Patent Description:
The use of immunological therapy for overcoming tumors has always been an important direction in the application of immunology in translational medicine. With the development of various omics (genomics, proteomics, etc.), tumor cells have been widely recognized due to their immunogenicity caused by mutations, which lays a theoretical foundation for tumor immunotherapy. At the same time, with the accumulation of tumor immunology research itself, tumor immunotherapy has recently made a great progress, and a series of new immunotherapy methods have gradually entered into the clinic. The current tumor immunology research has established the central position of T cell killing in tumor immunotherapy, and the chimeric antigen receptor T cell (CAR-T cell) is a tumor-killing cell which has combined the targeted recognition of antibody and the tumor-killing function of T cell, and been generated by artificial modification.

The concept of chimeric antigen receptor T cell was first proposed by Gross, Waks and Eshhar in <NUM>. They expressed TNP-recognizing antibodies on T cells, achieving antigen-specific, non-MHC-restricted T cell activation and enhanced effect, and proposed the concept of the application of CAR-T technology in tumor treatment. According to this principle, tumor-specific antibodies are embedded into T cells, which will give T cells new tumor-killing capabilities. After that, CAR-T technology was introduced into anti-tumor clinical trials, but the final clinical results of early CAR-T cells are not ideal since their intracellular signal transmission domain contains only the first signal, and the selected tumor type is a solid tumor. In <NUM>, the Fred Hutchison Cancer Institute and other institutions used CAR-T to treat B cell lymphoma, although the treatment results are not ideal, the key to this clinical trial is to demonstrate that CAR-T treatment with CD20-expressing B cells as the target is relatively safe. Subsequently, in <NUM>, NCI reported a case of successful treatment of B-cell lymphoma, using CAR-T targeting CD19, the patient's lymphoma was controlled, normal B cells were also eliminated, and serum Ig was significantly reduced, providing a theoretical and practical support for the effectiveness of CAR-T in the treatment of B cell-derived lymphomas. In <NUM>, a team led by Dr. Carl June of the University of Pennsylvania in the United States used CAR-T that specifically recognizes CD19 for the treatment of chronic lymphocytic leukemia derived from B cells, showing a "cure" effect. After that, clinical trials have also been launched in relapsed and refractory acute lymphoblastic cell leukemia, and good results have also been achieved. Due to this breakthrough progress and the development of other immune regulation methods, Science magazine ranked tumor immunotherapy as the number one scientific and technological breakthrough in <NUM>. This success has caused widespread influence in countries around the world, and countries have begun to carry out a large number of CAR-T-based scientific research and clinical trials of tumor treatment.

The structure of CAR consists of an extracellular antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular signal transduction domain. The extracellular antigen recognition domain generally consists of a single-chain antibody, which specifically recognizes membrane surface molecules of the tumor cell, or can be a ligand or receptor of certain tumor-specific antigens, etc. The extracellular hinge region is a spatial structure that separates the antigen recognition domain from the transmembrane domain, and its purpose is to provide a suitable spatial position, so that the extracellular antigen recognition domain can maintain the correct structure and transmit the intracellular signals before and after recognizing the antigen. The transmembrane domain is a domain for ensuring the positioning of the CAR molecule on the membrane surface. The intracellular signal transduction domain is a key part of mediating the CAR signal transduction, and is usually a combination of one or several first signals (for the recognition of TCR and MHC-I-peptide complex) and second signals (for the recognition of costimulatory receptor and costimulatory ligand). The first-generation CAR contains only the first signal, the second-generation CAR has one first signal and one second signal, and the third-generation CAR has one first signal and two second signal domains. Although CAR-T has achieved a great success in the treatment of leukemia derived from B cell, its relatively high recurrence rate and low effectiveness for solid tumors are important challenges currently. Therefore, there is an urgent clinic need of developing a new generation of high-efficiency CAR-T currently. In addition to the third-generation CAR-T, there are currently other new CAR-T design strategies, that is, new regulatory molecules independent of CAR are introduced on the basis of the second-generation CAR-T to further enhance the function of CAR-T.

The application of CAR-T targeting the B cell surface targeting molecules CD19 and CD20 prepared from the patient's own blood cells in the treatment of B cell leukemia has been relatively mature, but there are a large number of recurrences, even though the response rate is high. In addition, the treatment efficiency for solid lymphoma is relatively low, which is related to the immunosuppressive microenvironment in solid tumors.

