Chimeric Antigen Receptors (CAR) and uses thereof
阅读说明：本技术 嵌合抗原受体（car）及其应用 (Chimeric Antigen Receptors (CAR) and uses thereof ) 是由 张克礼 雷林均 于 2021-11-19 设计创作，主要内容包括：本发明涉及嵌合抗原受体(CAR)及其应用。本发明提供了一种ROR1特异性嵌合抗原受体,包括信号肽、抗人ROR1 scFv、铰链区、跨膜区、4-1BB胞内域和CD3ζ胞内域,其中抗人ROR1 scFv能够和目标抗原高亲和力结合,防止脱靶效益的发生,并且其热稳定和pH稳定性好,能够耐受极端环境；进一步的,在嵌合抗原受体中引入Fc片段,可提高肿瘤杀伤效果,防止免疫因子风暴产生,提高治疗的安全性和有效性；本发明中所提供嵌合抗原受体T细胞能够显著抑制乳腺癌、卵巢癌、肺癌等实体肿瘤细胞或组织生长,为相关药物的开发和肿瘤免疫治疗提供新的方案。(The present invention relates to Chimeric Antigen Receptors (CARs) and uses thereof. The invention provides a ROR1 specific chimeric antigen receptor, which comprises a signal peptide, an anti-human ROR1 scFv, a hinge region, a transmembrane region, a 4-1BB intracellular domain and a CD3 zeta intracellular domain, wherein the anti-human ROR1 scFv can be combined with a target antigen with high affinity to prevent off-target benefit from occurring, and has good thermal stability and pH stability and can tolerate extreme environment; furthermore, the Fc fragment is introduced into the chimeric antigen receptor, so that the tumor killing effect can be improved, the generation of an immune factor storm can be prevented, and the safety and the effectiveness of treatment can be improved; the chimeric antigen receptor T cell provided by the invention can obviously inhibit the growth of solid tumor cells or tissues such as breast cancer, ovarian cancer, lung cancer and the like, and provides a new scheme for the development of related medicaments and tumor immunotherapy.)
1. A ROR 1-specific chimeric antigen receptor characterized by: the chimeric antigen receptor comprises a signal peptide, an anti-human ROR1 scFv, a hinge region, a transmembrane region, a 4-1BB endodomain, and a CD3 zeta endodomain, wherein the anti-human ROR1 scFv comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively.
2. The ROR 1-specific chimeric antigen receptor of claim 1, wherein: the amino acid sequence of the heavy chain variable region in the scFv of the anti-human ROR1 is shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region in the scFv of the anti-human ROR1 is shown as SEQ ID NO: 8.
3. The ROR 1-specific chimeric antigen receptor of claim 1, wherein: the chimeric antigen receptor also comprises a human Fc domain, wherein the human Fc domain is selected from IgG1, and the amino acid sequence of the human Fc domain is shown as SEQ ID NO. 9.
4. The ROR 1-specific chimeric antigen receptor of claim 1, wherein: the amino acid sequence of the signal peptide is shown as SEQ ID NO. 10; the amino acid sequence of the hinge region is shown as SEQ ID NO. 11; the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 12; the amino acid sequence of the 4-1BB intracellular domain is shown as SEQ ID NO 13; the amino acid sequence of the intracellular domain of CD3 zeta is shown in SEQ ID NO. 14.
5. Nucleotides encoding the ROR 1-specific chimeric antigen receptor of any one of claims 1-4.
6. A recombinant vector comprising the nucleotides of a ROR 1-specific chimeric antigen receptor of claim 5.
7. A host cell comprising the recombinant vector of claim 6.
8. Use of the ROR 1-specific chimeric antigen receptor of any one of claims 1-4 in the preparation of an anti-tumor medicament.
9. Use according to claim 8, characterized in that: the tumor includes solid tumor and blood tumor.
10. Use according to claim 8 or 9, characterized in that: the tumor comprises breast cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, head and neck cancer, nasopharyngeal carcinoma and melanoma.
Technical Field
The invention belongs to the field of tumor immunotherapy and the field of biotechnology, and particularly provides a Chimeric Antigen Receptor (CAR) and application thereof.
