupts the formation of podosomes (Fig. 2B), promotes the migration of the macrophages in which they are located (Fig. 2C), and reduces the invasion capacity of the macrophages containing them.

[0088] In the tumor context, this invasive capacity of optoSRC can have a pro-tumorigenic effect: by increasing macrophage infiltration into the tumor microenvironment, these macrophages can adopt immunosuppressive functions or promote stromal remodeling, thus creating conditions conducive to tumor growth. Furthermore, SRC can also induce the secretion of pro-angiogenic or growth factors that support tumor cell proliferation.

[0089] Thus, optoSRC activation in macrophages not only facilitates their invasion of tumor tissue but also actively contributes to creating a microenvironment conducive to cancer progression. As we demonstrate in the spheroid model, macrophages expressing activated SRC significantly promote tumor growth.

[0090] Unlike optoSRC, optoHCK does not induce increased invasion or a marked increase in tumor growth, suggesting a lesser role in this context. We therefore use it as a functional control to demonstrate that each kinase in the Src family (SFK) possesses specific functions. Western blot analysis shows, however, that OH activation specifically induces STAT3 phosphorylation after 15 minutes of light stimulation, an effect abolished by the PP2 inhibitor treatment, whereas OS does not induce it (Fig. 2E). In macrophages, STAT3 phosphorylation is typically associated with immunosuppressive or pro-tumor polarization (M2-like).Nevertheless, despite this molecular activation, HCK does not appear sufficient to trigger a significant invasive or tumor response, highlighting that STAT3 phosphorylation alone is insufficient and that SFK functions depend on a set of signals and interactions specific to each family member. Through the third experiment conducted on T lymphocytes, in which OptoSrc complexes were activated, the inventors demonstrated that this optogenetic activation induces membrane relocalization of the probes and the formation of actin rings, characteristic of the activation of these cells (Fig. 2F). The inventors also demonstrated that the activation of OptoSrc complexes within these T lymphocytes leads to the phosphorylation of Zap70 (Fig.2G), showing the activation of a signaling pathway downstream of the engagement of the T cell receptor (TCR), which is a protein complex located on the surface of T cells, responsible for the specific recognition of antigens presented by other immune cells.

[0091] These results demonstrate that OptoSrc activation in T lymphocytes triggers cellular mechanisms characteristic of their functional activation. The observed membrane clustering and actin ring formation indicate cytoskeletal reorganization, essential for the formation of the immunological synapse. Phosphorylation of Zap70, a key kinase in T cell signaling, confirms that OptoSrc complexes induce efficient intracellular activation, thus reproducing a physiological activation signal. These observations highlight the potential of optogenetic systems to precisely control T lymphocyte activation, paving the way for therapeutic applications or targeted research in the immunological field.

[0092] These experiments demonstrate that a better understanding of the cellular processes of immune cells involving at least one of the SRC family kinases ultimately allows for the development and proposal of highly dynamic preventive and therapeutic treatment strategies based on dynamic cell therapies involving immune cells and the protein complexes according to the invention. As the inventors have demonstrated through the experimental results presented in Figures 1A-C and 2A-E, the use of different types of remotely activatable complexes makes it possible to induce distinct beneficial effects within the same type of immune cell, in this case, macrophages. Thus, they have shown that complexes including the Lyn kinase promote the differentiation of macrophages into LPS-dependent M1 macrophages with antitumor activity, leading to a reduction in tumor mass.However, although complexes containing Src kinase did not directly reduce tumor size in this initial study, their activation increased the invasive capacity of macrophages. This effect is of considerable interest for the treatment of various diseases, including cancers and infections.

[0093] Finally, the third series of experiments demonstrated that the complexes according to the invention could also activate T lymphocytes via remote activation. These results thus validate the possibility of implementing these complexes in different types of immune cells, confirming their broad applicability to the entire immune system.

