Patent Application
20240181096
Applicant claims priority under 35 U.S.C. § 119 of German Application No. 10 2022 132 082.0 filed Dec. 2, 2022, the disclosure of which is incorporated by reference.
The invention relates to a method for the loading of immunocompetent cells, especially of genetically transfected immunocompetent cells, with nanoparticles and/or a cytotoxic substance for the theranostic treatment of patient-specific genetically modified cells. In particular, these cells comprise viral genetically modified cells or patient-specific malignant tumors in an affected patient. The invention further relates to immunocompetent cells, immunocompetent cells for use in a method for the theranostic treatment of patient-specific genetically modified cells and a medical composition.
T cell therapy is a novel and proven method for the treatment of genetically modified cells, in particular viral genetically modified cells, including most recently Covid-19, or a patient-specific malignant tumor. Recent studies highlight the unique potential for new γδ-CAR T cell-based treatments (Mirzaei et al. (2016): “Prospects for chimeric antigen receptor (CAR) T cells: A potential game changer for adoptive T cell cancer immunotherapy”). These special cells have a specific T-cell receptor (TCR) on their surface. In their natural form, γδ T cells make up 2% to 10% of peripheral blood T lymphocytes. Among others, techniques developed for αβ T-cell therapies can also be applied.
A decisive advantage of γδ T cells over αβ T cells is the innate tumor recognition ability of γδ T cells, which enables the targeted killing of cancerous tissues. If genetically modified γδ CAR T cells are used, in which the specificity towards patient-specific genetically modified cells has been increased by a chimeric anti-gene receptor (CAR), the side effects of treatment can be kept to a minimum.
The particular cancer selectivity of genetically modified γδ CAR T cells is based on the ability of the chimeric antigen cell receptor (CAR) introduced into the cells by genetic engineering to recognize the isopentenyl pyrophosphate (IPP) antigen located on the surface of the cancer cell due to a disturbed isoprenoid signaling pathway in the body of the affected cell (Zeng, Jieming et al. (May 2019): “Derivation of mimetic T cells endowed with cancer recognition receptors from reprogrammed T cell”).
In response to the recognition of a cancer cell, the attacking γδ CAR-T cells activate inflammatory cytotoxic substances, which they inject into the malignant cell via the so-called “kiss-of-death” mechanism (Rozenbaum, Meir et al. (July 2020): “Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia”). In-vitro and clinical phase trials with γδ-T cells have already shown promising results and are increasingly establishing themselves in the treatment of leukemia, neuroblastoma, various carcinomas, and most recently, Covid-19 (Caron et al. (March 2021): “How to Train Your Dragon: Harnessing Gamma Delta T Cells Antiviral Functions and Trained Immunity in a Pandemic Era”). As the field of immunotherapy moves beyond the repeated iteration of established methods to more creative solutions, γδ CAR-T cells offer the opportunity to advance established cancer treatments and break new ground in oncology. These new methods are broad-based and have a much more favorable safety profile than conventional therapies, as the surrounding healthy cell tissue is spared during treatment due to the specificity of yo CAR-T cells.
In conventional therapy with αβ CAR-T cells, for example, two signals that stimulate the T cells are triggered by activating the chimeric antigen receptor (CAR) attached to the outside of the T cell. A disadvantage of αβ CAR-T cells is that they can also bind to healthy cells, which leads to the death of healthy cells after activation of the T cells. This phenomenon is referred to as “off-tumor toxicity” and causes strong side effects, such as those observed with classic chemotherapy.
The γδ CAR-T cells differ from other T cells in that they have a specific γδ T cell receptor (TCR) that only binds to antigens that are characteristic of impaired unhealthy cells. The TCR is a safety barrier against activation of the T cells in healthy cells. A genetically modified γδ CAR-T cell, which only has one co-stimulatory domain, only binds to one damaged cell. This reduces side effects, as healthy cells are not attacked. To obtain sufficient cell material, work is currently being carried out on allogeneic cell pools for which γδ T cells are taken from healthy donors, multiplied, and distributed worldwide via a distribution system.
The potential of γδ T cells is further enhanced by the recently developed method, which makes it possible to switch from individual autologous therapy (see U.S. Pat. No. 9,855,298 B2), which is associated with a wide range of side effects, to allogeneic therapy.
The allogeneic use of γδ T cells reduces graft-versus-host disease (GvHD), even if this initially appears to be a contradiction in terms. This is possible because the current γδ CAR-T cell technology allows us to overcome the restriction triggered by the major histocompatibility complex (MHC). Therefore, the risk of GvHD is minimized (Handgretinger et al. (Marcj 2018): “The potential role of T cells after allogeneic HCT for leukemia”). In addition, various studies have shown overall favorable outcomes of γδ CAR-T cell-based treatments.
The effect of a high γδ CAR-T cell immune reconstitution has been recorded, e.g., after hematopoietic stem cell transplantation (HSCT). In concomitant clinical studies, patients with an increased number of γδ CAR-T cells had a significantly higher overall survival rate and undetectable acute GvHD manifestations.
In addition, the likelihood of transplant rejection in patients with severe viral infections such as HIV, influenza, and COVID-19 is low, and corresponding advanced clinical trials have been ongoing since early 2022.
