CN116064397A

CN116064397A - Superdcs expressing immune checkpoint inhibitors and uses thereof

Info

Publication number: CN116064397A
Application number: CN202111289339.4A
Authority: CN
Inventors: 莫南德·阿德维希; 吴泽吉; 师传胤; 钱其军
Original assignee: Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Group Co Ltd
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2023-05-05
Also published as: CN118284689A; WO2023078305A1

Abstract

The present invention relates to antigen presenting cells expressing immune checkpoint inhibitors and uses thereof. In particular, the invention provides antigen presenting cells that secrete PD-1 binding molecules and CTLA-4 binding molecules or contain their coding sequences. The antigen presenting cells of the present invention can activate naive T cells, effectively stimulating the effector functions of activated T cells on cancer.

Description

Superdcs expressing immune checkpoint inhibitors and uses thereof

Technical Field

The present invention relates to the field of immunotherapy, in particular to antibody-loaded antigen presenting cells and uses thereof.

Background

As a novel tumor treatment mode, tumor immunotherapy mainly generates anti-tumor immunity by activating the immune system of an organism, thereby achieving the purpose of eliminating tumor cells, not only being capable of triggering durable anti-tumor immunity, but also having an important function of preventing postoperative recurrence. Dendritic Cells (DCs) are an important target for tumor immunotherapy as a primary link for the body to generate specific immune responses. DC vaccine adopts autologous or allogeneic tumor cell component sensitized patient peripheral blood derived mononuclear cells to induce differentiated DC, thereby directly inducing specific immune response.

The DC vaccine is to introduce in vitro cultured DC loaded with tumor antigen into human body, and the DC regulates proliferation and activation of tumor antigen specific Th1 cells through antigen presenting function and secretion cell factor, further promotes activation of NK cells and CTL, and mediates tumor killing. DC cells which can specifically identify tumor are induced or constructed in vitro, are infused back into a tumor patient, can activate the immune response of T cells to tumor, and express high levels of PD-1 and CTLA-4.

Therapeutic tumor vaccines targeting dendritic cells typically comprise three components. 1) Tumor associated antigen: the antigen may be a small molecule peptide, a recombinant protein. Tumor cells subjected to engineering modification, tumor antigens expressed by viral vectors and/or engineered bacterial vectors, and DNA or RNA. These differently expressed or prepared antigens may be taken up and processed by dendritic cells in vivo and expressed on the cell surface as MHC2, which is recognized by T cell CD 8. Antigens may also be expressed directly on dendritic cells. 2) Is a dendritic cell (in vivo, isolated and purified, or isolated in peripheral blood leukocytes). 3) Adjuvants or immunostimulants.

Although clinical studies have shown that DC vaccines induced by autologous monocytes of patients are well tolerated by patients and produce anti-tumor immune responses, tumors are not effectively treated, possibly due to lack of specific tumor antigens, low antigen loading efficiency, and activation of regulatory T cells following treatment, leading to immunosuppression. Tumor immune microenvironment and tumor immunosuppression mechanisms are critical to limit the use of DC vaccines.

Disclosure of Invention

In a first aspect, the invention provides antigen presenting cells that secrete PD-1 binding molecules and CTLA-4 binding molecules and/or contain their coding sequences.

In one or more embodiments, the cell contains and/or expresses the PD-1 binding molecule and CTLA-4 binding molecule.

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

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 a tumor-associated antigen or coding sequence thereof. Preferably, the antigen presenting cells express the tumor-associated antigen; the antigen presenting cells contain a coding sequence for a tumor-associated antigen. More preferably, the antigen presenting cells comprise mRNA encoding a tumor-associated antigen.

In one or more embodiments, the tumor-associated antigen comprises one or more of a cancer-testis antigen, an over-expressed antigen, a differentiation antigen, preferably one or more selected from the group consisting of: MAGE-A3, survivin, CEA.

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.

In a second aspect, the invention provides a method of producing an antigen presenting cell loaded with a tumor-associated antigen, comprising:

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

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

(3) Contacting the antigen presenting cells with a tumor associated antigen or a coding sequence thereof and a coding sequence for a PD-1 binding molecule and a CTLA-4 binding molecule.

