CN118652355A - Chimeric antigen receptor targeting PSMA, modified cell and application thereof - Google Patents

Abstract

The present invention discloses a Chimeric Antigen Receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a single chain variable fragment (scFv) that targets a Prostate Specific Membrane Antigen (PSMA). By integrating scFv targeting PSMA on chimeric antigen receptor, genetically engineered macrophages containing the CAR have the effect of targeting PSMA and prostate cancer cells, thereby promoting phagocytosis and killing effects of the macrophages on the prostate cancer cells.

Description

Chimeric antigen receptor targeting PSMA, modified cell and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a chimeric antigen receptor targeting PSMA and a modified cell thereof, and application of the chimeric antigen receptor in treating prostate cancer.

Background

Prostate Cancer (PCa) is the sixth most frequently occurring type of Cancer in men, with high morbidity and mortality, among the global Cancer incidences. The data indicate that almost all patients with advanced prostate cancer develop castration-resistant prostate cancer (CRPC) after endocrine treatment with a median survival of about 14 months. Despite the increase in existing therapies, the 5-year survival rate of metastatic castration resistant prostate cancer (mCRPC) patients remains low, and new therapies are needed.

The breakthrough of adoptive immune cell therapy changes the research and development pattern of tumor drugs, and 11 CAR-T (CHIMERIC ANTIGEN Receptor T cell) products are approved by FDA/NMPA to be marketed so far and used for treating hematological malignant lymphoma, and become an important milestone in clinical medical history of tumor. However, although CAR-T has shown good efficacy in hematological tumors, its efficacy in solid tumor treatment is still unsatisfactory. Macrophages have unique advantages as carriers for adoptive cell therapy given the specificity of the microenvironment of solid tumors.

Due to the high-proportion infiltration (50%) of macrophages in tumor microenvironments, the macrophages have the unique advantages when being used as adoptive cell therapy carriers: the solid tumor penetrating capacity is high, the phagocytic capacity is high, the tumor cells can be killed rapidly, the immune escape can be prevented, and the T cells can be activated as antigen presenting cells, so that the compound has the remarkable effects of remodeling immune cell interaction networks and reversing the immune suppression microenvironment. The chimeric antigen receptor macrophage is constructed aiming at the prostate cancer target, which is hopeful to meet the clinical requirement of solid tumor treatment mainly for malignant prostate tumor.

Disclosure of Invention

In view of the shortcomings of the prior art, the present disclosure aims to provide a chimeric antigen receptor targeting Prostate specific membrane antigen (PSMA, state-specific membrane antigen) and a method for constructing a macrophage targeting PSMA, so as to solve the problem of unsatisfactory effect of therapeutic means on Prostate cancer in the prior art.

According to one aspect of the present disclosure, there is provided a Chimeric Antigen Receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a single chain variable fragment (PSMA-scFv) that targets a Prostate Specific Membrane Antigen (PSMA).

In some embodiments, the extracellular domain comprises a CD8 a signal peptide and a PSMA-scFv, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane region, and the intracellular domain comprises an fcsr1γ intracellular region.

In some embodiments, the extracellular domain has an amino acid sequence of SEQ ID NO. 1 or 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 1 or 3, or an amino acid sequence of equivalent function formed by one or more amino acid additions, deletions, substitutions or modifications of the amino acid sequence of SEQ ID NO. 1 or 3.

In some embodiments, the transmembrane domain has an amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 5, or an amino acid sequence with equivalent function that is formed by one or more amino acid additions, deletions, substitutions or modifications to the amino acid sequence shown in SEQ ID NO. 5.

In some embodiments, the intracellular domain has an amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 7, or an amino acid sequence with equivalent function that is formed by one or more amino acid additions, deletions, substitutions or modifications to the amino acid sequence shown in SEQ ID NO. 7.

In some embodiments, the chimeric antigen receptor comprises a CD 8a signal peptide, a PSMA-scFv, a CD8 hinge region, a CD8 transmembrane region, and an fcsr1γ intracellular region, operably linked in sequence.

