Patent Application
20240173353
The present disclosure relates to chimeric antigen receptor T cells, especially universal chimeric antigen receptor T cells suitable for administration to non-specific patients. The present disclosure further relates to preparation methods and use of these chimeric antigen receptor T cells.
Adoptive immune cell therapy has shown great potential in the treatment of cancer, autoimmune diseases and infectious diseases. In recent years, the use of chimeric antigen receptor-expressing T cells (CAR-T cells) for treating cancer has received particular attention. At present, CAR-T cells are mainly prepared using the patient's own T cells. This involves isolating, modifying and expanding T cells for each patient, a time-consuming and expensive process. In addition, for patients such as newborns and the elderly, it is usually difficult to obtain T cells with good quality to produce specific CAR-T cells needed by patients.
One possible solution is to use T cells derived from healthy donors to generate universal CAR-T cells. Compared with autologous CAR-T cells, the universal CAR-T cells have many advantages:
Therefore, universal CAR-T products will be the main trend of CAR-T therapy in the future.
However, the development of universal CAR-T is also facing great challenges, and the following two problems need to be solved most:
At present, the first problem has been basically solved. The researchers knock out the TRAC gene encoding the T cell surface receptor (TCR) on 130-T cells through gene editing technology, effectively inhibiting CAR-T cells from indiscriminately attacking host cells by activating TCR, thereby avoiding the occurrence of GvHD.
In contrast, the second problem is more difficult to solve. In recent years, researchers have been exploring how to effectively expand universal CAR-T cells in the host. Currently, there are two mainstream solutions:
So far, researchers have optimized CAR-T cells with TRAC/B2M gene knockout in various ways, but there is still a high possibility of being cleared by host immunity.
In one aspect, provided herein is a cell comprising a mutant protein that causes the cell to be insensitive to an inhibitor affecting its activity and/or cytotoxicity.
In some embodiments, the mutant protein is a member of the tyrosine kinase family.
In some embodiments, the mutant protein is LCK.
In some embodiments, the LCK protein comprises T316 mutation.
In some embodiments, the LCK protein comprises T316I, T316A or T316M mutation.
In some embodiments, the inhibitor is a tyrosine kinase inhibitor.
In some embodiments, the inhibitor is dasatinib and/or ponatinib.
In some embodiments, the cell is a mammalian cell.
In some embodiments, the cell is a stem cell or immune cell.
In some embodiments, the immune cell is a NK cell or T cell.
In some embodiments, the cell expresses a chimeric antigen receptor (CAR).
In some embodiments, the mutant protein is formed by base editing, HDR and/or overexpression.
In some embodiments, the overexpression is achieved through transfection with lentivirus, retrovirus, adeno-associated virus and adenovirus.
In another aspect, provided herein is a method for introducing T316 mutation into the Lck gene of a cell, comprising introducing a base editor into the cell.
In some embodiments, the base editor is an ABE or CBE base editor.
In some embodiments, the method further comprises introducing sgRNA into the cell.
In some embodiments, the sgRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 5-8.
In some embodiments, the CEB base editor is an A3A-CBE3 fusion protein.
In some embodiments, the cell is a mammalian cell.
In some embodiments, the cell is a stem cell or immune cell.
In some embodiments, the immune cell is a NK cell or T cell.
In some embodiments, the cell expresses a chimeric antigen receptor (CAR).
In another aspect, provided herein is a cell expressing a chimeric antigen receptor, wherein the cell has cytotoxicity or is induced to have cytotoxicity, and the cell is engineered so that its cytotoxicity is insensitive to a cell activity inhibitor.
In some embodiments, the cell activity inhibitor is a T cell activity inhibitor.
In some embodiments, at least one bioactive molecule in the cell is engineered to be insensitive to the cell activity inhibitor, and the bioactive molecule can be inhibited by the cell activity inhibitor in a normal T cell.
In some embodiments, the bioactive molecule acts on a chimeric antigen receptor signal transduction pathway.
In some embodiments, the bioactive molecule is a protease.
In some embodiments, the protease is a protein tyrosine kinase.
In some embodiments, the protease is LCK protein.
In some embodiments, the Lck protein tyrosine kinase comprises T316 mutation.
In some embodiments, the Lck protein tyrosine kinase comprises T316I, T316A or T316M mutation.
In some embodiments, the engineering is accomplished by using a base editor.
In some embodiments, the base editor is an ABE or CBE base editor.
In some embodiments, the T316I mutation is obtained by introducing a base editor CBE and sgRNA into the cell, and the target sequence of the sgRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 5-8.
In some embodiments, the LCK protein in the cell comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-11.
In some embodiments, the cell is a T cell or NK cell.
In some embodiments, the cell is further engineered to eliminate or weaken the cytotoxicity generated via TCR on the cell surface thereof.
In some embodiments, a TCR-related gene of the cell is knocked out.
In some embodiments, the TRAC gene of the cell is knocked out.
In some embodiments, the β2m gene of the cell is knocked out.
In some embodiments, the CIITA gene of the cell is knocked out.
In some embodiments, the T cell activity inhibitor is a protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is an LCK protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is dasatinib and/or ponatinib.
In another aspect, provided herein is use of the above-mentioned cell in the preparation of a universal CAR-T cell.
In another aspect, provided herein is a method for preparing a CAR cell, comprising engineering the CAR cell so that its CAR-mediated cytotoxicity is insensitive to a T cell activity inhibitor.
In some embodiments, at least one bioactive molecule of the CAR cell is engineered to be insensitive to the T cell activity inhibitor, and the bioactive molecule can be inhibited by the T cell activity inhibitor in a normal T cell.
In some embodiments, the bioactive molecule acts on a signal transduction pathway of the CAR.
In some embodiments, the bioactive molecule is a protease.
In some embodiments, the protease is a protein tyrosine kinase.
In some embodiments, the enzyme is an LCK protein tyrosine kinase.
In some embodiments, the LCK protein tyrosine kinase comprises T316 mutation.
In some embodiments, the LCK protein tyrosine kinase comprises T316I mutation.
In some embodiments, the T316I mutation is obtained by introducing a cytosine base editor and sgRNA into the CAR cell, and the sgRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 5-8.
In some embodiments, the LCK protein tyrosine kinase in the CAR cell comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-11.
In some embodiments, the CAR cell is a T cells or NK cell.
In some embodiments, the CAR cell is further engineered to eliminate or weaken the cytotoxicity generated via TCR on the cell surface thereof.
In some embodiments, a TCR-related gene of the CAR cell is knocked out.
In some embodiments, the TRAC gene of the CAR cell is knocked out.
In some embodiments, the 132m gene of the CAR cell is knocked out.
In some embodiments, the CIITA gene of the CAR cell is knocked out.
In some embodiments, the T cell activity inhibitor is a protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is an LCK protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is dasatinib and/or ponatinib.
In some embodiments, during the preparation of the CAR cell from a T cell or NK cell, the T cell or NK cell is contacted with the T cell activity inhibitor.
In some embodiments, the T cell or NK cell is contacted with the T cell activity inhibitor when the T316I mutation is carried out; preferably, the concentration of the T cell activity inhibitor is 100 nM.
In some embodiments, the intracellular signaling domain of the CAR comprises:
In another aspect, provided herein is a method for treating a disease in a patient, comprising administering to the patient the above-mentioned cells in combination with a cell activity inhibitor.
In some embodiments, the cells are not derived from the patient
In some embodiments, the cells are T cells.
In some embodiments, the cell inhibitor is a T cell activity inhibitor.
In some embodiments, the cell activity inhibitor is a protein tyrosine kinase inhibitor.
In some embodiments, the cell activity inhibitor is an LCK protein inhibitor.
In some embodiments, the T cell activity inhibitor is dasatinib and/or ponatinib.
In some embodiments, the disease is a tumor.
In some embodiments, the method further comprises stopping the administration of the cell activity inhibitor, so that the patient's own T cells regain activity and clear the administered cells; or increasing the dose of the cell activity inhibitor to inhibit the cytotoxicity of the cells in the patient.
In another aspect, provided herein is a pharmaceutical kit or pharmaceutical combination comprising the above-mentioned cells and a cell activity inhibitor.
In some embodiments, the cells are T cells.
In some embodiments, the cell activity inhibitor is a T cell activity inhibitor.
In some embodiments, the cell inhibitor is a protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is an LCK protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is dasatinib and/or ponatinib.
In another aspect, provided herein is use of the above-mentioned cells in combination with a cell activity inhibitor in preparing an anti-tumor drug.
In some embodiments, the cells are T cells.
In some embodiments, the cell activity inhibitor is a T cell activity inhibitor.
In some embodiments, the T cell activity inhibitor is a protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is an LCK protein tyrosine kinase inhibitor.
In some embodiments, the T cell activity inhibitor is dasatinib and/or ponatinib.
The cells provided herein and the universal CAR-T cells prepared therefrom can be used for treating non-specific patients (allogeneic cell therapy), which overcome a series of problems caused by the need of existing CAR-T cells to be derived from the patients themselves.
Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.
“Chimeric antigen receptor (CAR)” is an engineered membrane protein receptor molecule that confers a desired specificity to immune effector cells, such as the ability to bind to specific tumor antigens. A chimeric antigen receptor generally consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen-binding domain is an scFv sequence responsible for recognizing and binding to a specific antigen. An intracellular signaling domain usually includes an immunoreceptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3z molecule, which is responsible for activating immune effector cells and producing cytotoxic effects. In addition, the chimeric antigen receptor may also comprise a signal peptide responsible for intracellular localization of the nascent protein at the amino terminus, and a hinge region between the antigen-binding domain and the transmembrane domain. In addition to signaling domain, intracellular signaling domain can also comprise costimulatory domains derived from, for example, 4-1BB or CD28 molecules.
“Bispecific chimeric antigen receptor” is intended to mean that the molecule includes at least two different antigen-binding sites in the extracellular antigen-binding domain, which respectively recognize and bind to different antigen molecules on target cells. For example, bispecific chimeric antigen receptors targeting both CD19 and BCMA are mentioned in the Examples herein.
“CAR cells” refers herein to cells that expresses CAR molecules on the cell surface. In most cases, the cells are immune cells, such as T cells, or NK cells. Accordingly, T cells expressing CAR are referred to herein as “CAR-T” or “CAR-T cells”. In addition, when referring to CAR-T cells herein, unless otherwise specified, it refers not only to the cells directly modified by CAR, but also to the daughter cells produced after the proliferation of these cells in vitro or in vivo.
“Universal CAR-T cells (UCAR-T)” herein refer to such cells that are not limited to CAR-T cells infused into a specific patient. In the prior art, in order to prevent GvHD and the host's rejection of the graft, cells (such as T cells) are usually collected from the patient, modified with CAR and infused back into the patient. This method is not only time-consuming and expensive, but also in some cases cannot obtain a sufficient number of T cells of the patient for CAR modification. In contrast, the universal CAR-T cells here mean that these cells are suitable for allogeneic transplantation, the same batch of CAR-T cells can be used in different patients, and these universal CAR-T cells are usually not derived from these patients.
The present invention is based, at least in part, on novel preparation methods and use strategies of universal CAR-T discovered by the inventors.
In order to prepare universal CAR-T cells (UCAR-T), in our design of universal CAR-T cells, we try to find a simple and easy treatment protocol, which has the following two key points:
In order to suppress the cytotoxicity of host T cells and NK cells, inhibitors with inhibitory activity against both of them can be used, or inhibitors with inhibitory activity against one of them separately can be used, such as small molecule inhibitors or inhibitory antibody molecules. These inhibitors may result in a decrease in T cells and/or NK cells or may not affect their numbers. In this case, in order to achieve point (2), CAR-T cells can be engineered to make them insensitive to these inhibitors (especially to make the CAR signal transduction pathway tolerant to the inhibitory effects of these inhibitors), namely in the presence of these inhibitors, these CAR-T cells can still survive, be activated by target cells, and have target cell cytotoxicity (even if the overall cytotoxicity is reduced). There are a variety of ways that can be used to carry out such engineering of CAR-T cells, including but not limited to, introducing genetic mutations into CAR-T cells, where the mutated gene products (such as proteins or enzymes) are not sensitive to these inhibitors and have the function of the unmutated gene product; introducing foreign genes into CAR-T cells, where the proteins or enzymes produced by the expression of these foreign genes in CAR-T cells are not affected by these inhibitors and can replace the target molecules of these inhibitors to function intracellularly; introducing binding partners of these inhibitors (such as exogenous proteins capable of binding to these inhibitors, such as intrabodies) into CAR-T cells in order to neutralize these inhibitors; overexpressing the target molecules of these inhibitors (such as proteases) in CAR-T cells so as to counteract the effects of these inhibitors, etc.
Methods for introducing genetic mutations into cells are known in the art, including, but not limited to, DNA homologous recombination, site-specific cleavage with endonucleases (such as ZFN and TALEN), CRISPR-based gene editing techniques, and various base editors (such as CBE, ABE and their various improved variants).