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

In one or more embodiments, (2) comprises introducing into the cell coding sequences, e.g., mRNA, for the PD-1 binding molecule and CTLA-4 binding molecule.

In one or more embodiments, loading the tumor-associated antigen comprises: 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 (e.g., maturation cocktail (maturation cocktails)) either before or after the antigen is loaded. The maturation composition comprises one or more selected from the group consisting of IFN-gamma, polyI C, R848, PGE 2.

In one or more embodiments, the tumor comprises a respiratory tumor, a digestive tumor, a urinary tumor, a nervous system tumor, a reproductive tumor, a skin tumor; preferably comprising one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, kidney cancer, bladder cancer and brain cancer.

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

The third aspect of the present invention also provides a pharmaceutical composition comprising an antigen presenting cell according to the first aspect of the present invention or produced by a method according to the second aspect of the present invention, and a pharmaceutically acceptable adjuvant.

In one or more embodiments, the pharmaceutical composition is for treating or preventing a tumor in a subject that expresses the tumor-associated antigen.

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

The invention also provides the use of a dendritic cell antigen presenting cell according to the first aspect of the invention or an antigen presenting cell produced by a method according to the second aspect of the invention in the manufacture of a medicament for preventing the occurrence or metastasis of a tumor in a subject or for inhibiting the growth or metastasis of a tumor in a subject, said tumor expressing said 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 of preventing or treating the occurrence, growth or metastasis of a tumor in a subject comprising administering a therapeutically effective amount of an antigen presenting cell according to the first aspect of the invention or produced by a method according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention.

In one or more embodiments, the occurrence of the tumor is prevented or the growth, metastasis of the tumor is reduced by the antigen presenting cells, and the antigen presenting cells are cultured ex vivo with a mature composition prior to administration, the antigen presenting cells bearing the tumor-associated antigen.

In one or more embodiments, the mature composition comprises one or more selected from IFN-gamma, polyI C, R848, PGE 2.

The invention has the advantages that: dendritic Cells (DCs) migrate to the lymph nodes after injection, where they encounter naive T cells and activate them. The activation mechanism is as follows: MHC interactions of dc surface with TCR of T cells; interactions between the B7 molecule of dcs and CD28 of T cells. The secreted PD-1 binding molecules and CTLA-4 binding molecules of the invention or DCs containing their coding sequences have the following advantages:

activating the activation of naive T cells by the following specific mechanisms: in lymph nodes, tregs express high levels of CTLA-4, and interaction with the B7 molecules of DCs reduces the ability of DCs to stimulate tumor-specific T cells. The DCs of the invention can secrete CTLA-4 binding molecules, block CTLA-4 of Tregs, relieve Tregs and reduce the capability of the DCs to stimulate tumor-specific T cells, so that the DCs of the invention can effectively activate naive T cells.

T cells, when activated, express high levels of PD-1 and CTLA-4, inhibiting tumor-specific T cell function at the tumor site by preventing them from killing cancer cells: A. immune cells such as tumor cells, macrophages express PD-L1, B.Treg expresses CTLA-4 at high level, and C.tumor cells express B7 molecules combined with T cell CTLA-4. The DCs of the invention can secrete PD-1 binding molecules and CTLA-4 binding molecules, so that PD-1 and CTLA-4 expressed by T cells can be blocked, namely, the CTLA-4 and PD-1 expressed by the T cells are inhibited before the T cells migrate to tumor sites, so that the effector functions of activated T cells on cancers can be effectively stimulated.

