WO2023078305A1

WO2023078305A1 - Super dc expressing immune checkpoint inhibitor and use thereof

Info

Publication number: WO2023078305A1
Application number: PCT/CN2022/129306
Authority: WO
Inventors: 莫南德阿德维希; 吴泽吉; 师传胤; 钱其军
Original assignee: 上海细胞治疗集团有限公司
Priority date: 2021-11-02
Filing date: 2022-11-02
Publication date: 2023-05-11
Also published as: CN116064397A

Abstract

Provided is an antigen presenting cell which secretes a PD-1 binding molecule and/or a CTLA-4 binding molecule or contains encoding sequences thereof. The antigen presenting cell can activate a naive T cell and effectively stimulate the effector function of the activated T cell against cancer.

Description

Super DCs expressing immune checkpoint inhibitors and uses thereof

technical field

The invention relates to the field of immunotherapy, in particular to antigen-presenting cells loaded with antibodies and applications thereof.

Background technique

As a new tumor treatment mode, tumor immunotherapy mainly generates anti-tumor immunity by activating the body's immune system, so as to achieve the purpose of eliminating tumor cells. It can not only trigger long-lasting anti-tumor immunity, but also play an important role in preventing postoperative recurrence. Dendritic cells (DCs), as the primary link in the body's specific immune response, are important targets for tumor immunotherapy. DC vaccines use autologous or allogeneic tumor cell components to sensitize patients' peripheral blood-derived monocytes to induce differentiated DCs, thereby directly inducing specific immune responses.

DC vaccine is to introduce DCs loaded with tumor antigens cultured in vitro into the body. These DCs regulate the proliferation and activation of tumor antigen-specific Th1 cells through antigen presentation and secretion of cytokines, and further promote the activation of NK cells and CTLs to mediate tumor killing. Induced or constructed in vitro DC cells that can specifically recognize tumors are injected back into tumor patients, which can activate the immune response of T cells to tumors and express high levels of PD-1 and CTLA-4.

Therapeutic tumor vaccines targeting dendritic cells typically consist of three components. 1) Tumor-associated antigen: the antigen can be a small molecule peptide or a recombinant protein. Tumor antigens expressed by engineered tumor cells, viral vectors and/or engineered bacterial vectors, and DNA or RNA. Antigens expressed or prepared in these different ways may be absorbed and processed by dendritic cells in vivo, and expressed on the cell surface as MHC2, which can be recognized by T cells CD8. Antigens can also be expressed directly on dendritic cells. 2) are dendritic cells (in vivo, isolated and purified, or isolated in peripheral blood leukocytes). 3) Adjuvants or immunostimulants.

Although clinical studies have shown that the DC vaccine induced by the patient's own monocytes can be well tolerated by the patient and can generate an anti-tumor immune response, it is still unable to effectively treat the tumor, which may be related to the lack of specific tumor antigens, antigen load The low efficiency is related to the activation of regulatory T cells after treatment, resulting in immunosuppression. Tumor immune microenvironment and tumor immunosuppressive mechanisms are the key to limit the use of DC vaccines.

The large size of mAbs limits their penetration and distribution in tumor tissues in certain clinical situations. Compared with monoclonal antibodies, the unique structure and biological activity of this small nanobody molecule make it an effective tool for successful immunotherapy. The bulky and complex structures of monoclonal antibodies limit their clinical applications. Nanobodies have a smaller molecular weight, endowing them with strong tissue penetrating power, they can quickly and specifically bind antigens, and unbound Nanobodies can be quickly cleared through renal excretion, resulting in a high target soon after administration - background signal. Thus, the introduction of nanobodies has demonstrated that they can overcome some of the shortcomings of monoclonal antibody-based immunotherapy and immunoimaging.

Contents of the invention

The first aspect of the present invention provides an antigen-presenting cell that secretes a PD-1 binding molecule and/or a CTLA-4 binding molecule and/or contains its coding sequence.

In one or more embodiments, the cells contain and/or express the PD-1 binding molecule and CTLA-4 binding molecule.

In one or more embodiments, the antigen presenting cells are from the peripheral blood of a mammal.

In one or more embodiments, the antigen presenting cells are from mammalian PBMCs.

In one or more embodiments, the antigen presenting cells are loaded with tumor associated antigens or coding sequences thereof. Preferably, the antigen-presenting cell expresses the tumor-associated antigen; the antigen-presenting cell contains the coding sequence of the tumor-associated antigen. More preferably, said antigen presenting cells comprise mRNAs encoding tumor-associated antigens.

In one or more embodiments, the tumor-associated antigens comprise one or more of cancer-testis antigens, overexpressed antigens, and differentiation antigens, for example, one or more selected from the group consisting of hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms tumor 1(WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A , Folate Receptorα(FR-α), HPV16/18-E6, HPV16/18-E7, Alpha Fetoprotein(AFP), Glypican3(GPC3), Prostate-Specific Antigen(PSA), Prostatic Acid Phosphatase(PAP), Prostate-specific membrane antigen(PSMA), Prostate stem cell antigen(PSCA), Six-transmembrane epithelial antigen of prostate 1(STEAP1), B-cell maturation antigen(BCMA), CMV pp65, gp100, PRAME, CA19-9, Synovial Sarcoma(SSX )-2; preferably comprising one or more selected from the group consisting of: MAGE-A3, survivin, CEA.

In one or more embodiments, the amount of anti-PD- ¹ nanobody secreted per 1×10 antigen-presenting cells within 24 hours is 80 ng or more, such as 85 ng or more, 90 ng or more, 95 ng or more, 100 ng or more, 105 ng or more, More than 110ng, more than 115ng, more than 120ng, more than 125ng, more than 130ng, more than 135ng, more than 140ng, more than 145ng, more than 150ng.

In one or more embodiments, the amount of anti-CTLA- ⁴ nanobody secreted per 1×10 antigen-presenting cells within 24 hours is 70 ng or more, such as 75 ng or more, 80 ng or more, 85 ng or more, 90 ng or more, 95 ng or more, More than 100ng, more than 105ng, more than 110ng, more than 115ng, more than 120ng, more than 125ng, more than 130ng, more than 135ng, more than 140ng, more than 145ng, more than 150ng.

In one or more embodiments, the amount of anti-PD-1 Nanobody secreted per 1×10 ⁶ antigen-presenting cells within 24 hours is 119.7 ng or more, and/or, the amount of secreted anti-CTLA-4 Nanobody is 85.9 ng or more.

In one or more embodiments, the PD-1 binding molecule is an anti-PD-1 antibody or antigen-binding fragment thereof.

In one or more embodiments, the CTLA-4 binding molecule is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

In one or more embodiments, the antigen presenting cells are mature or immature dendritic cells.

In one or more embodiments, the coding sequence of the PD-1 binding molecule is RNA, and/or, the coding sequence of the CTLA-4 binding molecule is RNA.

In one or more embodiments, the coding sequence for the tumor-associated antigen is RNA.

The second aspect of the present invention provides a method for producing antigen-presenting cells loaded with tumor-associated antigens, comprising:

(1) Loading the antigen-presenting cells described in the first aspect herein with tumor-associated antigens;

(2) causing antigen-presenting cells loaded with tumor-associated antigens to secrete PD-1 binding molecules and CTLA-4 binding molecules; or

(3) Expose the antigen-presenting cells to the tumor-associated antigen or its coding sequence and the coding sequences of the PD-1 binding molecule and the CTLA-4 binding molecule.

In one or more embodiments, (2) includes allowing the cell to express and secrete the PD-1 binding molecule and the CTLA-4 binding molecule.

In one or more embodiments, (2) includes introducing into the cell coding sequences, such as mRNA, for a PD-1 binding molecule and a CTLA-4 binding molecule.

In one or more embodiments, loading a tumor-associated antigen includes contacting or expressing a tumor-associated antigen, and/or contacting or introducing a coding sequence for a tumor-associated antigen.

In one or more embodiments, the dendritic cells are derived from monocytes.

In one or more embodiments, the dendritic cells are contacted with a maturation composition (eg, maturation cocktails) either before or after the antigen loading. The mature composition comprises one or more selected from IFN-γ, PolyI:C, R848, and PGE2.

In one or more embodiments, the tumors include respiratory system tumors, digestive system tumors, urinary system tumors, nervous system tumors, reproductive system tumors, skin tumors; preferably include one or more selected from the following: liver cancer, Gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulvar cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer , thyroid, gallbladder, kidney, bladder and brain cancers.

In one or more embodiments, the coding sequence for the binding molecule is RNA.

The third aspect of the present invention also provides a pharmaceutical composition, which comprises the antigen-presenting cells described in the first aspect of the present invention or the antigen-presenting cells produced by the method described in the second aspect of the present invention, and pharmaceutically acceptable excipients.

In one or more embodiments, the pharmaceutical composition is used to treat or prevent a tumor expressing the tumor-associated antigen in a subject.

In one or more embodiments, the pharmaceutical composition is a vaccine composition.

The present invention also provides the use of the dendritic cell antigen-presenting cells described in the first aspect of the present invention or the antigen-presenting cells produced by the method described in the second aspect of the present invention in the preparation of medicines, and the medicines are used to prevent the development of tumors in subjects. develop or metastasize, or inhibit the growth or metastasis of a tumor in a subject that expresses the tumor-associated antigen.

In one or more embodiments, the subject is a mammal, such as a human, canine, feline, equine, bovine or porcine.

In one or more embodiments, the subject has or is at risk of developing a tumor.

The present invention also provides a method for preventing or treating tumor occurrence, growth or metastasis in a subject, comprising administering a therapeutically effective amount of the antigen-presenting cell described in the first aspect of the present invention or the antigen produced by the method described in the second aspect of the present invention Presenting cells or the pharmaceutical composition described in the third aspect of the present invention.

In one or more embodiments, the antigen-presenting cells are used to prevent the occurrence of the tumor or reduce the growth and metastasis of the tumor, and the antigen-presenting cells are cultured in vitro with a mature composition before administration, the Antigen-presenting cells are loaded with said tumor-associated antigens.

In one or more embodiments, the maturation composition comprises one or more selected from IFN-γ, PolyI:C, R848, PGE2.

Advantages of the invention: After dendritic cells (DC) are injected, they migrate to the lymph nodes where they encounter naive T cells and activate them. The activation mechanism is as follows: 1. The interaction between MHC on the surface of DC and TCR of T cell; 2. The interaction between B7 molecule of DC and CD28 of T cell. The DCs secreting PD-1 binding molecules and/or CTLA-4 binding molecules or containing their coding sequences of the present invention have the following advantages:

Activation of naive T cell activation, the specific mechanism is: in lymph nodes, Tregs express high levels of CTLA-4, interact with the B7 molecule of DC, and reduce the ability of DC to stimulate tumor-specific T cells. The DCs of the present invention can secrete CTLA-4 binding molecules, block CTLA-4 of Tregs, and release Tregs from reducing the ability of DCs to stimulate tumor-specific T cells, so the DCs of the present invention can effectively activate naive T cells.

After T cells are activated, they will express high levels of PD-1 and CTLA-4. At the tumor site, the following three factors will inhibit the function of tumor-specific T cells and prevent them from killing cancer cells: A. Tumor cells, macrophages Immune cells such as cells express PD-L1, B. Treg expresses CTLA-4 at a high level, and C. Tumor cells express B7 molecules that bind to T cell CTLA-4. However, the DCs of the present invention can secrete PD-1 binding molecules and CTLA-4 binding molecules, so they can block PD-1 and CTLA-4 expressed by T cells, that is, they have been expressed on T cells before T cells migrate to the tumor site. Inhibition of CTLA-4 and PD-1 can effectively stimulate the effector function of activated T cells against cancer.

