CN111944760A - Immune cell for secreting bi-specific antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological cells, in particular to an immune cell for secreting a bispecific antibody and application thereof. The dual-specificity antibody is secreted to block two negative feedback ways of PD-1 and CTLA-4, thereby weakening the negative regulation and control of the tumor microenvironment on immune cells, reducing the exhaustion of the immune cells, enhancing the tumor killing function of the tumor microenvironment and reducing the inhibition of the tumor microenvironment on the immune cells.

Description

Immune cell for secreting bi-specific antibody and application thereof

Technical Field

The invention relates to the technical field of biological cells, in particular to immune cells capable of secreting antibodies and expressing CAR, which can optimize the killing function of the immune cells, reduce exhaustion and reduce the inhibition of tumor microenvironment on the immune cells.

Background

In recent years, tumor immunotherapy has been rapidly developed, especially Adoptive Cell Therapy (ACT), which refers to a method of isolating immune cells such as T cells and NK cells from a patient, amplifying the cells by in vitro modification, and then infusing the cells back into the patient for tumor treatment. In 2013, immunotherapy of tumors was evaluated as the first major breakthrough by Science impurities.

CAR-T and TCR-T are important components of adoptive cell therapy, in particular CAR-T therapy, with significant success in the treatment of hematological tumors, achieving a high remission rate, a typical CAR structure consisting of three parts, scFv, hinge and transmembrane domains, intracellular costimulatory signals and activation domains that recognize tumor antigens extracellularly. The first generation of CARs did not contain intracellular costimulatory signals, and CAR-T cells had lower killing activity and shorter survival time. Thus, second generation CARs began to add costimulatory signals such as CD28 and 4-1BB, and the CAR-T cells with different costimulatory signals also varied in their characteristics, with CD28 enhancing killing activity of CAR-T cells and 4-1BB enhancing killing activity of CAR-T cells while prolonging survival of CAR-T cells. Subsequently, a third generation CAR co-expressing two co-stimulatory signaling domains appeared, however its anti-tumor effect was not as good as the second generation CAR-T. Therefore, the clinical application is now primarily secondary CAR-T cells.

CAR-T therapy not only worked significantly on the treatment of hematological tumors, but also commercialized successfully, and the FDA officially approved two CAR-T drugs for marketing in 2017 in the united states. Although CAR-T cell therapy is very different in the treatment of hematological tumors, it has no good therapeutic effect on solid tumors, has low remission rate and is prone to off-target and other toxic and side effects. The solid tumor microenvironment is one of the important factors that CAR-T cells can not play a role effectively, PD-1 and CTLA-4 are two important immune negative regulation molecules, and in the tumor microenvironment, tumor cells, immune suppressor cells and the like inhibit immune cell functions through the two ways, so that immune cells are exhausted.

Disclosure of Invention

Therefore, it is necessary to provide an immune cell expressing CAR capable of secreting bispecific antibody, and the secreted bispecific antibody blocks two negative feedback pathways of PD-1 and CTLA-4, thereby weakening the negative regulation of tumor microenvironment on immune cells, reducing the exhaustion of immune cells, enhancing the tumor killing function thereof, and reducing the inhibition of tumor microenvironment on immune cells.

An immune cell that secretes a bispecific antibody, the immune cell expressing a chimeric antigen receptor and expressing a secreted bispecific antibody.

In one embodiment, the immune cell is a T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell, or other tumor killing cell.

In one example, genes expressing the chimeric antigen receptor and the secreted bispecific antibody are transferred into immune cells, and the genes are placed in expression cassettes.

In one embodiment, the expression cassette is a nucleic acid sequence comprising a chimeric antigen receptor or expresses an anti-PD-1& anti-CTLA-4 bispecific antibody.

In one embodiment, the gene expressing the chimeric antigen receptor and the secreted bispecific antibody is transferred by lentivirus, retrovirus, general plasmid vector, episomal vector, nano-delivery system, electrical transduction, transposon, or other delivery system.

In one embodiment, the secreted bispecific antibody is a scFv targeting PD-1 and/or a scFv targeting CTLA-4, linked in the middle by an Fc.