Methods for introducing exogenous genes into cells are known in the art and include, but are not limited to, electroporation, gene gun, microinjection, liposome introduction, viral transduction (e.g., using retrovirus, lentivirus, various improved viral vectors, such as adenovirus and adeno-associated virus vectors).
Methods for overexpressing certain proteins or enzymes in cells may include, for example, increasing the copy number of the encoding gene thereof, enhancing the function of the promoter of the encoding gene thereof.
The introduction of mutations, exogenous genes, or overexpression in cells can be short-term or transient, or permanent (e.g., integration of a mutated gene or an exogenous gene into a host cell chromosome). The introduction can be in the form of DNA or RNA, for example, through lentiviral transduction so that the foreign gene can be expressed in the host cells for a long time, or through the introduction of mRNA so that the host cells can express some foreign proteins or enzymes in the short term.
The “mutant protein” used herein refers to a protein with a change in the amino acid sequence relative to the wild type, or a change in expression level compared to the expression level in normal cells, such as increased expression level (or overexpression).
Those skilled in the art should understand that the above engineering can be performed before, during, or after modifying the original cells into CAR-T cells. For example, T cells can be engineered as described above to make them insensitive to T cell and NK cell inhibitors, T cell banks are cultured and prepared, and then CAR modification is carried out according to various treatment needs, so as to obtain universal CAR-T cells for various therapeutic purposes (such as various cancers).
The “original cells” here refer to cells of interest that have undergo or are about to undergo CAR modification, such as stem cells, and immune cells, T cells, and NK cells at various developmental stages. Considering that one of the purposes of the present application is to prepare universal CAR-T cells, the source of these cells has nothing to do with the patient to be treated. Therefore, the source of these original cells is basically unlimited, for example, they can come from blood banks, and healthy volunteers.
In some embodiments, the above-mentioned inhibitor is a tyrosine kinase inhibitor. In some embodiments, these tyrosine kinases act on the signal transduction pathway of the CAR. In some embodiments, these tyrosine kinases act on both the TCR signal transduction pathway and the CAR signal transduction pathway. In some embodiments, the tyrosine kinase is LCK kinase. In some specific embodiments, the tyrosine kinase is LCK kinase and the inhibitor is dasatinib (DS) and/or ponatinib (PN).
In a specific embodiment, the engineering of LCK kinase involves the mutation of its T316 site, such as T316I, T316M, T316A and other mutations, as long as the mutation makes the mutated product insensitive to the above-mentioned inhibitors (such as dasatinib and ponatinib) while having the protease function before mutation. Preferably, the mutation is T316I.
As far as the T316I mutation is concerned, the present disclosure provides an ingenious way to achieve this mutation. The method involves the use of cytosine base editors used in conjunction with sgRNAs, achieves, on the Lck gene, the base pair C G to T A transition. This base pair transition produces the T316I mutation in its expression product LCK, while the cytosine base editor also causes some other base pairs C G transitions near the target mutation site, but these other transitions happen to be nonsense mutations that do not produce amino acid changes in LCK. Those skilled in the art should understand that, although the preferred technical solution for forming T316I mutation is described in the specific Examples herein, it does not exclude other similar ways to form mutation at T316 site. For example, under the inspiration of the specific technical solution herein, gene editing technology can be used to form other mutations at the T316 site, or to change the specific sequence of the sgRNA (including length changes, target sequence changes), or even to generate multiple mutations (including T316 site) in the product LCK using these or other methods, as long as the formed product LCK can maintain its original function and is insensitive to the above inhibitors, then these changes should be included in the scope of the present invention.
In order to prevent CAR-T cells from attacking host normal cells (non-target cells), that is, GvHD, it may be considered to engineer the TCR-related genes on CAR-T cells to deactivate or reduce the activity of TCR, for example, by knocking out the TRAC gene encoding the α chain of the TCR receptor, and/or the TRBC gene encoding the 13 chain of the TCR receptor through gene editing or other methods.
Further, in order to prevent a few host T cells or NK cells from attacking CAR-T cells, it may be considered to reduce or avoid the expression of HLA-class I molecules on CAR-T molecules, for example, by knocking out the B2M gene encoding (32 microglobulin.
The universal CAR-T cells provided herein can be combined with cell activity inhibitors to treat patients (such as cancer patients). As already explained above, these cell activity inhibitors can be of various types, as long as they can make the immune system of the host (recipient of CAR-T cells) not cause the loss of the target cell cytotoxicity of the infused CAR-T cells. Generally, these inhibitors can prevent the host immune system (such as T cells, especially CD8+ T cells) from killing CAR-T cells. In this case, the infused CAR-T cells can be activated by their target cells to exert cytotoxic effects. At the same time, they can also proliferate in the host and exert long-term effects. Preferably, these inhibitors only suppress the function of the host's immune system without causing disruption of its function. In this case, when the inhibitor is cleared or the inhibitor is not administered, the host's immune system can restore its original function as soon as possible.
Therefore, in some embodiments, these inhibitors can be used as molecular switches to determine whether the universal CAR-T cells continue to perform their function. For example, the inhibitors can be administered to patients before the infusion of universal CAR-T cells to suppress or weaken the cytotoxicity of their immune system (especially T cells) on the universal CAR-T cells that are about to be infused. Next, universal CAR-T cells are infused to the patients, and then the inhibitors (same or different from previous inhibitors) continue to be regularly administered to patients as appropriate, so as to maintain the concentration of inhibitors in patients to a level that can continue to suppress the function of the patient's immune system. Since these infused universal CAR-T cells are engineered to be tolerant to the aforementioned inhibitors, they can recognize and kill their target cells (such as cancer cells). After the desired therapeutic effect (such as tumor regression or disappearance) is achieved, the administration of the inhibitor is stopped to allow the patient's own immune system to recover and clear the universal CAR-T cells in the body. During this process, the content and activity level of universal CAR-T in the patient as well as the disease status can be regularly detected to determine whether it is necessary to infuse universal CAR-T cells again. Using these inhibitors as molecular switches is also beneficial to drug safety. If the patient cannot tolerate the universal CAR-T cell therapy, the inhibitor can be stopped at any time so as to clear these universal CAR-T cells to avoid serious adverse reactions. In addition, by increasing the concentration of the inhibitor (for example, when ponatinib is used as the inhibitor, its concentration can be increased from 200 nM to 500 nM), the above-mentioned universal CAR-T cells that can tolerate the inhibitory effect of a certain dose of inhibitor become no longer tolerant to the action of the inhibitor and consequently cannot continue to proliferate and/or have cytotoxicity on target cells. Therefore, stopping the use of inhibitors or increasing the concentration of inhibitors can be used as a molecular switch to control the cytotoxicity of universal CAR-T cells in vivo.
As illustrated in the Examples below, in the case where dasatinib was used as an inhibitor, it can be administered to suppress or weaken the function of the patient's immune system at such a dose that the concentration of dasatinib in the patient is not lower than 10 nM, or not lower than nM, or not lower than 30 nM, or not lower than 50 nM, or not lower than 100 nM. In the case where ponatinib was used as an inhibitor, it can be administered at such a dose that the concentration of ponatinib in the patient is not lower than 100 nM, or not lower than 200 nM, or not lower than 300 nM.
The universal CAR-T cells provided herein can also be used in combination with other cancer therapeutic agents, that is, the patient is administered with other cancer therapeutic agents during, before or after administration of the inhibitor and the universal CAR-T cells to the patient. These other cancer therapeutic agents may include chemotherapy drugs, radiotherapy drugs, other biological therapeutic agents such as antibody therapeutic agents or cell therapeutic agents.
“Patient” as used herein refers to a subject to be treated and may include any mammal such as cats, dogs, sheep, cows, mice, rats, rabbits, humans, non-human primates, and the like. In addition, a patient can be any individual who already has a disease, is at risk of developing a disease, or has been treated for it. Correspondingly, the preparation method of the universal CAR-T and treatment method provided herein can be used in humans and non-human mammals. For example, the safety and effectiveness of the universal CAR-T cells provided herein can be first verified in an animal model, and then used in human clinical research and treatment.
In a specific embodiment, we have designed the following universal CAR-T treatment strategy (in the following, dasatinib is used as the T cell activity inhibitor as an example): (1) dasatinib and universal CAR-T are used in combination, the activity of host T cells and NK cells is inhibited by dasatinib to prevent them from killing the universal CAR-T; (2) since dasatinib can also inhibit the activity of the universal CAR-T, the universal CAR-T is designed and engineered to make it tolerant to dasatinib; (3) the engineering of the universal CAR-T is carried out using the CRISPR-Cas9-related cytosine base editor (CBE3) to make a point mutation on its Lck gene, resulting in T316I mutation of the LCK protein; and (4) the mutated UCAR-T T316I cells can show tolerance to dasatinib. Therefore, with the treatment with dasatinib, the host T cells and NK cells are inhibited and will not clear allogeneic cells, while UCAR-T can perform normal tumor cytotoxicity and expand normally; (5) In addition to editing Lck Genes aside, it still needs to edit the TRAC gene of CAR-T cells to avoid GvHD.
In the case where dasatinib effectively inhibits the activity of host T cells, the strategy mechanism of universal CAR-T is shown in
As shown in
In the case where dasatinib effectively inhibits host NK cells, the strategy mechanism of universal CAR-T is shown in
As in
Function of LCK in T Cell Signal Transduction
LCK is a member of the Src family of kinases. It generally binds to the intracellular region of the CD4 and CD8 co-receptor molecule of T cells and mediates the phosphorylation of the intracellular segment ITAMS of the T cell receptor molecule. Therefore, LCK is the most upstream tyrosine protein kinase in TCR signal transduction, mediating the transmission of the first signal after T cells receive antigen stimulation.
As shown in
Inhibition of T Cell Function by Dasatinib
Inhibition of T cell function by dasatinib has been an accepted event so far. Two documents in 2008 respectively elaborated on how dasatinib inhibits T cell function [1,2]. Stephen Blake et al. reported that dasatinib, as an inhibitor of Src/ABL kinase, can effectively inhibit many functions of normal human T lymphocytes in vitro, including that dasatinib can effectively block the transduction of TCR signal by binding to LCK, inhibit the activation, cytokine secretion and proliferation of T cells in vitro, but will not affect the viability of T cells [1]. Andrew E. Schade et al. reported similar experimental results and believed that dasatinib inhibits TCR-mediated signal transduction, cell proliferation, cytokine secretion, and cellular responses in vivo by inhibiting LCK phosphorylation. But this inhibition is reversible, when dasatinib is withdrawn, the function of T cells can be restored [2].
Inhibition of CAR-T Cell Function by Dasatinib
Since dasatinib can generally inhibit the phosphorylation of LCK to inhibit the function of TCR, will the signal transduction of CAR-T be inhibited by dasatinib? Two documents in 2019 respectively elaborated on how dasatinib inhibits CAR-T cell function [3,4]. Evan W. Weber et al. reported that dasatinib is a potential, rapid and reversible inhibitor of CAR-T cell function, which can inhibit the proliferation, cytokine secretion and tumoricidal activity of CAR-T cells in vivo [3]. Katrin Mestermann et al. reported that dasatinib can bind to LCK and inhibit the phosphorylation of CD3z and ZAP70, and therefore, dasatinib can inhibit the activation of CD28_CD3z or 4-1BB_CD3z in the molecular structure of CAR to inhibit the function of CAR molecules. Also, dasatinib will induce the functional resting state of CD8+ and CD4+ CAR-T, which can last for several days without affecting the viability of T cells. The data show that dasatinib can inhibit the secretion of cytokines and proliferation of CAR-T cells in vivo and in vitro, and this inhibition is reversible [4].
Inhibition of NK Cell Function by Dasatinib
In addition to its inhibitory effect on T cells and CAR-T cells, dasatinib can also inhibit the degranulation and cytokine release of primary human NK cells [5].
Relationship Between LCK Mutation and Tolerance to Dasatinib
It has been documented that the LCK-T316M mutation can show tolerance to dasatinib [6], the principle of which is that the T316 site is a key gatekeeper amino acid of the LCK kinase, and the T316M mutation greatly changes the structure of LCK. As shown in
As a small molecule inhibitor targeting ABL kinase, dasatinib has many analogues, such as imatinib and nilotinib. Among them, the most studied is the relationship between ABL mutation and tolerance to imatinib. The most common ABL mutations that can cause tolerance to imatinib are T315I, T315A, etc. [7,8,9]. We found that the activation domains are very similar between ABL kinase and LCK kinase, and as shown in
Design Protocol to Induce LCK Mutations on T Cells
CRISPR-Cas9, as one of the commonly used gene editing tools, can specifically and efficiently cleave the genome as mediated by single-stranded targeting RNA. Therefore, CRISPR-Cas9-related gene editing is very suitable for the induction of the LCK-T316 point mutation. We have designed the following two sets of CRISPR-related technical solutions, which can realize the mutation of the LCK-T316 site on T cells.