It is known that solid tumors are subject to further mutations over time, which also change the structure of the externally accessible receptors, which can currently only be recognized after a histological or histo-pathological examination, followed by more detailed examination methods.
Therapeutic approaches with immunocompetent cells can be found, for example, in WO 2022241151 A2, WO 2022238962 A1, WO 2022241036 A1, WO 2022147014 A2, and WO 2020109953 A1. Further therapeutic approaches can be found in WO 2019 181018 A1, WO 9817342 A2, CN 115215851 A, CN 115232129 A, FR 3120630 A1, CN 112675442 A, and JP 2016159107 A.
The disadvantage of previous treatment methods against patient-specific genetically modified cells, particularly against cancer, is that classic chemotherapy substances are administered that have a good toxic effect on the cancer cells but also kill healthy cells in a completely undirected manner. This often results in massive side effects and generally poor prospects of successful treatment because the administered substances do not reach the affected, particularly tumorous, tissue in a targeted manner.
Even those therapies that use αβ T cells transfected with a gene coding for a chimeric antigen receptor (CAR) do not offer the desired specificity of treatment, as the CAR also binds to surface proteins of cells that are not genetically modified, i.e. healthy. Although fewer side effects are observed with these approaches than with conventional chemotherapy, they do not offer the desired specificity and have been proven to be insufficient. In addition, these therapies only allow the progress of the therapy to be controlled and monitored in a roundabout way. Another disadvantage is that even if the therapy is successful, there is a high risk that cells of an affected tissue will survive, and the genetically modified cells will divide again. This can then trigger new viral infections or lead to a renewed flare-up of the infection and, in the case of tumor tissue, to (further) metastases.
In view of these disadvantages of prior art therapeutic approaches using immunocompetent cells like T cells the present invention has the object to improve such therapeutic approaches.
This object is solved by a method for loading immunocompetent cells with nanoparticles and/or a cytotoxic substance as it is described in claim 1. Preferred embodiments of the claimed method are outlined in the dependent claims. Furthermore, this object is solved by correspondingly loaded immunocompetent cells and by a medical composition comprising these loaded immunocompetent cells according to the further independent claims and, in preferred embodiments, in the further dependent claims.
Moreover, the object is solved by a method for preparation of genetically transfected immunocompetent cells loaded with nanoparticles and/or a cytotoxic substance, by correspondingly prepared immunocompetent cells and by a corresponding medical composition according to the described further aspects of the invention.
The method according to the invention is characterized by loading immunocompetent cells with nanoparticles and/or a cytotoxic substance for use in the theranostic treatment of patient-specific genetically modified cells, e.g. tumor cells or other viral genetically modified cells. The method comprises the following steps:
The invention provides a method for the production of immunocompetent cells, especially genetically transfected immunocompetent cells, wherein the immunocompetent cells are loaded with nanoparticles and/or a cytotoxic substance, resulting in improved immunocompetent cells for theranostic treatment approaches. The theranostic treatment is directed against cells of the patient which are patient-specific genetically modified, in particular viral genetically modified cells or other genetically modified cells like a patient-specific malignant tumor in an affected patient. The invention makes it possible to produce immunocompetent cells that counteract the disadvantages mentioned in the prior art and have higher immunocompetence than current patient-specific genetically modified cells. The loaded immunocompetent cells according to the invention have improved toxicity, and the treatment with them promises a higher chance of success.
Accordingly, the main point of the invention is that the immunocompetent cells, which interact specifically with certain altered cells of a patient, for example tumor cells, and can combat them, are loaded with nanoparticles and/or cytotoxic substances, wherein the nanoparticles and/or the cytotoxic substance are nuclides for radiotherapy. This loading of the immunocompetent cells can, on the one hand, considerably increase the efficiency in combating the altered cells. On the other hand, this approach according to the invention allows a controlled and controllable treatment of the patient, as described below in connection with the particularly preferred embodiments of the invention.
The term “nuclides for radiotherapy” comprises stable isotopes as well as radioisotopes which are useful for radiotherapy and nuclear medicine as will be outlined in more detail below.
In especially preferred embodiments the provided immunocompetent cells have been transfected with at least one gene whose gene product has a specificity for the immunocompetent cells to the patient-specific genetically modified cells.
In preferred embodiments the loading step b. comprises:
The invention is based on the finding that genetically modified immunocompetent cells, whose specificity with respect to patient-specific genetically modified cells has been increased and which already produce their own, i.e. native, cytotoxic substances and inject them into the patient-specific genetically modified cells, can be further improved by addition and loading of further substances in the form of nanoparticles and/or cytotoxic substances. The nanoparticles and/or cytotoxic substances have a direct or indirect cytotoxic effect on the patient-specific genetically modified cells. This increases the probability of successfully discharging the nanoparticles and/or the cytotoxic substance from the immunocompetent cells into the patient-specific genetically modified cells, which can also lead to the death of the corresponding cells.
The nanoparticles and/or the cytotoxic substances are nuclides for radiotherapy. Preferably, the nanoparticles and/or the cytotoxic substance is at least one stable isotope and/or at least one radioisotope from the following group of stable isotopes or radioisotopes: 10B, 11B, natB, 157Gd, 99mTc, 177Lu, 18F, and 211At or mixtures thereof. 10B and/or 157Gd are especially preferred.