Background
Because tumors have difficult healing, high mortality and low clinical manifestations after healing, the tumors become one of the most serious diseases threatening human health, and researchers try various tumor treatment means and technologies in sequence, including surgical treatment, radiotherapy, chemical drug therapy, gene therapy and the like, but no effective treatment means is found at present. With the intensive research of biotechnology and immunology, tumor immunotherapy and antitumor antibodies are receiving attention in the industry, and since the last 80 th century, there have been hundreds of antibodies worldwide and approved for tumor therapy, and a new generation of immunotherapy technology represented by immune checkpoint inhibitors and chimeric antigen receptor T cells has also been receiving attention and used for treating various malignant tumors.
Receptor tyrosine kinase-like orphan receptor1 (ROR 1) is one of the Receptor Tyrosine Kinase (RTKs) family members and has high homology with the tyrosine kinase domain of growth factor receptors. The human ROR1 molecule consists of an extracellular region including an immunoglobulin-like domain (Ig-like), two cysteine-rich frizzled domains (CRD or FZD) and a kringle (kng) domain; the intracellular domain contains a Tyrosine Kinase Domain (TKD), two serine/threonine-rich domains (Ser/ThrD), and a proline-rich domain (PRD). A great deal of research shows that ROR1 plays a key role in promoting the growth and metastasis of tumors, inducing tumor cell resistance, inhibiting apoptosis and the like, especially low-level expression in ROR1 normal tissues, but high expression in various malignant tumors or tissues, such as Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), breast cancer, ovarian cancer, melanoma, lung adenocarcinoma and the like. ROR1 is highly recognized in tumor tissues, and ROR1 is a novel tumor-specific marker and anti-tumor target based on the characteristic.
Against the ROR1 target, a variety of tumor immunotherapies have been developed, including: (1) the monoclonal antibody is an antibody which is highly uniform and only aims at a certain specific epitope and is generated by single B cell clone, has high sensitivity, strong specificity, less cross reaction and low preparation cost, and is the most widely applied antibody medicament at present. Researchers have developed various monoclonal antibodies against ROR1 target, such as JP2021522162A, WO2021202863a1, EP3842072a1, CN112384533A, etc. disclose corresponding anti-ROR1 monoclonal antibodies; (2) the bispecific antibody contains 2 specific antigen binding sites, can bridge between target cells and functional molecules (cells), stimulates directed immune response, further enhances the targeting of antibody therapy, and EP2984107A1 discloses the bispecific antibody aiming at ROR1 and CD3 targets, and can effectively resist leukemia, diffuse large B cell lymphoma, myeloma, breast cancer, lung cancer and other tumors; (3) an antibody-drug conjugate (ADC) is prepared by connecting a small molecular drug with bioactivity to a monoclonal antibody through a chemical link, and transporting the small molecular drug to a target cell by targeting the monoclonal antibody as a carrier. ADC drugs directed against ROR1 have also been reported, e.g., Peyman et al (Peyman B, Mozafar M, Ali Hakakian. anti-ROR1 scFv-EndoG as a novel anti-caner therapeutic drug, APJCP, 2017, 19 (1): 97-102) binding anti-ROR1 ScFv to immunotoxin can rapidly cause tumor cell apoptosis; (4) in addition to antibodies having classical structures such as monoclonal antibodies and bispecific antibodies, antibody derivatives such as single chain fragment variable antibodies (scFv) and Fab antibody fragments have been developed against ROR1, and have therapeutic effects.
Chimeric Antigen Receptor (CAR) T cells are an emerging tumor immunotherapy technology in recent years, and the technology combines high specificity of antibody molecules with high anti-tumor activity of T cells, and exhibits good therapeutic effects on tumors, especially refractory tumors and high-recurrence tumors, and is therefore valued by researchers in the industry. This technology has now evolved into several generations, where the first generation CARs mainly comprise an exoantigen binding domain (such as ScFv fragment) and an intracellular signal domain (such as CD3 ζ), and such CARs and their CART cells are difficult to form highly activated T cells, limiting the exertion of tumor killing effects; the second generation CAR is based on the first generation CAR, and co-stimulatory factors (also called co-stimulatory factors) located in cells are introduced, so that T cells can be effectively promoted to be activated and the anti-tumor effect can be fully exerted, and a plurality of second generation CART cells are approved to be on the market at present, including Kymriah (Novartis), Yescata (Gliead), Tectatus (Gliead) and the like; third generation CARs introduce two costimulators to create more efficient T cell activation; fourth generation CARs are the introduction of secretable cytokine genes, such as IL-2, IFN- γ, etc., into T cells.