A Strategy for Inducing LCK-T316M/I/a Mutations Based on CRISPR-Cas9-Mediated Homologous Recombination Repair
CRISPR-Cas9-mediated homologous recombination repair is a very effective technical means to achieve base/amino acid point mutations. Its principle is that after RNP is formed from Cas9 protein and sgRNA, it can be targeted by the sgRNA to a specific position in the genome, and cleaved at the first 3-4 nt position of the PAM sequence to induce double-stranded DNA to break. At this time, the introduction of homologous recombination carrying the LCK-T316 site mutation can enable the cells to repair with the template sequence, so as to obtain cells with endogenous LCK-T316 mutation. The template for this homologous recombination can be double-stranded DNA or single-stranded DNA. In the point mutation induction system, the efficiency of homologous recombination mediated by single-stranded DNA template is the highest.
Therefore, we initially designed 16 sgRNAs suitable for the Cas9 system with a length of 100 bp near the LCK-T316 site. As shown in
The off-target situation is shown in
The sequence of LCK-T316I protein is set forth in SEQ ID NO: 9, the sequence of LCK-T316A protein is set forth in SEQ ID NO: 10, and the sequence of LCK-T316M protein is set forth in SEQ ID NO: 11.
Another method to induce LCK-T316M/I/A mutations by CRISPR-Cas9-mediated homologous recombination is by cleavage with two sgRNAs plus repair with a single-stranded ssDNA template. Induction of the T316I/M/A mutation can also be achieved by combining sgRNA1 (SEQ ID NO: 14) with sgRNA11 (SEQ ID NO: 15), or sgRNA1 with sgRNA12 (SEQ ID NO: 18). Among them, the T316I/M/A mutant ssDNA template sequences required for combination of sgRNA1 and sgRNA11 are set forth in SEQ ID NOs: 28-30, respectively, and the T316I/M/A mutation ssDNA template sequences required for combination of sgRNA and sgRNA12 are set forth in SEQ ID NOs: 31-33, respectively.
Strategy to Induce LCK-T316I Mutation by CRISPR-Cas9-Based Cytosine Base Editor CBE3
Another tool capable of inducing LCK-T316 mutation is cytosine base editor based on CRISPR-Cas9 technique. The principle of CBE3 (cytosine base editor 3) is the fusion expression of Cas9n protein and APOBEC-3A, a protein that induces cytosine deamination (A3A-CBE3) to induce cytosine deamination to mutate when Cas9n cleaves the single strand of the target site. Cas9n makes mutation D10A in the RuvC1 domain of the Cas9 protein, making it a Cas9-nickase protein (Cas9n), which only retains the enzymatic activity of the HNH domain. Cas9n does not cause DNA double-strand break, but can only cleave DNA single strands complementary to sgRNA on the genome, thereby inducing base mismatch repair (BER). At this time, APOBEC-3A can induce the deamination of cytosine on the other DNA single strand to form uracil, and finally promote the mutation of cytosine to thymine (C->T mutation) in the presence of UGI protein.
The inventor cloned the A3A-CBE3 gene of the Escherichia coli expression system, purified and expressed the functional A3A-CBE3 protein through Escherichia coli, the protein sequence of which is set forth in SEQ ID NO: 34.
As shown in
Compared with the Cas9-mediated homologous recombination protocol, the advantages of this system are: (1) since the Cas9n protein is used in the CBE3 system, it will not cleave the double-stranded DNA of the genome, so the risk due to sgRNA off-target in this system is extremely low; (2) no template ssDNA is required, and the RNP formed by direct electroporation of CBE3 protein and sgRNA can realize gene editing; (3) the most important thing is that although the current CBE3 base editing is not yet accurate enough to achieve the site-specific targeting of a single base within the sgRNA coverage, near the LCK-T316 site, the mutation of any cytosine covered by the sgRNA except the T316 site will not cause amino acid mutations, that is, the CBE3 strategy only results in single amino acid mutation T316I.
This strategy still has two issues to be verified:
Based on this, as shown in Table 1, we optimized the lengths of sg12 and sg16, and designed a total of 7 sgRNAs for subsequent mutation induction and functional verification.
7) as the sgRNA for LCK-T316I mutation, but did not verify it by experiments. It is theoretically deduced that the sgRNA of 19 nt in length has functions similar or identical to those of 18 nt, 20 nt and 21 nt.
In addition, the inventors also designed two suitable sa-sgRNAs for saCas9-induced base editing, namely LCK-saCas9-sgRNA1 and sgRNA2, whose sequences are set forth in SEQ ID NOs: 12 and 13.
Functional Validation of sgRNA for Induction of LCK-T316I Mutation
sgRNA16 can Efficiently Induce LCK-T316I Mutation
We isolated CD3-positive T cells from cryopreserved PBMCs, activated the cells with CD3/CD28 DynaBeads for 48 hrs and then used them in electroporation of the RNP complex of CBE3 protein and sgRNA. T cell genomes were extracted 96 h after editing, and detected for LCK gene editing efficiency by PCR and Sanger sequencing.
As specifically described in the Examples below, for the T cells in the group electroporated with only CBE3 protein, no base mutation occurs in the Lck gene region of its genome, while all the RNPs formed by LCK-sgRNA16 and CBE3 proteins of different lengths can cause T316I mutation with a mutation efficiency of about 50%. Meanwhile, the first four cytosines at the T316 site also undergo C->T mutations, but none of them causes amino acid changes. Therefore, in T cells, these three LCK-sgRNA16 with different lengths can all induce LCK-T316I mutation efficiently. In order to reduce the impact of sgRNA off-target on editing, we prefer to use 21 nt sgRNA16 for subsequent experiments.
sgRNA12 can not Induce the LCK-T316I Mutation
Similar to the above gene editing experiment, we also tested the induction efficiency of sgRNA12 on T cell LCK-T316 mutation. Compared with the control group CBE3, LCK-sgRNA12 of different lengths basically did not edit the cytosine at the T316 site, and induced about 50% of C->T mutations on the four cytosines before the T316 site, but still failed to cause any changes in amino acids. Therefore, LCK-sgRNA12 cannot effectively induce LCK-T316I mutation, but it can be used as a negative control for sgRNA16, so we chose 21 nt LCK-sgRNA16 as a control for subsequent experiments.
Validation of the Relationship Between LCK-T316I Mutation and Inhibition of T/CAR-T Signal Activation by Dasatinib
Verification of the Functions of Dasatinib by Preliminary Experiments to Determine that Dasatinib can Efficiently Inhibit the Activation of T Cell TCR and the Translocation of CD107a in NK Cells
In order to verify the inhibition of TCR signal activation of T cells by dasatinib, we pre-treated T cells with dasatinib or DMSO for 5 hr, then activated T cells with CD3/CD28 DynaBeads for 24 hr, and then detected the expression of activation molecules CD25, CD69 and 4-1BB by flow cytometry. The results show that in the DMSO control group, after 24 hr of magnetic bead activation, the activation molecule CD25 was significantly up-regulated, and the CD69-positive or 4-1BB-positive cells were significantly grouped. However, after treatment with 100 nM/1000 nM dasatinib, there was no CD69/4-1BB-positive activated cell population after activation with CD3/CD28, suggesting that 100 nM dasatinib can effectively inhibit the activation of TCR signaling.
Next, in order to verify the inhibition of NK cell activation by dasatinib, NK cells isolated from human PBMCs were cultured and expanded in vitro. The cells were treated with different concentrations of dasatinib for 24 h, one group was not activated, and the other group was stimulated with target cells K562 for NK activation for 5 hr before CD107a detection, and then the CD107a translocation of NK cells was uniformly detected. The results show that for the NK cells in the non-activated group, after treatment with dasatinib, the basal CD107a translocation was also reduced to the baseline of 2%. After activation by adding K562 target cells, the CD107a translocation in the untreated group was 64.5%, while that in the treated group was reduced to the baseline of 2%, suggesting that 100 nM dasatinib is sufficient to inhibit the translocation of CD107a in NK cells and inhibit the activation of NK cells.
LCK-T316I can Make T Cells Tolerant to the Inhibition of TCR Activation by Dasatinib
In order to verify the tolerance of the LCK-T316I mutation to the function of dasatinib, we performed the following experiments on LCK-edited T cells. Three different treatments were set for each group of edited cells and MockT cells: in the first group, dasatinib was not added, and the cells were not activated with CD3/CD28 magnetic beads; in the second group, dasatinib was not added, but the cells underwent TCR activation with CD3/CD28 magnetic beads for 24 hr; in the third group, the cells were pretreated with 100 nM dasatinib for 5 hr, and then CD3/CD28 magnetic beads were added for TCR activation for 24 hr. The effect of gene editing on the inhibition of TCR activation by dasatinib was observed.
The results show that in the control group, the percentage of CD25/CD69 positive cells in the group with 100 nM dasatinib was significantly lower than that in the activation group, indicating that dasatinib inhibits the activation of TCR by CD3/CD28. Meanwhile, in the sg16 editing group, there was still a high percentage of CD25/CD69 positive cells in the group with dasatinib, and the same result could be obtained when 4-1BB was used as the activation marker. Therefore, a part of cells after sg16 editing could still be activated to become CD69/4-1BB positive after dasatinib treatment, which proves that they can tolerate the inhibition of T cell activation by dasatinib.
We further statistically analyzed the percentages of CD25, CD69, and 4-1BB single-positive cells in the control group, sg12 editing group, and sg16 editing group, and found that in the sg16 editing group, with the treatment with dasatinib and CD3/CD28 magnetic beads, the expression percentages of the three activation markers were significantly higher than those in the control group and the sg12 editing group. It is further suggested that T cells in the sg16 editing group (˜50% LCK-T316I), which show tolerance to dasatinib, can be activated by CD3/CD28.
LCK-T316I Makes Anti-CD19-CAR-T Cells Tolerant to the Inhibition of CD107a Translocation by Dasatinib
In order to verify that the LCK-T316I mutation can not only make T cells tolerant to the inhibition of TCR activation by dasatinib, but also make CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib during CAR activation, we designed the following experiment. First, we edited anti-CD19 CAR-T using CBE3 protein, CBE3+sg12 and CBE3+sg16. After 72 h of stabilization after editing, we pre-treated each group of CAR-T cells with 100 nM dasatinib for 12 hr, followed by stimulation with different target cells to activate CAR signals and mediate the release of CD107a in the target cells killed by CAR-T. 5 hr before detection, monensin and anti-CD107a antibody were added, and staining for CD8 and CAR positive was performed 5 hr later to detect the enrichment of CD107a translocation of CD8 positive CAR positive cells.
The sequence of the anti-CD19-CAR molecule used here is the same as that used in the subsequent examples, and the specific sequence is set forth in SEQ ID NOs: 35-56. The overall structure of this CAR molecule is a second-generation CAR molecule, which consists of anti-CD19 scFv (SEQ ID NOs: 35-38), plus a linker region (SEQ ID NOs: 39-40), plus a CD8a hinge region (SEQ ID NOs: 41-42), plus a CD8a transmembrane domain (SEQ ID NOs: 43-44), plus a CD28 intracellular domain (SEQ ID NOs: 45-46), plus a CD3z intracellular signaling transduction domain (SEQ ID NOs: 47-48), plus a T2A sequence (SEQ ID NOs: 49-50), plus a CSF2RA signal (SEQ ID NOs: 51-52), plus a tEGFR signal (SEQ ID NOs: 53-54). The total sequence of the final anti-CD19-CD28z-CAR is set forth in SEQ ID NOs: 55-56.
The results show that regardless of whether anti-CD19-CAR-T cells were edited or not, in the DMSO control group, the negative target cell K562 could not activate CAR-T for effective CD107a translocation, while the positive target cell Nalm6 could fully activate CAR-T to release CD107a with a positive rate of above 70%. With the 100 nM dasatinib treatment, the negative control group K562 hardly had any CD107a translocation enrichment, while under the positive target cell Nalm6 stimulation, only the sg16-LCK-T316I editing group showed 50% positive CD107a translocation, suggesting that the LCK-T316I mutation can indeed make anti-CD19-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib, and this tolerance has significant differences.
LCK-T316I Makes Anti-BCMA-CAR-T Cells Tolerant to the Inhibition of CD107a Translocation by Dasatinib
In order to verify that in addition to anti-CD19 CAR-T, LCK-T316I mutation can make other CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib, we performed three groups of editing of LCK on anti-BCMA CAR-T, i.e. CBE3, CBE3+LCK-sg12 and CBE3+LCK-sg16, and the experimental procedure is exactly the same as that described above. There were three groups of target cells used to stimulate anti-BCMA CAR-T, namely K562 negative target cells, U266-B1 positive target cells and RPMI-8226 positive target cells. The results show that in the DMSO-treated control group, the three groups of edited anti-BCMA CAR-T all had higher levels of CD107a translocation under the stimulation with positive target cells, while with the treatment with 100 nM dasatinib, only the LCK-T316I mutation group (CBE3+sg16 group) showed tolerance to dasatinib, with 39.6% and 44.7% positive CD107a release upon activation with the two positive target cells, respectively. Meanwhile, there was a significant difference in this degree of CD107a release compared to other edited controls, suggesting that the LCK-T316I mutation can make anti-BCMA-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib.