In solid tumors, there are a variety of immune cells, tumor cells and stromal cells, which together constitute the tumor microenvironment. The tumor microenvironment is usually immunosuppressive, and can inhibit endogenous anti-tumor T cell responses or adoptive T cells (such as CAR-T) at multiple levels, for example, leading to exhaustion of T cells and loss of tumor killing function, and eventually leading to the clearance of T cells. How to enhance the activation ability of CAR-T in solid tumors so that CAR-T can fight against the immune suppression in the tumor microenvironment is an important idea and direction for expanding CAR-T to solid tumor treatment.

The following documents relate to this overall concept:
<CIT> discloses a T cell expressing functional full length <NUM>-1BB costimulatory receptor and a CAR.

<CIT> discloses enhancing the antigen presenting ability of CAR-T cells by cointroducing of costimulatory molecules and methods of using the obtained T cells for treating diseases associated with the expression of disease-associated antigens.

<CIT> discloses immunoresponsive cells such as T cells comprising a second-generation chimeric antigen receptor and a chimeric costimulatory receptor which arrangement is referred to as a parallel chimeric activating receptor (pCAR).

<CIT> discloses a fusion protein that, when displayed on a cell, can convert a negative signal into a positive signal in the cell, wherein said fusion protein is a chimeric protein comprising at least two domains which two domains are either associated with a negative signal or with a positive signal, respectively. Such fusion proteins are expressed in CAR-T cells.

<NPL> discloses a CAR-T cell specific for CD19 and having a therapeutic activity against lymphomas.

However, the current CAR-T domains in clinical use still have insufficient tumor killing and expansion abilities, and have poor efficacy in controlling solid tumors/metastasis. Some CAR-T use novel regulatory molecules such as IL-<NUM>, <NUM> - 1BBL, etc. These molecules will also produce non-specific activation effects on other non-CAR-T cells in addition to affecting the CAR-T, which may cause immune side effects.

An object of the present invention is to address the defects in the prior art, provide a fusion protein comprising a chimeric antigen receptor including a co-stimulatory receptor as defined in the appended claims and use thereof, and provide a CAR-T cell constructed by a recombinant expression vector of the fusion protein. OX40 is an important co-stimulatory receptor which is primarily expressed in activated CD4 and CD8 T cells, and displays a variety of functions during the activation of T cells. They can promote the activation of T cells, exhibit more effector molecules, and reduce the expression of gene associated with apoptosis. Integrating the co-stimulatory receptor signal into the CAR-T has a potential effect-enhancing function.

To address the aforesaid object, the present invention utilizes the following technical solutions:
a first object of the present invention is to provide a fusion protein, which comprises, from N-terminus to C-terminus, (a) a chimeric antigen receptor (CAR) that specifically binds to a CD20, (b) a 2A peptide and (c) a OX40 protein wherein said CAR has a structure of scFv(X)-(Y)CD3zeta; the amino acid sequence of said scFv(X)-(Y)CD3zeta is as set forth in SEQ ID No.<NUM>; the amino acid sequence of said 2A peptide is as set forth in SEQ ID No.<NUM>, SEQ ID No.<NUM>, SEQ ID No.<NUM> or SEQ ID No.<NUM>; and the amino acid sequence of said OX40 protein is as set forth in SEQ ID No.<NUM>.

In said scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ having a sequence of SEQ ID No. <NUM>; said OX40 has a sequence of SEQ ID No.<NUM>, and said 2A has a sequence of SEQ ID No.<NUM>, SEQ ID No.<NUM>, SEQ ID No.<NUM> or SEQ ID No.<NUM>. Furthermore disclosed, but not according to the invention, are HVEM which has a sequence of SEQ ID No.<NUM>; ICOS which has a sequence of SEQ ID No.<NUM>; CD27 which has a sequence of SEQ ID No.<NUM>; <NUM>-1BB which has a sequence of SEQ ID No.<NUM>;.

Wherein the aforesaid sequences and other sequences disclosed herein are as follows:.

Also disclosed is a recombinant expression vector which encodes any one of the aforesaid fusion proteins.

A second object of the present invention is to provide a CAR-T cell constructed by a recombinant expression vector encoding any one of the aforesaid fusion proteins.