Although the CART technology is greatly developed, the existing CART cells mostly take CD19 as an anti-tumor target point, and the target point is mostly expressed in blood tumors such as lymphoma, leukemia, myeloma and the like, so that the CART cells also have good clinical curative effect. However, CD19 is also expressed in normal B cells, causing the targeted CART cells to attack normal cells, resulting in severe adverse events and even death of the patient. As mentioned above, ROR1 is much expressed in tumor cells, but is rarely expressed in normal cells, therefore ROR1 is considered to be one of the most suitable targets for constructing CART cells.
Therefore, researchers develop a plurality of CART cells taking ROR1 as targets, wherein, a plurality of chimeric antigen receptors with a classical second-generation CAR structure are used as targets, such as CN105924533A, WO2016115559A1, WO2016187216A1, WO2019090110A1, WO2020160050A1 and the like, the patent applications are mainly to screen and obtain a novel antibody structure aiming at the ROR1 target, and further to construct the chimeric antigen receptors, and the researchers also try to control the bioactivity of the CART cells in vivo and prevent serious side reactions by introducing a switch element, for example, CN107557337A discloses a caspase9 suicide gene safety switch, F36V-FKBP is dimerized under the action of AP1903 dimer, so that a homotypic signal pathway related to dimerization activation of caspase9 suicide gene is caused, and rapid death of target cells is induced; CN109837246A discloses knocking out PD-1 gene in CART cells, inhibiting the initiation of tumor escape mechanism.
The current CART therapy mainly takes CD19 as a target spot, the indications are mostly blood tumors, and the CART technology has poor curative effect in the treatment of solid tumors, so that a novel treatment technology which can play a role in inhibiting the solid tumors is urgently needed to be developed, CART cells with high specificity and strong tumor killing capacity to the solid tumors are screened and obtained, and meanwhile, the CART is also required to be prevented from activating the strong immune response to damage patients, the relevant adverse reactions are controlled within an acceptable range, and the life quality of the patients is improved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a ROR1 specific chimeric antigen receptor, which is characterized in that: the chimeric antigen receptor comprises a signal peptide, an anti-human ROR1 scFv, a hinge region, a transmembrane region, a 4-1BB endodomain, and a CD3 zeta endodomain, wherein the anti-human ROR1 scFv comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively.
Further, the amino acid sequence of the heavy chain variable region in the scFv of the anti-human ROR1 is shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region in the scFv of the anti-human ROR1 is shown as SEQ ID NO: 8.
The anti-human ROR1 scFv selected by the invention is derived from a novel antibody structure screened and obtained in a previous experiment by an applicant, has high affinity to a target antigen, also has good thermal stability and pH stability, can tolerate extreme environments in tumor tissues, has high inhibiting effect on solid tumors, and is suitable for developing drugs or immunotherapy aiming at the solid tumors.
Further, the chimeric antigen receptor also comprises a human Fc domain, wherein the human Fc domain is selected from IgG1, and the amino acid sequence of the human Fc domain is shown as SEQ ID NO. 9.
In the prior art, the Fc fragment is mostly used for combining with a target protein to form a fusion protein, thereby prolonging the in vivo half-life of related protein molecules and achieving the purpose of long-acting administration, such as combining the Fc fragment with GLP-2, IL-15 and the like. However, the invention surprisingly discovers that in the chimeric antigen receptor structure, after the Fc fragment is introduced, the binding activity of the antigen binding domain and a target antigen can be improved, and excessive secretion of certain inflammatory factors can be inhibited, so that serious adverse reactions can be prevented. The Fc segment comprises various types, such as IgG, IgM, IgA, IgD and the like, and the human IgG1 which is the most common human body and has the strongest physiological activity is selected as the binding segment in the invention, so that the rejection reaction is effectively reduced, and the action time of the CART cell is prolonged.
Further, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 10; the amino acid sequence of the hinge region is shown as SEQ ID NO. 11; the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 12; the amino acid sequence of the 4-1BB intracellular domain is shown as SEQ ID NO 13; the amino acid sequence of the intracellular domain of CD3 zeta is shown in SEQ ID NO. 14.
A nucleotide encoding a ROR 1-specific chimeric antigen receptor of the present invention is provided.
A recombinant vector is provided comprising the nucleotides of a ROR 1-specific chimeric antigen receptor of the present invention.
A host cell is provided, which is a recombinant vector described in the present invention.
Provides an application of ROR1 specific chimeric antigen receptor in preparing antitumor drugs.