In one embodiment, the chimeric antigen receptor comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising a costimulatory signaling region, which refers to a portion of the intracellular domain that comprises a costimulatory molecule that is a cell surface molecule required for the effective response of lymphocytes to an antigen, and a portion of the CD3 zeta chain.

In one embodiment, the chimeric antigen receptor comprises EGFR, GPC3, scFv of claudin 18.2, the hinge and transmembrane domain of CD28, the hinge and transmembrane domain of CD8, the costimulatory domain of CD28 or CD137 (4-1BB) or ICOS, and the intracellular activation signal CD3 ζ.

In one embodiment, the scFv is selected from one or more of a monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, a human antibody, a nanobody, and a synthetic antibody.

In one embodiment, both PD-1 antigen and CTLA-4 antigen are bound.

The invention also provides application of the immune cell secreting the bispecific antibody in preparation of a medicament for preventing and/or treating cancer or tumor, and the immune cell secreting the bispecific antibody can be prepared into a pharmaceutically acceptable carrier, diluent or excipient, which has a good forming effect and simultaneously keeps good medicinal effect.

The main advantages of the bispecific antibody secreting immune cells of the present invention include:

1. bispecific antibody secreting immune cells express a CAR, specifically recognizing tumor cells;

2. the immune cells secreting the bispecific antibody express the bispecific antibody targeting PD-1& CTLA-4, weaken or block the inhibition of an immune inspection point on T cells, reduce a tumor microenvironment, enhance the tumoricidal activity of CAR-T cells and reduce exhaustion;

3. the immune cells secreting the bispecific antibody can secrete target spots targeting two inhibition ways at the same time, so that the curative effect of the immunotherapy is enhanced;

4. the bispecific antibody has simple structure and small molecular weight, and is easy for preparing lentivirus;

5. the bispecific antibody of the invention is different from the structure of the traditional bispecific antibody, enhances the stability of the antibody, prolongs the half life of the antibody, and is beneficial to the long-term drug effect in vivo;

6. the secretion of the secretory bispecific antibody moves along with the movement of the CAR-T cells, has targeting property, reduces the damage to normal tissues, reduces the toxicity of the whole body, and avoids the problems of autoimmune diseases and the like caused by excessive antibody;

7. the secretory antibody is different from the combined treatment of PD-1& CTLA-4 antibody, the dosage of the antibody is low, and the cost is reduced.

Drawings

FIG. 1 shows the expression of PD-L1 in A549-PD-L1 of example 1;

FIG. 2 is a graph of example 1 in which Huh7-PD-L1 expresses PD-L1;

figure 3 is the CAR structure of the secreted bispecific antibody of example 1 and the structure of the CAR;

FIG. 4 is an expansion curve of bispecific antibody secreting immune cells of example 1;

FIG. 5 shows the expression of the CAR by three T cells of example 1 (DAY 6);

FIG. 6 shows the expression of the CAR by three T cells of example 1 (DAY 13);

FIG. 7 shows the expression of PD-1 by three T cells (DAY6) in example 1;

FIG. 8 shows the expression of PD-1 by three T cells (DAY8) in example 1;

FIG. 9 is an in vitro killing experiment of three T cells of example 1;

FIG. 10 shows the secretion of IL-2 by the bispecific antibody-secreting immune cells of example 1;

FIG. 11 shows the cytokine secretion by the bispecific antibody-secreting immune cells of example 1;

FIG. 12 is the expansion curve of CAR-T cells co-incubated with A540-PD-L1 in example 1;

FIG. 13 is the expansion curve of the CAR-T cells of example 1 incubated with Huh 7-PD-L1.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The invention provides an immune cell secreting a bispecific antibody, the immune cell expressing a chimeric antigen receptor and expressing a secreted bispecific antibody.

In one embodiment, the immune cell is a T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell, or other tumor killing cell.

Transferring genes expressing the chimeric antigen receptor and the secretory bispecific antibody into an immune cell to enable the genes to express the chimeric antigen receptor and the secretory bispecific antibody, wherein the genes can be placed in the same expression frame or two expression frames respectively for expression, the first expression frame comprises a nucleic acid sequence of the chimeric antigen receptor, and the second expression frame expresses the anti-PD-1& anti-CTLA-4 bispecific antibody. There may even be a third expression cassette, a fourth expression cassette, etc., to express other chemokines, other functional elements such as CARs, etc.