According to another aspect of the present disclosure, there is provided a nucleic acid molecule encoding a chimeric antigen receptor described in the present disclosure.

In some embodiments, the nucleic acid molecule has a nucleotide sequence set forth in SEQ ID NO. 9, or a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 9.

According to yet another aspect of the present disclosure, there is provided a recombinant vector comprising a nucleic acid molecule as described in the present disclosure.

According to yet another aspect of the present disclosure, there is provided a modified cell comprising a nucleic acid molecule as described in the present disclosure, or a recombinant vector as described.

In some embodiments, the cells comprise monocytes, macrophages or dendritic cells, preferably macrophages.

In some embodiments, the modified cell is genetically modified to express the chimeric antigen receptor.

In some embodiments, the modified cell has at least one up-regulated M1 marker and/or at least one down-regulated M2 marker.

In some embodiments, the modified cell has increased expression of CD80 and/or CD86.

In some embodiments, the modified cell has reduced expression of CD163.

According to yet another aspect of the present disclosure, there is provided a method of constructing a modified cell comprising introducing into a cell a chimeric antigen receptor, a nucleic acid molecule, or a recombinant vector as described in the present disclosure.

In some embodiments, the method comprises transducing the cell with a viral vector comprising a nucleic acid sequence encoding the chimeric antigen receptor.

In some embodiments, the cells comprise monocytes, macrophages or dendritic cells, preferably macrophages.

In some embodiments, the viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, preferably adenoviral vectors.

In some embodiments, the adenovirus vector has a multiplicity of infection (MOI) of 500-2000, which may be, for example, 500, 1000, 1500, 2000 or any value therebetween.

In some embodiments, the method comprises inducing the modified cell to exhibit an M1-like phenotype using GM-CSF.

In some embodiments, the concentration of GM-CSF is 10-200 ng/mL, which may be, for example, 10 ng/mL, 20 ng/mL, 50 ng/mL, 80 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL, or any value therebetween.

In some embodiments, the method comprises culturing the cells using X-VIVO10, X-VIVO15, or X-VIVO20 medium.

In some embodiments, the medium is supplemented with Fetal Bovine Serum (FBS).

In some embodiments, the medium is supplemented with Fetal Bovine Serum (FBS) and GM-CSF.

In some embodiments, the concentration of the fetal bovine serum is 8-15%, e.g., 8%, 10%, 12%, 15% or any value therebetween.

According to a further aspect of the present disclosure there is provided a pharmaceutical composition comprising a chimeric antigen receptor as described in the present disclosure, a modified cell as described or a modified cell obtained by the method as described, and a pharmaceutically acceptable carrier.

According to a further aspect of the present disclosure there is provided the use of a chimeric antigen receptor as described herein, the modified cell obtained by the method, or the pharmaceutical composition, in the manufacture of a medicament for the treatment of prostate cancer.

In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer.

According to a further aspect of the present disclosure there is provided a method of treating a disease or condition associated with prostate cancer comprising administering to a subject in need thereof a therapeutically effective amount of a chimeric antigen receptor described in the present disclosure, the modified cell obtained by the method, or the pharmaceutical composition.

In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer.

The present disclosure provides a Chimeric Antigen Receptor (CAR) targeting PSMA; by integrating the scFv targeting PSMA on the chimeric antigen receptor, the CAR and modified cells containing the CAR, especially macrophages, have the effect of targeting PSMA and prostate cancer cells, thereby promoting phagocytosis and killing effects of the macrophages on the prostate cancer cells. The present disclosure further enhances the anti-tumor ability of CAR-M by optimizing CAR-M transfection and culture systems such that CAR-M exhibits an M1 phenotype. The present disclosure provides PSMA-targeted CAR-M cell immunotherapy for the first time, and experiments prove the effects of anti-PSMA CAR-M cell immunotherapy on alleviating disease progression and prolonging survival time, which is of great significance for the treatment of advanced prostate cancer.