Some embodiments of the invention also provide methods of introducing exogenous RNA that can elicit innate and adaptive immune responses in an organism to produce a DC vaccine. By utilizing the mechanism in tumor treatment, exogenous RNA is introduced in the form of vaccine, which can promote the release of inflammatory cytokines and interferon, activate NK, macrophages and effector T cells, change cold tumor without immune cell infiltration into hot tumor, promote the killing effect of immune system on cancer cells, and also synergistically improve the curative effect of immune checkpoint inhibitor antibody, namely 'stepping on accelerator' while 'releasing brake' for immune system, so as to further enhance anti-tumor immune response. Compared with the traditional vaccine, the mRNA has more advantages in safety, can not be inserted into gene mutation, can be degraded by normal cells, and can change half-life and the like through regulating sequence modification and delivery vectors. Much evidence suggests that mRNA not only mediates better transfection efficiency and longer protein expression times, but also has greater advantages over DNA, including: (1) mRNA functions without entering the nucleus. Reaching the cytoplasm, mRNA initiates protein translation. In contrast, DNA needs to be first nucleated and then transcribed into mRNA. This process makes DNA less efficient than mRNA because its function depends on the disruption of the nuclear envelope during cell division. (2) In contrast to DNA and viral vectors, mRNA is not inserted into the genome, but only transiently expresses the encoded protein, and therefore, it provides an excellent safety option for researchers and pharmaceutical companies due to its low insertion risk. (3) mRNA is readily synthesized by In Vitro Transcription (IVT) processes. This procedure is relatively inexpensive and can be rapidly applied to a variety of therapies. Moreover, mRNA is theoretically capable of expressing any protein, and thus can be used to treat almost all diseases. This solves the problems of expensive DC vaccine and complicated preparation process.

Drawings

FIG. 1 shows the amount of antibody secreted at different times by electrotransfer of different concentrations of anti-PD-1/anti-CTLA-4 mRNA (1194-2 VHH). A: the comparative electric rotation amounts are 40ug, 60ug, 80ug, 100 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ Cell secretion of PD-1 antibody (a-PD-1) at 24h, 48 h; b: the comparative electric rotation amounts are 40ug, 60ug, 80ug, 100 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ CTLA-4 antibody (a-CTLA-4) secretion amount of cells at 24h and 48 h.

FIG. 2 shows the antibody secretion of mRNA of MAGE-A3 antigen at various times at various concentrations of a-PD-1/a-CTLA-4mRNA (1194-2 VHH) by electrotransformation. The comparison electric rotation amounts are 40ug, 60ug and 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ a-PD-1 secretion amount of 24h, 48h and 72 h; b: the comparative electric rotation amounts are 40ug, 60ug, 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ Cell secretion of a-CTLA-4 at 24h, 48h, 72 h.

FIG. 3 shows the antibody secretion of survivin antigen mRNA at different times at different concentrations of a-PD-1/a-CTLA-4mRNA (1194-2 VHH) by electrotransformation. The comparison electric rotation amounts are 40ug, 60ug and 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ a-PD-1 secretion amount of 24h, 48h and 72 h; b: the comparative electric rotation amounts are 40ug, 60ug, 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ Cell secretion of a-CTLA-4 at 24h, 48h, 72 h.

FIG. 4 shows the antibody secretion of CEA antigen mRNA at different times at different concentrations of a-PD-1/a-CTLA-4mRNA (1194-2 VHH) by electrotransformation. The comparison electric rotation amounts are 40ug, 60ug and 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ a-PD-1 secretion amount of 24h, 48h and 72 h; b: the comparative electric rotation amounts are 40ug, 60ug, 80 ug/2.5X10 respectively ⁶ When the cells are taken up, 1X 10 ⁶ Cell secretion of a-CTLA-4 at 24h, 48h, 72 h.

FIG. 5, antibody secretion of electrotransferred a-PD-1/a-CTLA-4mRNA (1194-2 VHH) and three antigen mRNAs at different times. The amounts of the comparative electrotransformation were respectively (antigen mRNA:5 ug/2.5X10) ⁶ Cells) and (1194-2 VHH:60 ug/2.5X10 ⁶ Cells), 1×10 ⁶ Cell secretion of a-PD-1 and a-CTLA-4 at 24h, 48h and 72 h.