The gene transfer means for expressing the chimeric antigen receptor and the secreted bispecific antibody is by lentivirus, retrovirus, general plasmid vector, episomal vector, nano delivery system, electrical transduction, transposon or other delivery system.

The secretory bispecific antibody is a scFv targeting PD-1 and/or a scFv targeting CTLA-4, and is linked by Fc which is modified to mutate NK cell recognition sites so that the NK cell recognition sites cannot cause ADCC reaction.

A chimeric antigen receptor includes an extracellular domain including an antigen binding domain, a transmembrane domain, and an intracellular domain including a costimulatory signaling region, which refers to a portion of the intracellular domain that includes a costimulatory molecule, a cell surface molecule required for an effective response by lymphocytes to an antigen, and a portion of the CD3 zeta chain.

The chimeric antigen receptor comprises EGFR, GPC3, scFv of Claudin18.2, the hinge and transmembrane domain of CD28, the hinge and transmembrane domain of CD8, the co-stimulatory domain of CD28 or CD137 (4-1BB) or ICOS, and the intracellular activation signal CD3 ζ.

The scFv is selected from one or more of monoclonal antibody, chimeric monoclonal antibody, humanized monoclonal antibody, human antibody, nano antibody and synthetic antibody.

Simultaneously bind PD-1 antigen and CTLA-4 antigen.

The present invention typically describes immune cells of the present invention in detail, taking CAR-T cells as an example. The immune cells of the invention are not limited to the CAR-T cells described above and below, and the immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cells, NKT cells, TIL, gamma-delta T cells are identical to T cells (or T cells can replace NK cells).

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term CAR: the design of chimeric antigen receptors CARs goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, the development is carried out to the third generation CARs and the fourth generation CARs, and a double CAR or a multi CAR, etc. which can target 2 targets or a plurality of targets, are expressed on the same cell

The Chimeric Antigen Receptor (CAR) of the present invention comprises an extracellular domain including an antigen binding domain, a transmembrane domain, and an intracellular domain including a costimulatory signaling region, which refers to a portion of the intracellular domain including a costimulatory molecule, a cell surface molecule required for an effective response of lymphocytes to an antigen, and a CD3 zeta chain portion.

In a preferred embodiment, the extracellular domain of a CAR provided by the invention comprises an antigen binding domain that targets GPC 3. When expressed in T cells, the CARs of the invention are capable of antigen recognition based on antigen binding specificity or protein receptor binding. When it binds to its cognate antigen, CAR-T cells will target tumor cell lysis, resulting in a reduction or elimination of the tumor burden in the patient. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the CD3 zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of the CD28/4-1BB/ICOS signaling domain, and the CD3 zeta signaling domain.

For the hinge and transmembrane regions (transmembrane domains), regulatory amino acid sequences are added before or after the transmembrane domain, so that the expression of the CAR varies depending on the concentration of the target antigen. In some instances, the transmembrane domain may be selected, or modified by amino acid substitution.

The intracellular domains in the CAR of the invention include the signaling domain of CD28/4-1BB/ICOS and the signaling domain of CD3 ζ.

The term "expression box": in one embodiment, the expression cassette comprises a first expression cassette comprising a nucleic acid sequence encoding said CAR and a second expression cassette expressing an anti-PD-1& anti-CTLA-4 bispecific antibody. In one embodiment, the expression cassette further comprises a third expression cassette, expressing another CAR, i.e. a dual CAR with two transmembrane regions, or a multiple CAR. In one embodiment, the first expression cassette, the second expression cassette and the third expression cassette each comprise a promoter, and the first expression cassette and the second expression cassette may be located on the same or different vectors. Preferably, the first expression cassette, the second expression cassette and the third expression cassette are located in the same vector.

The term "vector", a vector of an expression cassette is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof. Preferably, the vector is a viral vector.