Drawings

FIG. 1 shows the results of the prepared PBMac detection of the expression of macrophage markers CD11B (A) and CD68 (B) using flow cytometry.

FIG. 2 shows the results of PBMac expression of CD80 (A) and CD163 (B) obtained during PBMac induction with or without addition of GM-CSF.

FIG. 3 shows the killing effect of PBMac on target cell 22Rv1 obtained by adding M-CSF (A) or GM-CSF (B) during induction PBMac.

FIG. 4 shows the results of cell viability (A) and phagocytic capacity (B) of different culture systems of PSMA-targeted chimeric antigen receptor macrophages (PBMac).

Figure 5 shows transfection efficiency results (a) for PSMA-targeted chimeric antigen receptor macrophages (PBMac) and expression of CAR molecules on cell membranes (B).

Fig. 6 shows tumor phagocytosis results (a) and in vitro killing results (B) of PSMA-targeted chimeric antigen receptor macrophages (PBMac).

Figure 7 shows the in vitro cytokine release results of PSMA-targeted chimeric antigen receptor macrophages (PBMac) for CAR-M.

Fig. 8 shows the results of drug efficacy studies of PSMA-targeted chimeric antigen receptor macrophages (PBMac) in tumor-bearing immunodeficient mice, where a is an image of the live tumor-bearing immunodeficient mice during treatment, B is a statistical result of tumor tissue remaining in the tumor-bearing immunodeficient mice at the experimental endpoint, C is a quantitative statistical result of the live tumor-bearing immunodeficient mice during treatment, and D is a statistical result of survival time of the tumor-bearing immunodeficient mice.

FIG. 9 shows the results of safety evaluation of PSMA-targeted chimeric antigen receptor macrophages (PBMac) in tumor-bearing immunodeficient mice, wherein A is the pathological change of organs of the tumor-bearing immunodeficient mice, and B is the blood biochemical detection result of serum of the tumor-bearing immunodeficient mice.

Detailed Description

Prostate specific membrane antigen (PSMA, prostate-specific membrane antigen) is a transmembrane glycoprotein expressed on the cell membrane. PSMA exhibits a specific high expression pattern in prostate cancer (PCa) compared to normal prostate tissue and other parts of the body, and its expression level is highly correlated with the aggressiveness of PCa.

The present disclosure designs a chimeric antigen receptor nucleotide sequence targeting PSMA; by using adenovirus transfection mode, the high-efficiency expression of the CAR molecule in macrophages is realized, GM-CSF is added in the induction process of the CAR-M, the polarization of the CAR-M to M1 is promoted, and the killing and phagocytic functions of the CAR-M to tumor cells are enhanced. In an animal model, the curative effect of the CAR-M is evaluated, and the CAR-M can obviously inhibit the proliferation of prostate cancer tumor, delay the progress of tumor and improve the survival time of tumor-bearing mice.

The present disclosure provides a method of constructing a PSMA-targeted chimeric antigen receptor macrophage comprising the following sequentially performed steps:

step 1: an adenovirus expression vector integrated with a Chimeric Antigen Receptor (CAR) targeting PSMA, the nucleotide sequence of which is shown as SEQ ID NO. 9, is constructed, and adenovirus loaded with the CAR sequence targeting PSMA is obtained through packaging.

Step 2: adenovirus was used to infect macrophages, and PSMA-targeted CAR-M cells were obtained by screening.

Further, in step 1, the extracellular domain of the chimeric antigen receptor targeted to PSMA includes a CD8 alpha signal peptide having an amino acid sequence as shown in SEQ ID NO: 1 and a scFv targeted to PSMA; the transmembrane domain of the chimeric antigen receptor targeting PSMA comprises a hinge region and a transmembrane region of CD8 with the amino acid sequence shown as SEQ ID NO. 5; the intracellular domain of the chimeric antigen receptor targeting PSMA also includes an Fc epsilon R1 gamma intracellular region having the amino acid sequence shown in SEQ ID NO. 7.