Further, the tumor includes a solid tumor and a hematological tumor.
Further, the tumor comprises breast cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, head and neck cancer, nasopharyngeal cancer and melanoma.
Advantageous effects
The invention provides a novel ROR1 specific chimeric antigen receptor, which has a brand-new antigen binding domain, can be highly specifically bound with a target antigen, and effectively prevents off-target benefits. The Fc fragment is introduced into the chimeric antigen structure, so that the affinity and the targeting property with a target antigen can be improved, the occurrence of serious adverse reactions is inhibited, the occurrence of immune factor storm is prevented, and higher safety is provided. The chimeric antigen receptor T cell provided by the invention can inhibit various solid tumors, promote the secretion of cancer suppressor factors and activate the immune system in vivo. The chimeric antigen receptor T cell can obviously inhibit the growth of tumor in vivo and in vitro experiments, and plays a strong role in resisting tumor.
Drawings
FIG. 1: FIG. 1 is a diagram of a chimeric antigen receptor structure; FIG. 1A a second generation CAR structure; FIG. 1B is an Fc-CAR structure;
FIG. 2: killing effect of CART cells on solid cells;
FIG. 3: effect of CART cells on mouse model tumor volume;
FIG. 4: effect of CART cells on mouse SKOV3 model cytokines;
FIG. 5: effect of CART cells on mouse BT-549 model cytokines.
Detailed Description
Example 1: chimeric antigen receptor molecular design
The inventor screens an antibody targeting human ROR1 protein in advance to obtain a plurality of monoclonal antibodies, screens antibody molecules with higher affinity from the monoclonal antibodies, selects ScFv fragments containing heavy chains and light chains as antigen binding domains of the chimeric antigen receptor disclosed by the invention, and the antigen binding domains can be combined with target antigens at a high level and the affinity can reach 2.20E-09 nM. And can maintain stronger antigen binding capacity under the conditions of 4-42 ℃ and pH 2-12, can tolerate extreme environment in the microenvironment of the solid tumor, and is favorable for playing the role of inhibiting the solid tumor.
To obtain safe and effective CART cells, two chimeric antigen receptor structures are provided in the present invention, as shown in figure 1, the structure shown in figure 1A is a classical second generation CAR structure comprising a signal peptide, an anti-human ROR1 scFv, a hinge region, a transmembrane region, a 4-1BB endodomain and a CD3 zeta endodomain; an Fc fragment is introduced into the structure shown in figure 1B, an Fc-CAR structure is constructed, the Fc-CAR structure comprises a signal peptide, an Fc fragment (specifically a human IgG1 fragment), an anti-human ROR1 scFv, a hinge region, a transmembrane region, a 4-1BB intracellular domain and a CD3 zeta intracellular domain, and by introducing the Fc fragment, the tumor microenvironment is improved, the escape effect of the tumor on T cells is inhibited, the CART treatment safety is improved, and the bioavailability is improved.
The anti-human ROR1 scFv was stored by the laboratory and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively. Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
The Fc fragments have more types, including IgG, IgM, IgA, IgD and the like, and the difference among different species is larger, in order to reduce the rejection reaction in the in vivo application process, the human IgG1 fragment with the most abundant content in the human body is selected in the invention, and the amino acid sequence is shown as SEQ ID NO. 9.
Obtaining the sequence structures of other related elements through molecular biology and bioinformatics retrieval, wherein the amino acid sequence of the signal peptide is shown as SEQ ID NO. 10; the amino acid sequence of the hinge region is shown as SEQ ID NO. 11; the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 12; the amino acid sequence of the 4-1BB intracellular domain is shown as SEQ ID NO 13; the amino acid sequence of the intracellular domain of CD3 zeta is shown in SEQ ID NO. 14.
Example 2: CART cell preparation
According to the amino acid sequence of the element, the nucleotide sequence is obtained by means of reverse transcription, nucleic acid amplification and the like, and the corresponding gene fragment is inserted into a lentivirus expression vector pLent-EF1a-FH-CMV-RFP-P2A-Puro vector. The above expression vector was introduced into DH5 α e.coli by electrotransformation, and after correct sequencing, the plasmid was extracted and purified using a plasmid extraction kit (purchased from QIAGEN).