The nucleic acid sequence of the vector encoding the desired molecule may be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the expression cassette of the present invention is inserted. A vector derived from a retrovirus such as lentivirus, characterized by long-term, stable integration of a gene of interest into a cell; transducible non-proliferating cells, such as hepatocytes; low immunogenicity; the safety is high. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression vector may be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence, another example is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. A preferred method for introducing the polynucleotide into the host cell is lipofection. The nucleic acid can be associated with a lipid, which can be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising a lipid, which is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

The invention provides a pharmaceutical composition comprising the immune cells as described above and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³ -1×10⁸Individual cells/ml, more preferably 1X 10⁴~1×10⁷Individual cells/ml. In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

The invention includes therapeutic applications of cells (e.g., T cells) transduced with lentiviral vectors encoding the nucleic acid constructs of the invention. The transduced T cells can elicit CAR-mediated T-cell responses. The injected cells are capable of killing the recipient's tumor cells, and the CAR-T cells are capable of replicating in vivo, resulting in long-term persistence that can lead to sustained tumor control. The CAR-T can secrete anti-PD-1& CTLA-4 bispecific antibody, so that the activity durability of CAR-T cells is improved, the CAR-T cells are not easy to exhaust, and the anti-tumor activity is maintained for a long time

In one embodiment, the immune cells of the invention can undergo robust T cell expansion in vivo and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, GPC3 CAR-T cells elicited a specific immune response against cells expressing GPC 3.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The immune cells of the invention, the ex vivo procedure for modifying T cells, at least one of the following occurs in vitro prior to administration of the cells into a human: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR and an account encoding the bispecific antibody into the cell, and/or iii) cryopreserving the cell. Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells isolated from human peripheral blood are used to express the CARs and bispecific antibodies disclosed herein. The modified cells can be administered to a recipient to provide a therapeutic benefit. The secondary immune cells may be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

The modified T cells of the immune cells of the invention may be administered alone or in combination with other drugs, pharmaceutical compositions, diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition prepared using the immune cells of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and by the clinical protocol. When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). Can be used for dredgingIt is often pointed out that: pharmaceutical compositions comprising T cells described herein may be at 1 × 10⁴~1×10⁹Dosage of 1X 10 cells/kg body weight, preferably⁵ ~1×10⁶The dose of individual cells/kg body weight was administered. The T cell composition may also be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular patient may be determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the formulations of the invention may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment,the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery. The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶ 1 to 10¹⁰ The individual cells are administered to the patient, for example, by intravenous infusion.

The main advantages of the immune cells of the invention include: 1. the immune cell expresses a CAR, specifically recognizing the tumor cell; 2. the immune cells express bispecific antibodies targeting PD-1& CTLA-4, weaken or block the inhibition of immune checkpoints on T cells, reduce the tumor microenvironment, enhance the tumoricidal activity of CAR-T cells and reduce exhaustion; 3. the immune cells of the invention secrete target spots targeting two inhibition ways at the same time, so that the curative effect of the immunotherapy is enhanced; 4. the bispecific antibody has simple structure and small molecular weight, and is easy for preparing lentivirus; 5. the bispecific antibody of the invention is different from the structure of the traditional bispecific antibody, enhances the stability of the antibody, prolongs the half life of the antibody, and is beneficial to the long-term drug effect in vivo; 6. the secretion type bispecific antibody disclosed by the invention has the advantages that the secretion moves along with the movement of CAR-T cells, the targeting property is realized, the damage to normal tissues is reduced, the systemic toxicity is reduced, and the problems of autoimmune diseases and the like caused by excessive antibody are avoided; 7. the secretory antibody is different from the combined treatment of PD-1& CTLA-4 antibody, the dosage of the antibody is low, and the cost is reduced.

Example 1

The preparation method and the functional verification of the immune cells comprise the following steps:

the method comprises the following steps: isolation of peripheral blood PBMC and culture of T cells

Separating monocytes from donor peripheral blood, performing density gradient centrifugation using ficol, and enriching T cells using a T cell sorting kit (CD3 MicroBeads, human-lymphotized, 130-; t cells were obtained by culturing all cells in a 5% CO2 incubator at 37 ℃ in a Medium containing 10% FBS, 2mM L-glutamine, and 100IU/ml rhIL2 using TexMACS GMP Medium (Miltenyi Biotec, 170-.

Step two: cell line culture

Cell line expressing GPC 3: huh-7 (human hepatoma cells), purchased from ATCC.

Cell line not expressing GPC 3: a549 (human non-small cell lung cancer cells) purchased from ATCC.

Packaging cells: 293T (human embryonic kidney cell line) purchased from ATCC.