LCK-T316I Makes Anti-CD19/BCMA Dual-Target UCAR-T Cells Tolerant to the Inhibition of CD107a Translocation by Dasatinib
In addition, basically consistent with the above experiment, we further verified that the LCK-T316I mutation makes the CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib in anti-CD19 and BCMA dual-target UCAR-T cells (with TRAC knockout). The results show that under normal circumstances, the negative target cell K562 basically do not activate dual UCAR-T, and the two positive target cells U266 and Raji can activate dual UCAR-T. With the treatment with 100 nM dasatinib, only the sg16 editing group could show tolerance to the inhibition of the translocation of CD107a by dasatinib under target cell activation, which is completely consistent with the previous results.
Validation of the Relationship Between LCK-T316I Mutation and Inhibition of CAR-T Cytotoxicity by Dasatinib
LCK-T316I Makes Anti-BCMA CAR-T Cells Tolerant to the Inhibition of CAR-T Cytotoxicity by Dasatinib
As mentioned above, the mutation of LCK-T316I can make T cells tolerant to the inhibition of TCR activation by dasatinib, and can also make CAR-T cells tolerant to the inhibition of CAR activation and CD107a translocation by dasatinib. We wanted to further explore whether this tolerance can be reflected in the function of T cells, so we designed the following experiments to explore the relationship between the LCK-T316I mutation and the inhibition of CAR-T cytotoxicity by dasatinib.
We performed three different editing treatments on anti-BCMA CAR-T: CBE3 protein, CBE3+sg12 and CBE3+sg16. After the editing was stabilized for 72 hr, we pretreated each group of CAR-T cells with 100 nM dasatinib for 12 hr. Next, the cells were co-incubated with different luciferase-positive target cells (luciferase+) at an effector-to-target ratio of 1:1 for 24 hr. After the incubation, the cytotoxicity of CAR-T cells on target cells was evaluated by detecting the activity of luciferase. The lower the luciferase value, the fewer surviving target cells and the stronger the cytotoxicity of CAR-T. The results show that in the DMSO control group, anti-BCMA CAR-T could reduce the luciferase value of RPMI-8226 positive target cells, but did not affect the luciferase value of negative target cells Nalm6, proving the normal cytotoxicity of anti-BCMA CAR-T. With the treatment with 100 nM dasatinib and under the stimulation with positive target cells RPMI-8226, the luciferase value in the CBE3 and CBE3+sg12-LCK unediting groups was significantly higher than that in the DMSO group, proving that with the treatment with dasatinib, the cytotoxicity of anti-BCMA CAR-T was significantly inhibited. The luciferase value in the CBE+sg16-LCK editing group was significantly lower than that in the CBE3 and CBE3+sg12-LCK unediting groups, proving that the anti-BCMA CAR-T after the LCK-T316I mutation developed tolerance to inhibition of CART cytotoxicity by dasatinib.
In addition, we repeatedly edited another batch of anti-BCMA CAR-T cells, and obtained results consistent with the above experiment. In this replicate, we used two other negative target cells, K562-luciferase and Raji-luciferase. Luciferase results show that in the DMSO control group, all the edited anti-BCMA CAR-T cells would not kill K562 and Raji negative target cells, but would target RPMI-8226 positive target cells, with their luciferase value basically decreased to the baseline, suggesting that anti-BCMA CAR-T has normal specific cytotoxicity. Subsequently, with 100 nM dasatinib treatment, high luciferase values could still be detected in the CBE3 and CBE3+sg12 groups in the positive target cell groups, suggesting that the cytotoxicity of CAR-T was still inhibited. The luciferase value of CBE3+sg16-LCK-T316I editing group decreased significantly, which once again demonstrates the tolerance of LCK-T316I mutation to the inhibition of CAR-T cytotoxicity by dasatinib.
The CAR molecule sequence of the anti-BCMA-CAR-T cells used here is the same as that used in the subsequent examples, and the specific sequence is set forth in SEQ ID NOs: 77-78.
Validation of the Relationship Between LCK-T316I Mutation and Inhibition of T/CAR-T Cell Proliferation by Dasatinib
LCK-T316I Makes Anti-BCMA CAR-T Cells Tolerant to the Inhibition of Cell Proliferation by Dasatinib
Since LCK-T316I can produce tolerance to the functions of inhibiting TCR/CAR activation and cytotoxicity of CAR-T cell of dasatinib, we would like to further explore the inhibition of T/CAR-T proliferation in vitro by dasatinib reported in the literature to see whether it can be resisted in CAR-T cells with LCK-T316I mutation. Therefore, we designed the following experiments to verify CAR-T proliferation in vitro. Firstly, the anti-BCMA CAR-T cells were edited with CBE3, CBE3+sg12 and CBE3+sg16. After the editing was stabilized for 72 hr, the target cells U266 were added at an effector-to-target ratio of 10:1 for stimulation every 3 days, and at the same time the total number of cells and CAR positive rate were detected once every three days. During the experiment, the cells were divided into two groups treated with DMSO and 100 nM dasatinib. As shown in
Preliminary Brief Data Summary
We have found a method for site-directed mutation of LCK-T316I, that is, cytosine base editor CBE3 in combination with LCK-sgRNA16 to achieve efficient T cell LCK-T316I mutation through electroporation;
In addition, our further research found that:
Ponatinib at a concentration of 200 nM can effectively inhibit the activation of NK cells in vitro;
Finally, by LCK-T316I editing of the universal CAR-T, combined with dasatinib/ponatinib treatment, the activation and cytotoxicity of host T and NK cells are inhibited, so that the UCAR-T with LCKT316I could not be cleared by the immune system of the host, and the cells have normal tumor-killing function, and can effectively expand and function in the body.
The present invention will be further described below through specific examples.
Methods
1.1 Sorting and Activation of CD3+ T Cells
Frozen healthy donor PBMCs were resuscitated, totally 1.0×108 cells per tube, thawed quickly and resuspended in 8 mL of preheated Rinsing buffer. A small amount of cell suspension was taken for cell counting. The PBMC suspension was centrifuged at 400 g for 10 min, with an increasing speed setting of 8 and a decreasing speed setting of 8 (hereinafter referred to as ↑8 ↓8). After centrifugation, the supernatant was discarded, and 20 ul/107 CD3 microbeads were added. The system was mixed well, and then incubated in a 4° C. refrigerator for 20 min, during which the tube wall was flicked several times every 10 min to avoid cell sedimentation. After the incubation, the cells were rinsed with Rinsing buffer once, then centrifuged (400 g 10 min ↑8 ↓8), and then resuspended with 500 μl of Rinsing buffer. At the same time, an LS sorting column was placed on a Miltenyi magnetic sorting rack, rinsed with 2 ml of Rinsing buffer once, to which 500 μl of cell suspension was added. After the cell suspension was added dropwise, 2 ml of Rinsing buffer was added twice onto the LS column. The cells of interest were rinsed out from the LS column with 5 mL of Rinsing buffer and collected, and counted after appropriate dilution. About 1×105 cells were taken and the purity of the sorted T cells was determined by flow cytometry. Then, the cell suspension was centrifuged at 300 g for 10 min, the cell density was adjusted to 1×106 with fresh T cell culture medium, and anti-CD3/-CD28 antibody magnetic beads were added at a concentration of 10 ul/106 cells for activation. The cells were seeded into a 12-well plate at 4 mL/well, and cultured in a 37° C., CO 2 incubator.
1.2 T Cell CAR-Positive Lentiviral Transduction
CD3-positive T cells were activated with anti-CD3/CD28 magnetic beads for 24-48 hr and then underwent viral transduction. Viability detection and cell counting were performed on the cell suspension. The cells were centrifuged at 300 g for 10 min and collected. The cell density was adjusted to 1×106/mL, lentivirus was added at a volume of 1 mL per well in a 24-well plate, the MOI was adjusted to 3, and 800 ng/uL PolyBrene and 1 ug/uL DEAE were added to assist in the transduction. After mixing well, the cells were further cultured in a 37° C. incubator. After 24 hr, the virus was removed by centrifugation at 300 g for 10 min, and the T cells were cultured.
1.3 T Cell Gene Knockout
Taking TRAC and CD5 knockout as an example: T cells were activated with anti-CD3/anti-CD28 antibody magnetic beads for 24 hr, then the magnetic beads were removed, the cells were counted and the cell viability was determined. Several portions of cell suspensions, 2×106/portion, were taken and placed in centrifuge tubes and centrifuged at 100 g for 10 min After centrifugation, the culture medium was completely removed and the cells were resuspended in Lonza electroporation buffer. At the same time, for each portion, RNP complexes of 35 pmol Cas9 protein+75 pmol TRAC-sgRNA; 35 pmol Cas9 protein+60 pmol CD5-sgRNA were prepared and incubated for 15 min at room temperature. Then the cell suspension and RNP complex were well mixed and added to the Lonza 16-well electroporation cuvette, and placed in the 4D-Nucleofector™ X unit for electroporation with the EH-115 program. To the electroporation cuvette, pre-culture at 80 ul/well was added to incubate at 37° C. for 15 min, then the cells were transferred to a 48-well plate for further culturing with 500 uL of T cell complete medium.
1.4 LCK-T316I gene editing (introduction of Km editing) T cells were taken, put as per 2×106/cell suspension in a centrifuge tube for each group, and centrifuged at 100 g for 10 min After centrifugation, the culture medium was completely removed and the cells were resuspended in Lonza electroporation buffer. At the same time, RNP complexes (per poriton) were prepared for the above groups: the RNP complexes of {circle around (1)} 60 pmol CBE3 protein; {circle around (2)} 60 pmol CBE3 protein+100 pmol K-sgRNA12; {circle around (3)} 60 pmol CBE3 protein+100 pmol K-sgRNA16 were respectively incubated at room temperature 15 min. Then the cell suspension and RNP complex were well mixed and added to the Lonza 16-well electroporation cuvette, and placed in the 4D-Nucleofector™ X unit for electroporation with the EH-115 program. To the electroporation cuvette, 80 ul of pre-culture was added to incubate at 37° C. for 15 min, then the cells were transferred to a 48-well plate for further culturing with 500 uL of T cell complete medium.
1.5 Detection of Gene Knockout Efficiency
Taking TRAC and CD5 knockout as an example: 72 hr after T cells were electroporated to knock out TRAC and CD5 genes, the efficiency of TRAC and CD5 gene knockout was detected by flow cytometry. The specific steps are as follows: about 2×105 cells were taken and put in a 1.5 mL centrifuge tube, washed twice with PBS+2% fetal bovine serum buffer. The supernatant was completely discarded. The cells were resuspended with 100 μL of buffer, 1 μL of each of PE-anti-human-TRAC and APC-anti-human-CD5 antibodies were added, the cells were mixed well, incubated at 4° C. in the dark for 30 min, washed once with buffer, and tested on the machine.
1.6 CAR Positive Rate Detection
The CAR positive rate was detected by flow cytometry 5 days after infection of T cells by lentivirus. The specific steps are as follows: Two portions of about 1×105 cells were taken, and placed in a 1.5 mL centrifuge tube, washed twice with PBS+2% fetal bovine serum buffer, and the supernatant was completely discarded. For one portion, the cells were resuspended with 100 μl of buffer, and 5 μl of FITC-labeled-human-target antigen protein was added. For another portion, the cells were resuspended with 100 μl of buffer, and 1 μL of APC-anti-human-EGFR (transduction marker) antibody was added (if there is EGFR expression). The cells were mixed well and incubated at 4° C. in the dark for 30 min, washed once with buffer, and then tested on the machine.
1.7 Detection of Editing Efficiency of LCK-T316I Gene Editing
The T316I mutation rate was detected 72 hr after Km editing of T cells or CAR-T cells. About 1×105 cells in each group were taken for genomic DNA extraction, and the genomic DNA was used as a template to amplify a DNA fragment covering the Km region and including about 200 bp upstream and downstream sequences by PCR. The base sequence map was obtained by Sanger sequencing. The .ab1 format file of Sanger sequencing in the EditR web version was uploaded and the 20 bp sgRNA sequence were input, DataQC and Predict Editing were clicked to view the analysis results, and the results were downloaded through Download Report.
The main reagent materials used are listed in Table 2.
Example 2: using single base editor A3A-CBE3 to efficiently induce human LCK mutation (Km for short) in T cells
The inventors used the single-base cytosine editor A3A-CBE3[10] based on CRISPR-Cas9 technique to induce T316I mutation in the human LCK protein, that is, the position 316 amino acid of the human LCK protein was mutated from threonine (T) to Isoleucine (I), referred to as Km for fort.