Further, in step 2, granulocyte-macrophage colony-stimulating factor (GM-CSF) is used to induce CAR-M to exhibit an M1-like phenotype, with high expression of CD80 and CD86.

Further, in step 2, the working concentration of GM-CSF is 10-200 ng/mL.

The disclosure also provides the use of a PSMA-targeted chimeric antigen receptor macrophage in the manufacture of a medicament for treating prostate cancer, primarily for treating PSMA-positive prostate cancer, particularly PSMA-positive advanced prostate cancer, more preferably advanced metastatic castration-resistant prostate cancer.

The present disclosure also provides a composition for treating metastatic castration-resistant prostate cancer. In some embodiments, the composition comprises PSMA-targeted CAR-M cells.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide that is different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes referred to as "chimeric receptor", "T-body (Tbody)" or "Chimeric Immune Receptor (CIR)". An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that signals to cause activation or inhibition of biological processes within a cell. "transmembrane domain" refers to any oligopeptide or polypeptide known to span the cell membrane and which is capable of acting to connect an extracellular domain to a signaling domain. The chimeric antigen receptor can optionally include a "hinge" domain that serves as a linker between the extracellular domain and the transmembrane domain.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, DNA genomes, cdnas, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemical or biochemically modified, non-natural or derivatized nucleotide bases.

The term "encoding" when applied to a nucleic acid sequence refers to a polynucleotide stated to "encode" a polypeptide, either in its native state or when manipulated by methods well known to those of skill in the art, which can be transcribed and/or translated to produce mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be derived therefrom.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different hosts, including, but not limited to, plasmids, viruses, cosmids, phages, BACs, YACs, and the like. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, the viral vectors may be made from baculoviruses, retroviruses, adenoviruses, AAV, and the like, according to techniques known in the art. In one embodiment, the viral vector is an adenovirus vector.

As used herein, the term "modification" means a change in the state or structure of a molecule or cell of the invention. The molecules may be modified in a variety of ways, including chemical, structural and functional modifications. Cells may be modified by nucleic acid introduction.

The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A cell that is "transfected" or "transformed" or "transduced" is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and their progeny.

Non-limiting exemplary polynucleotide sequences encoding components of each domain, for example: "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not taken into account. Also, since the target sequence nucleotide or amino acid is counted, and the nucleotide or amino acid from the reference sequence is not counted, gaps in the reference sequence are not counted.

Percent sequence identity can be calculated by the following procedure: determining the number of positions in which the same amino acid residue or nucleobase occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information, BLAST website (BLAST. Ncbi. Lm. Nih. Gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to make a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, needle, stretcher, water or Matcher, part of the EMBOSS suite of bioinformatics programs, and are also available from European Bioinformatics Institute (EBI) on www.ebi.ac.uk/Tools/psa.

Macrophages are phenotypically heterogeneous immune cells that play an important role during inflammation (initiation and regression). Macrophages can be polarized to two phenotypes upon stimulation: (1) One is the classical activation (inflammatory) phenotype M1, which can be induced by Lipopolysaccharide (LPS) or interferon gamma (IFN-gamma) etc., to produce pro-inflammatory cytokines such as TNF alpha, IL-1 beta etc.; (2) Another alternative is the alternate activation (wound healing) phenotype M2, which can be induced by IL-4, IL-13, etc., to produce anti-inflammatory cytokines such as IL-10, IL-13, arg1, etc. The balance of M1/M2 macrophage polarization determines the fate of an organ in inflammation or injury. M1 exerts a pro-inflammatory effect against stimulation in the early stages of inflammation, but persists to cause tissue damage; m2 exerts an anti-inflammatory effect, promoting tissue repair and revascularization.

The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to human and veterinary subjects, such as humans, animals, non-human primates, dogs, cats, sheep, mice, horses, and cattle. In some embodiments, the subject is a human.