Moreover, the PD-1 binding molecules and/or CTLA-4 binding molecules in the present invention are nanobodies or antigen-binding fragments thereof, which have the following advantages: small molecular weight, low immunogenicity; high stability; high solubility; high affinity and Cavity binding; strong tissue penetration; high expression yield; easy modification and functional modification, so it can better inhibit CTLA-4 and PD-1.

Some embodiments of the present invention also propose the method of introducing exogenous RNA to make DC vaccine, and the exogenous RNA can trigger the body's innate immunity and acquired immune response. Utilizing this mechanism in tumor therapy, introducing exogenous RNA in the form of a vaccine can promote the release of inflammatory cytokines and interferons, activate NK, macrophages, and effector T cells, and treat "cold tumors" without immune cell infiltration. "Turn into a "hot tumor", promote the killing effect of the immune system on cancer cells, and can also synergistically improve the efficacy of immune checkpoint inhibitor antibodies, that is, "step on the accelerator" while "releasing the brakes" on the immune system, thereby further enhancing Anti-tumor immune response. Compared with traditional vaccines, the safety of mRNA is more advantageous. It will not insert gene mutations, can be degraded by normal cells, and its half-life can be changed by regulating sequence modification and delivery vectors. Many evidences show that mRNA can not only mediate better transfection efficiency and longer protein expression time, but also has greater advantages than DNA, these advantages include: (1) mRNA can function without entering the nucleus. Once in the cytoplasm, the mRNA initiates protein translation. Instead, DNA needs to enter the nucleus and then be transcribed into mRNA. This process makes DNA less efficient than mRNA, since its function depends on the disruption of the nuclear envelope during cell division. (2) Compared with DNA and viral vectors, mRNA does not insert into the genome, but only expresses the encoded protein transiently, so it provides an excellent safe choice for researchers and pharmaceutical companies due to its low insertion risk. (3) mRNA is easily synthesized by in vitro transcription (IVT) process. The process is relatively inexpensive and can be quickly applied to a variety of therapies. Moreover, mRNA can theoretically express any protein, so it can be used to treat almost any disease. This solves the problem of expensive DC vaccine and cumbersome preparation process.

Description of drawings

Figure 1, the amount of antibody secreted by electroporation of different concentrations of anti-PD-1/anti-CTLA-4 mRNA (1194-2VHH) at different times. A: Comparing the PD-1 antibody (a-PD-1) secretion of 1×10 ⁶ cells at 24h and 48h when the electroporation amount was 40ug, 60ug, 80ug, and 100ug/2.5×10 ⁶ cells respectively; B: Comparing electroporation When the amount is 40ug, 60ug, 80ug, 100ug/2.5×10 ⁶ cells, the amount of CTLA-4 antibody (a-CTLA-4) secreted by 1×10 ⁶ cells at 24h and 48h.

Figure 2, the antibody secretion of different concentrations of a-PD-1/a-CTLA-4 mRNA (1194-2VHH) and MAGE-A3 antigen mRNA at different times by electroporation. A: Compare the a-PD-1 secretion of 1×10 ⁶ cells at 24h, 48h, and 72h when the electroporation amounts are 40ug, 60ug, and 80ug/2.5×10 ⁶ cells; B: Compare the electroporation amounts of 40ug and 60ug respectively , 80ug/2.5×10 ⁶ cells, 1×10 ⁶ cells in 24h, 48h, 72h a-CTLA-4 secretion.

Figure 3, the antibody secretion of different concentrations of a-PD-1/a-CTLA-4 mRNA (1194-2VHH) and survivin antigen mRNA at different times by electroporation. A: Compare the a-PD-1 secretion of 1×10 ⁶ cells at 24h, 48h, and 72h when the electroporation amounts are 40ug, 60ug, and 80ug/2.5×10 ⁶ cells; B: Compare the electroporation amounts of 40ug and 60ug respectively , 80ug/2.5×10 ⁶ cells, 1×10 ⁶ cells in 24h, 48h, 72h a-CTLA-4 secretion.

Figure 4, the antibody secretion of different concentrations of a-PD-1/a-CTLA-4 mRNA (1194-2VHH) and CEA antigen mRNA at different times by electroporation. A: Compare the a-PD-1 secretion of 1×10 ⁶ cells at 24h, 48h, and 72h when the electroporation amounts are 40ug, 60ug, and 80ug/2.5×10 ⁶ cells; B: Compare the electroporation amounts of 40ug and 60ug respectively , 80ug/2.5×10 ⁶ cells, 1×10 ⁶ cells in 24h, 48h, 72h a-CTLA-4 secretion.

Figure 5, the amount of antibody secreted by electroporation of a-PD-1/a-CTLA-4 mRNA (1194-2VHH) and three antigen mRNAs at different times. Compare the a-PD-1 of 1×10 ⁶ cells at 24h, 48h, and 72h when the amount of electroporation is (antigen mRNA: 5ug/2.5×10 ⁶ cells) and (1194-2VHH: 60ug/2.5×10 ⁶ cells) and a-CTLA-4 secretion.

Figure 6. Four groups of DC streaming assays. A: Control DCs (immature DCs, iDCs), mature DCs (mDCs), mature DCs were electroporated with three antigen mRNAs (mDC/Ags-mRNA), mature DCs were electroporated with three antigen mRNAs and antibody mRNAs (mDC/[Ags-mRNA] +1194-2VHH]-mRNA, also known as super DC (Super DC) HLA-ABC positive cell MFI comparison. B: Control DC, mature DC, mature DC were electroporated three antigen mRNA, mature DC electroporated three antigen mRNA Comparison of MFI of HLA-DR positive cells with antibody mRNA. C: MFI comparison of CD80 positive cells of control DC, mature DC, mature DC electroporated with three antigen mRNAs, mature DC electroporated with three antigen mRNAs and antibody mRNA. D: control DCs, mature DCs, mature DCs were transfected with three antigen mRNAs, mature DCs were transfected with three antigen mRNAs and antibody mRNA, and MFI of CD86 positive cells was compared. E: Control DC, mature DCs, mature DCs were transfected with three antigen mRNAs, mature DCs Comparison of MFI of CD40 positive cells in which DCs were electroporated with three antigen mRNAs and antibody mRNAs. F: MFI of control DCs, mature DCs, mature DCs electroporated with three antigen mRNAs, and mature DCs electroporated with three antigen mRNAs and antibody mRNAs Compare.

Figure 7, detection of cytokines secreted by DCs. A: Comparison of IL-6 secretion of control DC, mature DC, and mature DC electroporated with three antigen mRNAs, and mature DC electroporated with three antigen mRNAs and antibody mRNA. B: comparison of IL-12 secretion of control DC, mature DC, three antigen mRNAs electroporated by mature DC, three antigen mRNAs and antibody mRNA electroporated by mature DC. C: Comparison of TNF-α secretion of control DC, mature DC, three antigen mRNAs electroporated by mature DC, three antigen mRNAs and antibody mRNA electroporated by mature DC. D: Comparison of IL-10 secretion of control DC, mature DC, three antigen mRNAs electroporated by mature DC, three antigen mRNAs and antibody mRNA electroporated by mature DC.

Figure 8, Immunocytochemical analysis of the expression of MAGE-A3 (A), Survivin (B) and CEA (C) in Super DC.

Figure 9. Four groups of DC flow detection. A: Comparison of control DCs, mature DCs, mature DCs electroporated with three antigen mRNAs, mature DCs electroporated with three antigen mRNAs and 1194-2VHH CCR7 positive cells. B: Comparison of control DCs, mature DCs, mature DCs electroporated with three antigen mRNAs, mature DCs electroporated with three antigen mRNAs and 1194-2VHH CXCR4 positive cells.

Figure 10, the migration function detection of DCs in four groups [control DCs, mature DCs, mature DCs electroporated with three antigen mRNAs, and mature DCs electroporated with three antigen mRNAs and 1194-2VHH]. A: Subgroups to CCL19; B: Subgroups to CCL21; Figure C: Subgroups to CCL19 and CCL21.

Fig. 11, super DC:T co-cultured at different times for antibody secretion and flow cytometric detection of T cell surface markers. A: Super DC: T co-culture 24h, 48h, 96h a-PD-1, a-CTLA-4 antibody secretion; B: Super DC: T co-culture 96h flow cytometry to compare T cells and Super DC: T group CTLA- 4. PD-1 expression percentage.

Fig. 12, detection of T cell surface markers after super DC: T cell co-culture. A: Control DC/T cells, mature DC/T cells, mature DCs electroporated with three antigen mRNAs/T cells, mature DCs electroporated with three antigen mRNAs and antibody mRNAs/T cells expressing CD3 ⁺ CD25 ⁺ percentage comparison. B: Control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNAs/T cells, mature DC electroporated with three antigen mRNAs and antibody mRNA/T cells expressing CD3 ⁺ CD69 ⁺ percentage comparison. C: Control DC/T cells, mature DC/T cells, mature DCs electroporated with three antigen mRNAs/T cells, mature DCs electroporated with three antigen mRNAs and antibody mRNAs/T cells expressing CD3 ⁺ HLA-DR ⁺ percentage comparison. D: Control DC/T cells, mature DC/T cells, mature DCs electroporated with three antigen mRNAs/T cells, mature DCs electroporated with three antigen mRNAs and antibody mRNAs/T cells expressing CD3 ⁺ CD62L ⁺ percentage comparison. E: Control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNA/T cells, mature DC electrotransfected with three antigen mRNA and antibody mRNA/T cell expression CD3 ⁺ CD137 ⁺ percentage comparison. F: Control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNA/T cells, mature DC electroporated with three antigen mRNA and antibody mRNA/T cell expression CD8 ⁺ CD107a ⁺ percentage comparison.

Fig. 13, the amount of secreted cytokines was detected after co-culture of DC:T cells. A: Comparison of control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNAs/T cells, and mature DC electroporated with three antigen mRNAs and antibody mRNA/T cells IFN-γ secretion. B: Comparison of control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNAs/T cells, and mature DC electroporated with three antigen mRNAs and antibody mRNA/T cells TNF-α secretion. C: Comparison of control DC/T cells, mature DC/T cells, mature DCs electroporated with three antigen mRNAs/T cells, and mature DCs electroporated with three antigen mRNAs and antibody mRNAs/T cells for IL-6 secretion. D: Comparison of control DC/T cells, mature DC/T cells, mature DC electroporated with three antigen mRNAs/T cells, and mature DC electroporated with three antigen mRNAs and antibody mRNA/T cells for IL-10 secretion.

Figure 14, control DC/T cells, mature DC/T cells, mature DCs were electroporated with three antigen mRNAs/T cells, mature DCs were electroporated with three antigen mRNAs and 1194-2VHH (super DC)/T cells proliferated.

Fig. 15 , comparison of secretion levels of a-PD-1 (A) and a-CTLA-4 (B) after fresh DCs of the present invention and frozen DCs of the present invention were cultured for 48h, 48h, and 72h.

Detailed ways

The inventors found that antigen-loaded antigen-presenting cells (such as DCs) that secrete immune checkpoint inhibitors can induce stronger anti-tumor immune responses. Antigen-loaded cells activate antigen-presenting cells. Activated antigen-presenting cells are injected into the body to trigger an immune system response in the body. Taking DC as an example, mature DC activates naive T cells and is at the center of initiating, regulating, and maintaining immune responses. Activated DC cells can enter the draining lymph nodes, secrete immune checkpoint inhibitors, promote DC to activate T cells and strengthen the killing effect of T cells.