The A3A-CBE3 protein is a nickase fusion protein of APOBEC3A and Cas9-D10A mutations, which can be targeted to a specific position in the genome by a sgRNA of a specific sequence, and mediates the mutation of cytosine to thymine within the targeting range of the sgRNA, thereby realizing base editing and protein mutation. Cas9-D10A makes the D10A mutation on the RuvC1 domain of the Cas9 protein, making it a Cas9-nickase protein (Cas9n), which only retains the enzymatic activity of the HNH domain, so that Cas9n will not cause DNA double-strand to break, but can only cleave the DNA single strand complementary to sgRNA on the genome, thereby inducing base mismatch repair (BER). At this time, APOBEC3 can induce the deamination of cytosine on the other DNA single strand to form uracil, and finally promote the mutation of cytosine to thymine (C->T mutation) in the presence of UGI protein.
The inventors designed the sgRNA for inducing LCKT316I mutation.
As shown in
The inventors verified the function and efficiency of sgRNA on the induction of LCKT316I mutation. LCK-sgRNA16 can efficiently induce LCK-T316I mutation, while LCK-sgRNA12 cannot induce LCK-T316I mutation.
The inventors isolated CD3-positive T cells from frozen human PBMCs, and the sorting positive rate was over 95%. The sorted CD3 positive T cells were stimulated and activated with anti-CD3/CD28 DynaBeads magnetic beads for 48 hr and then used for mutation induction. After the A3A-CBE3 protein and LCK-sgRNA16 of different lengths were co-incubated to form RNP complexes in vitro, the activated T cells were electroporated by using LONZA Nucleofector-4D electroporation instrument and P3 Primary Cell 4D-Nucleofector™ X Kit with electroporation program of EH-115. After electroporation, the culture was continued for 96 hr, the T cell genome was extracted, and the efficiency of lck gene editing was detected by PCR and Sanger sequencing.
As shown in
At the same time, the four cytosines before the T316 site also underwent C->T mutation, but since these four cytosines are all in the third degenerate codon position of the amino acid, the main mutations except the mutated cytosine at the T316 site did not result in amino acid changes.
As shown in
The inventors verified that LCK-sgRNA12 can not induce LCK-T316I mutation.
Similar to the above gene editing experiment, the inventors also tested the induction efficiency of LCK-sgRNA12 on T cell LCK-T316 mutation. As shown in
Dasatinib, as an inhibitor of Src/ABL kinase, can effectively inhibit many functions of normal human T lymphocytes in vitro, including: dasatinib can effectively block the transduction of TCR signal by binding to LCK, inhibit the activation, cytokine secretion and proliferation of T cells in vitro, but will not affect the viability of T cells [1]. It is known that dasatinib inhibits TCR-mediated signal transduction, cell proliferation, cytokine secretion and cellular response in vivo by inhibiting LCK phosphorylation. But this inhibition is reversible, and when dasatinib is withdrawn, the function of T cells can be restored [2]. Meanwhile, dasatinib is also a potential, rapid and reversible inhibitor of CAR-T cell function, which can inhibit the proliferation, cytokine secretion and in vivo tumor killing activity of CAR-T cells [3], can inhibit the secretion of cytokines and proliferation of CAR-T cells in vivo and in vitro, and this inhibition is reversible [4].
In order to verify that the LCKT316I mutation can make universal CART cells resistant to the inhibition of T cell activation by dasatinib, the inventors first repeated that dasatinib can effectively inhibit TCR signals to inhibit T cell activation.
The inventors pre-treated T cells with dasatinib or DMSO for 5 hr, then activated T cells with anti-CD3/CD28 DynaBeads for 24 hr, and then detected the expression of activation molecules CD25, CD69 and 4-1BB by flow cytometry. As shown in
In order to verify the tolerance of the LCK-T316I mutation to the function of dasatinib, the inventors carried out experimental verification on the T cells with LCK edited with different sgRNA lengths prepared in Example 2. Three groups of different treatments were set for each group of electroporated T cells and MockT cells without electroporation: as shown in
In this example, the T cells were edited and grouped as follows: the MockT group was T cells without electroporation, the 01CBE3 group was T cells electroporated with only CBE3 protein; groups 02-05 were T cells electroporated with RNPs formed by CBE3 protein and LCK-sgRNA12 of different lengths, which can be used as negative controls for LCK editing because sgRNA will not cause any amino acid mutation of LCK protein; groups 06-08 were T cells electroporated with RNPs formed by CBE3 protein and LCK-sgRNA16 of different lengths, these T cells had LCK-T316I mutation with mutation efficiency of about 50%. The data are shown in
The inventors proved that LCK-T316I mutation can make T cells tolerant to the inhibition of TCR activation by dasatinib.
As shown in
The inventors further statistically analyzed the data, set groups MockT and 01-CBE3 as blank control groups, set groups 02-05 as sgRNA12 editing groups (nonsense mutation groups), and set groups 06-08 as sgRNA16 editing groups (LCK-T316I mutation groups), and statistically analyzed the percentages of positive cells expressing CD25, CD69 and 4-1BB under the three treatment conditions, respectively. As shown in
In addition, however, compared with the cells in the sgRNA16 group directly activated by anti-CD3/CD28 magnetic beads without dasatinib treatment, the expression of activation molecules in the dasatinib treatment group was significantly reduced (about 20%-50%,
Based on the fact that LCK-T316I mutation can make T cells tolerant to the inhibition of TCR activation by dasatinib, which had been verified, in order to verify that LCK-T316I mutation can not only make T cells tolerant to the inhibition of TCR activation by dasatinib, but also make CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib during CAR activation, the inventors designed and carried out the following experiments to prove that LCK-T316I mutation makes anti-CD19-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib.
First, the inventors edited the prepared anti-CD19 CAR-T by electroporation with the CBE3 protein, the RNP complex of CBE3 and LCK-sgRNA12, and the RNP complex of CBE3 and LCK-sgRNA16 respectively, to achieve three different editing treatments. 72 hr after editing, the mutation efficiency was detected, and the LCK-T316I mutation reached about 43% (
By analyzing the data of flow cytometry, in the gating strategy shown in
The inventors performed statistical analysis on the two replicates of the above experiment. According to the results shown in
In Example 5, the inventors had demonstrated that LCK-T316I mutation makes anti-CD19-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib. In order to strengthen this conclusion, in this example, the inventors verified that it can still be observed in BCMA-targeted CAR-T that LCK-T316I mutation makes BCMA-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib.
First, the inventors edited the prepared anti-BCMA CAR-T by electroporation with CBE3 protein, the RNP complex of CBE3 and LCK-sgRNA12, and the RNP complex of CBE3 and LCK-sgRNA16 respectively, to achieve three different editing treatments. 72 hr after editing, the mutation efficiency was detected, and the LCK-T316I mutation reached about 42% (
By analyzing the data of flow cytometry, the CD107a translocation enrichment was analyzed in CD8 and CAR double-positive cells. The results are shown in
The inventors performed statistical analysis on the two replicates of the above experiment. According to the results shown in
In Example 5 and Example 6, the inventors had demonstrated that LCK-T316I mutation makes anti-CD19-CAR-T cells and anti-BCMA-CAR-T cells tolerant to the inhibition of CD107a translocation by dasatinib. In order to strengthen this conclusion, in this example, the inventors further verified that it can still be observed in CD19 and BCMA bispecific CAR-T cells that LCK-T316I mutation resists the inhibition of CD107a translocation by dasatinib.
The inventors edited the prepared CD19/BCMA dual-target CAR-T by electroporation with the CBE3 protein, the RNP complexes (18 nt and 21 nt) of CBE3 and LCK-sgRNA12, and the RNP complexes (18 nt and 21 nt) of CBE3 and LCK-sgRNA16 respectively, to achieve three different groups of editing treatments. The inventors pretreated the CAR-T cells in each group for 12 hr with 100 nM dasatinib, and then co-incubated the anti-BCMA-CAR-T cells with different target cells to verify the activation of CAR signaling by different target cells and the release of CD107a. These target cells include U266B1 (BCMA positive), Raji (CD19 positive), K562 (negative control), allogeneic T cells (negative control) and blank Buffer control (negative control). 5 hr before detection, monensin and anti-CD107a antibodies were added, and staining for CD8 and CAR positive was performed 5 h later to detect the enrichment of CD107a translocation of CD8 positive CAR positive cells.
The results are shown in
The above Example 4 demonstrates that the mutation LCK-T316I can make T cells tolerant to the inhibition of TCR activation by dasatinib, and Examples 5-7 demonstrate that the mutation LCK-T316I can make CAR-T cells tolerant to the inhibition of CAR activation and CD107a translocation by dasatinib. In order to further prove that the LCK-T316I mutation can make CAR-T cells resist the inhibition of the cytotoxicity of CAR-T by dasatinib, the inventors carried out the following experiments on anti-BCMA CAR-T cells.
The inventor's experiment proved that the anti-BCMA CAR-T after LCK-T316I mutation becomes tolerant to the inhibitory function of dasatinib on CAR-T cytotoxicity.
The inventors conducted experiments using the edited anti-BCMA CAR-T cells in Example 6, wherein the LCK-T316I mutation efficiency was 42% (
The results are shown in
The inventor's repeated experiments proved that the anti-BCMA CAR-T after LCK-T316I mutation becomes tolerant to the inhibitory function of dasatinib on CART cytotoxicity.
The inventors performed repeated killing experiments on the above-mentioned LCK-edited and non-edited anti-BCMA CAR-T cells. The experimental procedure was consistent with
The results are shown in
In the above examples, the inventors proved that dasatinib at a concentration of 100 nM can effectively inhibit the activation of TCR and CAR signals, that is, at this concentration, the activation of normal T cells and CAR-T cells is inhibited. At this point, the LCK-T316I mutation can make T cells tolerant to the inhibition of its activation by dasatinib, that is, the UCAR-T cells with the LCK-T316I mutation can retain the ability of being activated by and killing target cells in the environment of 100 nM dasatinib.
Based on this, the strategy proposed in the present invention to realize universal CAR-T is to use dasatinib to pretreat patients to inhibit the activation function of host T cells, and then infuse back the UCAR-T cells with LCK-T316I mutation. In this case, the host T cells are unable to clear UCAR-T cells due to the inhibition of TCR activation by dasatinib; at the same time, UCAR-T cells are able to resist the inhibition of CAR signal activation by dasatinib due to the LCK-T316I mutation, and target malignant cells and mediate tumor clearance.
The inventors have demonstrated in Example 3 that 100 nM dasatinib is sufficient to inhibit the activation of TCR signals in T cells. In order to further explore the concentration threshold of dasatinib to inhibit T cell activation and guide clinical medication, the inventors further explored lower concentrations of dasatinib.
The inventors isolated CD3-positive T cells from human peripheral blood and divided them into eleven portions for processing, with 1e5 cells in each portion. One of these portions was not treated with dasatinib, anti-CD3/CD28 DynaBeads were not added to activate TCR, and this portion was as the initial control group; the other 10 portions of cells were pretreated with different concentrations of dasatinib for 4 hr, ten concentrations ranging from 0 nM to 50 nM were set, and then anti-CD3/CD28 DynaBeads were added to activate TCR signals overnight. On the second day, the expression of CD25, CD69 and 4-1BB activation signals was detected, and the activation of TCR signals was detected. Two replicates were set for each group of experiments.
As shown in
Meanwhile, as shown in
The above data show that dasatinib at a concentration of 25 nM can effectively inhibit the activation function of T cells in vitro. It can basically guide the minimum in vivo concentration of dasatinib required to inhibit TCR signal activation in clinical medication (25 nM). It is speculated that when the concentration of dasatinib in vivo is above 25 nM, it can inhibit the activation of host T cells.
In Example 9, the inventors proved that dasatinib at a concentration of 25 nM can effectively inhibit the activation function of T cells in vitro, so as to guide the concentration used in vivo. According to the existing pharmacokinetic data of dasatinib [12], the inventors found that compared with other tyrosine kinase inhibitors, the half-life of dasatinib in vivo is extremely short, 3-4 hr. Meanwhile, for patients with CML and ALL in different stages, the dosage of dasatinib is 100 mg or 140 mg per day, or twice a day with halved dose. Therefore, the metabolism of dasatinib in vivo is relatively rapid, and it is very likely that there will be several hours in the interval of administration when its concentration is lower than 25 nM. In order to fully consider fluctuations in the concentration of dasatinib in vivo, the inventors designed a dynamic concentration experiment of dasatinib in vitro to detect whether its inhibition on T cell activation is still effective with dynamic concentration changes.