A third object of the present invention is to provide a method of preparing the aforesaid CAR-T cell comprising the following steps:.

For further optimizing the method of preparing the aforesaid CAR-T cell, the technical means used in the present invention further includes: incorporating the nucleic acid encoding 2A peptide between the nucleic acid encoding the CAR and the nucleic acid encoding the OX40 protein by overlap PCR to form the nucleic acid encoding the fusion protein, and adding restriction sites to both ends of the nucleic acid encoding the fusion protein to clone a lentiviral vector; subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with the lentiviral packaging plasmid; after a predetermined time period, collecting a supernatant, filtering, and centrifuging to concentrate the virus to obtain a virus comprising the nucleic acid encoding the fusion protein.

Still further, the specific steps of the construction of lentiviral vector and production of virus are as follows: incorporating 2A sequence between scFv(X)-(Y)CD3zeta and OX40 by overlap PCR, adding EcoRI and SalI restriction sites to both ends of the fusion protein to clone the pCDH-MSCVEF vector, subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid into 293X; after <NUM> and <NUM> hours, collecting the supernatant, filtering it by a <NUM> filter and performing centrifugation at 25000RPM for <NUM> hours to concentrate the viruses to obtain the scFv(X)-(Y)CD3zeta-2A-OX40 virus.

Further, the specific steps of the preparation of scFv(X)-(Y)CD3zeta-2A-OX40 CAR-T cell include: isolating human PBMC for purification, inoculating into a culture plate under suitable stimulation conditions, culturing for a predetermined period of time, infecting said PBMC with the scFv(X)-(Y)CD3zeta-2A-OX40 virus obtained in Step <NUM>, and subjecting it to cell expansion under suitable stimulation conditions, after <NUM> rounds of expansion under stimulation, the obtained cells are the scFv(X)-(Y)CD3zeta-2A-OX40 CAR-T cells.

Further, the stimulation conditions for culturing the isolated and purified human PBMC are anti-hCD3 and anti-hCD28; and the stimulation conditions for cell expansion are stimulation by use of artificial antigen presenting cell or anti-hCD3/<NUM> every <NUM> days.

Still further, the specific steps of preparing the scFv(X)-(Y)CD3zeta-2A-OX40 CAR-T cell are as follows: purifying human PBMC by a Stemcell T cell isolation kit, inoculating into a <NUM>-well culture plate coated by anti-hCD3 and anti-hCD28. After <NUM> days, infecting the cells with the scFv(X)-(Y)CD3zeta-2A-OX40 virus at MOI=<NUM>-<NUM>. After <NUM> day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/<NUM> every <NUM> days. After <NUM> rounds of stimulation, the obtained cells are scFv(X)-(Y)CD3zeta-2A-OX40 CAR-T cells.

Said scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ having a sequence of SEQ ID No. <NUM>; said OX40 has a sequence of SEQ ID No.<NUM>; and said 2A has a sequence of SEQ ID No.<NUM>.

Further, the lentiviral packaging plasmid in Step <NUM> includes VSV-g, pMD Gag/Pol, RSV-REV, and the centrifugation is performed with Beckman ultracentrifuge and SW28 head.

A fourth object of the present invention is to provide a formulation including the aforesaid CAR-T cell or the CAR-T cell prepared by the aforesaid preparation method. Further, the formulation also includes a pharmaceutically diluents or excipient.

A fifth object of the present invention is to provide the aforesaid fusion protein, the aforesaid CAR-T cell or the CAR-T cell prepared by the aforesaid preparation method in preparation for use as a medicament for treating or preventing tumor.

Further, said tumors are solid tumors. Examples of said solid tumors include, but are not limited to, lymphomas, renal tumors, neuroblastoma, germ cell tumor, osteosarcoma, chondrosarcoma, soft tissue sarcoma, liver tumor, thymoma, pulmonary blastoma, pancreatoblastoma, hemangioma, etc..

As compared with the prior art, the present invention has the following beneficial effects:
the CAR-T cell of the present invention significantly increases the tumor killing ability and expansion ability, and exhibits a greatly increased solid/metastasis tumor killing ability. The CAR-T cell of the present invention includes a co-stimulatory receptor, instead of a conventionally used ligand or excreted factor, and works only on the CAR-T cell, thereby reducing the risk of causing an immune side effect.