5% CO at 37 ℃2Culturing 293T cells in an incubator at constant temperature to ensure that the growth density of the 293T cells reaches 70-80% before transfection, and replacing a fresh culture medium for later use; diluting the liposome and the mixture of the purified plasmid and pLP/VSVG, pLP1 and pLP2 plasmid respectively by using serum-free DMEM-HG culture medium, then uniformly mixing the two, and standing at room temperature for 15min to form a liposome/DNA complex; adding the liposome/DNA complex into prepared 293T cells, mixing, culturing at 37 deg.C for 12-18 hr, replacing fresh culture medium, culturing for 48 hr, centrifuging, and collectingThe titer of the recombinant virus is determined to reach 3.35 multiplied by 106And pfu/mL or more meets the experimental requirements.
10mL of peripheral blood from fresh healthy volunteers were collected, and human T cells were extracted using RosetteSep T cell enrichment Cocktail (available from Stemcell) and Ficoll-Paque PLUS (available from GE Healthcare), according to the protocol. Adding the virus collected in the previous step into a T cell culture medium according to the ratio of 2:1, and transferring into 5% CO2And cultured in an incubator at 37 ℃ for 48 hours. The positive rate of the CAR modified T cells is detected by using a flow cytometer, and the result shows that the positive rate of the CAR modified T cells reaches over 90 percent. For ease of differentiation, T cells provided in the present invention that have chimeric antigen receptors of the two structures as described in example 1 are designated ROR1-CART and Fc-ROR1-CART, respectively.
Example 3 in vitro killing Effect of CART cells on tumor cells
According to the previous experimental results, the monoclonal antibody containing the CDR regions such as HCDR shown in SEQ ID NO. 1-3 and LCDR shown in SEQ ID NO. 4-6 provided by the invention has stronger recognition capability and killing effect on hematopoietic system tumor cells and ovarian cancer representative solid tumor cells, the range of the solid tumor is expanded in the embodiment, and the inhibition effect of the related CART cells on the solid tumor cells is verified.
ROR1 is reported to be highly expressed in various solid tumor tissues, SKOV3 (ovarian cancer cell line), BT-549 (breast cancer cell line), A549 (lung cancer cell line) and MGC-803 (gastric cancer cell line) are selected as research objects in the invention, and the killing effect is detected by a CCK-8 method. The specific method comprises the following steps:
(1) inoculating the tumor cells into a culture dish, and replacing a fresh culture medium for later use after the cells grow to a logarithmic growth phase;
(2) culturing ROR1-CART cells and Fc-ROR1-CART cells, and replacing fresh culture medium for later use after the cells are in a logarithmic growth phase;
(3) tumor cells and modified T cells were collected by centrifugation separately, as described in 1: mixed at a ratio of 1, and then seeded in a 96-well plate and cultured in a cell incubator at 37 ℃ for 48 hours.
(4) Adding CCK-8(Dojindo, Japan) reagent (20. mu.L/well) to each well, incubating the mixture in a cell incubator at 37 ℃ for 2 hours, and measuring the absorbance at 450 nm;
(5) according to the CCK-8 kit (Dojindo, Japan) instructions, the number of living cells was counted and the killing efficiency was estimated: the killing rate [ ((T + E) -T & E)/T ]. times.100%
Wherein T represents the number of viable target cells; e represents the number of viable effector cells; t + E equals the total number of viable target and effector cells; t & E represents the number of viable cells after killing of the target cells by effector cells.
As shown in fig. 2, the chimeric antigen receptor modified T cell provided by the present invention has inhibitory effect on various solid tumor cells: for SKOV3 cells, the inhibition capacity of Fc-ROR1-CART is slightly stronger than that of ROR1-CART, and the difference is significant (P < 0.01); for the BT-549 cells, the inhibition capacities of the two CART cells are similar, and no significant difference is found; for A-549 cells, the inhibitory capacity of Fc-ROR1-CART also seems to be stronger than ROR1-CART (P <0.05), but the difference in cell killing rate is not as obvious as in SKOV3 cells; for MGC-803 cells, no significant difference is found between the two cells, and the killing capacity of the cells is obviously weaker than that of the other three cells, which indicates that the CART cells provided by the invention have general killing capacity on gastric cancer cell lines represented by MGC-803.
The experimental results show that the chimeric antigen receptor T cell provided by the invention can inhibit the growth of various solid tumor cells, has obvious effect particularly on breast cancer, ovarian cancer and lung cancer cells, and can be used as a treatment means of solid tumors.