Establishment of a tumor cell line overexpressing PD-L1: cloning a base sequence expressing PD-L1 into a PHBVV lentiviral vector skeleton, placing the PHBVV-EF 1 alpha-PD-L1 under a promoter of EF1 alpha (EF-1 alpha) to form PHBVV-EF 1 alpha-PD-L1, and transferring three plasmids of PHBVV-EF 1 alpha-PD-L1, a lentiviral envelope Plasmid pMD2, G (Addgene, Plasmid #12259) and a lentiviral packaging Plasmid psPAX2(Addgene Plasmid #12260) into 293T by using Lipofectamine3000 to prepare a lentiviral complete expression vector; viral supernatants were collected at 48h and 72h, concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used for infecting Huh-7 and A549 to finally obtain Huh-7 and A549 cell lines of over-expressed PD-L1, which are named as Huh-7-PD-L1 and A549-PD-L1. FIG. 1 and FIG. 2 are the detection graphs of the expression of PD-L1 by Huh-7-PD-L1 and A549-PD-L1 cells.

Culture in a culture medium: huh-7, A549 and 293T are cultured by using a DMEM medium. All media were supplemented with 10% (v/v) fetal bovine serum.

Step three: CAR structural design and lentiviral packaging

GPC3-CAR structure, i.e. CAR structure targeting GPC3 (glypican 3):

the methods of the invention construct the bispecific antibody and the second generation CAR in one expression cassette, linked with P2A. The core structure of the CAR includes a secretion signal peptide sequence; scFv of an antibody derived from anti-GPC3 (patent No.: US 2007/0190599A 1); the CD8 transmembrane region; intracellular segment stimulation signal 4-1BB-CD3 ζ, designated PCGPC3 CAR, and GPC3 CAR with only the expression cassette of the CAR as control, as shown in figure 2.

Cloning an expression frame into a PHBV lentiviral vector skeleton, placing the expression frame under a promoter of EF1 alpha (EF-1 alpha) to form PHBVV-EF 1 alpha-GPC 3-CAR and PHBVV-EF 1 alpha-PCGPC 3-CAR, and transferring three plasmids of PHBVV-EF 1 alpha-GPC 3-CAR or PHBVV-EF 1 alpha-PCGPC 3-CAR, a lentiviral envelope Plasmid pMD2, G (Addgene, Plasmid #12259) and a lentiviral packaging Plasmid psPAX2(Addgene Plasmid #12260) into 293T by using Lipofectamine3000 to prepare a lentiviral complete expression vector; viral supernatants were collected at 48h and 72h, concentrated by ultracentrifugation (Merck Millipore); the concentrated virus is ready for infecting T cells.

Step four: CAR-T cell preparation

4.1 Lentiviral infection

After the primary T cells separated and purified in the step 1 are activated for 1 day, the lentivirus vectors of 2 lentiviruses packaged in the step three are infected according to MOI (1-10), and the lentivirus vectors are transferred to a cell culture flask and cultured in a constant-temperature incubator at 37 ℃ and 5% CO 2.

4.2 cell proliferation and CAR Positive Rate detection

Cell numbers were measured daily on days 6, 9, 11, and 13 post-infection, CAR positivity was measured on days 6 and 9, respectively, and PD-1 expression was measured on days 6, 7, and 8, respectively. The culture medium is subcultured and supplemented every 1-2 days.

2 CAR-T cells, designated PCGPC3 CAR T and GPC3 CAR T, were successfully constructed using 2 lentiviral vectors, with no lentiviral-infected T cells as controls (NT)

As shown in fig. 4: proliferation rates of 3 CAR-T cells, the insertion of the sequence secreting bispecific antibody had no significant effect on the proliferation rate of the cells.

As shown in fig. 5 and 6: the expression profiles of 3T cell CARs at day6 and day 9, respectively. Secretion of bispecific antibodies was seen to have no significant effect on CAR positivity.

As shown in fig. 7 and 8, PD-1 expression was observed in 3T cells. The expression of PD-1 was much reduced for CAR-T secreting bispecific antibodies. The reason is that the secreted bispecific antibody can block the binding site of PD-1 and PD-L1, while the site of the fluorescent antibody that detects PD-1 binding to PD-1 is also this site and therefore undetectable.