The present invention provides antigen-presenting cells (APCs) secreting PD1-binding molecules and CTLA4-binding molecules or coding sequences thereof, and vaccine compositions comprising the cells. Antigen-presenting cells refer to cells that can deliver the antigen information they carry to lymphocytes (such as T cells) to trigger an immune response, including macrophages, B cells and dendritic cells (DC cells or DC). Activated antigen-presenting cells are injected into the body to trigger an immune system response in the body. Taking DC as an example, mature DC can induce naive T cells, and is at the center of initiating, regulating, and maintaining immune response. Activated DC cells can enter the draining lymph nodes, secrete PD1-binding molecules and CTLA4-binding molecules, promote DC to activate T cells and enhance the killing effect of T cells.

Herein, DCs may be DCs differentiated from DC precursor cells isolated from the subject's autologous blood, such as CD34+ hematopoietic precursor cells from umbilical cord blood or CD14+ monocytes from peripheral blood. After the autologous DCs obtained after being isolated, cultured, expanded and differentiated from the subject are activated, matured and loaded with the antibodies described herein, a cell mixture preparation containing DCs is obtained. The culture method for DC precursor cells to differentiate into DC can be a method known in the art or any other method that can differentiate DC precursor cells into DC, such as adding cytokines GM-CSF and IL-4 to the medium for differentiation culture . The cell mixture preparation is reinfused into the subject as a DC vaccine, and the subject's autologous mature DC presents the antigen, and activates specific T cells to cause an immune response against the antigenic epitope in the body. In other embodiments, the DC may be obtained from an immortalized DC precursor cell line that is expanded and cultured in vitro and then differentiated and cultured. The immortalized DC precursor cell line can be a cell line known in the art or has been published, such as the MUTZ3 cell line, or an immortalized DC precursor cell line prepared by the method described in CN201810368646.3 . The immortalized DC precursor cell line can be expanded in large quantities in vitro to form DCs through differentiation culture, and the method of differentiation culture can be the aforementioned method. After the DCs obtained from the immortalized DC precursor cell line are expanded and differentiated and cultured, activated, matured, and loaded with the antibodies described herein, a DC-containing cell mixture preparation is obtained, and the cell mixture preparation is injected into the subject as a DC vaccine , and then activate specific T cell responses by presenting the antigen information loaded by DC.

Herein, the antigen-presenting cells may be derived from mammalian peripheral blood (eg, from PBMCs), eg, by cell separation methods. Or the antigen-presenting cells are artificially constructed antigen-presenting cell lines (such as DC cell lines) or antigen-presenting cell precursor cell lines (such as DC precursor cell lines) that can be immortalized, expanded and cultured in vitro.

Antigen-presenting cells are activated by loading with tumor-associated antigens. As used herein, "loading" refers to causing an antigen-presenting cell to contain (capture) a tumor-associated antigen in such a way that the antigen is processed and presented to other immune cells. Taking DC cells as an example, the loading can be carried out in a variety of ways to contact the antigen or its coding sequence, such as incubation with recombinant, synthetic or purified tumor antigen peptides or proteins, incubation with tumor cell lysates, incubation with apoptotic or necrotic The tumor cells are incubated or the cells are made to express the antigen. Expressing an antigen by a cell can be achieved by contacting (eg, co-incubating) the cell with nucleic acid (DNA or RNA) encoding a tumor antigen or introducing (eg, electroporation) the nucleic acid into the cell (eg, by electroporation of RNA). Introduction of a DNA coding sequence into a cell generally involves nucleic acid (DNA) constructs, such as expression vectors and integrating vectors, comprising the DNA sequence together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. Alternatively, the antigen's RNA coding sequence (eg, mRNA) can be introduced directly into cells to express the antigen. Depending on the tumor to be targeted, the antigen can be contacted and loaded with the corresponding tumor antigen or its encoding nucleic acid (such as mRNA). Tumor-associated antigens include, but are not limited to, cancer testis antigens, overexpressed protein/antigens, and differentiation antigens. Tumor-associated antigens exemplarily used herein include: hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms tumor 1 (WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A, Folate Receptorα(FR-α), HPV16/18-E6, HPV16/18-E7, Alpha Fetoprotein(AFP), Glypican3(GPC3), Prostate -Specific Antigen (PSA), Prostatic Acid Phosphatase (PAP), Prostate-specific membrane antigen (PSMA), Prostate stem cell antigen (PSCA), Six-transmembrane epithelial antigen of prostate 1 (STEAP1), B-cell maturation antigen (BCMA ), CMV pp65, gp100, PRAME, CA19-9, Synovial Sarcoma (SSX)-2. The means of antigen loading are known in the art, such as incubation, cell transformation (such as electroporation of DNA or mRNA) and the like. Herein, the antigen or its coding sequence is in a soluble form or the antigen or its coding sequence is linked to a solid carrier. The solid support may include polystyrene beads. The solid phase carrier is biodegradable.

Antigen-presenting cells (eg, DCs) are induced to mature by exposure to maturation compositions (maturation cocktails). The mature composition exemplarily used herein comprises one or more selected from IFN-γ, PolyI:C, R848, PGE2. The dendritic cells are contacted with the maturation composition for at least 10 hours, at least 20 hours, at least 30 hours, or at least 40 hours.

The sequence of activation (antigen-loading) and maturation of antigen-presenting cells is generally not particularly limited, ie, cells can be loaded with antigen and then exposed to a cytokine composition, or antigens can be exposed to a cytokine composition and then loaded with antigen. This is within the knowledge of those skilled in the art.

The antigen presenting cells herein contain, express, and/or secrete PD1 binding molecules and CTLA4 binding molecules. In this paper, "PD1" and "PD-1" can be used in common, both refer to programmed cell death protein 1 (programmed cell death protein 1); "CTLA4" and "CTLA-4" can be used in common, both refer to cytotoxic T lymphocytes Associated protein 4 (cytotoxic T-lymphocyte-associated protein 4). Herein, "PD1-binding molecules" and "CTLA4-binding molecules" are proteins that specifically bind to PD1 and CTLA4, respectively, including, but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affinity receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), Diabodies and single chain molecules, as well as antibody fragments, especially antigen binding fragments, eg, Fab, F(ab')2 and Fv). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH1, CH2, CH3 and CH4) constant domain (CH) and the hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH and CL is aligned with the first constant domain (CH1) of the heavy chain. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic and Clinical Immunology, Eighth Edition, edited by Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, pp. 71 and 6 chapter. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

A "heavy chain antibody" as described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibodies, the heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of variable region (VHH) and other constant regions. The variable region passes through a structure similar to the hinge region linked to the constant region. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous heavy chain antibody contains 1 variable region and 5 Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc.

As used herein, the terms "single domain antibody", "anti-mesothelin single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", "Nanobody" are used interchangeably and refer to A single domain antibody that specifically recognizes and binds to mesothelin. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Single domain antibodies are the smallest functional antigen-binding fragments. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

In one or more embodiments, the anti-PD-1 antibody of the present invention is a Nanobody, having any of SEQ ID NO: 1, 4-39, 320 in CN202011582908.X (preferably the SEQ ID NO in this patent application Any of: 19, 36-39, 320, i.e. any of CDR1, SEQ ID NO: 2, 40-75 shown in the SEQ ID NO: 1-6 of the sequence listing attached hereto (preferably in this patent application Any of SEQ ID NO:53, 55, 72-75, i.e. CDR2 shown in SEQ ID NO:7-12 of the sequence listing attached hereto, and any of SEQ ID NO:3, 76-183 (preferably Any one of SEQ ID NO:87,108-183 in this patent application, i.e. the CDR3 shown in SEQ ID NO:13-89 of the sequence listing attached hereto. The VHH of the anti-PD-1 antibody is shown in any one of SEQ ID NO: 184-319 in CN202011582908.X (ie, SEQ ID NO: 90-225 in the sequence listing attached hereto). Exemplarily, the CDR1-3 of the anti-PD-1 nanobody (1194-NLA) is respectively shown in the sequence listing SEQ ID NO: 1, 10, and 13 attached hereto; the anti-PD-1 nanobody (1194-NLA) VHH is as shown in the sequence listing SEQ ID NO:137 attached hereto.

In one or more embodiments, the anti-CTLA4 antibody of the present invention is a nanobody, having SEQ ID NO: 1, 4-10 in CN202111152925.4 (preferably any of SEQ ID NO: 4-10 in this patent application , that is, CDR1, SEQ ID NO:2, 11-18 shown in the SEQ ID NO:226-232 of the sequence listing attached hereto (preferably any one of SEQ ID NO:11-18 in the patent application, that is, any one of the herein described The CDR2 shown in the SEQ ID NO:233-240 of the attached sequence listing, and SEQ ID NO:3,19-26 (preferably any one of the SEQ ID NO:19-26 in this patent application, namely the sequence listing attached hereto CDR3 shown in SEQ ID NO:241-248). The VHH of the anti-CTLA4 antibody is shown in any one of SEQ ID NO: 27-73 in CN202111152925.4 (ie, SEQ ID NO: 249-295 in the sequence listing attached hereto). Exemplarily, the CDR1-3 of the anti-CTLA4 nanobody (Z12) is shown in the sequence listing SEQ ID NO:230, 238, and 246 attached hereto; the VHH of the anti-CTLA4 nanobody (Z12) is shown in the sequence listing SEQ ID NO:246 attached hereto. ID NO:258.

A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificities is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies through a linker. The linker usually consists of 1-15 amino acids selected from G and S.

Herein, heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to the similarity in the structures of the two, the following structural descriptions for antibodies are also applicable to heavy chain antibodies except for the light chain.

"Variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.

The term "variable" refers to the fact that certain segments of the variable domains vary widely among antibody sequences. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across all amino acids spanned by a variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 (heavy Chain antibodies may be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3 of the light chain variable region. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation connected by the formation of loops and in some cases forming β-sheet structures Part of three HVR connections. The HVRs in each chain are held together in close proximity by the FR regions and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site. Generally, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the involvement of antibodies in antibody-dependent cell-mediated cytotoxicity.

"Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including those located in the immune system. Binding to Fc receptors on various cells (eg, effector cells), or to the first component (Clq) of the classical complement system. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and/or variable region of an intact antibody. Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; Or any fragment that should be able to increase the half-life by incorporation into liposomes. Digestion of antibodies with papain yields two identical antigen-binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name reflecting its ability to readily crystallize. The Fab fragment consists of the complete light chain and the variable domains of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody yields a larger F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of the two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) protrude from the fold of these two domains, contributing the amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower avidity than the full binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen-binding structure.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation, ), the individual antibodies constituting the population were identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody has acquired characteristics from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and the use of antibodies that have part or all of the human immunoglobulin loci or encode human immunoglobulin loci. A technique for producing human or human-like antibodies from the genes of immunoglobulin sequences, and single-cell sequencing.

Monoclonal antibodies also include herein "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remaining portions are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity.

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Thus, "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions have been exchanged with sequences found in human antibodies. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by or is identical to such an antibody (except for the CDRs) by a polynucleotide of human origin. CDRs, some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the β-sheet framework of human antibody variable regions to produce antibodies whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies and the like all include humanized variants of each of these antibodies.

A "human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or has been produced using any of the techniques disclosed herein for the production of human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

In some embodiments, the invention also provides a single domain antibody, heavy chain antibody, antibody or antigen thereof that binds to the same epitope of PD-1 (or CTLA4) as any anti-PD-1 antibody (or anti-CTLA4 antibody) of the invention Binding fragments, that is, single domain antibodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof capable of cross-competing with any antibody of the invention for binding to PD-1 (or CTLA4).

The binding molecules described herein may be monovalent or multivalent antibodies (monovalent or multivalent antibody single domains), heavy chain antibodies or antigen-binding fragments thereof comprising one, two or more antibodies described herein. The heavy chain antibody also comprises a heavy chain constant region, such as that of a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.