Example 4 in vivo inhibitory Effect of CART cells on hematological tumors
In order to further verify the antitumor effect of the CART cells provided by the invention, SKOV3 (ovarian cancer cell line) and BT-549 (breast cancer cell line) are selected to construct animal models and study the in vivo antitumor effect.
4.1 animal model preparation and treatment
C57BL/6 mice, 6-8 weeks old, were female and weighed 18-23 g. The experimental animals are raised in an SPF-level constant temperature and humidity room for 5 days, and are adaptive to the environment; 5% CO at 37 ℃2Culturing SKOV3 or BT-549 cells in the environment, subculturing every 2-3 days, and adjusting the cells to logarithmic phase; centrifuging to collect cells, resuspending the cells in sterile physiological saline, adjusting the concentration to 1 × 107one/mL, the right flank hairs of the C57BL/6 mice were shaved off, and 100. mu.L of the cell suspension was injected subcutaneously into the right anterior flank of the mice. Tumor growth was observed daily and subsequent experiments were performed when tumor diameters reached between 3mm and 5 mm.
After successful modeling, experimental animals were randomly divided into three groups, and ROR1-CART cells (2X 10 cells) were injected each week6One/one), Fc-ROR1-CART cells (2X 10)6One/one) and an equal volume of saline, tail vein bleeding after 4 weeks of treatment, blood samples retained, and mice sacrificed.
4.2 tumor volume
The change in tumor volume was measured 4 weeks after administration to the experimental animals, and the size of the tumor, the tumor volume (L x W) was measured using a vernier caliper2) The/2 estimate, where L is the length or longest dimension and W is the width of the tumor.
The results are shown in fig. 3, after 4 weeks of treatment, CART cells were able to significantly inhibit the tumor growth rate in mice, and the tumor volume was greatly reduced compared to the control group. In the SKOV3 cell model, the inhibition abilities of Fc-ROR1-CART and ROR1-CART are similar, and the two can effectively inhibit the growth of tumor tissues without significant difference; in the BT-549 cell model, the killing capability of Fc-ROR1-CART is obviously stronger than that of ROR 1-CART. The Fc fragment can prolong the half-life of the fusion protein, improve the bioavailability, have a certain immunoregulation function, further strengthen the tumor immune effect and other factors, and influence the response capability of the CART due to different tumor microenvironments of different tumor tissues.
4.3CART affects cytokine expression in hematological tumor models
The occurrence and development of blood tumor are influenced by various factors, and the anti-tumor effect of the antibody is also related to various factors such as cytokine secretion, immune cell activation, hypoxia environment formation, tumor microenvironment change and the like, wherein in the antibody treatment process, the expression level of the immune factor is changed, a series of immune mechanisms can be mediated, and the synergistic anti-tumor effect is exerted. Therefore, in order to preliminarily discuss the action mechanism of the CART cells, the invention detects the changes of IFN-gamma, IL-2 and IL-6 levels in the blood plasma of the mice after treatment.
After 4 weeks of treatment, tail vein blood is taken, serum is collected by centrifugation, and the concentration of IFN-gamma, IL-2 and IL-6 in plasma is detected by using an ELISA method, as shown in figure 4, in an SKOV3 cell model, CART cell treatment can improve the expression of cell factors, and the concentration of IFN-gamma, IL-2 and IL-6 is improved, which indicates that CART cells can stimulate the immune response in animals and play a synergistic anti-tumor role; specifically, there is no difference in the induction of IFN- γ expression between ROR1-CART cells and Fc-ROR1-CART cells, but the Fc-ROR1-CART cells can significantly inhibit the expression of interleukin factors such as IL-2 and IL-6.
Similar results were seen in the BT-549 cell model, as shown in fig. 5, ROR1-CART cell and Fc-ROR1-CART cell induced high expression of IFN- γ, and there was no significant difference between the two; however, in both IL-2 and IL-6 expression, Fc-ROR1-CART cells can cause interleukin factors to be at relatively low expression levels.
The results show that the chimeric antigen receptor T cell provided by the invention can effectively inhibit the growth of solid tumors such as ovarian cancer, breast cancer, lung cancer and the like, induce the expression of immune cytokines, introduce Fc fragments into the chimeric antigen receptor structure, and possibly obviously inhibit the over-expression of interleukin immune factors, prevent the occurrence of cytokine storm and improve the safety of treatment.
While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Sequence listing
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Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 10
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210> 11
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 12
<211> 69
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys
65
<210> 13
<211> 42
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 14
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110