The term "pharmaceutical composition" or "composition" generally refers to a combination of an active agent (e.g., a compound or composition) and a naturally occurring or non-naturally occurring carrier that is inert, e.g., a detectable agent or label, or is active, e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, di-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc., and polysaccharides or sugar polymers), which may be present alone or in combination, individually or in combination, comprise 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin (e.g., human Serum Albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, and the like. Representative amino acid/antibody components that also have buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended to be within the scope of the present technology, examples of which include, but are not limited to: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrins, glucans, starches, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and inositol.

An "effective amount" means that amount of the agent or combined amounts of two or more agents, when administered to treat a mammal or other individual, is sufficient to effect such treatment of the disease. The "effective amount" will vary depending on the agent, the disease and its severity and the age, weight, etc., of the subject to be treated.

The present invention will be further described in detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

The reagents and/or kits used in the following examples are all commercially available or can be synthesized by known methods.

Examples

EXAMPLE 1 construction of expression plasmid and packaging of adenovirus

This example constructs a chimeric antigen receptor plasmid targeting PSMA and packages into adenovirus.

The chimeric antigen receptor comprises a CD 8a signal peptide, a scFv targeting PSMA, a CD8 hinge and transmembrane region, and an fcsr1γ cytoplasmic domain, wherein the sequence information is shown in table 1 below.

TABLE 1

Connecting a gene fragment with a sequence shown as SEQ ID NO. 9 to a multiple cloning site of a universal adenovirus expression vector Ad5/F35 (purchased from a Nanoboat organism) to obtain a CAR molecule over-expression vector for expressing a targeted PSMA, which can be used for expressing the CAR molecule; the CAR sequence is obtained by connecting CD8 alpha, PSMA-scFv-1, CD8 hinge region, CD8 transmembrane region and Fc epsilon R1 gamma intracellular region sequence fragments.

Adenovirus plasmid linearization: the adenovirus plasmid was digested with restriction enzyme Pac I at 37℃and the linearized adenovirus plasmid was dephosphorylated to prevent vector self-ligation, 30 min at 37℃and then placed at 4 ℃.

Adenovirus packaging: 293A cells were seeded at a density of 3X 10 ⁵/mL in 10cm cell culture dishes and incubated overnight at 37℃in a 5% CO ₂ incubator. During transfection, the plasmid solution and the transfection reagent jetPRIME: transfection Reagent (purchased from Polyplus) were mixed uniformly, left at room temperature for 10 minutes, and the mixture was slowly added dropwise to the culture broth of 293A cells, mixed uniformly, and incubated in a 5% CO ₂ incubator at 37 ℃.16 After h, the original medium was discarded, and pre-warmed 10% FBS DMEM medium (available from Gibco) was slowly added to the wall and placed in an incubator.

Virus liquid concentration and purification: after 293A cells are diseased and the culture medium turns yellow, the culture is continued by supplementing 3% FBS DMEM culture medium, and the culture medium is supplemented slowly and lightly to prevent the cells from falling off. After the cells are completely diseased and dead, virus supernatant is collected, frozen in a refrigerator at the temperature of minus 80 ℃ and repeatedly frozen and thawed for 3 times. Centrifuging at 4deg.C and 10000g for 15min to remove cell debris; the supernatant was filtered through a 0.45 μm filter in a 40mL ultracentrifuge tube; centrifuging at 4deg.C for 2 hr at 25000g, removing supernatant after centrifuging, removing residual liquid as much as possible, adding PBS, and lightly and repeatedly blowing to resuspend; after sufficient dissolution, a virus concentrate is obtained and the virus titer is detected.

EXAMPLE 2 construction of PSMA-targeting chimeric antigen receptor macrophages

Chimeric antigen receptor macrophages (CAR-M) were prepared using PSMA-targeting adenoviruses constructed by the method in example 1.