In especially preferred embodiments the nuclides are intended to be activated later by supportive radiotherapy, e.g. in connection with boron neutron capture therapy (BNCT).
It is very advantageous if the poration is carried out under very mild and preferably controlled conditions, as the immunocompetent cells are very sensitive.
Preferably, the poration is carried out by sonoporation or electroporation or laser poration. Sonoporation is especially preferred.
In especially preferred embodiments the loading of the immunocompetent cells with nanoparticles and/or a cytotoxic substance is performed in the presence of microbubbles (also called microsomes), like liposomes. When performing the poration and especially the sonoporation in the presence of microbubbles, the loading is especially effective. Preferably the diameter of the microbubbles is 0.5 μm to 4 μm, preferably 1 μm to 3 μm, more preferably about 2 μm.
Preferably, the poration step is a sonoporation step and
A further advantage arises if the step of coupling the nanoparticles and/or the cytotoxic substance with the microbubbles is carried out before or after the nanoparticles or the cytotoxic substance is applied to or mixed with the microbubbles together with the immunocompetent cells.
It is also favorable if the diameter of the microbubbles is 0.5 μm to 4 μm, preferably 1 μm to 3 μm, preferably about 2 μm.
The term “immunocompetent cells” comprises different cells which may be naturally occurring or artificially designed.
In especially preferred embodiments the immunocompetent cells are T cells., and especially γδ T cells are preferred.
Moreover, it is principally possible to use other immunocompetent cells, even artificial cells without DNA, which have specific receptors comparable to T cells.
In preferred embodiments the immunocompetent cells are genetically transfected cells that have been transfected with at least one gene whose gene product confers specificity for the immunocompetent cells with respect to the patient-specific genetically modified cells.
It is advantageous when the immunocompetent cells comprise at least one gene for the specificity of the immunocompetent cells in view of the patient-specific genetically modified cells. Preferably, the immunocompetent cells have been transfected with a set of genes encoding at least a portion of a chimeric antigen receptor (CAR), wherein the CAR comprises at least an extracellular specificity-mediating scFv antibody domain, an intracellular T cell activation domain, and a protein in the cell membrane connecting the scFv antibody domain and the T cell activation domain and causing the protein to be expressed in the cell membrane with the specificity-mediating gene coding for the scFv antibody domain.
Advantageously, the immunocompetent cells are CAR-T cells, preferably γδ CAR-T cells.
Allogeneic immunocompetent cells are especially preferred, in particular allogeneic γδ T cells, preferably allogeneic γδ CAR T cells.
Preferably, in order to control the poration step, in particular to control the permeabilization of the cell membranes, a stationary or a non-stationary electric or electromagnetic field is applied to the cell solution, especially in connection with sonoporation. This measure is carried out in particular to control and monitor the resealing or healing of the cell membranes after poration.
In order to provide a sufficient amount of loaded immunocompetent cells for the therapeutic applications a cultivation and proliferation of the cells is useful.
Preferably, the following step is carried out before the loading step:
It is also advantageous if the following step is carried out after the loading step of the immunocompetent cells:
Preferably, the growth of the loaded immunocompetent cells, as well as the distribution of the nanoparticles and/or the cytotoxic substance in the immunocompetent cells is controlled during the cultivation step, by, e.g., ionizing particle radiation emanating from the loaded immunocompetent cells being detected and visualized for this purpose. For stable isotopes this can be induced by thermal neutron radiation (nth) or, in the case of the isotope 11B, by proton radiation. Radioactive payloads or tracers can be detected by radiation detectors.
Advantageously, before the step of providing the immunocompetent cells, a mutation is determined that is specific for the patient-specific genetically modified cells. This specific mutation is located in particular in a coding gene that codes for an antigen that is presented on the outside of these cells, for example the tumor cells. Furthermore, a gene that confers specificity for this mutation is provided for transfection of the immunocompetent cells and the immunocompetent cells are transfected with this gene so that the immunocompetent cells can specifically attack the patient-specific genetically modified cells of the patient.
According to a further aspect of the invention, the above-mentioned object is solved by immunocompetent cells, in particular T cells, in particular γδ T cells, in particular, theranostic γδ CAR-T cells, which are loaded with nanoparticles and/or a cytotoxic substance according to the method described herein. The specific process may include some or all of the features of the method as described above.
According to another aspect of the invention, the above object is further solved by immunocompetent cells having some or all of the features mentioned with respect to immunocompetent cells for use in a method for any theranostic therapy of patient-specific genetically modified cells, in particular viral genetically modified cells or a patient-specific malignant tumor. Preferably, the immunocompetent cells are for use in the treatment of cancer and/or malignant tumors.
According to an additional aspect of the invention, the above object is further solved by a medical composition comprising immunocompetent cells having some or all of the features mentioned above with respect to said immunocompetent cells. The medical composition further comprises a carrier, preferably a liquid carrier, in which these cells are present. The carrier liquid is intended to be administered to a patient in need thereof. The medical composition allows the immunocompetent cells to be infused to the patient in the carrier liquid which is specifically designed to be administered to a patient as it will be apparent to the skilled person. In particularly preferred embodiments, the medical composition is used in the treatment of cancer.