And 5: cell killing experiment in vitro

In vitro killing experiments were performed on the 3-T cells obtained in step 4. The LDH method (promega: G1780) detects the killing effect of the CAR-T cells, the target cells and the effector cells are incubated for 6h, the killing efficiency is obvious, and the secretion of the bispecific antibody has no significant influence on the killing activity of the CAR-T. As shown in fig. 8.

Step 6: cytokine release assay

CAR-T cells (obtained in step 4) and target cells (a549, Huh-7) were treated at 1: mixing the mixture with the effective target ratio of 1, placing the mixture in an RPMI culture medium, co-culturing for 24h, collecting supernatant, centrifuging the supernatant, taking the supernatant to detect the release levels of the cytokines IFN-gamma and IL2, and detecting the release levels by using an Elisa kit (abbkine, KET6011 and KET 6014).

The results are shown in FIG. 10, and the CAR-T cells released large amounts of IFN- γ and IL-2 after coculture with GPC3+ target cells, and secreted only small amounts of IFN- γ and IL-2 after coculture with GPC 3-target cells. Indicating that CAR-T can be efficiently and specifically activated by the tumor surface GPC3 antigen.

And 7: long time in vitro killing experiment.

Different CAR-T cells and target cells were co-cultured at a 3:1 ratio, cell counting was performed every 5 days, the ratio of CAR-T cells was flow-detected, target cells were supplemented again, the ratio of CAR-T cells and target cells was made 3:1, and the total number of CAR-T cells expanded was 25 days as shown in fig. 11 and 12 below. FIG. 11 is a graph of T cell expansion when CAR-T cells were incubated with A549-PC. FIG. 12 is a T cell expansion curve when CAR-T cells were incubated with Huh 7-PC. It is thus clear that secretion of bispecific antibodies can effectively counteract immunosuppression by PD-1.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express one embodiment of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (11)

1. An immune cell that secretes a bispecific antibody, wherein the immune cell expresses a chimeric antigen receptor and expresses a secreted bispecific antibody.

2. The bispecific antibody-secreting immune cell according to claim 1, wherein said immune cell is a T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell or other tumor killer cell.

3. The bispecific antibody secreting immune cell according to claim 2, wherein the gene expressing said chimeric antigen receptor and the secreted bispecific antibody is transferred into said immune cell, said gene being placed in said expression cassette.

4. The bispecific antibody secreting immune cell of claim 3, wherein said expression cassette is a nucleic acid sequence comprising said chimeric antigen receptor or expresses anti-PD-1& anti-CTLA-4 bispecific antibody.

5. The bispecific antibody secreting immune cell according to claim 3, wherein said gene expressing said chimeric antigen receptor and said secreted bispecific antibody is transferred by lentivirus, retrovirus, general plasmid vector, episomal vector, nano delivery system, electrical transduction, transposon or other delivery system.

6. The bispecific antibody secreting immune cell of claim 1, wherein said bispecific antibody secreting is a PD-1 targeting scFv and/or a CTLA-4 targeting scFv, said bispecific antibody secreting middle is linked by an Fc.

7. The bispecific antibody secreting immune cell of claim 1, wherein said chimeric antigen receptor comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain and an intracellular domain comprising a costimulatory signaling region, which refers to a part of the intracellular domain comprising a costimulatory molecule which is a cell surface molecule required for an effective response of lymphocytes to an antigen, and a CD3 zeta chain part.

8. The bispecific antibody secreting immune cell of claim 7, wherein said chimeric antigen receptor comprises EGFR, GPC3, scFv of Claudin 18.2, the hinge and transmembrane domain of CD28, the hinge and transmembrane domain of CD8, the costimulatory domain of CD28 or CD137 (4-1BB) or ICOS, and the intracellular activation signal CD3 ζ.

9. The bispecific antibody secreting immune cell according to claim 6 or 8, wherein said scFv is selected from one or several of the group consisting of monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, human antibodies, nanobodies and synthetic antibodies.

10. The bispecific antibody secreting immune cell of claim 1, wherein said binding binds PD-1 antigen and CTLA-4 antigen simultaneously.

11. Use of bispecific antibody secreting immune cells according to any of claims 1 to 10 for the preparation of a medicament for the prevention and/or treatment of cancer or tumor.

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