Sorting peripheral blood mononuclear cells: peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh peripheral blood (from syngeneics) using a lymphocyte separation solution (from the next holothurian) by density gradient centrifugation. PBMCs were resuspended in low adhesion 50mL centrifuge tubes and monocytes were sorted using CD14 magnetic beads (purchased from Miltenyi Biotec). 400 Centrifugation at 10 min g, discarding the supernatant, adding MACS buffer (from miltenyi), adding 80. Mu.L of MACS buffer per 1X 10 ⁷ of cells, gently blowing off, adding 20. Mu.L of CD14 beads per 1X 10 ⁷ of cells, gently blowing on, incubating at 4℃at 15 min. 1-2 mL MACS buffer was added per 1X 10 ⁷ of cells and centrifuged at 400 g for 10 min. The separation column was loaded to magnetic force and washed with 3 mL MACS buffer, 500 μl MACS buffer was added per 1×10 ⁸ of cells, the cell suspension was added to the separation column, after the cell suspension had completely passed through the separation column, the centrifuge tube was washed with 3 mL MACS buffer and added to the separation column, and after elution 3 mL MACS buffer was added directly to the separation column and repeated. The cells adsorbed by the magnetic beads were pushed into a centrifuge tube by adding 5 mL MACS buffer, counted, spread in a 10 cm dish according to the cell amount of 5×10 ⁶ cells/dish, and cultured in a 5% CO ₂ incubator at 37 ℃.

Adenovirus infection of peripheral blood derived monocytes: after 5 days of sorting, virus concentrate was added to peripheral blood-derived monocytes at MOI of 1000, and the culture medium was 10% FBS+ XVIVO (purchased from Lonza) +GM-CSF (50 ng/mL) (purchased from offshore protein) and placed in a 5% CO ₂ incubator at 37℃for further culture for 2 days. Chimeric antigen receptor macrophages targeting PSMA were obtained (PBMac).

Example 3 purity detection of PSMA-targeting chimeric antigen receptor macrophages (PBMac)

PBMac obtained in example 2 was used to detect macrophage markers CD11b and CD68 expression using flow cytometry using anti-human CD68 (from Biolegend) and anti-human CD11b (from Biolegend) flow antibodies.

The results of A and B in FIG. 1 show that the purity of macrophages reaches over 80%.

Example 4 detection of the CAR-M phenotype of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

PSMA-targeted chimeric antigen receptor macrophages were constructed in the same manner as in example 2, except that when adenovirus infects peripheral blood-derived monocytes, the medium used was one without GM-CSF (10% fbs+ XVIVO 15), i.e., either with or without GM-CSF during PBMac induction.

PBMac obtained with/without GM-CSF during PBMac induction was used to detect macrophage polarization markers CD80 and CD163 of CAR-M using flow cytometry using anti-human CD80 (purchased from Biolegend) and anti-human CD163 (purchased from Biolegend) flow antibodies.

The results of A and B in FIG. 2 show that the use of GM-CSF can increase CD80 expression while attenuating CD163 expression, indicating that GM-CSF can promote M1 polarization of CAR-M.

Example 5 optimization of Induction System of PSMA-targeting chimeric antigen receptor macrophages (PBMac)

PSMA-targeted chimeric antigen receptor macrophages were constructed in the same manner as in example 2, except that when adenovirus infects peripheral blood-derived monocytes, macrophage colony stimulating factor (M-CSF, purchased from an off-shore protein) was used in the medium instead of GM-CSF, i.e., macrophage colony stimulating factor (M-CSF) was added during PBMac induction instead of GM-CSF.

After PBMac obtained by adding M-CSF or GM-CSF during PBMac induction was co-cultured with Luciferase-expressing target cells 22Rv1 (purchased from Ke Bai) in 96-well plates at different target ratios (1:1, 1:2, 1:5, 1:10) for 48 hours, the following procedure was performed with reference to Steady-Glo Luciferase Assay kit (purchased from Promega): adding 100ml of Steady-Glo cube Luciferase Assay Buffer into 1 bottle of Steady-Glo cube Luciferase Assay Substrate (lyophilized) and uniformly mixing; 50 mu L of prepared Steady-Glo Luciferase Assay reagent is added into each well of a 96-well plate to be detected, after 5 minutes of reaction, the reader reads on an enzyme-labeled instrument, and the smaller the value is, the stronger the killing capacity is.