Fig. 6, four sets of DC flow detection. A comparison of MFI of HLA-ABC positive cells of control DCs (immature DCs, iDCs), mature DCs (mDCs), mature DCs which were electroporated with three antigen mRNAs (mDC/Ags-mRNA), mature DCs which were electroporated with three antigen mRNAs and antibody mRNAs (mDC/[ Ags+1194-2VHH ] -mRNA, also known as super DCs). B, comparison of MFI of HLA-DR positive cells of control DC, mature DC electrically transformed into three antigen mRNAs and antibody mRNAs. Comparison of MFI of CD80 positive cells against DC, mature DC electrotransformed with three antigen mrnas and antibody mrnas. D, comparison of the MFI of CD86 positive cells of control DC, mature DC, and mature DC, which were electroporated with three antigen mRNAs and antibody mRNAs. E, comparison of MFI of CD40 positive cells of control DCs, mature DCs were electroporated with three antigen mrnas, and antibody mrnas. F, comparison of MFI of CCR7 positive cells of control DC, mature DC electrically transformed into three antigen mRNAs and antibody mRNAs.

FIG. 7, the amount of DC secreted cytokines was measured. A, control DC, mature DC is electrotransformed into three antigen mRNA and IL-6 secretion of antibody mRNA is compared. And B, comparing the IL-12 secretion amount of the control DC, the mature DC and the mature DC which are electrically transformed into three antigen mRNA and antibody mRNA. C, comparing the TNF-alpha secretion of the control DC, the mature DC and the mature DC which are electrically transformed into three antigen mRNA and antibody mRNA. D, comparing the IL-10 secretion amount of the control DC, the mature DC and the mature DC which are electrically transformed into three antigen mRNA and antibody mRNA.

FIG. 8, super DC T co-culture for different time antibody secretion and T cell surface marker flow assay. Super DC, T co-cultured for 24h, 48h, 96h a-PD-1, a-CTLA-4 antibody secretion; superDC:T co-culture 96h flow assay compares the percentage expression of CTLA-4 and PD-1 in T-groups for T cells and SuperDC:T.

FIG. 9, super DC-detection of T cell surface markers after T cell co-culture. A, control DC/T cells, mature DC is electrically transformed into three antigen mRNA and antibody mRNA/T cells to express CD3 ⁺ CD8 ⁺ Percentage comparison. B, control DC/T cells, mature DC is electrically transformed into three antigen mRNA and antibody mRNA/T cells to express CD3 ⁺ CD4 ⁺ Percentage comparison. C control DC/T cell, mature DC is electrically transformed into three antigen mRNA and antibody mRNA/T cell to express CD3 ⁺ CD62L ⁺ Percentage comparison. D, control DC/T cells, mature DC is electrically transformed into three antigen mRNA/T cells, and mature DC is electrically transformed into three antigen mRNA and antibody mRNA/T cells to express CD3 ⁺ CD25 ⁺ Percentage comparison. E control DC/T cells, mature DC is electroporated with three antigen mRNA and antibody mRNA/T cells to express CD3 ⁺ CDHLA-DR ⁺ Percentage comparison. F, control DC/T cell, mature DC is electrically transformed into three antigen mRNA and antibody mRNA/T cell to express CD3 ⁺ CD69 ⁺ Percentage comparison.

FIG. 10 measurement of the amount of secreted cytokines after T cell co-culture. A, comparing the secretion amount of IFN-gamma of the control DC/T cell, the mature DC/T cell and the mature DC which are electrically transformed into three antigen mRNA/T cells and the mature DC which are electrically transformed into three antigen mRNA/antibody mRNA/T cell. And B, comparing the secretion amount of TNF-alpha of the control DC/T cells, the mature DC/T cells and the mature DC by the three antigen mRNA/antibody mRNA/T cells. C, comparing the IL-6 secretion amount of the control DC/T cell, the mature DC/T cell and the mature DC which are electrically transformed into three antigen mRNA/T cells. And D, comparing the IL-10 secretion amount of the control DC/T cells, the mature DC/T cells and the mature DC by the three antigen mRNA/antibody mRNA/T cells.

FIG. 11 comparison of secretion levels of a-PD-1 (A) and a-CTLA-4 (B) after fresh and cryopreserved DCs of the invention for 48h, 72 h.