The present invention also includes said antibody derivatives and analogs. "Derivatives" and "analogues" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present invention. Derivatives or analogs of the present invention may be (i) polypeptides having substituent groups in one or more amino acid residues, or (ii) mature polypeptides in combination with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol A polypeptide formed by fusion of diol), or (iii) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify this polypeptide, or with a 6His tag formed fusion protein). Such derivatives and analogs are within the purview of those skilled in the art from the teachings herein.

Under the premise of not substantially affecting the activity of the antibody, those skilled in the art can change one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) of the sequence of the present invention. Multiple) amino acids to obtain variants of the antibody or functional fragment sequence thereof. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. In this field, conservative substitutions with amino acids with similar or similar properties usually do not change the function of the protein. Amino acids with similar properties are substituted eg in the FR and/or CDR regions of the variable region. Amino acid residues that may be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the protection scope of the present invention. Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and those capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions The protein encoded by DNA, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention. In some embodiments, the sequence of the variant described herein may be at least 95%, 96%, 97%, 98%, or 99% identical to its source sequence. Sequence identity according to the invention can be measured using sequence analysis software. For example the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The present invention also includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein .

Antibodies or their coding sequences can be introduced into antigen-presenting cells in the form of protein, RNA or DNA. For example, DNA vectors expressing antibodies are constructed and antigen-presenting cells are transformed. Accordingly, the invention also includes polynucleotides (in DNA or RNA form) encoding the antigens or antibodies described herein, or fragments thereof, as well as nucleic acid constructs (eg, expression vectors and integrating vectors) comprising these polynucleotides. The vectors described herein generally contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively referred to in certain embodiments as "flanking sequences") typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing and the complete intron sequence of the acceptor splice site, sequence encoding the leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, polylinker for insertion of nucleic acid encoding the antibody to be expressed zone and optional marker elements.

The polynucleotide introduced into antigen-presenting cells of the present invention may be in the form of DNA or RNA (eg, mRNA). The inventors found that compared with traditional vaccines, mRNA has more safety advantages, for example, it will not insert gene mutations, can be degraded by normal cells, and its half-life can be changed by regulating sequence modification and delivery vectors, etc. Methods for introducing mRNA into antigen-presenting cells (eg, DC cells) are well known in the art, eg, by electroporation. In an exemplary embodiment, an exemplary coding sequence of an anti-PD-1 antibody is shown in SEQ ID NO: 296 63-434; an exemplary coding sequence of an anti-CTLA-4 antibody is shown in SEQ ID NO: 296 573- 950 bits shown.

The coding sequence of the antibody may also have a signal peptide to direct antibody secretion, and those skilled in the art know signal peptides that can be used in the present invention. Exemplary signal peptides include: human kappa chain signal peptide coding sequence (eg, SEQ ID NO: 296, 3-62), human immunoglobulin light chain signal peptide (eg, SEQ ID NO: 296, 510-572).

The present invention provides a method for preparing the DC cells, including enabling dendritic cells to have the ability to secrete PD-1 binding molecules (such as antibodies) and CTLA-4 binding molecules (such as antibodies) and to load tumor-associated antigens. The method may comprise the steps of: (1) loading dendritic cells capable of secreting PD-1 binding molecules and CTLA-4 binding molecules with tumor-associated antigens or coding sequences thereof; or (2) loading dendritic cells loaded with tumor-associated antigens The dendritic cells are capable of secreting PD-1 binding molecules and CTLA-4 binding molecules; or (3) exposing dendritic cells to tumor-associated antigens or coding sequences thereof and coding sequences of PD-1 binding molecules and CTLA-4 binding molecules. The antigen-loading method is as described above, for example, by contacting the cells with the antigen or its encoding nucleic acid (DNA or RNA) (e.g. co-incubation) or introducing (e.g. electroporation) the antigen or its encoding nucleic acid (e.g. by electroporation of RNA).

Commonly used methods for making cells secrete antibodies or have the ability to secrete antibodies include expressing the antibodies. The process of expressing an antibody in a cell is known in the art, for example, introducing nucleic acid encoding the antibody (eg, DNA or RNA) into the cell. Introduction of a DNA coding sequence into a cell generally involves nucleic acid (DNA) constructs, such as expression vectors and integrating vectors, comprising the DNA sequence together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. Exemplary vectors can be found in CN105154473A and CN111206043A, which are incorporated herein by reference in their entirety. Alternatively, the antibody's RNA coding sequence (eg, mRNA) can be introduced directly into cells to express the antibody.

In the present invention, the DC cells containing the coding sequence of the anti-PD-1 nanobody and/or the coding sequence of the anti-CTLA-4 nanobody can express the nanobody at a high level. ^{For example, when the mRNA of anti-PD-1 nanobody and/or anti-CTLA-4 nanobody is electrotransferred into cells at an amount of 60ug/2.5×10 6} ^cells , the a-PD- 1 (Anti-PD-1 nanobody) secretion is 80ng or more, such as 85ng or more, 90ng or more, 95ng or more, 100ng or more, 105ng or more, 110ng or more, 115ng or more, 120ng or more, 125ng or more, 130ng or more, 135ng or more, 140ng or more Above, above 145ng, above 150ng, preferably above 119.7ng; a-CTLA-4 (anti-CTLA-4 nanobody) secretion is above 70ng, such as above 75ng, above 80ng, above 85ng, above 90ng, above 95ng, above 100ng , 105 ng or more, 110 ng or more, 115 ng or more, 120 ng or more, 125 ng or more, 130 ng or more, 135 ng or more, 140 ng or more, 145 ng or more, 150 ng or more, preferably 85.9 ng or more.

The RNA coding sequence of the antibody can be synthesized by a gene company or obtained by in vitro transcription. Those skilled in the art are aware of methods for preparing RNA sequences by in vitro transcription. An exemplary in vitro transcription method includes the steps of constructing a transcription template DNA vector and incubating in a transcription system. Transcription system and incubation conditions are well known in the art, such as transcription system: transcription buffer (including but not limited to Tris-HCl, MgCl2, DTT, spermidine), NTP, RNase inhibitor, RNA polymerase, etc.; incubation conditions such as 37 °C for at least 2 hours.

The coding sequences of the respective antibodies can be located on separate nucleic acid constructs or combined in a suitable manner on the same nucleic acid construct. In the example of expressing an antibody by mRNA, the mRNA sequences encoding each antibody can be combined and expressed separately on the same nucleic acid construct by linking the mRNA sequences encoding each antibody. The linker includes, but is not limited to: the coding sequence of the Furin cleavage site, the coding sequence of 2A (such as F2A, T2A), the IRES sequence and the like. Exemplarily, the linker is Furin-GSG-T2A, and its coding sequence is shown in positions 435-509 of SEQ ID NO:296. The coding sequence of an antibody usually also contains a stop codon (such as TGATAA), and may contain a restriction site for genetic engineering manipulation.

In addition, in the embodiment of expressing tumor-associated antigens and binding molecules (such as PD-1 and CTLA-4 binding molecules, such as antibodies), the coding sequences of tumor-associated antigens and binding molecules can be introduced into cells separately or simultaneously. Similarly, the coding sequences of the tumor-associated antigen and the respective binding molecules can be located on separate nucleic acid constructs or combined in a suitable manner on the same nucleic acid construct.

Specific steps for preparing DC cells For example, antigen presenting cells (APC) such as DC are obtained from a subject, such as a patient suffering from cancer or at risk of developing cancer, using leukapheresis. The purified dendritic cells are cultured in the presence of the maturation composition to obtain mature DC cells. Mature DC cells are loaded with antigens (such as MAGE-A3, survivin, CEA), for example, by electroporation of mRNA encoding antigens, and then mature DCs and DC vaccines containing antigens are obtained. Before, at the same time or after antigen loading, DC cells can express the PD-1 binding molecules and CTLA-4 binding molecules described herein, such as electroporation of mRNA of anti-PD-1 antibody and anti-CTLA-4 antibody. The antigen-loaded and activated DCs are then administered to the patient. An exemplary course of treatment includes 3 administrations of DC over a period of 4 weeks.

Herein, the culture medium and culture conditions required in the process of preparing DC cells can adopt the conditions for conventionally culturing DC cells. Exemplary media and culture conditions are shown in the Examples.

Antigen presenting cells (eg, DC cells) described herein can be used in the preparation of pharmaceutical compositions, eg, DC vaccines, for the prevention or treatment of various conditions and diseases described herein. The conditions and diseases are primarily the occurrence, growth and/or metastasis of tumors (cancers) including, but not limited to: lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer Cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, mesothelioma, colorectal cancer, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma, bone cancer, Pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, skin or intraocular melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, Non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, bile duct cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, urothelial cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, cancer of the kidney or ureter, cancer of the renal pelvis, central nervous system ( CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, Hodgkin's lymphoma, epidermal carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, various leukemias and lymphomas, and various precancerous lesions. Especially for tumors sensitive to anti-PD-1 antibody and anti-CTLA-4 antibody.

The pharmaceutical composition of the present invention may be different antigen-presenting cells loaded with different antigens, or one antigen-presenting cell loaded with multiple antigens. In an exemplary embodiment, the pharmaceutical composition comprises three antigen-presenting cells (such as DC cells) expressing the PD1-binding molecule and the CTLA4-binding molecule described herein loaded with MAGE-A3, survivin, and CEA, respectively. The concentration and ratio of various antigen-presenting cells in the pharmaceutical composition can be adjusted by those skilled in the art as needed. Exemplarily, the above three antigen-presenting cells are included in the pharmaceutical composition in equal proportions.

In addition to the DC cells described herein, the pharmaceutical compositions herein also contain pharmaceutically acceptable adjuvants, including but not limited to diluents, carriers, solubilizers, emulsifiers and/or preservative adjuvants. The excipient is preferably nontoxic to recipients at the dosages and concentrations employed. Such excipients include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, a pharmaceutical composition may contain ingredients for improving, maintaining or retaining, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of a substance. These substances are known from the prior art. The optimum pharmaceutical composition will be determined by the intended route of administration, mode of delivery and desired dosage.

Adjuvants in pharmaceutical compositions also include vaccine adjuvants. The adjuvant can be a compound small molecule, biomacromolecule, composition, complex or extract known in the art that can enhance the effect of immune response. In one or more embodiments, the adjuvant comprises an adjuvant selected from aluminum adjuvant (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, Alpha interferon, Corynebacterium pumilus, lipopolysaccharide, cytokines, oil-in-water emulsion, water-in-oil emulsion, nanoemulsion, microparticle delivery system, liposome, microsphere, biodegradable microsphere, plaque virion, protein Liposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro/nanoparticles, polymeric lamellar substrate particles (PLSP), Microparticle resins, nanolipogels, synthetic/biodegradable and biocompatible semi-synthetic or natural polymers or dendrimers (such as PLG, PLGA, PLA, polycaproic acid ester, silicone polymer, polyester, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyltrimethylammonium chloride, polystyrene divinylbenzene resin, polyphosphazene, poly -[di-(carboxyacetylphenoxy)phosphazene (PCPP), poly-(methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and its derivatives, chitosan and its derivatives, Polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (such as polyamino acids, poly-(γ-glutamic acid), poly-arginine-HCl , poly-L-lysine, polypeptide, biopolymer), cationic dimethyl di(octadecyl) ammonium (DDA), α-galactoside ceramide and its derivatives, archaeal lipids and One or more of derivatives, lactams, gallen, glycerides, phospholipids and spirochetes.