In this example, CD5-UCAR-T cells with double knockout of CD5 and TRAC were used, and edited by CBE3+LCK-sgRNA16 or CBE3+sgRNA12 at the same time to prepare UCAR-T cells with LCK-T316I mutation and control UCAR-T cells. Subsequently, they were treated overnight with dasatinib at a concentration of 50 nM or 25 nM, respectively. The next day, the concentration of dasatinib was reduced, as shown in
The results show that the sg12 control group CD5-UCART cells with LCK nonsense mutation were pretreated with 50 nM dasatinib, then the concentration was reduced to 0 nM, and the cells were activated by Jurkat target cells for 4 hr to produce 12% positive CD107a release (
Consistent with the experimental results of 50 nM dasatinib pretreatment, the inventors proved that after 25 nM dasatinib pretreatment, adjusting its concentration to 10 nM is enough to inhibit the CD5-UCAR-T cells in the control group from being activated by the target cells and releasing CD107a (
The above experiment proved that the inhibition of T cell activation function by dasatinib has an extended effect. Dasatinib can inhibit the activation function of T cells with the dynamic concentration changes in vitro from 50 nM to 15 nM or from 25 nM to 10 nM, and LCK-T316I mutation can resist this inhibition.
In order to repeatedly verify that the inhibition of T cell activation function by dasatinib has an extended effect, the inventors repeated the above experiment. CD5-CAR-T cells were pretreated with 50 nM or 25 nM dasatinib overnight, the drug concentration was adjusted and decreased the next day, the cells were co-incubated with positive or negative target cells, and blocker monensin and anti-CD107a antibody were also added. After 4 hr of co-incubation, CD107a release from CD8/CAR double-positive cells was detected.
As shown in
As shown in
Summarizing the above two experiments, it is basically proved that the inhibition of T cell activation function by dasatinib has an extended effect. Dasatinib can inhibit the activation function of T cells with the dynamic concentration changes in vitro from 50 nM to 10-15 nM or from 25 nM to 10 nM, and LCK-T316I mutation can resist this inhibition.
The sequence of the anti-CD5-CAR molecule used here is the same as that used in the following examples, and it is a CAR molecule related to the single domain antibody sequence. See SEQ ID NOs: 57-76 for the specific sequence. The overall structure of this CAR is a second-generation CAR molecule, which consists of two anti-CD5 single-domain heavy chains (SEQ ID NOs: 57-60), plus a linker region (SEQ ID NOs: 61-62), plus a CD8a region (SEQ ID NOs: 63-64), plus a CD28 intracellular domain (SEQ ID NOs: 65-66), plus a CD3z intracellular signaling domain (SEQ ID NOs: 67-68), plus a T2A sequence (SEQ ID NOs: 69-70), plus a CSF2RA signal (SEQ ID NOs: 71-72), plus a tEGFR signal (SEQ ID NOs: 73-74). The total sequence of the final anti-CD19-CD28z-CAR signal is set forth in SEQ ID NOs: 75-76.
In addition to its inhibitory effect on the activation of T cells and CAR-T cells, dasatinib can also inhibit the degranulation and cytokine release of primary human NK cells [5]. Being cleared by functional host T and NK cells is the main reason why UCAR-T is difficult to expand in the host. Therefore, in the present invention, the treatment with dasatinib in the UCAR-T therapy can enhance the survival of UCAR-T cells in the host not only by inhibiting the activation of host T cells but also by inhibiting the activation of host NK cells.
The inventors first verified that dasatinib can efficiently inhibit the activation of NK cells.
The inventor used Miltenyi human NK sorting magnetic beads to separate NK cells from human peripheral blood, and used DAKEWE NK medium to culture and expand them in vitro. On Day 8, a population of NK cells with more than 95% having positive CD56 expression could be obtained. These NK cells were taken out and treated with 0 nM, 100 nM and 200 nM dasatinib respectively for 24 hr, and then divided into two groups, one of which was not activated, and the other group was stimulated with MHC-class I molecule-deficient K562 cells for 5 hr. Then, the CD107a release of NK cells were detected in both of them.
As shown in
In the above examples, the inventors proved that dasatinib at a concentration of 100 nM can effectively inhibit the activation of NK cells. In order to further explore the concentration threshold of dasatinib to inhibit NK cell activation and guide clinical medication, the inventors further explored lower concentrations of dasatinib.
The inventors isolated NK cells from human PBMCs, and then cultured and differentiated them in NK medium to obtain CD56-positive NK cells with a purity higher than 95%. The exploration of the threshold of NK activation with dasatinib was carried out with seven concentrations in the range of 0-50 nM, namely 0, 10, 20, 25, 30, 40 and 50 nM, and the treatment was carried out overnight. The next day, the cells were resuspended with NK medium and activated with K562. Lymphocyte activator was used as a positive control for activation, and a part of NK cells that were not activated by K562 were also kept. Two replicates were set for each group. anti-CD107a antibody and monensin were added while carrying out activation, and after co-incubation for 6 hr, the CD107a release of CD56-positive NK cells was detected by flow cytometry.
As shown in
The above data can basically guide the minimum in vivo concentration of dasatinib required to inhibit NK cells in clinical medication (30 nM). It is speculated that when the concentration of dasatinib in vivo is above 30 nM, it can inhibit the activation of host NK cells.
In Example 12, the inventors proved that dasatinib at a concentration of 30 nM can effectively inhibit the activation function of NK cells in vitro, so as to guide the concentration used in vivo. As mentioned in Example 10, compared with other tyrosine kinase inhibitors, the half-life of dasatinib in the body is extremely short, its metabolism in the body is fast, and it is very likely that there will be several hours in the interval of administration when its concentration will be lower than 30 nM [12]. In order to fully consider the fluctuations in the concentration of dasatinib in vivo, the inventors designed a dynamic concentration experiment of dasatinib in vitro to detect whether its inhibition of NK cell activation is still effective with dynamic concentration changes.
The inventors isolated NK cells from human PBMCs, and then cultured and differentiated them in NK medium to obtain CD56-positive NK cells with a purity higher than 95%. Three pretreatments were done respectively (1) absence of dasatinib pretreatment; (2) pretreatment with nM dasatinib; (3) pretreatment with 50 nM dasatinib, and the cells were cultured overnight. The next day, the concentration of dasatinib was reduced, and the concentration was adjusted to 0, 7.5, 12.5, 17.5 and 25 nM respectively. At the same time, K562 cells were used as positive target cells to activate NK cells, the cells were co-incubated at an effector-to-target ratio of 1:1, and CD107a and monensin were added at the same time. Two replicates were set for each group. After 5 hr of co-incubation, CD107a release in CD56 positive NK cells was detected.
As shown in
As shown in
The above experiment proved that the inhibition of NK cell activation function by dasatinib has an extended effect. Dasatinib can inhibit the activation function of NK cells with the dynamic concentration changes in vitro from 25 nM or 50 nM pretreatment to a reduced concentration of 7.5 nM.
In order to repeatedly verify that the inhibition of NK cell activation function by dasatinib has an extended effect, the inventors repeated the above experiment. NK cells were pretreated with 50 nM, 25 nM and 10 nM dasatinib overnight, the drug concentration was adjusted and decreased the next day, the cells were co-incubated with positive K562 or negative Buffer, and blocker monensin and anti-CD107a antibody were also added. After 4 hr of co-incubation, CD107a release from CD56 positive NK cells was detected.
As shown in
As shown in
Summarizing the above two experiments, it is basically proved that the inhibition of NK cell activation function by dasatinib has an extended effect. Dasatinib can inhibit the activation function of NK cells with the dynamic concentration changes in vitro as the concentration was reduced from 50 nM to 10 nM or from 25 nM to 10 nM.
In-vitro mixed lymphocyte reaction was carried out using enough CD5-UCART and BCMA-UCART prepared, so as to simulate whether the combination of LCK-T316I-UCART (hereinafter referred to as Km-UCART) and dasatinib can obtain living space and even proliferation advantages in the host T environment.
The present invention relates to the preparation of universal CAR-T cells by knocking out the TRAC gene. Given that the CD5 antigen is expressed on chronic B lymphocytic leukemia cells, the universal CAR-T cells involved in the following embodiments target CD5. However, it is known that all peripheral blood T cells and a very small part of B cells also express CD5, and UCART cells targeting CD5 will recognize and kill autologous or allogeneic T cells. Therefore, the present invention uses gene editing technology to simultaneously knock out the CD5 gene and the TRAC gene in T cells to prepare CD5-UCART cells.
CD5-UCART cells were prepared from healthy donors in a small number. In the cell samples prepared in this batch, the inventors measured by flow cytometry that the knockout efficiency of TRAC gene was >90%; the knockout efficiency of CD5 gene was >99%; and the T cells expressing CAR were about 35% as determined by detecting the EGFR transduction marker. The inventor carried out further gene editing on the successfully constructed CD5-UCART cells that can be used for in vitro testing. This gene editing used the CBE3 base editor to cause the T316I mutation at the LCK site on the CD5-UCART cells: the CBE3 group was CD5-UCAT cell control group; the sg12 group was the CD5-UCAT cell control group with nonsense mutation of LCK; the sg16 group (Km group) was the CD5-UCAT cell experimental group with LCK-T316I edited.
The present invention is intended to carry out LCK-T316I on UCART cells to construct UCART tolerant to the inhibition of dasatinib, then in the host T cell environment where dasatinib exists, the host T cells have their cytotoxicity (almost) completely inhibited as they do not receive genetic engineering for tolerating the inhibitory effect of dasatinib. At this time, the UCART in the present invention can get rid of the immune rejection originally mediated by the host T cells and maintain a normal proliferation rate.
The inventors used a mixed lymphocyte reaction (MLR) model to evaluate the ability of Km-CD5-UCART cells to resist immune rejection mediated by T cells in vitro and to clear allogeneic T cells (host T cells are also the target cells of CD5-UCART cells because of the absence of endogenous CD5 gene knockout).
The method comprises the steps of:
The effector cells in this mixed lymphocyte reaction were divided into 3 groups: (1) the CBE3 group was CD5-UCAT cell control group; (2) the sg12 group was the control group of CD5-UCAT cells with nonsense mutation of LCK; (3) the sg16 group (Km group) was the experimental group of CD5-UCAT cells with LCKT316I edited. Only allogeneic T cells from the same donor source were used as the target cells in this mixed lymphocyte reaction. Before the start of the MLR assay, dasatinib was added to the complete medium of T cells, that is, the simulated host T cells (target cells) and CD5-UCART cells (effector cells) were pretreated with dasatinib for 12 hr.
This example was carried out at a 1:5 effector-to-target ratio. What needs to be explained here is that the inventors define the effector-to-target ratio as the ratio of the absolute number of CD5-UCART cells expressing CAR molecules (the CAR positive rate in this example is 35%) to the absolute number of simulated host T cells. The 3 groups of effector cells were separately plated in two wells in a flat-bottomed 96-well plate at 8.09×104, 5.20×104, and 1.26×104 cells (100 μl volume), and the target cells were plated at 1.42×105, 9.10×4, 2.20×104 cells (100 μL volume) respectively and mixed with the effector cells, the total volume in each well was 200 μL, and all were cultured in T cell complete medium. At present, there were 3 groups of mixed wells of effector cells and target cells, two wells each, one of which was used as the experimental well to which 100 nM Dasatinib was added, and the other well was the control well to which the same volume of DMSO was added (the solvent for dasatinib in this example was DMSO). The plate was placed in a 37° C. incubator to culture.
In this test, the inventors predict that the effector cells in the sg16 group could exhibit a growth advantage over other groups in the dasatinib treatment groups. Therefore, the inventors need to collect the following data during the mixed lymphocyte reaction: cell count at different time points; percentage and absolute number of effector cells and target cells at different time points; CAR positive rate of effector cells.
In order to obtain the above data, the inventors took part of the cells from each well on day 2 and day 10 of the co-culture (the day of plating was defined as day 0) for cell counting. It needs to determine the percentage of the cells taken out relative to the overall cells in the well, and the absolute number of cells in each well is calculated by using this percentage and the counting result of the cells taken out. The cells taken out also need to be used for flow cytometric analysis to determine the percentages of effector cells and target cells and the CAR positive rate of effector cells.
The inventor combined the results of cell counting and flow cytometry analysis at each time point to calculate the indicators of interest.
As for the changes of host-T in the overall system, as shown in
Meanwhile, as shown in
This example is a supplement and optimization of Example 14. With the more extreme amount of allogeneic T, it aims to evaluate the ability of Km-CD5-UCART cells to resist T cell-mediated immunological rejection in vitro and the ability to clear allogeneic T cells through the mixed lymphocyte reaction (MLR) model.
The method comprises the steps of:
The effector cells in this mixed lymphocyte reaction were divided into 2 groups: (1) the Ctrl group was the control group of CD5-UCAT cells with nonsense mutation of LCK; (2) the Km group was the experimental group of CD5-UCAT cells with LCKT316I editing. Only homologous host T cells from the same donor source were used as the target cells in this mixed lymphocyte reaction. Before the start of the MLR assay, dasatinib was added to the complete medium of T cells, that is, the simulated host T cells (target cells) and CD5-UCART cells (effector cells) were pretreated with dasatinib for 24 hr.