Detailed Description

The inventors have found that antigen-loaded antigen presenting cells (e.g., DCs) that secrete immune checkpoint inhibitors can induce a stronger anti-tumor immune response. The loaded antigen may activate antigen presenting cells. The activated antigen presenting cells are injected into the human body to elicit an immune system response in the body. Taking DCs as an example, mature DCs activate naive T cells, in the central link in the initiation, regulation, and maintenance of immune responses. The activated DC cells can enter drainage lymph nodes, secrete immune checkpoint inhibitors, promote the DC to activate T cells and strengthen the killing effect of the T cells.

The present invention provides Antigen Presenting Cells (APCs) that secrete PD1 binding molecules and CTLA4 binding molecules or their coding sequences, and vaccine compositions comprising the same. Antigen presenting cells refer to cells that can transfer antigen information carried by them to lymphocytes (e.g., T cells) to elicit an immune response, including macrophages, B cells, and dendritic cells (DC cells or DCs). The activated antigen presenting cells are injected into the human body to elicit an immune system response in the body. Taking DC as an example, mature DC can induce naive T cells, in the central link of initiation, regulation, and maintenance of immune responses. The activated DC cells can enter drainage lymph nodes, secrete PD1 binding molecules and CTLA4 binding molecules, promote DC to activate T cells and strengthen the killing effect of the T cells.

Herein, the DCs may be DCs differentiated from DC precursor cells derived from separation in autologous blood of a subject, hematopoietic precursor cells such as cd34+ derived from umbilical cord blood, or monocytes derived from cd14+ of peripheral blood. Autologous DCs obtained after isolation, culture, expansion and differentiation from a subject are activated, matured, loaded with antibodies as described herein to obtain a cell mixture preparation containing DCs. The method of differentiating the DC precursor cells into DC may be a method known in the art or any other method capable of differentiating the DC precursor cells into DC, such as differentiation culture by adding the cytokines GM-CSF and IL-4 to a medium. The cell mixture preparation is returned to the body of a subject as a DC vaccine, the antigen is presented by the autologous mature DC of the subject, and specific T cells are activated so as to cause an immune response against an epitope of the antigen in the body. In other embodiments, the DCs may be cells obtained from an immortalized DC precursor cell line that has been expanded in vitro and then subjected to differentiation culture. The immortalized DC precursor cell line may be a cell line known in the art or publicly reported, such as the MUTZ3 cell line, or an immortalized DC precursor cell line prepared by the method described in CN 201810368646.3. The immortalized DC precursor cell line may be expanded in vitro in large amounts and then subjected to a differentiation culture to form DCs, which may be the method described above. After the DC obtained by the differentiation culture after the amplification of the immortalized DC precursor cell line is activated, matured and loaded with the antibody described herein, a DC-containing cell mixture preparation is obtained, which is inputted as a DC vaccine into a subject, and the antigen information loaded by the DC is presented to activate a specific T cell response.

Herein, the antigen presenting cells may be derived from mammalian peripheral blood (e.g., from PBMCs), for example, by cell separation. Or the antigen presenting cells are artificially constructed antigen presenting cell lines (e.g., DC cell lines) or antigen presenting cell precursor cell lines (e.g., DC precursor cell lines) that can be immortalized and cultured in vitro.

Antigen presenting cells are activated by loading with tumor-associated antigens. As used herein, "load" refers to the process of causing antigen presenting cells to contain (capture) tumor-associated antigen in a manner such that the antigen is processed and presented to other immune cells. In the case of DC cells, the loading can be carried out in a variety of ways in contact with the antigen or its coding sequence, for example by incubation with recombinant, synthetic or purified tumor antigen peptides or proteins, incubation with tumor cell lysates, incubation with apoptotic or necrotic tumor cells or expression of the antigen by the cells. Expression of the antigen by the cell may be achieved by contacting (e.g., co-incubating) the cell with a nucleic acid (DNA or RNA) encoding the tumor antigen or introducing (e.g., electroporation) the nucleic acid into the cell (e.g., by electrotransfer RNA). Introduction of a DNA coding sequence into a cell typically involves a nucleic acid (DNA) construct, such as an expression vector and an integration vector, comprising the DNA sequence and an appropriate promoter or control sequence. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Alternatively, the RNA coding sequence (e.g., mRNA) of an antigen can be introduced directly into a cell to express the antigen. Depending on the tumor to be targeted, the antigen may be contacted and loaded with the corresponding tumor antigen or nucleic acid encoding it (e.g., mRNA). Tumor-associated antigens include, but are not limited to, cancer-testis antigens (cancer testis antigen), over-expressed antigens (overexpressed protein/anti), differentiation antigens (differentiation antigen). Tumor-associated antigens illustratively used herein include MAGE-A3, survivin, CEA. The manner in which the antigen is loaded is known in the art, e.g., incubation, cell transformation (e.g., electrotransport DNA or mRNA), etc. Herein, the antigen or its coding sequence is in soluble form or the antigen or its coding sequence is attached to a solid support. The solid support may comprise polystyrene beads. The solid support is biodegradable.