Pharmaceutical compositions for in vivo administration are generally presented as sterile preparations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, this method can be used for sterilization either before or after lyophilization and reconstitution. Pharmaceutical compositions of the invention may be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliary agents. Parenteral compositions are usually presented in containers with sterile access ports, eg, intravenous solution strips or vials with a hypodermic needle-punctureable stopper. Alternatively, compositions may be selected for inhalation or delivery through the alimentary tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating various other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads and depot injections, are also known to those skilled in the art.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals or as dehydrated or lyophilized powders. The formulations can be stored in a ready-to-use form or reconstituted (eg, lyophilized) before administration. The invention also provides kits for producing single dosage administration units. The kits of the invention may each contain a first container with a dry protein and a second container with an aqueous formulation. In certain embodiments of the invention, kits containing single and multi-lumen prefilled syringes (eg, liquid syringes and lyophilized syringes) are provided.

The present invention also provides methods of treating a patient, especially a patient with a mesothelin-related disease, by administering a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof. Herein, the terms "patient", "subject", "individual", "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog, cat , rabbits, etc.), and most preferably humans. "Treatment" refers to the subject's use of the treatment regimens described herein to achieve at least one positive therapeutic effect (e.g., a decrease in cancer cell number, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth. ). "Prevention" refers to the use of the treatment regimens described herein in a subject at risk to at least one effect prevent the occurrence of a disease or symptom. Therapeutic regimens that effectively treat or prevent a patient may vary depending on a variety of factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

The therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the invention to be employed will depend, for example, on the extent and goal of the treatment. Those skilled in the art will appreciate that appropriate dosage levels for therapy will depend in part on the molecule being delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. conditions) vary. In certain embodiments, the clinician can titrate the dose and vary the route of administration to achieve optimal therapeutic effect. For example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.

The frequency of dosing will depend on the pharmacokinetic parameters of the binding molecule in the formulation used. The clinician typically administers the composition until a dosage is reached to achieve the desired effect. The composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is according to known methods, such as oral, injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Either by a sustained release system or by an implanted device. In one or more embodiments, the pharmaceutical composition as a vaccine may be administered to the inguinal segment by intranodal injection. Alternatively, depending on the target of the vaccine, the vaccine may be administered subcutaneously or intradermally to the extremities of cancer patients undergoing treatment. Other routes of administration, such as intramuscular injection or blood injection can also be used.

Depending on the type of pharmaceutical composition (e.g. vaccine) being prepared, the pharmaceutical composition can be scaled up, if desired, by culturing cells in bioreactors or fermentors or similar vessels and apparatus suitable for bulk growth of cells. to expand. In one or more embodiments, according to the present invention, the device or composition containing the vaccine or antigen produced or recovered is suitable for sustained or intermittent release, and can be implanted in the body or locally administered at the corresponding location in the body. administered to achieve a slow and timed release of these materials into the body.

The present invention also provides a method for treating and/or preventing cancer, the method comprising administering effective doses of one or more of the aforementioned cells and pharmaceutical compositions to a subject. The method includes at least one of both therapeutic and prophylactic effects. In one or more embodiments, the method of the present invention is for prophylactic purposes, and one or more of the cells and pharmaceutical compositions of the present invention are administered to the subject before cancer or precancerous lesions occur. In some cases, the pharmaceutical composition is administered to the individual subject after the onset of one or more of the above cancers, in order to prevent further symptoms from appearing or worsening of existing symptoms. Prophylactic administration of one or more of the cells and pharmaceutical compositions described herein is intended to prevent or alleviate any subsequent symptoms. In one or more embodiments, the method of the present invention is for therapeutic purposes, and one or more of the cells and pharmaceutical compositions of the present invention are administered to the subject when or after cancer occurs, aiming at alleviating Symptoms of cancer that has developed.

The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples, unless otherwise stated, are conventional materials and methods in the art.

Example

experimental method

Antibody mRNA preparation

1194-NLA of anti-PD1 VHH (positions 63-434 of SEQ ID NO:296) and Z12 of anti-CTLA4 VHH (positions 573-950 of SEQ ID NO:296) were ligated by Furin-GSG-T2A with 5' addition of Nco I Restriction site and human κ chain signal peptide sequence, human immunoglobulin light chain signal peptide is added between Z12 of CTLA4 VHH and Furin-GSG-T2A, and two stop codons TGATAA and Sal I restriction site are added at 3', Entrust Jinweizhi Company to synthesize the sequence 1194-NLA-T2A-Z12 (SEQ ID NO: 296). Self-constructed EGFP transcription template vector pT7-m5U-eGFP, containing T7 promoter, 5'UTR sequence of HBB mRNA (NM_000518), EGFP sequence, HBB mRNA3'UTR sequence and polyA sequence (SEQ ID NO: 297), through Nco I and The 2118bp backbone fragment was recovered by double digestion with Sal I, and the 956bp target fragment recovered by the same digestion with the above synthetic sequence was ligated to obtain the transcription template pT7-m5U-1194-2VHH.

The in vitro transcription reaction produces mRNA. The in vitro transcription system includes: template cDNA 1.0μg, 10×transcription buffer (400mM Tris-HCl pH 8.0, 190mM MgCl2, 50mM DTT, 10mM spermidine) 2.0μl, NTP (25mM each) 7.2μl, RNase inhibitor 20U , T7 RNA polymerase 3000U, add dH20 to 20.0μl.

Incubate at 37°C for 3 hours to 5 hours. The crude IVT mixture can be stored overnight at 4°C for purification the next day. The original template was removed by 1 U RNase-free DNase digestion at 37°C for 15 minutes, and mRNA was purified using Ambion's MEGACLEAR(TM) kit (Austin, TX) following the manufacturer's instructions. This kit can purify up to 500μg of RNA. After purification, the RNA was quantified using NanoDrop and analyzed by agarose gel electrophoresis to confirm that the RNA was of the appropriate size and that the RNA was not degraded.

Antigen mRNA preparation

Antigen mRNA was prepared in a similar manner to antibody mRNA.

DC cell culture and induction

(1) Isolation of PBMC

1) Use a syringe to draw blood from the blood bag into a 50ml centrifuge tube, wash the blood bag with the same volume of PBS (branded as HyClone), and mix with the blood taken out (1:1 dilution);

2) Take a 50ml centrifuge tube (with 15ml of Ficoll Lymphocyte Separation Medium (the brand is GE)) and slowly add the blood and PBS mixture in step 1 along the tube wall, then centrifuge at 800g, 20min, at a speed up of 1, and a speed down of 0 .

3) Carefully pipette the white cell layer into another 50ml centrifuge tube, add PBS, and centrifuge at 400g/min for 10min with a ramp rate of 9.

4) Pour off the waste liquid without pouring it out, add DPBS, wash again, and centrifuge (same as step 3).

5) Pour off the waste liquid, add culture medium AIM-V (the brand is gibco) to suspend the cells, add it into a culture flask, and stick to the wall overnight.

(2) DC cell generation

Monocyte-derived DCs are generated from peripheral blood mononuclear cells (PBMCs) by standard Ficoll density centrifugation to isolate PBMCs from patient leukapheresis samples. PBMC were seeded in serum-free AIM-V medium and allowed to adhere to culture flasks with 0.22 μm filter caps. After 2 hours, non-adherent cells were removed, and adherent monocytes were subsequently cultured in AIM-V containing 50 ng/ml rhIL-4 and 100 ng/ml rhGM-CSF for 6 days. On day 3, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. A maturation mixture consisting of 100 IU/ml IFN-γ, 30 μg/ml poly(I:C), 5 μg/ml R848, and 1 μg/ml PGE2 was used to induce DC maturation for 24 hours.

(3) DC cell transformation

Prepare the mRNA encoding anti-PD-1 and anti-CTLA-4 nanobody sequences (hereinafter referred to as antibody mRNA) required for the preparation of the electroporation reagent. According to the instructions of the LONZA electrotransfer kit, prepare an electrotransfer reagent with an antibody mRNA content of 60ug/2.5*10e6cells. Collect DCs induced and matured from mononuclear sources. After electroporation according to the instructions of the LONZA electroporation kit, transfer the cells to a 24-well plate and add AIM-V medium for culture, so that the cell density in the well plate is 1*10e6/ml. The volume is 1 ml. After culturing for 3 days, the cell supernatants were collected at 24h, 48h, and 72h to detect the amount of anti-PD-1 and anti-CTLA-4 nanobodies by ELISA.

Prepare the antibody mRNA and three kinds of mRNAs encoding antigen sequences (hereinafter referred to as MAGEA-3 mRNA, CEA mRNA, Suvivin mRNA) required for the preparation of electroporation reagents, among which the NCBI Reference Sequence of MAGEA-3 mRNA: NM_005362.4, and the NCBI Reference of CEA mRNA Sequence: NM_004363.6, NCBI Reference Sequence of Suvivin mRNA: NM_001168.3). According to the instructions of the LONZA electrotransfer kit, prepare electrotransfer reagents corresponding to MAGEA-3 mRNA; CEA mRNA; Suvivin mRNA at 5ug/2.5*10e6cells. Then prepare electrotransfer reagents for antibody mRNA+MAGEA-3 mRNA; antibody mRNA+CEA mRNA; antibody mRNA+Suvivin mRNA (antibody mRNA is 60ug/2.5*10e6cells, antigen mRNA is 5ug/2.5*10e6cells). Collect DCs induced and matured from mononuclear sources and divide them into 6 parts. According to the instructions of the LONZA electroporation kit, electroporation corresponding to the above electroporation solution was carried out, and the cells transfected with 3 kinds of antigen mRNA were mixed; Mix the antigen + antibody mRNA cells, transfer the cells to a 24-well plate and add AIM-V medium for culture, so that the cell density in the well plate is 1*10e6/ml, the volume is 1ml, and set immature DC and mature DC as an experimental control. After culturing for 3 days, DCs were collected at 24 hours to detect the DC function after electroporation. Cell supernatants were collected at 24h, 48h, and 72h to detect anti-PD-1 and anti-CTLA-4 nanobodies by Elisa method; detect cytokine IL-12 by Elisa method; detect cytokine IL-6, IL-10, TNF-alpha.

Prepare the antibody mRNA and three mRNAs encoding antigen sequences (hereinafter referred to as MAGEA-3 mRNA, CEA mRNA, Suvivin mRNA) required for the preparation of electroporation reagents. According to the instructions of the LONZA electrotransfer kit, prepare electrotransfer reagents corresponding to MAGEA-3 mRNA; CEA mRNA; Suvivin mRNA at 5ug/2.5*10e6cells. Then prepare electrotransfer reagents for antibody mRNA+MAGEA-3 mRNA; antibody mRNA+CEA mRNA; antibody mRNA+Suvivin mRNA (antibody mRNA is 60ug/2.5*10e6cells, antigen mRNA is 5ug/2.5*10e6cells). Collect DCs induced and matured from mononuclear sources and divide them into 6 parts. According to the instructions of the LONZA electroporation kit, electroporation corresponding to the above electroporation solution was carried out, and the cells transfected with 3 kinds of antigen mRNA were mixed; Mix the antigen + antibody mRNA cells, transfer the cells of each group to a 12-well plate and add 1640 medium for culture, so that the number of DC cells in the well plate is 2*10e5, and the volume is 1ml, and set immature DC and mature DC as Experimental control. After 2 hours, add unactivated T cells and activated T cells to the grouped DCs (placed in a CD3\CD28-coated culture plate and culture for 48 hours), so that the DC in the well plate: T=1:10=2* The number of 10e5:2*10e6 cells, the volume is 2ml, cultured for 3 days, and collected T cells at 48 hours to detect T cell function by flow cytometry. Cell supernatants were collected at 24h, 48h, and 72h, respectively, for detection of anti-PD-1 and anti-CTLA-4 nanobodies by Elisa method; and detection of cytokines IFN-γ, TNF-α, IL-6, and IL-10 by CBA method.

flow cytometry

The basic steps of streaming detection are as follows:

(1) Add 1×10 ⁶ cells to each tube.