It is advantageous if the loaded immunocompetent cells are designed to distribute themselves within the patient's body within a maximum of 60 minutes, preferably within a maximum of 30 minutes, in such a way that their distribution in the patient's body can be detected with spatial resolution.
In addition, it is advantageous if the loaded immunocompetent cells can be detected in a spatially resolved manner in that they have a visualizable marker and/or ionizing particle radiation emanating from them can be detected, in the case of stable isotopes (e.g. 10B) after prior exposure to thermal neutron radiation (nth) or, in the case of 11B, to proton radiation. Therefore, in preferred embodiments, in the case of 10B an exposure to thermal neutron radiation is applied and in the case of 11B a proton beam is applied.
A further aspect of the invention relates to a method for the preparation of genetically transfected immunocompetent cells loaded with nanoparticles and/or a cytotoxic substance for the theranostic treatment of patient-specific genetically modified cells, in particular of virally genetically modified cells or of a patient-specific genetically modified cell, malignant tumors in an affected patient, comprising the following steps:
Further details and preferred embodiments of this aspect of the invention correspond to the details and preferred embodiments as described above.
In particularly preferred embodiments, the nanoparticles and/or the cytotoxic substance is at least one stable isotope and/or at least one radioisotope, wherein preferably the at least one stable isotope and/or the at least one radioisotope is selected from the following group of stable isotopes or radioisotopes: 10B, 11B, natB, 157Gd, 99mTc, 177Lu, 18F and 211At or mixtures thereof. In a particularly preferred manner, 10B and/or 157Gd is used for loading the immunocompetent cells.
In preferred embodiments of this aspect of the invention the immunocompetent cells were transfected in addition to at least one gene for the specificity of the immunocompetent cells towards the patient-specific genetically modified cells with a set of genes which is responsible for at least part of a chimeric antigen Receptor (CAR). Therein, the CAR comprises at least one extracellular specificity-imparting scFv antibody domain, an intracellular T cell activation domain, and a transmembrane domain connecting the scFv antibody domain and the T cell activation domain and anchoring the protein in the cell membrane, wherein the specificity-conferring gene encodes the scFv antibody domain.
Preferably, the immunocompetent cells are T cells, in particular γδ T cells, in particular theranostic γδ CAR-T cells.
The loading step b. is preferably carried out by sonoporation and, preferably, sonporation is performed in the presence of microbubbles. In preferred embodiments with respect to the microbubbles the following features are present:
Preferably, the step of coupling the nanoparticles and/or the cytotoxic substance with the microbubbles is carried out before or after the application or mixing of the nanoparticles or the cytotoxic substance with the immunocompetent cells.
In especially preferred embodiment of this aspect of the invention the charge of the loaded immunocompetent cells per unit volume is determined by the nanoparticles and/or the cytotoxic substance and the load is distributed evenly to the daughter cells at each cell division of the cells during culturing and proliferation of the cells.
In an especially preferred embodiment of this aspect of the invention, prior to the step of providing the immunocompetent cells, a method for preparing the immunocompetent cells for the patient is carried out as already outlined above. This encompasses the use of a specific mutation in a coding gene of an antigen presented on the cell membrane of these cells. The latter is determined, and a gene mediating specificity for this mutation is provided for transfection of the immunocompetent cells, and the immunocompetent cells are transfected with this gene.
According to a further aspect of the invention, the above-mentioned object is solved by immunocompetent cells which are loaded with nanoparticles and/or a cytotoxic substance and which can be produced according to said method described above. The immunocompetent cells are in particular T cells, preferably γδ T cells. CAR-T cells are particularly preferred, especially theranostic γδ CAR-T cells.
The invention further relates to a medical composition which comprises said loaded and immunocompetent cells. Said loaded and immunocompetent cells are obtained by said method for the preparation of genetically transfected immunocompetent cells loaded with nanoparticles and/or a cytotoxic substance as described above.
In preferred embodiments of said medical composition the loaded immunocompetent cells can be detected with temporal and spatial resolution in that they have a visualizable marker and/or ionizing particle radiation emanating from them that can be detected. In the case of stable isotopes like 10B after prior exposure to thermal/epithermal neutron radiation (nth) or in the case of 11B to proton radiation.
According to a further aspect of the invention, the above object is solved by immunocompetent cells having some or all of the features mentioned with respect to the immunocompetent cells for use in a method for the theranostic treatment of patient-specific genetically modified cells, in particular viral genetically modified cells or a patient-specific malignant tumor, in an affected patient. Preferably, the immunocompetent cells loaded according to the invention are intended for the treatment of cancer diseases.
Further features and advantages of the invention result from the following description of further embodiments in connection with the drawings. The individual features here can each be implemented individually or in combination with each other.
Other objects and features of the invention will become apparent from the following detailed description considered in connection with the accompanying drawings. It is to be understood, however, that the drawings are designed as an illustration only and not as a definition of the limits of the invention.
In the drawings,
In the following, embodiments of the invention are explained in more detail with reference to the drawings.
The figures show embodiments of the invention, and the following explanations relate to these embodiments. It is clear to a skilled expert that the explanations of the specific embodiments of the invention are in no way restrictive of the general idea of the invention.