Antigen presenting cells (e.g., DCs) induce cell maturation by contact with a maturation composition (maturation cocktail (maturation cocktails)). The maturation composition illustratively used herein comprises one or more selected from the group consisting of IFN-. Gamma., polyI: C, R848, PGE 2. 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 order of activation (antigen-loading) and maturation of antigen-presenting cells is generally not particularly limited, i.e., the cells may be loaded with antigen prior to contacting the cytokine composition, or the antigen may be contacted with the cytokine composition prior to loading with antigen. This is within the knowledge of a person skilled in the art.

The antigen presenting cells herein contain, express, and/or secrete PD1 binding molecules and CTLA4 binding molecules. Herein, "PD1" and "PD-1" may be used interchangeably, both referring to programmed death receptor 1 (programmed cell death protein 1); "CTLA4" and "CTLA-4" are used interchangeably and refer to cytotoxic T lymphocyte-associated protein4 (cytotoxin T-lymphocyte-associated protein 4). Herein, "PD1 binding molecule", "CTLA4 binding molecule" is a protein that specifically binds to PD1 and CTLA4, respectively, including, but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen binding fragments, e.g., fab, F (ab') 2, and Fv. The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

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 (CH 1, CH2 and CH3 for each alpha and gamma chain) and four (CH 1, CH2, CH3 and CH 4) constant domains (CH) for the mu and epsilon isoforms and a Hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL and VH are aligned together, while CL and the first constant domain of the heavy chain (CH 1) are aligned together. 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, daniel p.sties, abba i.terr and Tristram g.Parsolw editions, appleton & Lange, norwalk, CT,1994, pages 71 and chapter 6. Immunoglobulins may be assigned to different classes or isotypes depending on their heavy chain constant domain (CH) amino acid sequence. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM have heavy chains called α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes can be further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1 and IgA2.

The "heavy chain antibody" as referred to herein is an antibody derived from a camelidae or cartilaginous fish organism. In contrast to the 4-chain antibodies described above, the heavy chain antibody lacks the light and heavy chain constant regions 1 (CH 1), comprising only 2 heavy chains consisting of variable regions (VHH) and other constant regions, which are linked to the constant regions by hinge-like structures. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). 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 to 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. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

In one or more embodiments, the anti-PD-1 antibodies of the invention are nanobodies having a CDR1 as shown in any one of SEQ ID NOS.1, 4-39, 320 in CN202011022908. X (preferably any one of SEQ ID NOS.19, 36-39, 320 in the patent application), a CDR2 as shown in any one of SEQ ID NOS.2, 40-75 (preferably any one of SEQ ID NOS.53, 55, 72-75 in the patent application), and a CDR3 as shown in any one of SEQ ID NOS.3, 76-183 (preferably any one of SEQ ID NOS.87, 108-183 in the patent application). VHH of anti-PD-1 antibodies is as shown in any one of SEQ ID NOs 184-319 in CN202011022908. X. Illustratively, CDR1-3 of the anti-PD-1 nanobody is as shown in SEQ ID NO 1-3, respectively, of the sequence Listing appended hereto; VHH of the anti-PD-1 nanobody is shown in a sequence table SEQ ID NO:4 attached hereto.