(2) Add 1ml PBS phosphate buffered saline to wash twice, centrifuge at 400g for 5min, discard

Supernatant; add 100 μL PBS phosphate buffer for resuspension;

(3) Add the flow antibody to be detected, mix well, place in the refrigerator at 2-8°C, and incubate in the dark for 30 minutes;

Set up a group of blank controls, without adding reagents or adding corresponding isotypes;

(4) Add 1ml of PBS phosphate buffered saline, centrifuge at 400g for 5min, wash twice, discard the supernatant,

Draw 400 μl of PBS phosphate buffered saline to resuspend the cells, use a flow cytometer to detect, and set up all cells

Collect 1×10 ⁴ cells. Data were analyzed using Kaluza Analysis software.

The detection is as shown in the above steps, and the different antibodies added are shown in the table below:

DC detection

检测项目抗体Detection Item Antibody	发光信号luminous signal
CD80(Biolegend)CD80 (Biolegend)	FITCFITC
CD83(Biolegend)CD83 (Biolegend)	APCAPCs
CD86(Biolegend)CD86 (Biolegend)	APCAPCs
CD40(Biolegend)CD40 (Biolegend)	AF700AF700
HLA-ABC(Biolegend)HLA-ABC (Biolegend)	PEPE
HLA-DR(Biolegend)HLA-DR (Biolegend)	PE-Cy7PE-Cy7
CD197(CCR7)(Biolegend)CD197(CCR7)(Biolegend)	PE-Cy7PE-Cy7

T detection

检测项目抗体Detection Item Antibody	发光信号luminous signal
CD3(Biolegend)CD3 (Biolegend)	FITCFITC
CD4(Biolegend)CD4 (Biolegend)	FITCFITC
CD8(Biolegend)CD8 (Biolegend)	PEPE
CD279(PD-1)(BD)CD279(PD-1)(BD)	BV421BV421
CD152(CTLA-4)(BD)CD152(CTLA-4)(BD)	PEPE
CD62L(BD)CD62L(BD)	PEPE
CD25(BD)CD25(BD)	APCAPCs
CD69(Biolegend)CD69 (Biolegend)	BV421BV421
HLA-DR(Biolegend)HLA-DR (Biolegend)	PE-Cy7PE-Cy7

Multiple cytokine CBA method detection

According to the operation steps in the manual, firstly dilute the standard product serially, after resuspending the standard product in 2mL, take out 9 flow-type sample tubes and mark the multiples of serial dilution 1:2, 1:4, 1:8, 1:16 respectively , 1:32, 1:64, 1:128, 1:256. In order to ensure that each experimental tube contains 6 kinds of microspheres, 10 μl of each capture microsphere is required, and the volume of the mixed microspheres is 60 μl. This ratio configures the microspheres. For example, there are 8 samples to be tested, 9 standard products, and 1 negative control, totaling 18 tubes. Therefore, the required amount of each microsphere is 180 μl. After mixing 6×180 μl, add 50 μl of diluted sample/standard (50 μl) to each tube, then add 50ul PE detection reagent and incubate at room temperature for 3 hours in the dark.

Before going on the machine, it is necessary to calibrate and adjust the voltage of the instrument in combination with magnetic beads, and adjust the compensation with the control magnetic beads. After incubation, use the CBA analysis software FCAP combined with the standard curve analysis to calculate the concentration of the detection sample.

ELISA detection

The basic steps of ELISA detection of anti-PD-1 antibody and anti-CTLA-4 antibody secretion are as follows:

(1) Antigen coating plate: prepare for coating antigen. Dilute the antigen with the coating solution, coat the enzyme-labeled reaction plate with 100ul/well, and leave overnight at 4°C. After overnight, wash 5 times with PBST, 200ul/well, 3 minutes each time, and pat dry with absorbent paper.

(2) Sealing: add 300ul of blocking solution to each well, and incubate for 2 hours in a biochemical incubator at 37°C. Wash 5 times with PBST, 200ul/well, 3 minutes each time, and pat dry with absorbent paper.

(3) Adding samples: Dilute the sample and standard with diluent, gradiently dilute the standard, set 7 gradients and 0ng/ml, and dilute the sample according to the actual situation. Add samples and standards, 100ul/well, and set up duplicate wells and control wells. Incubate for 1 hour at 37°C in a biochemical incubator. Wash 5 times with PBST, 200ul/well, 3 minutes each time, and pat dry with absorbent paper.

(4) Add secondary antibody: dilute the secondary antibody proportionally with blocking solution, 100ul/well, and incubate at 37°C in a biochemical incubator. Wash 5 times with PBST, 200ul/well, 3 minutes each time, and pat dry with absorbent paper.

(5) Color development: add color development solution TMB (the brand is Abcam), 100ul/well, and develop color at room temperature for 5-15min in the dark.

(6) Termination: 50ul/well of stop solution was added to terminate the reaction. Take an immediate on-board reading.

The detection is as shown in the above steps, and the different parameters are shown in the table below:

参数parameter	PD-1PD-1	CTLA-4CTLA-4
包被抗原浓度Coating antigen concentration	1ug/mL(品牌为Arco)1ug/mL (the brand is Arco)	2ug/mL(自产)2ug/mL (self-produced)
标准品最高梯度Standard Highest Gradient	12.5ng/mL(自产)12.5ng/mL (self-produced)	20ng/mL(自产)20ng/mL (self-produced)
标准品及样品孵育时间Standard and sample incubation time	1h1h	1.5h1.5h
二抗稀释比例Secondary Antibody Dilution Ratio	1:20000(品牌为Abcam)1:20000 (the brand is Abcam)	1:10000(品牌为GenScript)1:10000 (branded as GenScript)
二抗孵育时间Secondary antibody incubation time	30min30min	1h1h

Example 1

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Anti-PD-1/anti-CTLA-4 antibody mRNA was electroporated into mature DC by electroporation. The amount of electroporation was 40ug, 60ug, 80ug, 100ug/2.5×10 ⁶ cells respectively. Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After 24h and 48h, the supernatant was collected to detect the levels of a-PD-1 and a-CTLA4 secreted by 1×10 ⁶ cells by ELISA. The test results are as follows: Electroporation of different concentrations of a-PD-1 and a-CTLA-4 affects the secretion of a-PD-1 and a-CTLA-4. The secretion of a-PD-1 and a-CTLA-4 after electroporation was higher at 24h than at 48h; 100ug>80ug>60ug>40ug group.

The result is shown in Figure 1:

A. The secretion of a-PD-1 in the 100ug group was 145.3ng in 24h and 72.2ng in 48h; the secretion of a-PD-1 in the 80ug group was 133.7ng in 24h and 61.4ng in 48h; the secretion of a-PD in 60ug in 24h -1 secretion was 122.2ng, 48h secretion was 56.6ng, 40ug group had 24h a-PD-1 secretion of 93.8ng, 48h secretion was 38.9ng.

B. The secretion of a-CTLA-4 in the 100ug group was 60.9ng in 24h and 24.3ng in 48h; the secretion of a-CTLA-4 in the 80ug group was 56.5ng in 24h and 20.9ng in 48h; the secretion of a-CTLA in the 60ug group was 24h -4 secretion was 52.1ng, 19.2ng at 48h, 40.9ng at 24h and 15.1ng at 48h in the 100ug group.

Example 2

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Anti-PD-1/anti-CTLA-4 antibody mRNA and MAGE-A3 antigen mRNA were electroporated into mature DC by electroporation. The amount of electroporation was (antigen mRNA: 5ug/2.5×10 ⁶ cells) and (antibody mRNA: 40ug, 60ug, 80ug/2.5×10 ⁶ cells). After 24h, 48h, and 72h, the supernatant was collected and detected by ELISA 1× 10 ⁶ cells secreted a-PD-1 and a-CTLA4 content. The test results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electroporation decreased over time. Higher levels of a-PD-1 and a-CTLA4 can be secreted at 24h.

The result is shown in Figure 2:

A: The 24h a-PD-1 secretion in the 80ug group was 120.5ng, the 48h secretion was 41.2ng, and the 72h secretion was 16.9ng; the 24h a-PD-1 secretion in the 60ug group was 89.1ng, and the 48h secretion was 24.0ng , 72h secretion is 12.8ng; 40ug group 24h a-PD-1 secretion is 72.3ng, 48h secretion is 21.1ng, 72h secretion is 13.0ng;

B: 24h a-CTLA-4 secretion of 80ug group was 75.9ng, 48h secretion was 36.8ng, 72h secretion was 16.1ng; 24h a-CTLA-4 secretion of 60ug group was 62.5ng, 48h secretion was 29.1ng , 72h secretion is 12.0ng; 40ug group 24h 24h a-CTLA-4 secretion is 55.8ng, 48h secretion is 27.3ng, 72h secretion is 9.1ng.

Example 3

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Anti-PD-1/anti-CTLA-4 antibody mRNA and survivin antigen mRNA were electroporated into mature DC by electroporation. The amount of electroporation was (antigen mRNA: 5ug/2.5×10 ⁶ cells) and (antibody mRNA: 40ug, 60ug, 80ug/2.5×10 ⁶ cells). Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After 24h, 48h, and 72h, the supernatant was collected to detect the levels of a-PD-1 and a-CTLA4 secreted by 1×10 ⁶ cells by ELISA. The test results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electroporation decreased over time. Higher levels of a-PD-1 and a-CTLA4 can be secreted at 24h.

The result is shown in Figure 3:

A: 24h a-PD-1 secretion of 80ug group was 106.3ng, 48h secretion was 31.2ng, 72h secretion was 14.9ng; 24h a-PD-1 secretion of 60ug group was 96.0ng, 48h secretion was 35.0ng , 72h secretion is 13.1ng; 40ug group 24h a-PD-1 secretion is 76.5ng, 48h secretion is 27.4ng, 72h secretion is 11.5ng;

B: The secretion of a-CTLA-4 in the 80ug group was 69.1ng in 24h, 28.5ng in 48h, and 16.9ng in 72h; the secretion of a-CTLA-4 in the 60ug group was 57.3ng in 24h and 23.4ng in 48h , 72h secretion was 12.5ng; 40ug group 24h a-CTLA-4 secretion was 45.6ng, 48h secretion was 18.1ng, 72h secretion was 10.1ng.

Example 4

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Anti-PD-1/anti-CTLA-4 antibody mRNA and CEA antigen mRNA were electroporated into mature DC by electroporation. The amount of electroporation was (antigen mRNA: 5ug/2.5×10 ⁶ cells) and (antibody mRNA: 40ug, 60ug, 80ug/2.5×10 ⁶ cells). Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After 24h, 48h, and 72h, the supernatant was collected to detect the levels of a-PD-1 and a-CTLA4 secreted by 1×10 ⁶ cells by ELISA. The test results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electroporation decreased over time. Higher levels of a-PD-1 and a-CTLA4 can be secreted at 24h.

The result is shown in Figure 4:

A: The 24h a-PD-1 secretion of 80ug group is 139.6ng, 48h secretion is 39.7ng, 72h secretion is 19.8ng; 24h a-PD-1 secretion of 60ug group is 93.0ng, 48h secretion is 25.5ng , 72h secretion is 9.3ng; 40ug group 24h a-PD-1 secretion is 75.0ng, 48h secretion is 22.7ng, 72h secretion is 8.9ng;

B: The secretion of a-CTLA-4 in the 80ug group was 62.3ng in 24h, 26.2ng in 48h, and 13.4ng in 72h; the secretion of a-CTLA-4 in the 60ug group was 55.7ng in 24h and 23.3ng in 48h , 72h secretion is 11.6ng; 40ug group 24h 24h a-CTLA-4 secretion is 48.1ng, 48h secretion is 19.5ng, 72h secretion is 7.2ng.