[0094] In this sense, the inventors highlighted the following correlations between the different members of the SRC kinase family and the associated biological processes:

[0095] - Src kinase: macrophage-dependent activation of an anti-inflammatory and pro-regenerative tissue response, control of bone degradation,

[0096] - Fyn kinase: activation of NK cells (notably to induce their cytolytic and inflammatory activities), activation of pre-B cell receptors leading to NFkB activation (which participates in B cell maturation and antibody production), increased pro-tumorigenic response of dendritic cells (notably plasmacytoid dendritic cells (pDCs)),

[0097] - kinase Yes: regulation of the differentiation and self-renewal properties of stem cells,

[0098] - Hck kinase: regulation of neutrophil activation (notably increased penetrance and pro-inflammatory activity of neutrophils in tumors),

[0099] - Kinase Fgr: regulation of lipid metabolism via macrophages, increased penetrance and pro-inflammatory activity of neutrophils in tumors,

[0100] - Kinase Blk: regulation of proliferation, differentiation and antibody secretion by B cells, involvement in autoimmune disease processes, activation of pre-B cell receptors leading to NFkB activation and antibody production,

[0101] - Lyn kinase: regulation of B cell functions and activation of pre-B cell receptors leading to NFkB activation and antibody production, activation of an antitumor action of macrophages, regulation of mast cells, negative regulation of autoimmunity and inflammatory processes, activation of NK cells (notably to induce their cytolytic and inflammatory activities), increased penetrance and pro-inflammatory activity of neutrophils in tumors, increased pro-tumor response of dendritic cells (notably plasmacytoid dendritic cells (pDCs)),

[0102] - Lck kinase: regulation and activation of T lymphocytes to induce a CD4 or CD8 response downstream of the T lymphocyte receptor (TCR), which is a protein complex located on the surface of T cells, responsible for the specific recognition of antigens presented by other immune cells (and thus induce their cytolytic and pro-inflammatory activities).

Claims

DEMANDS 1. Immune cell comprising a protein complex, said protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe for use as a drug.

2. Immune cell for use according to claim 1, wherein the stimulus-activatable probe is a photosensitive and / or chemosensitive probe.

3. Immune cell for use according to any one of claims 1 or 2, said immune cell being selected from: macrophage, T lymphocyte, NK cell, B lymphocyte, plasma cell, mast cell, dendritic cell, osteoclast, megakaryocyte, basophil, eosinophil and neutrophil.

4. Immune cell for use according to the preceding claim, wherein the SRC family kinase is selected from: - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a macrophage, - Lck, Fyn, Lyn and Src, when said immune cell is a T lymphocyte. - Fyn, Src, Lck and Lyn, when said immune cell is an NK cell, - Blk, Lyn and Fyn, when said immune cell is a B lymphocyte or a plasma cell, - Src and Lyn, when said immune cell is a mast cell, - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a dendritic cell - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is an osteoclast, - Src, Fyn, Lyn, Hck and Fgr, when said immune cell is a megakaryocyte - Lyn, when said immune cell is a basophil, - Lyn, Hck, Fgr and Lck, when said immune cell is an eosinophil, or - Fgr, Hck and Lyn, when said immune cell is a neutrophil.

5. Immune cell for use according to any one of the preceding claims, wherein said protein complex comprises or consists of an amino acid sequence selected from sequences SEQ ID NO: 1 to 48 or comprises or consists of a sequence having at least 65% identity with an amino acid sequence selected from sequences 1 to 48.

6. Immune cell for use according to any one of the preceding claims, comprising several protein complexes, each protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe.

7. Immune cell for use according to any one of the preceding claims, said immune cell being a macrophage comprising at least one protein complex comprising a Lyn kinase and a photosensitive probe and at least one protein complex comprising a Src kinase and a photosensitive probe.

8. Immune cell for use according to any of the preceding claims, in the prevention or treatment of tumor cells.

9. Combination of at least two immune cells as defined in any of the preceding claims for use as a medicinal product.

10. Combination for use according to the preceding claim, comprising a macrophage, which includes a protein complex comprising a Lyn kinase and a photosensitive probe, and a T lymphocyte, which includes a protein complex comprising a Src kinase and a photosensitive probe, in the prevention or treatment of tumor cells.

11. Protein complex comprising a kinase of the SRC family and at least one stimulus-activatable probe comprising or consisting of an amino acid sequence selected from sequences 2 to 48 or comprising or consisting of a sequence exhibiting at least 65% identity with an amino acid sequence selected from sequences 2 to 48.

12. Nucleic acid molecule comprising or consisting of a nucleic acid sequence selected from sequences 50 to 96 or comprising or consisting of a sequence having at least 65% identity with a nucleic acid selected from sequences 50 to 96, said nucleic acid molecule encoding a protein complex according to claim 11.

13. Expression vector comprising a nucleic acid molecule according to claim 12.

14. Host cell comprising a protein complex according to claim 11, a nucleic acid molecule according to claim 12 or an expression vector according to claim 13.

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