In one or more embodiments, the anti-CTLA 4 antibodies of the invention are nanobodies having CDR1 as shown in SEQ ID NO 1, 4-10 (preferably any one of SEQ ID NO 4-10 in the patent application), CDR2 as shown in SEQ ID NO 2, 11-18 (preferably any one of SEQ ID NO 11-18 in the patent application), and CDR3 as shown in SEQ ID NO 3, 19-26 (preferably any one of SEQ ID NO 19-26 in the patent application) of CN 202111152925.4. The VHH of the anti-CTLA 4 antibody is shown in any one of SEQ ID NOs 27-73 in CN 202111152925.4. Illustratively, CDRs 1-3 of the anti-CTLA 4 nanobody are shown in the sequence Listing SEQ ID NOS 5-7, respectively, appended hereto; the VHH of the anti-CTLA 4 nanobody is shown in the attached sequence table SEQ ID NO. 8.

The binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; binding molecules comprising two or more different specific single domain antibodies are multispecific single domain antibodies. Multivalent or multispecific single domain antibodies connect multiple single domain antibodies via linkers. The linker generally consists of 1-15 amino acids selected from G and S.

Herein, heavy chain antibodies and antibodies are intended to distinguish between different combinations of antibodies. Because of the similarity in structure, the following structural descriptions for antibodies are applicable to heavy chain antibodies as well as to light chains.

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

The term "variable" refers to the case where certain segments in the variable domain differ widely in antibody sequence. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domains. Instead, it focuses on three segments called hypervariable regions (HVRs), both in the light and heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 for the heavy chain variable region (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 for the light chain variable region, respectively. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR 4) that mostly take on a β -sheet conformation, linked by three HVRs that form a loop linkage and in some cases form part of the β -sheet structure. 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 antigen binding site of the antibody. Typically, the light chain variable region is of the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4 and the heavy chain variable region is of the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cell-mediated cytotoxicity.

"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1 q) of the classical complement system. As used herein, the Fc region may 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 regions of an intact antibody. The antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; an scFv-Fc fragment; multispecific antibodies formed from antibody fragments; and any fragment that should be capable of increasing half-life by chemical modification or by incorporation into liposomes. Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of the complete light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH 1). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of antibodies produced a larger F (ab') 2 fragment, roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F (ab ') 2 antibody fragments were initially generated as pairs of Fab ' fragments with hinge cysteines between the Fab ' fragments. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also the region recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The 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 heavy and light chains) are highlighted from the fold of these two domains, contributing to the antigen-binding amino acid residues and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less avidity than the complete binding site. "Single chain Fv" may also be abbreviated "sFv" or "scFv" and 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 such that the sFv forms the desired antigen-binding structure.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being 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 culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention may be generated by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for producing human or human-like antibodies from animals having a portion or the entire human immunoglobulin locus or gene encoding a human immunoglobulin sequence, single cell sequencing methods.

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

"humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged for sequences found in a human antibody. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are grafted into the β -sheet framework of the human antibody variable region to produce antibodies, the specificity of which 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 include humanized variants of each of the antibodies.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing a human antibody. This definition of human antibodies specifically excludes humanized antibodies that comprise 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 binding fragment thereof that binds to the same epitope of PD-1 (or CTLA 4) as any anti-PD-1 antibody (or anti-CTLA 4 antibody) of the invention, i.e., a single domain antibody, heavy chain antibody, antibody or antigen binding fragment thereof that is capable of cross-competing with any antibody of the invention for binding to PD-1 (or CTLA 4).

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 further comprises a heavy chain constant region, for example a constant region of a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

The invention also includes the antibody derivatives and analogs. "derivatives" and "analogs" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The derivative or analogue of the invention may be (i) a polypeptide having a substituent in one or more amino acid residues, or (ii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, for example polyethylene glycol, or (iii) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader or secretory sequence or a sequence for purification of the polypeptide or a pro-protein sequence, or a fusion protein with a 6His tag). These derivatives and analogs fall within the scope of the teachings herein, as known to those skilled in the art.