Example 5

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. The mature DC cells were divided into three groups for electroporation. The first group: anti-PD-1/anti-CTLA-4 antibody mRNA and MAGE-A3 antigen mRNA; the second group: anti-PD-1/anti-CTLA-4 antibody mRNA and survivin antigen mRNA; the third group: anti-PD-1 /anti-CTLA-4 antibody and CEA antigen mRNA). The amount of electroporation was (antigen mRNA: 5ug/2.5×10 ⁶ cells) and (antibody mRNA: 60ug/2.5×10 ⁶ cells). After electroporation, the three groups of cells were mixed together, and the cells were cultured in a 24-well plate (1×10 ⁶ cells/ml). After 24h, 48h, and 72h, the supernatant was collected to detect the levels of a-PD-1 and a-CTLA4 secreted by 1×10 ⁶ cells by ELISA. The test results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electroporation decreased over time. Higher levels of a-PD-1 and a-CTLA4 can be secreted at 24h.

The results are shown in Figure 5: 24h a-PD-1 secretion was 119.7ng, 48h secretion was 36.9ng, 72h secretion was 13.7ng; 24h a-CTLA-4 secretion was 85.9ng, 48h secretion was 32.1 ng, 72h secretion is 15.3ng.

Example 6

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On day 6, mature DC cells were divided into three groups for electroporation. The second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD- 1/anti-CTLA-4 antibody mRNA. Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After culturing for 24 hours, the expressions of HLA-ABC, HLA-DR, CD80, CD86, CD40 and CCR7 in the four groups were compared by flow cytometry.

The result is shown in Figure 6:

A: The MFI of HLA-ABC positive cells in the first group of DCs is 4611, the second group of DCs is 12746 MFI, the third group of DCs is 13186 MFI, and the fourth group of DCs is 15112 MFI. B: The MFI of HLA-DR positive cells in the DCs of the first group was 1761, the DCs of the second group were 2786 MFI, the DCs of the third group were 2898 MFI, and the DCs of the fourth group were 2999 MFI. C: The CD80 positive cell MFI of the DCs in the first group is 2809, the DCs in the second group are 8666 MFIs, the DCs in the third group are 9210 MFIs, and the DCs in the fourth group are 12015 MFIs. D: The CD86-positive cell MFI of the DCs in the first group was 3722, the DCs in the second group were 6357 MFIs, the DCs in the third group were 9771 MFIs, and the DCs in the fourth group were 10634 MFIs. E: The CD40-positive cell MFI of the DCs in the first group was 2674, the DCs in the second group were 4221 MFI, the DCs in the third group were 6084 MFI, and the DCs in the fourth group were 6884 MFI. F: The MFI of CD83-positive cells in the first group of DCs was 918, the second group of DCs was 1960.5 MFI, the third group of DCs was 2316 MFI, and the fourth group of DCs was 2567.5 MFI. From the above results, it can be seen that mRNA transfection has a positive effect on the maturation and activation of DC.

Example 7

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On day 6, mature DC cells were divided into three groups for electroporation. The second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD- 1/anti-CTLA-4 antibody mRNA. Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After 24 hours of culture, the supernatant was collected, and the secretion of IL-6, TNF-α and IL-10 was detected by CBA flow cytometry. The secretion of IL-12 was detected by ELISA method.

The result is shown in Figure 7:

A: The IL-6 secretion of DC in the first group was 64.4pg/ml, the DC in the second group was 128.2pg/ml, the DC in the third group was 277.31pg/ml, and the DC in the fourth group (super DC) It was 414.3 pg/ml. B: The IL-12 secretion of the DCs of the first group was 17.6pg/ml, the DC of the second group was 91.2pg/ml, the DC of the third group was 120.1pg/ml, and the DC of the fourth group (super DC) It was 129.7 pg/ml. C: The TNF-α secretion of the DCs of the first group is 17.1pg/ml, the DC of the second group is 103.9pg/ml, the DC of the third group is 131.4pg/ml, the DC of the fourth group (super DC) It was 155.3 pg/ml. D: the IL-10 secretion of the DC of the first group is 31.2pg/ml, the DC of the second group is 34.0pg/ml, the DC of the third group is 31.5pg/ml, and the DC of the fourth group is 32.2pg/ml ml. From the above results, it can be seen that super DC secrete the highest levels of pro-inflammatory cytokines, which are important in T cell responses.

Embodiment 8, the expression of antigen

On day 6, mature DC cells were electroporated with three antigen mRNAs (MAGE-A3, survivin and CEA) and 1194-2VHH. Immunocytochemical staining (ICC) was used to detect the expression of electroporated antigenic protein in DC cells.

Chemotaxis Assay: Immunocytochemistry. Super DCs were seeded into 4-chamber slides (NuncLab-Tek Chamber Slide System). Cells were then incubated overnight. After 24 hours, cells were rinsed with PBS, fixed with 3.7% w/v paraformaldehyde (Sigma), rinsed with PBS and permeabilized in 0.5% TritonX-100 (Sigma). Nonspecific immunoglobulin binding was blocked with 5% normal goat serum and 0.5% NP-40 (Sigma). Primary antibodies recognizing MAGE-A3, survivin and CEA (Abcam) were diluted 1:100 in blocking solution. After incubation with primary antibodies, cells were washed with 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) in PBS, and then incubated with secondary antibodies for 1 hour at room temperature. Stained with 3,3'-diaminobenzidine (DAB) and observed under a light microscope. Figure 8, A, B and C reveals the strong presence of MAGE-A3, Survivin and CEA proteins in the cytoplasm.

Embodiment 9, migration function

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On the 6th day, the mature DC cells were divided into three groups of electroporation, the second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): Three antigen mRNAs (MAGE-A3, survivin and CEA) and 1194-2VHH. Cells were cultured in 24-well plates (1×10 ⁶ cells/ml). After 24 hours of culture, the expressions of CCR7 and CXCR4 in the four groups were compared by flow cytometry.

As shown in Figure 9, A: the CCR7 positive cell percentage of the DCs of the first group is 17.9%, the DC of the second group is 89.6%, the DC of the third group is 92.0%, and the DC (super DC) of the fourth group is 97.9%. B: The CXCR4-positive cells of the DCs of the first group were 11.1%, the DCs of the second group were 89.2%, the DCs of the third group were 90.6%, and the DCs (super DCs) of the fourth group were 94.7%.

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On the 6th day, the mature DC cells were divided into three groups of electroporation, the second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): Three antigen mRNAs (MAGE-A3, survivin and CEA) and 1194-2VHH. The migration function of different groups of DC cells towards chemokine ligands (CCL19, CCL21 or both) was tested using chemotaxis assays. Chemotaxis assay: 24-well culture plates with polycarbonate membrane-coated Transwell ^™ permeable inserts (5 μm pore size; Costar) were used. The lower plate chamber was filled with 600 μL of DC medium per well. The CCR7 ligand CCL19/CCL21 (R&D Systems) was used as a chemoattractant and was added to the lower well at an optimal concentration of 100 ng/mL. Next, DCs (1.0 × ¹⁰⁵ cells) were seeded onto each Transwell ^TM insert in a total volume of 100 μL of DC medium and incubated in a humidified 37 °C/5% _CO2 incubator (chemokines) mid-migration to the lower compartment for 180 min-driven migration). Parallel control experiments were performed in the absence of chemokine ligands to assess spontaneous cell migration (negative control) or to transfer all cells (1.0 x ¹⁰⁵ ) to the lower well to determine the maximum possible DC yield (positive control). Cells from each lower well were collected, centrifuged and concentrated to a final sample volume of 200 µL. Cells were counted in duplicate at a fixed flow rate by flow cytometry analysis over a defined time period of 60 seconds (counts per minute; cpm). DC migration is expressed using the following equation: % Migrated Cells = [(cpm _{Tested Sample} - cpm _{Negative Control} )]/cpm _{Positive Control}

As shown in Figure 10, A: Migration ability of DC subsets shown in Transwell chemotaxis assay to CCL19. The migration ability of DCs in the first group was 4.0%, that of the second group was 36.7%, that of the third group was 40.6%, and that of the fourth group (super DC) was 43.4%. B: Migration capacity of the indicated DC subsets towards CCL21 in a Transwell chemotaxis assay. The migration ability of DCs in the first group was 5.1%, that of the second group was 38.0%, that of the third group was 41.2%, and that of the fourth group (super DC) was 44.6%. C: Migration capacity of the indicated DC subsets towards CCL19 and CCL21 in a Transwell chemotaxis assay. The migration ability of DCs in the first group was 7.5%, that of the second group was 43.3%, that of the third group was 46.5%, and that of the fourth group (super DC) was 50.7%. These results indicated that the electroporation process did not negatively affect the migratory function of DCs.

Example 10

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Anti-PD-1/anti-CTLA-4 antibody mRNA and three antigen mRNAs (MAGE-A3, survivin and CEA) were electroporated into mature DC by electroporation. After electroporation for 4 hours, it was co-cultured with T cells at a ratio of 1:10 (DC: 2×10 ⁵ cells, T cells: 2×10 ⁶ ), and the supernatants were taken at 24 hours, 48 hours, and 96 hours for ELISA detection of a-PD-1 and a -CTLA-4, and cells were collected at 96h for flow cytometric detection. The test results are shown in Figure 11: A is the ELISA test result, the secretion of a-PD-1 at 24h, 48h, and 96h is 29.2ng, 14.6ng, and 5.2ng; the secretion of a-CTLA-4 at 24h, 48h, and 96h is 17.5 ng, 10.44ng, 3.8ng. B is 96h flow cytometric detection of T cell surface markers CTLA-4 and PD-1. The analysis method is CD3+(CTLA-4), CD3+(PD-1). From Figure 11, the upper panel of B, it can be seen that CTLA-4 on the surface of T cells is 35.05%, and that of super DC: CTLA-4 on the surface of T cells in the T group is 23.83%; from Figure 11, the lower panel of B, it can be seen that the surface of T cells PD-1 was 16.07%, super DC: PD-1 on the surface of T cells in the T group was 5.05%. It can be seen that super DC can secrete a-CTLA-4 and a-PD1, which reduces the expression of CTLA-4 and PD-1 on the surface of T cells.

Example 11

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On the 6th day, the mature DC cells were divided into three groups of electroporation, the second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): Three antigen mRNAs (MAGE-A3, survivin and CEA) and 1194-2VHH. After 4 hours of electroporation, co-culture with T cells at a ratio of 1:10 (DC: 2×10 ⁵ cells, T cells: 2×10 ⁶ cells). After 96 hours of culture, T cells were harvested for detection of surface markers (CD3 ⁺ CD25 ⁺ , CD3 ⁺ CD69 ⁺ , CD3 ⁺ HLA-DR ⁺ , CD3 ⁺ CD62L+, CD3 ⁺ CD137 ⁺ ^, CD8 ⁺ CD107a ⁺ ).