Specifically shown in the figures is a functional design based on genetically modified immunocompetent cells as allogeneic γδ CAR-T cells, but autologous applications are not excluded where necessary.
An effective expansion and distribution method for allogeneic transfer is known, for example, from WO 2016005752 A1, and has led to positive results in clinical studies. The procedure described there begins with the initial collection of γδ T cells taken from a healthy first donor or, for example, umbilical cord blood. An enrichment to 99% of γδ T cells is then achieved before multiplication by several orders of magnitude in this process.
Quantities of 2.5×1010 cells can be obtained from a single donor within 10 to 18 days. The optimum time for a loading with nanoparticles and/or a cytotoxic substance is agreed upon by clinicians after thorough testing with the clinics in which the expansion and enrichment process of the γδ CAR-T cells is carried out.
An international network of dedicated allogeneic supply chains in Europe, the US, and Asia, which includes cryogenic facilities, will enable the storage of γδ CAR-T cells, which can be used for therapies for up to two to three weeks after exponential multiplication.
The loading of the nanoparticles and/or a cytotoxic substance, according to the invention, represents an advantageous extension of the variety of emerging γδ CAR-T cell therapies. For example, the present embodiments utilize the unique cancer recognition capability of yo-CAR-T cells, which can be tuned to target only specific cancer cells. With the support cytokines (for example, IL-2 or IL-12), the cancer cells are targeted so that the side effects caused by an attack on the patient's healthy cells are greatly reduced using the cells according to the invention and the manufacturing process described.
Patient-specific genetically modified cells in the form of malignant tumor cells in the embodiments depicted are initially attacked by T-cell cytotoxic substances. They can then be further attacked by releasing the loaded nanoparticles or the loaded cytotoxic substance. The nanoparticles may, for example, be nanoparticles that can be activated later by supportive radiotherapy.
Nanoparticle technologies have a significant impact on the development of therapeutic and diagnostic agents (theranostics). At the interface between treatment and diagnosis, clinically effective formulations have been developed. Most of these new developments see nanoparticles as carriers of drugs or contrast agents. Various contrast agents have been developed in clinical examinations to observe their interactions with the specific disease. Most commonly, optically active small molecules, metals and metal oxides, ultrasound contrast agents, and radionuclides are used. Theranostics is of great importance for active substances that act on molecular biomarkers of disease and will, therefore, contribute to personalized medicine for efficient treatments.
The focus for these nanoparticles is on specific isotopes relevant to cancer treatment, with 10B, which is used in boron neutron capture therapy (BNCT) (Malouff et al. (February 2021): “Boron Neutron Capture Therapy: A Review of Clinical Applications”). The most important feature of the nanoparticles described is that they can simultaneously loaded with other nanoparticles and/or a cytotoxic substance but also can be loaded separately or together with radiomarkers. This enables the monitoring and controlling of the density and distribution of the γδ CAR-T cell loading. Related times can be measured and optimized, e.g. the total time needed for the treatment to be most effective. This can also applied before any irradiation, for example, the aforementioned neutron radiation.
The nanoparticles can be any nanoparticles that are currently relevant in nuclear medicine. At present, as already mentioned, 10B is primarily used for BNCT. Gadolinium 157Gd is very interesting for a similar procedure; the so-called GdNCT in reference to BNCT, as this isotope has the highest neutron capture cross-section in nature; a much lower level of irradiation is required as part of a GdNCT. Therefore, even very sensitive regions of the body can be treated. Both isotopes are not radioactive by nature but are only excited to decay when they are activated by an external neutron source, for example, by an accelerator or a nuclear reactor. The maximum effective cross-section for triggering the decay is in the epithermal to thermal energy range of the neutrons, corresponding to a kinetic energy of about 0.025 eV.
The BNCT utilizes the fission reaction that occurs when non-radioactive 10B is irradiated with thermal or epithermal neutrons nth. The tissue of the body is transparent to such neutrons so that they penetrate the tissue unhindered.
However, the epithermal neutrons lose a small amount of energy when they penetrate tissue, so they become thermal neutrons - they are “thermalized”.
10B +nth→4He+7Li+2.8 MeV (1)
According to Formula 1, a high-energy α-particle and a 7Li-ion are generated, both of which lose kinetic energy determined by the Q value (2.8 MeV) of the reaction through linear energy transfer (LET) as they pass through the cell. The 7Li and the lighter 4He-ions penetrate for a maximum range of ˜4 μm for 7Li and ˜9 μm for 4He inside (a single) cell. Since these lengths correspond to the diameter of a human cell, the lethality of the capture reaction is only limited to the cell itself, and/or cells in the immediate vicinity of the boron nanoparticles. The success of BNCT, therefore, depends on the selective delivery of sufficiently high quantities of 10B to the tumor cells themselves, whereby only small quantities are localized in the surrounding healthy tissue. This is one of the core objectives of the treatment with the genetically modified and loaded immunocompetent cells of the present invention.