The results of a and B in fig. 3 show that CAR-M obtained using GM-CSF culture has significantly enhanced ability to kill 22Rv1 and has a significant dose-response relationship.

Example 6 optimization of culture System of PSMA-targeting chimeric antigen receptor macrophages (PBMac)

PSMA-targeted chimeric antigen receptor macrophages were constructed in the same manner as in example 2, except that DMEM medium was used instead of XVIVO medium or 10% fbs was not added to the culture medium used when adenovirus infects peripheral blood-derived monocytes, i.e., DMEM medium or XVIVO medium was used during PBMac induction, with/without 10% fbs.

The induced cell viability of PBMac was examined using a cytometer, and it was found from the results of A in FIG. 4 that using XVIVO15+10% FBS culture PBMac significantly increased the cell viability of PBMac harvested.

The phagocytic capacity of PBMac harvested under different culture conditions was tested. After PBMac was incubated with fluorescent-labeled phagocytic microspheres (purchased from Sigma) for 3 hours, the fluorescence intensity in PBMac was detected in flow. The results of B in FIG. 4 indicate that XVIVO15+10% FBS culture PBMac has the strongest phagocytic capacity.

Example 7 detection of the Positive Rate of CAR-PBMac of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

Using PBMac obtained in example 2, flow cytometry was performed to detect the expression of CAR molecules using human PSMA antigen (purchased from ACROBiosystems). The results of A in FIG. 5 indicate that the transfection efficiency reached 86.12%.

Using DIR and DAPI fluorochromes to label cell membranes and nucleic acids, human PSMA antigen labels CAR molecules, confocal microscopy observed that CAR molecules were expressed on cell membranes, as shown in B in fig. 5.

Example 8 in vitro killing assessment of PSMA-targeted chimeric antigen receptor macrophages (PBMac) by CAR-M

After PBMac (CAR-M) obtained in example 2 was co-cultured with fluorescent-labeled target cells 22Rv1 in a ratio of 1:2 for 4 hours, phagocytic capacity of CAR-M was examined using flow cytometry, and the stronger fluorescence in macrophages indicated the stronger phagocytic capacity of macrophages. The results in figure 6, a, show that CAR-M (42%) exhibits greater tumor phagocytic capacity compared to Mac without adenovirus transfection (UTD, 19%).

PBMac is co-cultured with target cells 22Rv1 expressing luciferase according to different target ratios (3:1, 1:1, 1:3, 1:5, 1:10) for 48 hours, and then luciferase substrate is added, and a fluorescence value is detected by an enzyme-labeling instrument, wherein the smaller value is the stronger the killing capacity. The results of B in fig. 6 demonstrate that CAR-M has significantly enhanced ability to kill 22Rv1 with a significant dose-response relationship.

Example 9 in vitro cytokine release by PSMA-targeted chimeric antigen receptor macrophages (PBMac) of CAR-M

After PBMac (CAR-M) obtained in example 2 was incubated with PSMA-positive tumor cells (purchased from kobai) (1:1), cell culture supernatants were collected and assayed for the levels of various cytokines by ELISA.

The results in FIG. 7 show that the levels of IFN-gamma, IL-2, IL-6 and TNFα secretion by the M1-type cytokines are increased and the levels of IL-10 and IL-4 secretion by the M2-type cytokines are decreased compared to the control group.

Example 10: use of PSMA-targeted chimeric antigen receptor macrophages (PBMac) for the treatment of prostate cancer

Human prostate cancer cells 22Rv1 (purchased from the family herborist) were resuspended, the cell density was adjusted to 5×10 ⁷ cells/mL, male immunodeficiency NCG mice (purchased from the collectable drug) for 6-8 weeks were intraperitoneally injected with 200 μl of cell suspension (the total amount of injected cells was 1×10 ⁷ cells), after molding for 6 hours, mice intraperitoneally injected with PBS buffer were PBS groups, mice intraperitoneally injected with 1×10 ⁷ UTD cells or CAR-M cells (cell density 5×10 ⁷ cells/mL) were UTD groups and CAR-M groups, respectively, 5 mice per group. Tumor burden was continuously observed and recorded during treatment, tumor tissue was collected for histological treatment 25 days after treatment, and survival curves were recorded.