The result is shown in Figure 12:

A: The CD3 ⁺ CD25 ⁺ positive cell expression percentage of the T cells in the first group was 8.4%, the T cells in the second group were 19.9%, the T cells in the third group were 24.3%, and the T cells in the fourth group (super DC group) was 29.6%. B: The CD3 ⁺ CD69 ⁺ positive cell expression percentage of the T cells of the first group was 16.8%, the T cells of the second group were 40.8%, the T cells of the third group were 43.2%, and the T cells of the fourth group (super DC group) was 50.5%. C: the CD3 ⁺ HLA-DR ⁺ positive cell expression percentage of the T cells of the first group is 4.7%, the T cells of the second group are 14.5%, the T cells of the third group are 24.9%, the T cells of the fourth group ( super DC group) was 27.0%. D: The CD3 ⁺ CD62L ⁺ positive cell expression percentage of the T cells of the first group was 66.8%, the T cells of the second group were 60.0%, the T cells of the third group were 51.1%, and the T cells of the fourth group (super DC group) was 43.2%. E: The CD3 ⁺ CD137 ⁺ positive cell expression percentage of the T cells of the first group was 2.9%, the T cells of the second group were 3.5%, the T cells of the third group were 5.6%, and the T cells of the fourth group (super DC group) was 7.8%. F The CD8 ⁺ CD107a ⁺ positive cell expression percentage of T cells in the first group was 1.1%, T cells in the second group were 2.2%, T cells in the third group were 3.7%, T cells in the fourth group (super DC group) was 6.7%. From the above results, it can be seen that super DCs can effectively activate T cells.

Example 12

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On day 6, mature DC cells were divided into three groups for electroporation. The second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD- 1/anti-CTLA-4 antibody mRNA. After 4 hours of electroporation, co-culture with T cells at a ratio of 1:10 (DC: 2×10 ⁵ cells, T cells: 2×10 ⁶ cells). After culturing for 48 hours, the cell supernatant was taken to detect the secretion of IFN-γ, TNF-α, IL-6 and IL-10 by CBA.

The result is shown in Figure 13:

A: The IFN-γ secretion of the first group is 265.0pg/ml, the second group is 413.6pg/ml, the third group is 695.2pg/ml, and the fourth group (super DC) is 1037.7pg/ml ml. B: The secretion amount of TNF-α of the first group is 155.3pg/ml, that of the second group is 252.9pg/ml, that of the third group is 307.1pg/ml, and that of the fourth group (super DC) is 506.5pg/ml ml. C: The IL-6 secretion of the first group was 100pg/ml, that of the second group was 178pg/ml, that of the third group was 226pg/ml, and that of the fourth group (super DC) was 422pg/ml. D: The secretion amount of IL-2 of the first group is 102.2pg/ml, that of the second group is 240.0pg/ml, that of the third group is 323.9pg/ml, and that of the fourth group (super DC) is 453.2pg/ml ml. E: The secretion amount of IL-10 of the first group is 26.2pg/ml, the second group is 28.5pg/ml, the third group is 27.4pg/ml, the fourth group (super DC) is 25.7pg/ml ml. From the above results, it can be seen that super DCs can effectively promote the activity of T cells and enhance the secretion of type 1 and type 2 cytokines.

Example 13

On day 5, DC cells were untreated (group 1: immature DC-iDC-) or matured with a maturation cocktail (IFN-γ, PolyI:C, R848, PGE2) for 24 h (mature DC-mDC-). On the 6th day, the mature DC cells were divided into three groups of electroporation, the second group: no electroporation; the third group: three antigen mRNAs (MAGE-A3, survivin and CEA); the fourth group (super DC): Three antigen mRNAs (MAGE-A3, survivin and CEA) and 1194-2VHH. After 4 hours of electroporation, co-culture with T cells at a ratio of 1:10 (DC: 2×10 ⁵ cells, T cells: 2×10 ⁶ cells). T cell proliferation was assessed by counting cells after 96 hours of co-culture.

The result is shown in Figure 14. The T cell expansion times of the first group were 2.0 times, the T cell expansion times of the second group were 2.5 times, the T cell expansion times of the third group were 5.7 times, and the T cell expansion times of the fourth group (super DC group) is a multiple of 9.6.

Example 14

Mature DC can be obtained from immature DC stimulated by IFN-γ, PolyI:C, R848, and PGE2 for 24 hours. Three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNA were electroporated into mature DC (super DC) by electroporation. The electroporation concentration is 60ug/2.5×10 ⁶ cells. After electroporation, the cells were divided into two parts: 1) the cells were cultured [24-well plate (1×10 ⁶ cells/ml)] for 24h, 48h, and 72h, and then the supernatant was collected; 2) the cells were cultured [24-well plate (1×106 cells/ml)] 10 ⁶ cells/ml)], the cells were collected and frozen after 4 hours. After 4 weeks of frozen storage, the cells were recovered and cultured in 24-well plates (1×10 ⁶ cells/ml). The supernatant was collected after 24h, 48h and 72h. The contents of a-PD-1 and a-CTLA4 secreted by 1×10 ⁶ cells were detected by ELISA in the supernatant.

The result is shown in Figure 15:

A: After 24 hours, the secretion of a-PD-1 in fresh cells was 70.3ng, and the secretion of a-PD-1 in frozen cells was 65.2ng. After 48h, the secretion of a-PD-1 in fresh cells was 25.0ng, and the secretion of a-PD-1 in frozen cells was 20.7ng. After 72 hours, the secretion of a-PD-1 in fresh cells was 11.9ng, and the secretion of a-PD-1 in frozen cells was 10.2ng.

B: 24h later, the secretion of a-CTLA-4 in fresh cells was 48.9ng, and the secretion of a-CTLA-4 in frozen cells was 45.1ng. After 48 hours, the secretion of a-CTLA-4 in fresh cells was 22.8ng, and the secretion of a-CTLA-4 in frozen cells was 21.0ng. After 72 hours, the secretion of a-CTLA-4 in fresh cells was 11.0 ng, and the secretion of a-CTLA-4 in frozen cells was 10.1 ng.

From the results of ELISA experiments, it can be seen that the cryopreserved DC cells after electroporation can still maintain the ability to secrete high levels of a-PD-1 and a-CTLA-4 after recovery.

Claims

An antigen-presenting cell capable of secreting a PD-1 binding molecule and/or a CTLA-4 binding molecule, the PD-1 binding molecule being an anti-PD-1 Nanobody or an antigen-binding fragment thereof, and/or, the CTLA-4 binding The molecule is an anti-CTLA-4 Nanobody or an antigen-binding fragment thereof.
The antigen-presenting cell according to claim 1, wherein,

The CDR1 of the anti-PD-1 nanobody is as shown in any one of SEQ ID NO:1-6, the CDR2 is as shown in any one of SEQ ID NO:7-12, and the CDR3 is as shown in any one of SEQ ID NO:13-89 One item shows,

CDR1 of the anti-CTLA4 nanobody is as shown in any one of SEQ ID NO:226-232, CDR2 is as shown in any one of SEQ ID NO:233-240, and CDR3 is as shown in any one of SEQ ID NO:241-248 shown.
The antigen-presenting cell according to claim 2, wherein the CDR1, CDR2, and CDR3 of the anti-PD-1 nanobody are respectively shown in SEQ ID NO: 1, 10, and 13, and the CDR1, CDR2, and CDR3 are shown in SEQ ID NO:230, 238, 246 respectively.
The antigen-presenting cell according to claim 2 or 3, wherein the VHH of the anti-PD-1 nanobody is as shown in any one of SEQ ID NO:90-225, and the VHH of the anti-CTLA4 nanobody is as SEQ ID NO: any of 249-295,

Preferably, the VHH of the anti-PD-1 nanobody is shown in SEQ ID NO:137, and the VHH of the anti-CTLA4 nanobody is shown in SEQ ID NO:258.
The antigen-presenting cell according to any one of claims 1-4, wherein the antigen-presenting cell is selected from macrophages, B cells or dendritic cells.
The antigen-presenting cell according to any one of claims 1-5, wherein the antigen-presenting cell contains a coding sequence of a PD-1 binding molecule and a coding sequence of a CTLA-4 binding molecule;

Preferably, the coding sequence of the PD-1 binding molecule is RNA, and/or, the coding sequence of the CTLA-4 binding molecule is RNA.
The antigen-presenting cell according to any one of claims 1-6, wherein the antigen-presenting cell is loaded with a tumor-associated antigen; preferably, the antigen-presenting cell expresses the tumor-associated antigen; more preferably , the tumor-associated antigen is selected from one or more of the following: hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms tumor 1 (WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A, Folate Receptorα(FR-α), HPV16/18-E6, HPV16/18-E7, Alpha Fetoprotein(AFP), Glypican3( GPC3), Prostate-Specific Antigen(PSA), Prostatic Acid Phosphatase(PAP), Prostate-specific membrane antigen(PSMA), Prostate stem cell antigen(PSCA), Six-transmembrane epithelial antigen of prostate 1(STEAP1), B-cell maturation antigen (BCMA), CMV pp65, gp100, PRAME, CA19-9, Synovial Sarcoma (SSX)-2; further preferably, the tumor-associated antigens include MAGE-A3, survivin and CEA.
An antigen-presenting cell, characterized in that it comprises a coding sequence of a tumor-associated antigen; preferably, the coding sequence of the tumor-associated antigen is RNA, and/or the antigen-presenting cell is a dendritic cell; more preferably, The tumor-associated antigen is selected from one or more of the following: hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms tumor 1 ( WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A, Folate Receptorα (FR-α), HPV16/18-E6, HPV16/18-E7, Alpha Fetoprotein (AFP), Glypican3 (GPC3 ), Prostate-Specific Antigen (PSA), Prostatic Acid Phosphatase (PAP), Prostate-specific membrane antigen (PSMA), Prostate stem cell antigen (PSCA), Six-transmembrane epithelial antigen of prostate 1 (STEAP1), B-cell maturation Antigen (BCMA), CMV pp65, gp100, PRAME, CA19-9, Synovial Sarcoma (SSX)-2; further preferably, the tumor-associated antigens include MAGE-A3, survivin and CEA.
A method of producing antigen-presenting cells loaded with tumor-associated antigens, comprising:

(1) loading the antigen-presenting cell according to any one of claims 1-7 with a tumor-associated antigen;

(2) enabling antigen-presenting cells loaded with tumor-associated antigens to secrete PD-1 binding molecules and CTLA-4 binding molecules; or

(3) contacting antigen-presenting cells with tumor-associated antigens or coding sequences thereof and coding sequences of PD-1 binding molecules and CTLA-4 binding molecules,

Preferably, the PD-1 binding molecule is an anti-PD-1 antibody or an antigen-binding fragment thereof, and/or, the CTLA-4-binding molecule is an anti-CTLA-4 antibody or an antigen-binding fragment thereof.
The method according to claim 9, wherein (2) comprises introducing coding sequences of PD-1 binding molecules and CTLA-4 binding molecules into antigen-presenting cells;

Preferably, the coding sequence of the PD-1 binding molecule is RNA, and/or, the coding sequence of the CTLA-4 binding molecule is RNA.
The method according to claim 9 or 10, wherein loading tumor-associated antigens comprises: contacting or expressing tumor-associated antigens, and/or contacting or introducing coding sequences of tumor-associated antigens,

Preferably, the coding sequence of said tumor-associated antigen is RNA.
The method of any one of claims 9-11, wherein the antigen-presenting cells are contacted with the maturation composition before or after the tumor-associated antigen loading.
A pharmaceutical composition, comprising the antigen-presenting cell according to any one of claims 1-8 or the antigen-presenting cell produced by the method according to any one of claims 9-12, and a pharmaceutically acceptable auxiliary material,

Preferably, said pharmaceutical composition is used to treat or prevent a tumor expressing said tumor-associated antigen in a subject.
Use of the antigen-presenting cell according to any one of claims 1-8 or the antigen-presenting cell produced by the method according to any one of claims 9-12 in the preparation of a medicament for preventing tumor growth in a subject develop or metastasize, or inhibit the growth or metastasis of a tumor in a subject that expresses the tumor-associated antigen.