This example was planned to be carried out with an effector-to-target ratio of 1:50-100. The 2 groups of effector cells were separately plated in 6 wells in a flat-bottomed 96-well plate at 2.5×105 (250 μl volume), and the target cells were plated at 1.5×106 cells (250 μL volume) respectively and mixed with the effector cells, the total volume in each well was 500 μL, and all were cultured in T cell complete medium. There were currently 2 groups of mixed wells of effector cells and target cells, 6 wells for each, and 3 treatments were given respectively, and 2 technical replicates were set for each treatment: treatment 1 was DMSO, treatment 2 was 25 nM dasatinib, treatment 3 was 50 nM dasatinib. The plate was placed in a 37° C. incubator to culture. Dasatinib and DMSO in the culture system were supplemented every two days.
In this test, the inventor predicted that the effector cells in the Km group could show a growth advantage over other groups in the dasatinib treatment groups, and based on the data obtained in the above examples, the inventor speculated that the Km group treated with 50 nM dasatinib has more growth advantages than the Km group treated with 25 nM dasatinib, that is, the growth advantage obtained by Km-CD5-UCART under dasatinib treatment was dose-dependent on dasatinib. To verify the above hypothesis, the inventors needed to collect the following data during the mixed lymphocyte reaction: cell count at different time points; percentage and absolute number of effector cells and target cells at different time points; CAR positive rate of effector cells.
In order to obtain the above data, the inventors took part of the cells from each well on day 2, 5, 8, 11 and 13 of the co-culture (the day of plating was defined as day 0) for cell counting. It needs to determine the percentage of the cells taken out relative to the overall cells in the well, and the absolute number of cells in each well is calculated by using this percentage and the counting result of the cells taken out. The cells taken out also need to be used for flow cytometric analysis to determine the percentages of effector cells and target cells and the CAR positive rate of effector cells.
In this mixed lymphocyte reaction, the inventor combined the results of cell counting and flow cytometry analysis at each time point to calculate the indicators of interest. The actual number of plated cells on day 0 is obtained by cell counting and flow cytometry after plating, not by the amount of cells to be added in the experimental design. Because the error caused by adding samples will cause the actual plated volume to be inconsistent with the planned plated cell volume, and secondly, because technical repetitions are set, counting the actual plated volume makes it easier to perform biostatistical analysis on the results. The actual CAR positive rate of effector cells was about 10% during the test, and the actual effector-to-target ratio obtained by combining the results of cell counting and flow cytometry was 1:50-1:100. After the test, according to the indicators obtained at each time point, the inventors carried out exhaustive statistical analysis on the dynamic changes in the number of effector cells (
As shown in
In addition, from the analysis of the percentage of effector cells relative to the overall cells, as shown in
Regarding the dynamic changes of host T cells, as shown in
The inventors believed that in this test, the real-time effector-to-target ratio is helpful to reflect the dynamic relationship between the effector components and the simulated host T cells in the system. As shown in
In addition, by further analyzing the genome sequences of each group of cells in the mixed lymphocyte reaction on D11, the inventors amplified the LCK-T316 region of the remaining cells on D11 in the entire reaction, and analyzed the enrichment level of LCK-T316I mutation of the remaining cells by Sanger sequencing. The results are shown in
In summary, the inventors confirmed that in order to prevent UCART from immune rejection mediated by host T cells, it is feasible to combine Km editing with dasatinib.
The previous examples basically clarify the strategy and principle of the invention. There are two core inventions: (1) special treatment with drugs: the inventors introduce the tyrosine kinase inhibitor dasatinib into the universal CAR-T pretreatment in that dasatinib inhibits the activation of host T and NK cells, enabling the universal CAR-T to survive in the host cell environment; (2) introduction of LCK-T316I mutation: the inventors use the single base editing technology A3A-CBE3 system to introduce LCK-T316I (Km) mutation into universal CAR-T cells, making them tolerant to inhibition of CAR signaling by dasatinib. Finally, with dasatinib treatment, Km-edited UCAR-T can be normally activated to target and kill tumor cells, and its clearance by host T and NK cells is also avoided.
The tyrosine kinase inhibitor (TKI) used in the present invention is not only dasatinib. The inventors found that ponatinib can also be used as a special treatment drug to realize the universal CAR-T strategy in the present invention.
In the study of ponatinib, the inventors first used the Miltenyi CD3-positive magnetic bead sorting kit to obtain CD3-positive T cells from donor PBMCs, and identified the purity and efficiency of the sorted CD3-positive cells by a flow cytometer. Then, the sorted CD3-positive T cells were divided into three groups, namely the control group electroporated with CBE3 protein (CBE3), the nonsense mutation control group electroporated with the complex of CBE3 protein and sgRNA12 (Sg12), and the LCK-T316I mutation group electroporated with the complex of CBE3 and sgRNA16 (Sg16-Km). The inventors used the Lonza electroporation instrument to carry out single-base editing on the LCK gene. The editing efficiency is shown in
The experimental procedure is shown in
The results of flow cytometry (
In order to further determine the concentration range in which ponatinib inhibits T cell activation in vitro and the inhibition is tolerant for the T316I mutation, the inventors tested the inhibition of T cell activation with treatment with higher concentrations of ponatinib.
The inventors conducted research using two groups of T cells (CBE3 control group and Km edited group) in the above experiments. The experimental procedure is basically the same as the above-mentioned experiment (
The results of flow cytometry (
In addition, with the treatment with 500 nM ponatinib, the activation of T cells in the CBE3 control group was completely inhibited, and only 2.42% of CD25 and 4-1BB double-positive cells and 6.75% of CD25 and CD69 double-positive cells were found in the Km edited group (
Based on the above, the inventors believed that 200 nM ponatinib can effectively inhibit the activation of T cells. For T cells with LCK-T316I mutation, the mutation can tolerate the inhibition of T cell activation by ponatinib at a concentration of 200 nM, but when the concentration of ponatinib rises to 500 nM, this tolerance is significantly reduced. Ponatinib at 500 nM can block TCR signal activation in Km-edited T cells. Therefore, ponatinib can be used as a pretreatment solution for the universal CAR-T strategy in the present invention, and the function of ponatinib at a concentration of 200 nM is consistent with that of dasatinib, i.e. inhibiting host T from killing UCAR-T while ensuring that LCK-T316I-UCAR-T cells still have normal functions, and can be activated and mediate the killing of targeted tumor cells. When the concentration of ponatinib increases to 500 nM, it can act as a molecular switch to inhibit the activation of LCK-T316I-UCAR-T cells.
In the above examples, the inventors proved that ponatinib at a concentration of 200 nM can effectively inhibit the activation of T cells. The inhibition of NK cell activation by ponatinib and the concentration threshold are further explored, to determine that ponatinib can effectively inhibit the activation of both T cells and NK cells and determine the concentration threshold.
The inventors used CD3-negative cells in human PBMCs, which contained more than 5% of CD56-positive NK cells. The inventors used treatments with ponatinib of four concentrations of 0, 50 nM, 200 nM and 500 nM respectively to study the effects of different concentrations on NK activation. The procedure is as follows: The cells were treated with different concentrations of ponatinib overnight. The next day, the cells were resuspended with NK medium and activated with K562. Lymphocyte activator was used as a positive control for activation, and a part of NK cells that were not activated by K562 were also kept. Two replicates were set for each group. anti-CD107a antibody and monensin were added while carrying out activation, and after co-incubation for 6 hr, the CD107a release of CD56-positive NK cells was detected by flow cytometry.
As shown in
Therefore, the above data further show that ponatinib can be used as the pretreatment protocol of the universal CAR-T strategy in the present invention, and ponatinib at a concentration of 200 nM can inhibit the activation of T and NK cells, thus avoiding the killing of UCAR-T by host T and NK cells. At the same time, the LCK-T316I mutation can make UCAR-T cells tolerant to the inhibition of T cell activation by ponatinib, and can be activated normally and mediate the killing of targeted tumor cells.
In Example 16, the inventors proved that ponatinib at a concentration of 200 nM can effectively inhibit the activation function of T cells in vitro, so as to guide the concentration used in vivo. Considering the drug metabolism of ponatinib in vivo, the inventors designed a dynamic concentration experiment of ponatinib in vitro to detect whether its inhibition of T cell activation is still effective with dynamic concentration changes.
In this example, the prepared wild-type anti-BCMA CAR-T cells were used for experiments. Treatment with ponatinib at a concentration of 0 nM, 200 nM or 500 nM was carried out, respectively, and the concentration was decreased the next day. In
The results are shown in
After pretreatment with 200 nM ponatinib, the concentration was adjusted to 0 nM the next day, and the percentage of CD107a positive cells was reduced from about 40% to about 16%, which proved that the inhibition of T cell activation by ponatinib has an extended effect (
After pretreatment with 500 nM ponatinib, the concentration was adjusted to 0 nM the next day, and the percentage of CD107a positive cells was reduced from about 40% to about 3%, which proved once again that the inhibition of T cell activation by ponatinib has an extended effect, and the higher the concentration of pretreatment, the more obvious the extended effect of inhibition (
As shown in
In Example 17, the inventors proved that ponatinib at a concentration of 200 nM can effectively inhibit the activation function of NK cells in vitro, so as to guide the concentration used in vivo. In order to fully consider the fluctuations in the concentration of ponatinib in vivo, the inventors designed a dynamic concentration experiment of ponatinib in vitro to detect whether its inhibition of NK cell activation is still effective with dynamic concentration changes.
The inventors isolated NK cells from human PBMCs, and then cultured and differentiated them in NK medium to obtain CD56-positive NK cells with a purity higher than 95%. Four pretreatments were done respectively: (1) absence of ponatinib pretreatment; (2) pretreatment with 50 nM ponatinib; (3) pretreatment with 100 nM ponatinib; (4) pretreatment with 200 nM ponatinib, and the cells were cultured overnight. The next day, the concentration of ponatinib was reduced, and the concentration was adjusted to 0, 50, 100 and 200 nM respectively. At the same time, K562 cells were used as positive target cells to activate NK cells, the cells were co-incubated at an effector-to-target ratio of 1:1, and CD107a and monensin were added at the same time. Two replicates were set for each group. After 5 hr of co-incubation, CD107a release in CD56 positive NK cells was detected.
As shown in
As shown in
In Example 16, the inventors proved that ponatinib at a concentration of 200 nM can effectively inhibit the activation function of T cells in vitro, and for T cells with LCK-T316I mutation, the mutation can tolerate the inhibition of T cell activation by ponatinib at the concentration of 200 nM, so as to guide the concentration used in vivo. When the concentration of ponatinib increases to 500 nM, ponatinib can block TCR signal activation in Km-edited T cells. Therefore, it can act as a molecular switch to inhibit the activation of LCK-T316I-UCAR-T cells. In Example 18, the inventors proved that with dynamic concentration changes, the inhibition of T cell activation by ponatinib has an extended effect, and the higher the drug concentration, the more obvious the extended effect. However, when the concentration of ponatinib increases to 500 nM, this tolerance is significantly reduced.
In order to verify that LCK-T316I mutation can not only make T cells tolerant to the inhibition of TCR activation by ponatinib, but also make CAR-T cells tolerant to the inhibition of CD107a translocation by ponatinib during CAR activation, and at the same time, fully considering the fluctuation of ponatinib concentration in vivo, the inventors designed and carried out the following experiments to prove that CD19-CAR-T cells with LCK-T316I mutation can tolerate the inhibition of CD107a translocation by ponatinib at a concentration of 200 nM. However, when the concentration of ponatinib increased to 500 nM, ponatinib can also block the CD107a translocation of CD19-CAR-T cells with LCK-T316I mutation. Therefore, the combination of LCKT316I mutation and ponatinib can be used as a switch for CD19-UCART therapy.
In this example, the inventors used the prepared CD19-UCART and Km-edited CD19-UCART cells in the experiments. Cells in both groups were treated with 200 nM or 500 nM ponatinib overnight (
As shown in
As shown in
To sum up, the inventors proved that CD19-UCART cells with LCK-T316I mutation are tolerant to the inhibition of CD107a translocation by ponatinib at a concentration of 200 nM during CAR activation. Moreover, with the dynamic concentration change, the ability of CD19-UCART cells with LCK-T316I mutation to tolerate the inhibition of CD107a translocation by ponatinib during CAR activation has an extended effect. However, pretreatment with ponatinib at a concentration of 500 nM basically completely inhibits the activation of CD19-UCART cells by target cells, while CD19-UCART cells with LCK-T316I mutation are basically unable to be activated by Raji target cells with pretreatment with ponatinib at a concentration of 300 nM. Therefore, the inventors believe that ponatinib at a concentration of 200 nM and LCK-T316I mutation can be combined for the activation of CD19-UCART, while ponatinib at a concentration of 500 nM can shut down the activation of CD19-UCART with LCK-T316I mutation.