(1) Chinese medicinal effect research of CAR-M in tumor-bearing immunodeficiency mice

In the treatment process, a living body imaging technology is continuously applied, and the tumor load conditions of three groups of mice are analyzed by taking the fluorescence intensity as a judgment basis. The results are shown as a and C in fig. 8.

After some mice were sacrificed at the experimental endpoint time, tumor tissue remaining in the mice was collected and weighed to count tumor tissue mass size. The results are shown as B in fig. 8.

Another part of mice continue to be raised until the mice die or reach the euthanasia standard, and the survival time of the mice is counted. The results are shown as D in fig. 8.

The results of a-D in fig. 8 show that CAR-M injection produces significant inhibition of tumor growth, significantly extending the survival time of mice, compared to PBS and UTD groups during experimental observations.

(2) Safety assessment of CAR-M in tumor-bearing immunodeficiency mice

At the end of the experiment, the main organs (heart, liver, spleen, lung and kidney) of the mice were collected and fixed, and then HE staining was performed, and histopathological examination was performed to observe pathological changes of the organs. The results are shown as a in fig. 9.

Collecting a mouse serum sample for blood biochemical detection: glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), total Bilirubin (TBIL), serum Total Bile Acid (TBA), albumin (ALB), alkaline phosphatase (ALP), UREA nitrogen (UREA), creatinine (CREA). The results are shown as B in fig. 9.

The results of A-B in FIG. 9 show that no abnormality related to administration was found in the experimental results of organ pathology and blood biochemical detection in tumor-bearing immunodeficient mice.

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (13)

1. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a CD8 a signal peptide and a single chain variable fragment that targets a prostate specific membrane antigen, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane region, and the intracellular domain comprises an fcsr1γ intracellular region.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular domain has an amino acid sequence shown in seq id No. 1 or 3; and/or

The transmembrane domain has an amino acid sequence shown in SEQ ID NO. 5; and/or

The intracellular domain has the amino acid sequence shown in SEQ ID NO. 7.

3. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1 or 2.

4. A recombinant vector comprising the nucleic acid molecule of claim 3.

5. A modified cell comprising the nucleic acid molecule of claim 3, or the recombinant vector of claim 4.

6. The modified cell of claim 5, wherein the cell comprises a monocyte, macrophage or dendritic cell; and/or

The modified cells have at least one up-regulated M1 marker and/or at least one down-regulated M2 marker.

7. The modified cell of claim 5, wherein the modified cell has increased expression of CD80 and/or CD86; and/or the modified cell has reduced expression of CD163.

8. A method of constructing a modified cell according to any one of claims 5 to 7, comprising introducing the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the recombinant vector according to claim 4 into a cell.

9. The method of claim 8, comprising inducing the modified cell to exhibit an M1-like phenotype using GM-CSF; and/or

The method comprises culturing the cells using X-VIVO10, X-VIVO15 or X-VIVO20 medium; fetal bovine serum is added to the culture medium.

10. The method of claim 9, wherein the GM-CSF is at a concentration of 10-200 ng/mL; and/or

The concentration of the fetal bovine serum is 8-15%.

11. A pharmaceutical composition comprising the chimeric antigen receptor of claim 1 or 2, the modified cell of any one of claims 5-7, or the modified cell obtained by the method of any one of claims 8-10, and a pharmaceutically acceptable carrier.

12. Use of the chimeric antigen receptor of claim 1 or 2, the modified cell of any one of claims 5-7 or the modified cell obtained by the method of any one of claims 8-10, or the pharmaceutical composition of claim 11, in the manufacture of a medicament for the treatment of prostate cancer.

13. The use according to claim 12, wherein the prostate cancer is metastatic castration-resistant prostate cancer.