As of today, in the best case, the boron load of a patient-specific genetically modified cell, in particular a cancer cell, can be increased by only three to four times compared to the neighboring healthy surrounding cells. According to the invention, this ratio can be enhanced by four to six orders of magnitude while at the same time reducing side effects with the presented method. The limitation of the previous methods is disadvantageous because normal tissue tolerates the flow of slow neutrons due to the relatively low nuclear capture reactions that occur with hydrogen and nitrogen. In comparison, the undesirable radiation doses associated with thermal and epithermal neutron irradiation, to which healthy tissue is exposed, are minimized by the methods of the invention. For BNCT to be effective, only a single fission of a 10B isotope is sufficient to disrupt a patient-specific genetically modified cell, in particular a tumor-like malignant cell.
The isotope 10B isotope makes up around 20% of the low-priced natural boron (natB) thus, nanoparticles carry also a large proportion of 11B, which is also stable and can be used for radiotherapy. 11B nanoparticles have a large effective radius for proton radiation with an energy of approx. 600 keV.
This, in turn, means that, i.e., immunocompetent cells loaded with them in the form of γδ CAR-T cells can also be used for proton radiation therapy, although most of the benefits for BNCT will be lost.
In addition to the non-radioactive isotopes mentioned above, other radioisotopes and/or isomers can be considered for cancer theranostics, for example, e.g., 99mTc, 177Lu, 18F, and 211At. All other isotopes and/or isomers of current medical relevance for cancer or other treatments that target patient-specific genetically modified cells that have a potentially pathogenic potential are included in the proposed method. In general, these are either commonly used radiological isotopes or those currently being used in advanced stages of clinical trials, where they promise to treat various malignant diseases.
For the isotopes described in the previous paragraph, the radioactive activity differs as a function of time A(t), which is referred to here for the described biological activity of the γδ CAR-T cells.
The activity A(t) describes the decay of a radioactive sample per second and is defined by
A(t)=λ×N(t) (2)
where N(t)=N0×exp(−×t) is the number of isotopes in the sample, λ is the decay constant for a particular isotope, and N0 is the number of isotopes at an arbitrarily chosen reference time t0.
Clinicians can determine the time frame and the number of nanoparticles of the above-mentioned isotopes with comparatively high precision. In contrast to A(t), biological activation does not follow such a simple exponential rule. Instead, it depends on various controllable external factors, such as cell growth, survival rate, and cryopreservation for later “revival”.
The dose rates required to activate a therapeutic amount of radioactive decay are calculated based on continuous or pulsed sources currently available at specially enhanced nuclear reactor sites, radio frequency (RF) accelerators, or tandem accelerators. Furthermore, there will soon be modern laser-driven accelerators available for clinical use, delivering comparatively high temporal beam doses and thus minimizing the total time for patient irradiation.
The activities A(t) of the theranostic sample are adjusted accordingly for each treatment to determine the optimal dose rate:
D=dE/dm=(1/ρ)dE/dV (3)
where dE describes the energy released within a mass segment dm in the patient's body with a specific density of ρ, which extends over a volume element of dV.
For precise calculations, the heterogeneous structures of the tissue would have to be divided into a number of inhomogenity groups Di, before they are summed up to D to form a treatment plan advised by clinicians. The dose rate D is expressed in microgray per hour [μGy/h] and converted into the equivalent dose rate HT, given in Sieverts per hour [Sv/h] for one treatment.
In the following, reference is made to the figures and the reference symbols shown therein, whereby the figures only show specific embodiments of the invention are described below. Reference signs are generally only assigned once in each the following. However, it will be readily apparent that the same reference signs identify the same structures in the figures, unless another reference sign has been explicitly assigned.
Genetically modified immunocompetent cells in the form of cultured γδ CAR-T cells (1), e.g. vγ9vδ2 T cells, which have a co-stimulatory chimeric antigen receptor (CAR) (2) inserted into their cell membrane by genetic transfection and a native γδ-T-cell receptor (γδ-TCR) (3) can recognize the endogenous isopentenyl pyrophosphate (IPP) antigens on the cell surface of cancer cells. They are removed at an early stage of their expansion process in suitable batch sizes and placed on a Petri dish.
Their cell membrane (5) contains cytotoxins (4) (see
The nanoparticles and/or the cytotoxic substance can be added as a layer of free atoms or molecules, whereby they can also, as can be seen in
The nanoparticles or the cytotoxic substance can, therefore, be freely present. They can also be present in the interior of the microbubbles (6), preferably chemically bound to each other or, for example, in the form of a chelate complex (7), in microbubble membranes, in particular mechanically embedded (9) or covalently bound to the microbubbles (10) from the outside. A preferred diameter of a microbubble is approx. 2 μm (preferably less than 3 μm). Preferably, the nanoparticles and/or the cytotoxic substance and the immunocompetent cells are added to the Petri dish in layers. It is also advantageous to keep a diameter tolerance of the microbubbles low by appropriate pre-filtration, i.e. that all microbubbles have essentially the same size.
In the present embodiment, as soon as the load samples are distributed, the Petri dish will get reversed, so that the γδ CAR-T cells are placed at the top of the arrangement.
The superimposed layers of this preparation are then compacted using ultrasonic pressure so that the distances between the components of the layers are brought to a predetermined and comparatively small average distance.
The pressure P(t) applied by the ultrasonic transducer drives the nanoparticles, the cytotoxic substance and/or the loaded microbubbles into the vicinity of the γδ CAR-T cells up to an optimum distance from the γδ CAR-T cells so that the microbubbles can release their charge to the γδ CAR-T cells.