Considering that it has been proved in the above examples that dasatinib can effectively inhibit the activation of T cells and NK cells, and CAR-T cells carrying LCK-T316I mutation can effectively resist the inhibition of the activation of CAR molecules by dasatinib, so as to normally target tumor cells for activation and killing. Moreover, the inventors have demonstrated in Examples 14 and 15 that the version of CD5-UCART with B2M not knocked out can effectively resist the killing by allogeneic T cells in the in-vitro mixed lymphocyte reaction with allogeneic T cells to gain the advantage of expansion. Therefore, in order to further verify whether the B2M-knockout version of UCART can effectively resist the clearance by NK cells and gain the advantage of expansion based on the combination with dasatinib, the inventors designed the following experiments.
The inventor isolated CD3-positive T cells from PBMCs, knocked out B2M and TRAC genes after activation, and infected them with anti-CD19 CAR molecule through lentivirus infection (see SEQ ID Nos: 35-56 for specific sequences). As shown in
Subsequently, the inventors used these prepared anti-CD19 UCART cells to conduct an in-vitro mixed lymphocyte reaction test. The inventors removed CD3-positive T cells from fresh PBMCs from donors different from UCAR-T, and kept CD3-removed PBMCs (mainly containing CD56-positive NK cells and CD19-positive B cells) to undergo mixed lymphoid reaction with UCART. The effector-to-target ratio (allogeneic cells:UCART) was set as four gradients of 20:1, 10:1, 5:1 and 2.5:1. There were two types of CD19 UCART cells tested: LCK edited group (Km) and LCK unedited group, which were respectively treated with DMSO or 50 nM dasatinib, cultured in NK medium, and 24 hr (D1), 48 h (D2) and 96 hr (D4) after co-incubation, cell counting and flow cytometry for detection of the percentage of each component were carried out, and the molecules detected by flow cytometry included CD19, CAR molecules, TRAC and CD56, etc.
The results are shown in
Similarly, as shown in
In addition, as shown in
Finally, as shown in
The method comprises the steps of:
The target cells in this mixed lymphocyte reaction were divided into 2 groups: (1) the Ctrl group was the control group of CD19-UCAT cells with same-sense mutation of LCK; (2) the Km group was the experimental group of CD19-UCAT cells with LCKT316I editing. Only homologous host PBMC cells from the same donor source were used as the effector cells in this mixed lymphocyte reaction.
This example was planned to be carried out with an effector-to-target ratio of 10:1. The 2 groups of target cells were separately plated in 6 wells in a flat-bottomed 48-well plate at 1.5×105 CAR positive cells (500 μl volume), and the effector cells were plated at 1.5×106 cells (500 μL volume) respectively and mixed with the effector cells, the total volume in each well was 1000 μL, and all were cultured in T cell complete medium. There were currently 2 groups of mixed wells of effector cells and target cells, 6 wells for each, and 2 treatments were given respectively, and 2 technical replicates were set for each treatment: treatment 1 was DMSO, treatment 2 was 50 nM dasatinib. The plate was placed in a 37° C. incubator to culture. Dasatinib and DMSO in the culture system were supplemented every two days.
In this test, the inventors predict that the effector cells in the Km group could exhibit a growth advantage over other groups in the dasatinib treatment groups. To verify the above hypothesis, the inventors needed to collect the following data during the mixed lymphocyte reaction: cell count at different time points; percentage and absolute number of effector cells and target cells at different time points; CAR positive rate of effector cells.
In order to obtain the above data, the inventors took part of the cells from each well on day 4, 9, 12, and 16 of the co-culture (the day of plating was defined as day 0) for cell counting. It needs to determine the percentage of the cells taken out relative to the overall cells in the well, and the absolute number of cells in each well is calculated by using this percentage and the counting result of the cells taken out. The cells taken out also need to be used for flow cytometric analysis to determine the percentages of effector cells and target cells and the CAR positive rate of effector cells.
In this mixed lymphocyte reaction, the inventor combined the results of cell counting and flow cytometry analysis at each time point to calculate the indicators of interest. The actual number of plated cells on day 0 is obtained by cell counting and flow cytometry after plating, not by the amount of cells to be added in the experimental design. Because the error caused by adding samples will cause the actual plated volume to be inconsistent with the planned plated cell volume, and secondly, because technical repetitions are set, counting the actual plated volume makes it easier to perform biostatistical analysis on the results. The actual CAR-positive rate of effector cells was about 18% (Ctrl group) and 25% (Km group) during the test. After the test, according to the indicators obtained at each time point, the inventors carried out exhaustive statistical analysis on the dynamic changes in the number of target cells (
As shown in
For the dynamic changes of host T cells and NK cells, as shown in
Considering that the UCAR-T cells with LCK-T316I mutation prepared in the above examples have different LCK-T316I mutation efficiencies, in order to maintain high-efficiency LCK-T316I mutation in the application process, the inventors optimized the preparation process of UCAR-T with LCKT316I mutation, and confirmed the preparation method that can ensure the high mutation rate of LCK-T316I.
The final preparation process we confirmed is as follows:
On Day 0, Miltenyi's Pan T Cell Isolation Kit, human kit was used to sort T cells from PBMCs; the T cells was cultured in a cytokine-free medium; after resting for 5 hr, a Celetrix electroporation instrument was used to perform resting electroporation, while Cas9 protein was used to knock out TRAC and B2M genes, and CBE3 protein was used to edit Km gene; 50 nM-100 nM dasatinib was immediately added for treatment after editing; and 2 hr later, Miltenyi's Human CD2/CD3/CD28 activator was used for T cell activation at 25 ul/ml by volume to stimulate cells for 2 days;
The inventors isolated CD3-positive T cells from PBMCs from three different donors using PanT kits, knocked out the B2M and TRAC genes, and performed LCK-T316I editing on the cells. Then the cells were divided into two portions, to one portion, 100 nM DS was added (experimental group), and to the other portion, a corresponding amount of DMSO was added as a control (control group). After 2 hr, to the experimental group and the control group, T cell activators (CD2/CD3/CD28, 25 ul/1M/mL) were added. After 48 hr of activation, the cells were infected with the anti-CD19 CAR molecule through lentivirus infection, and the medium was replaced 24 hr after the virus infection, and DS or DMSO was removed at the same time.
As shown in
A sufficient number of the prepared CD19-UCART containing different third signal structures and Raji cells treated with mitomycin-C underwent repeated antigen stimulation to simulate the process where UCART cells continuously killed target cells and continuously expanded in the environment of repeated contact between target cells and UCART cells in vivo.
The present invention uses gene editing technology to simultaneously knock out the TRAC gene and the B2M gene in T cells to prepare CD19-UCART cells.
CD19-UCART cells were prepared from healthy donors in a small number. In the cell samples prepared in this batch, the inventors measured by flow cytometry that the knockout efficiency of TRAC gene was >90%; and the expression of CAR in different UCART cells was about 10-50% as determined by detecting CD19 antigen marker.
The present invention intends to measure the proliferation and cytotoxicity characteristics of UCAR-T cells with different third signal elements through repeated stimulation of target cell antigens, so that the UCAR-T carrying third signal element LCKT316I mutation is preferred. A total of two experiments were carried out, which were distinguished as Experiment I and Experiment II.
The method comprises the steps of:
The flow chart of this example is shown in
This example was carried out with a 2:1 effector-to-target ratio. What needs to be explained here is that the inventors define the effector-to-target ratio as the ratio of the absolute number of CD19-UCART cells expressing CAR molecules to the absolute number of simulated host T cells. The effector cells with a total cell number of 2M in each group were plated in two wells of a flat-bottomed 12-well plate (1.5 ml volume), and the target cells were respectively plated with a cell number of (number of effector cells*CAR+ percentage/2) (1.5 ml volume) and mixed with effector cells, with a total volume of 3 ml per well and a culture density of effector cells of 0.67M/ml. The effector cells were fixed to a total cell number of 2M each time the target cells were replenished repeatedly. If the effector cells were less than 2M after the previous stimulation, all of them were used for the next plating.
When patients take immunosuppressants, a series of immune cells are suppressed and their ability to secrete cytokines is reduced. Perhaps, a certain type of pretreatment lymphodepletion drugs can also cause the decrease of macrophages in patients, resulting in poor secretion of cytokines, such as etoposide. In order to compare whether there is any difference in the expansion of various UCART cells containing the third signal in a cytokine-poor environment and a cytokine-rich environment, The inventors set the co-culture conditions into two groups, namely M1 and M2. Among them, M1 was CTS basal T cell medium without supplementing any cytokine, and M2 medium was CTS basal T cell medium+200 U/ml IL2. In addition, for each group of effector cells, a group without target cells was set up under the corresponding medium conditions to observe their basal proliferation.
In this test, the inventors predicted that the proliferation ability of UCART cells containing the third signal would be stronger than that of the control group, but it is necessary to confirm the UCART cells with the best expansion ability. Therefore, the inventors needed to collect the following data during the repeated antigen stimulation experiment: cell counts at different time points; the CAR positive rate of effector cells at different time points and the cytotoxicity of different effector cells after repeated antigen stimulation.
In order to obtain the above data, the inventors took part of the cells from each well on day 3, day 6, day 9, day 12 and day 15 of the co-culture (the day of plating was defined as day 0) for cell counting. It needs to determine the percentage of the cells taken out relative to the overall cells in the well, and the absolute number of cells in each well is calculated by using this percentage and the counting result of the cells taken out. The cells taken out also need to be used for flow cytometric analysis to determine the CAR positive rate of effector cells.
The inventor combined the results of cell counting and flow cytometry analysis at each time point to calculate the indicators of interest.
The previous experiments had proved that 2759 and 2661 had the advantage of CAR+ expansion after repeated antigen stimulation. In order to further verify whether the cells of 2759 and 2661 have the advantage of cytotoxicity after antigen stimulation, the inventors co-cultured the effector cells and Raji-luc cells after four rounds of repeated stimulation in Experiment I in the complete culture medium of T cells at an effector-to-target ratio of 1:1, and co-cultured the effector cells and Raji-luc cells after four rounds of repeated stimulation in the complete culture medium of T cells at an effector-to-target ratio of 2:1 and 0.5:1, and then detected the luminosity of luciferase at 24 hr and 48 hr, so as to judge the c of different effector cells on Raji cells. The inventors converted the luminosity of luciferase each time into the corresponding percentage of cytotoxic cells and displayed the cytotoxicity of different effector cells in
In order to further verify whether 2759 and 2661 cells have advantages in cytotoxicity after repeated antigen stimulation, in Experiment II, the inventors co-incubated UCART cells after four rounds of repeated antigen stimulation with different target cells (positive target cells Raji expressing CD19 antigen and negative target cells CCRF that do not express CD19 antigen) and different groups of CD19-CAR-T cells to stimulate the activation of CAR signals, mediate CAR-T to kill target cells and accumulation of CD107a in CAR-T. 5 hr before detection, monensin (1:1000) and anti-CD107a-PEcy7 antibody (1:100) were added, and staining for CD8 and CAR positive was performed 5 hr later to detect the enrichment of CD107a translocation of CD8 positive CAR positive cells. The statistical results are shown in
The aforementioned experiments have proved that 2759 and 2661 have CAR+ expansion advantages and cytotoxicity advantages after repeated antigen stimulation. In order to further verify whether the cells 2759 and 2661 have the advantage of cytokine secretion after antigen stimulation, the inventors collected the cell culture supernatants after four rounds of repeated antigen stimulation in the aforementioned experiments I and II, quickly stored them in a refrigerator at −80° C., and frozen and thawed the supernatants for cytokine detection within one week. Experiment I detected TNF-alpha, as shown in
The inventors used the human IL2 kit, human TNF-alpha kit, and human IFN-gamma kit from Cisbio to detect the cytokine supernatant after freezing and thawing. Because the supernatant concentration of IFN-gamma is generally too high, the inventors diluted the cytokine supernatant by 20 times and then tested for IFN-gamma. The detection process was operated in full accordance with the instruction manual.
It can be found that under the M1 and M2 culture environment, TNF-alpha and IFN-gamma of 2759 had obvious secretion advantages, followed by 2661. The IL2 secretion of 2661, 2759, and 2842 did not exceed that of the control group (1175). The IL2TNF-alpha and IFN-gamma secretion of 2758 was lower than that of the control group.
Based on the above experimental results, the inventors have proved that part of the third signaling structure can indeed promote the expansion ability of UCART cells, and make them still retain a considerable degree of cytotoxicity after massive expansion. The inventors finally selected 2661 and 2759 as the third signal elements that need to be carried by UCAR-T to further improve the LCKT316I mutation.
Some of the amino acid or nucleotide sequences mentioned herein are as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110280575.3 | Mar 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/081289 | 3/16/2022 | WO |