The present invention first utilizes the co-stimulatory receptor in the construction of CAR-T. As compared with the current CAR-T technology in clinic use, the present invention significantly increases the activation ability and survival ability of CAR-T cell in tumors, and controls the ability of solid/metastatic tumors, thereby improving the therapeutic effect of the CAR-T cell to get a more superior anti-tumor therapeutic effect.

The present invention provides a fusion protein comprising achimeric antigen receptor including a co-stimulatory receptor specifically binding to a CD20 and having a structure of scFv(X)-(Y)CD3zeta of SEQ ID No. <NUM>, a 2A peptide and an OX protein. The present invention also relates to a CAR-T cell constructed by a recombinant expression vector of any one of the aforesaid fusion proteins and a preparation method therefor, a formulation including the CAR-T cell, and a use of the CAR-T cell.

Hereinafter the embodiments of the present invention are further described with reference to the accompanying drawings and examples. The following examples are only for more clearly illustrating the technical solutions of the present invention, but not for limiting the protective scope of the present invention.

The chimeric antigen receptor (CAR) molecules including a co-stimulatory receptor used in the following examples are BBZ-2A-OX40 as an inventive example, and BBZ-2A-HVEM, BBZ-2A-ICOS, BBZ-2A-CD27, BBZ-2A-<NUM>-1BB, respectively, as comparative examples. Their structures are shown in <FIG>.

The preparation of the 20BBZ-2A-OX40 CAR-T cell in this example includes the following steps:.

The preparation of the 20BBZ-2A-HVEM CAR-T cell in in this example includes the following steps:.

The preparation of the 20BBZ-2A-ICOS CAR-T cell in this example includes the following steps:.

The preparation of 20BBZ-2A-CD27 CAR-T cell in this example includes the following steps:.

The preparation of the 20BBZ-2A-<NUM>-1BB CAR-T cell in this example includes the following steps:.

20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell prepared in Step <NUM> of Example <NUM> were continuously cultured for <NUM> days, and stimulated with artificial antigen presenting cell once every <NUM> days. The cells were counted, and the results are shown in <FIG>. It can be seen from the figure that 20BBZ-2A-OX40 CAR-T cell has enhanced proliferation ability as compared with 20BBZCAR-T cell.

20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell obtained in Step <NUM> of Example <NUM>, 20BBZ-2A-ICOS CAR-T cell obtained in Step <NUM> of Example <NUM>, and 20BBZ-2A-CD27 CAR-T cell obtained in Step <NUM> of Example <NUM> were inoculated into a <NUM>-well plate, and Raji tumor cells were added at a CAR-T:tumor cell ratio of <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>. After <NUM> and <NUM> hours, the survival rates of tumor cells were compared, and the results are shown in <FIG>. It can be seen from the figure that 20BBZ-2A-OX40/ICOS/CD27 CAR-T cell has similar tumor killing ability as compared with 20BBZ CAR-T cell, and some CAR-T including the co-stimulatory receptor has a stronger tumor killing ability.

<NUM><NUM> Nalm-<NUM> tumor cells were intravenously inoculated into B-NDG mice, which were treated with <NUM><NUM> 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells after <NUM> days. The mice were observed for their survival rates, and some mice were detected for the level of tumor cells and CAR-T cells in their marrow on Day <NUM>. The results are shown in <FIG>, respectively. It can be seen from the figure that 20BBZ-2A-OX40 CAR-T cell, as compared with 20BBZ CAR-T cell, significantly prolongs the survival of mice, and expanded more in vivo.

Claim 1:
A fusion protein, which comprises, from N-terminus to C-terminus, (a) a chimeric antigen receptor (CAR) that specifically binds to a CD20, (b) a 2A peptide and (c) a OX40 protein wherein;
said CAR has a structure of scFv(X)-(Y)CD3zeta; the amino acid sequence of said scFv(X)-(Y)CD3zeta is as set forth in SEQ ID No.<NUM>;
the amino acid sequence of said 2A peptide is as set forth in SEQ ID No.<NUM>, SEQ ID No.<NUM>, SEQ ID No.<NUM> or SEQ ID No.<NUM>; and
the amino acid sequence of said OX40 protein is as set forth in SEQ ID No.<NUM>.