P(t) and the time duration ttotal of the effect of the ultrasonic pressure on the cell solution are partly based on empirical data derived from laboratory work, considering radioactive and biological activation. The exact process settings are specific to the microbubbles and the specialized γδ CAR-T cells to be used in treatment.
The implosion of a microbubble, which is the process for delivering the payload, is determined by the Rayleigh-Plesset formula, which determines the change in the bubble radius R(t) over time:
where ρL is the density of the surrounding liquid, vL is the kinematic viscosity of the surrounding liquid, γ is the surface tension of the microbubble-liquid interface, γP(t)=P∞(t)−PB (t) is the pressure difference between the uniform pressure inside the bubble PB (t) and P∞ (t) is the external pressure “infinitely” far from the bubble. Formula 4 makes it possible to optimize the transducer's trigger signal in relation to the time of disintegration of a microbubble, whereby the free nanoparticles or the nanoparticles bound in microbubbles or the cytotoxic substance enter the T cells.
In order to be able to carry out at least partial permeabilization of the cell membranes of the γδ CAR-T cells used in the present example in a gentle and controlled manner, it is advantageous in the present embodiment if the following steps are carried out.
All components involved in the sonoporation process can be subjected to precise tolerance control of the dimensions, as these have a significant influence on the intrinsic modes of the set-up. In particular, this requirement concerns the diameter of the microbubbles used, so that they are brought to a ±10% tolerance by a micropore filter film before use.
Any expert understands that the device settings are precisely maintained and documented and that electrical or electromagnetic fields are preferably used to control the reclosure of the cell membranes; for example, in a closed cell membrane, a feedback loop can be used. Since P(t) in Formula 4 is periodic in nature: P(t′)=P(t+tperiod), and even if a sinusoidal ultrasonic waveform provides good results, an optimized pulse profile shape with constant period may be more favorable for optimal sonoporation in terms of the set goals. Standard microbubbles produced industrially and available on the market are assumed.
P(t) is optimized with respect to the amplitude P0 (t), the period tperiod and the total duration of the sonoporation process (ttotal). For each specific variety of γδ CAR-T cells, cellular responses such as cell membrane permeability and cytoskeletal fragmentation are to be investigated with respect to the dependence on nuclear parameters such as acoustic driving pressure P(t) and the distances between the emitting microbubbles.
For a given bubble diameter of ˜2 μm and a cell with dimensions of a few um, a two-dimensional simulation model according to Formula 4 leads to optimal values of P0 (t)˜400 hPa. A typical time for the endocytosis of the contents of the microbubbles into the immunocompetent cells is less than 1 μs. One aim of the simulations and the design is to make the carefully prepared γδ CAR-T cells from unnecessary stress and thus optimize the effectiveness of the manufacturing process.
The sonoporation process, as can be seen in
In the immediate vicinity of the microbubbles, the application of ultrasound pressure via the ultrasound transducers leads to an indentation (13) with the depth dt of the cell membranes of the γδ CAR-T cells. The size of it depends on the distance ds between the microbubbles and the γδ CAR-T cells; see the reference sign (14). Both parameters dt and ds are functions of the applied pressure P(t), see the reference sign 15, and thus implicitly of the time t, see the reference sign (16).
The nanoparticles and/or the cytotoxic substance, see the reference sign (17) in
This completes the loading process for the γδ CAR-T cells. After the loading of the γδ CAR-T cells, the nanoparticles and/or the cytotoxic substance are located inside the γδ CAR-T cells, see the reference sign (20) in
Residual microbubbles are filtered using standard molecular biological methods. The loading depends on the radiological and biological activity of the patient-specific genetically modified cells in the patient, which is determined in advance for the treatment. It is assumed that the loading process takes place at an earlier stage of cell culture growth. Therefore, the intended payload per cell should not exceed the biologically acceptable, i.e. non-lethal, threshold. For boron loading, this would correspond to a payload of approximately 0.13 picograms [pg] per cell. This corresponds to 3×1010 atoms of natural boron, per cell.
For radioisotopes, the dose exposure for the treating person should be taken into account. Patient-specific exposure is also determined by factors such as the proliferation of γδ CAR-T cells in a patient's body and a period of two to four days during which they usually accumulate around the patient-specific genetically modified cells, such as tumor cells.
For a more straightforward description, the γδ CAR-T cells loaded with the nanoparticles and/or the cytotoxic substance and used here are referred to as “T-ninjas”. As illustrated in
In addition, all the T-ninjas described can also be used with additional substances at the same time, which will enable better monitoring and control of the manufacturing process and also the treatment of patients in certain phases of therapy.
Ultrasonic pulses with a total duration of 20 s to 40 s are only applied in several cycles, for example, in order to increase the achievable payload of the γδ CAR-T cells under constant observation and to determine the actual own modes of the set-up. The microbubbles themselves are located in a phosphate buffer solution, for example. A total quantity of six million microbubbles per ml is considered the ideal concentration for the sonoporation process.
After a total number of 100,000 ultrasonic periods, a loading efficiency of the microbubbles of at least 10% can be assumed if the sound pressure profile, the duration of the sound pressure periods, and the transducer geometry have been optimized.
As can be seen from
The latter provides a power of >200 W at frequencies of up to several MHZ, see the reference sign (33), with a focal spot of >18 mm. The ultrasonic transducer is driven from a waveform generator (36), which is amplified by a class A broadband amplifier (37) to provide a linear output in the frequency range from 10 KHz to 12 MHz for any waveform.
A typical setting comprises a curve with a constant period of P(t) with an average value of <P∞(t) and >=400 kPa at a frequency of >1 MHz. The sonoporation process can be monitored using a microscope setup with a monochromatic light source (34) and a CCD camera (35).
An expert will be aware that the loading of the immunocompetent cells essentially depends on increasing the permeability of the cell membranes so that the nanoparticles that may be coupled to the microbubbles or the cytotoxic substance coupled to the microbubbles can enter the cell lumen of the immunocompetent cells. Therefore, instead of sonoporation, electroporation or laser poration may be used, whereby sonoporation is comparatively advantageous, as explained in detail above.
As can be seen in
Clinical needs and ongoing monitoring will inform the optimal timing and extent of cell proliferation and determine the final average payload of engineered T-ninja cells based on the Formula 5. In the case of radioisotopes, it is advantageous if the loading of the T-ninja cells is carried out immediately before the infusion of the T-ninja cells into the patient's body, as the T-ninja cells do not survive for long due to the ionizing particle radiation of the radioisotopes.
As can be seen from
In the present case, an activating electron, neutron, proton, or γ beam (41) for a stable isotope such as 10B or 157Gd is used for this purpose, with which T-ninja-S cells (42) of the cell culture are loaded, but preferably a thermal or an epithermal neutron beam is used. Under the influence of the activation beam, a fission reaction according to Formula 1 is initiated in the T-ninja-S cells, which can be monitored with a detector (43). T-ninja-R cells, which are not illustrated here, and which are loaded with a cytotoxic substance in the form of a radioisotope such as 18F, do not require such an activation beam, as they already emit ionizing particle radiation. However, they can be detected but can be observed in exactly the same way with the same detectors. The detector (43) can be represented by a high-purity germanium detector (HPGe) or a lanthanum bromide detector (LaBr3). However, detectors with better spatial and temporal resolution, which are currently under development, are preferred for use in this method.
In addition, suitable measurements of the density and distribution of the nanoparticles and/or the cytotoxic substance in the cell culture can be undertaken.
The core vehicles in a therapy against patient-specific genetically modified cells, for example a tumor or cells genetically modified by a viral infection, are, as already mentioned, summarized as T-ninja cells. In the following, reference will be made to
Due to the TCR and the CAR, T-ninja cells have an exceptionally high specificity for corresponding patient-specific genetically modified cells and “search” for the affected body cells (46). They attach themselves to these, see the reference sign (50), mediated by the specificity of the T-ninjas, for example, for a biological “stress signal” (48) and a target antigen (49).
The approach process of the T-ninja cells to the patient-specific modified cells can be divided into six sequences (52) to (57), displayed in
The sequence (52) shows the last stage of the recognition process of a cancer cell (46), whereby the reference sign (51) indicates the direction of the T-ninja movement. Sequence (53) shows an initial docking phase of the T-ninja cells, which is caused by the high specificity of the T-ninja cells with respect to the cancer cells. The sequence (54) shows an interaction of the CAR-T-cell receptor and the γδ T-cell receptor (γδ TCR) with the target antigens of the cancer cell as well as an excitation of two independent trigger signals for cytokine release of the T-ninja cells (58), (59).
The combination of the two intracellular signals activated by the CAR-T and the γδ T cell receptor “switch” the T-ninja cells into a cytokine-enhanced attack mode. These two signals induce a cellular response from the T-ninja cells, which is also known as the “kiss of death”. This is illustrated in sequences (55) and (56).
Sequence (55) shows how the cell membrane of the cancer cell is opened by the T-ninja cell. The reference sign (56) refers to the actual “kiss-of-death”, in which the T-ninja cells release their own, i.e. native, cytotoxic substances as well as charged nanoparticles and/or a cytotoxic substance as described above through osmotic pressure into the patient-specific genetically modified cells.
The reference sign (57) refers to a detachment process of the T-ninja cells. The T-ninja cells will then continue their path (63) in order to find further patient-specific genetically modified cells, such as cancer cells in the present case, of the same type. The unhealthy cell remains with the nanoparticles and/or the cytotoxic substance enclosed inside its body (64).
Reference is made below to
The first part of the radiological therapy is preferably carried out with radioisotopes, and the second part of the therapy begins only after the activation beam has been switched on at a time determined during the therapy; in any case after the first part of the therapy and depending on the success achieved by the latter. The nanoparticles can be used in separate T-ninja cultures, or together in a single one, so that T-ninja-SR cells are used instead of T-ninja-R and T-ninja-S cells.
An example of an embodiment of the immunocompetent cells according to the invention and an example of the application according to the invention in a theranostic therapy against patient-specific genetically modified cells in the affected patient following the production can be summarized as follows:
Although only a few embodiments of the present invention have been shown and described, it is to be understood that many changes and modifications may be made thereunto without departing from the spirit and scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2022 132 082.0 | Dec 2022 | DE | national |