One skilled in the art can alter the sequences of the invention by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequences thereof without substantially affecting the activity of the antibody. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. Conservative substitutions with amino acids of similar or similar properties generally do not alter the function of the protein in the art. Amino acids having similar properties are substituted, for example, in the FR and/or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. They are all considered to be included within the scope of the present invention. Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention. In some embodiments, the sequences of the variants of the invention may have at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which they were derived. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The invention also includes those molecules having antibody heavy chain variable regions with CDRs, provided that the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

Antibodies or coding sequences thereof may be introduced into antigen presenting cells in the form of proteins, RNA or DNA. For example, construction of DNA vectors expressing antibodies and transformation of antigen presenting cells. Thus, the invention also includes polynucleotides (in DNA or RNA form) encoding the antigens or antibodies or fragments thereof described herein, and nucleic acid constructs (e.g., expression vectors and integration vectors) comprising these polynucleotides. The vectors described herein generally contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) 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 complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting nucleic acid encoding an antibody to be expressed, and optional marker elements.

The polynucleotide introduced into an antigen presenting cell of the present invention may be in the form of DNA or RNA (e.g., mRNA). The inventors found that mRNA safety is more advantageous than conventional vaccines, e.g., it is not mutated by insertion, it can be degraded by normal cells, its half-life can be altered by regulatory sequence modification and delivery vehicles, etc. Methods for introducing mRNA into antigen presenting cells (e.g., DC cells) are well known in the art, such as by electrotransformation. In exemplary embodiments, exemplary coding sequences for anti-PD-1 antibodies are set forth in SEQ ID NO. 9 at positions 63-434; exemplary coding sequences for anti-CTLA-4 antibodies are shown at positions 576-953 of SEQ ID NO. 9.

The coding sequence of an antibody may also have a signal peptide to direct secretion of the antibody, the signal peptide useful in the present invention being known to those skilled in the art. Exemplary signal peptides include: human kappa chain signal peptide coding sequence (e.g., SEQ ID NO:9 at positions 3-62), human immunoglobulin light chain signal peptide (e.g., SEQ ID NO:9 at positions 510-575).

The invention provides methods of making the DC cells, comprising providing dendritic cells with the ability to secrete PD-1 binding molecules (e.g., antibodies), CTLA-4 binding molecules (e.g., antibodies), and to load tumor-associated antigens. The method may comprise the steps of: (1) Loading a tumor-associated antigen or a coding sequence thereof onto a dendritic cell capable of secreting a PD-1 binding molecule and a CTLA-4 binding molecule; or (2) enabling the dendritic cells loaded with the tumor associated antigen to secrete the PD-1 binding molecule and CTLA-4 binding molecule; or (3) contacting the dendritic cells with a tumor associated antigen or a coding sequence thereof and a coding sequence for a PD-1 binding molecule and a CTLA-4 binding molecule. The method of loading an antigen as described herein before can be accomplished, for example, by contacting (e.g., co-incubating) the cell with the antigen or its encoding nucleic acid (DNA or RNA) or introducing (e.g., electroporation) the antigen or its encoding nucleic acid into the cell (e.g., by electroRNA).

Common methods for allowing cells to secrete or have the ability to secrete antibodies include expression of the antibodies. Processes for expressing antibodies in cells are known in the art, such as introducing into a cell a nucleic acid (e.g., DNA or RNA) encoding an antibody. Introduction of a DNA coding sequence into a cell typically involves a nucleic acid (DNA) construct, such as an expression vector and an integration vector, comprising the DNA sequence and an appropriate promoter or control sequence. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Exemplary vectors can be found in CN105154473a and CN111206043a, which are incorporated herein by reference in their entirety. Alternatively, the RNA coding sequence (e.g., mRNA) of an antibody can be introduced directly into a cell to express the antibody.

The RNA coding sequences of antibodies may be synthesized by gene companies or obtained by in vitro transcription. Methods for preparing RNA sequences by in vitro transcription are known to those skilled in the art. An exemplary in vitro transcription method includes the steps of constructing a transcription template DNA vector and incubating in a transcription system. Transcription systems and incubation conditions are well known in the art, transcription systems such as: transcription buffers (including but not limited to Tris-HCl, mgCl2, DTT, spermidine), NTP, rnase inhibitors, RNA polymerase, etc.; incubation conditions are for example 37℃for at least 2 hours.

The coding sequences for each antibody may be located on separate nucleic acid constructs or combined on the same nucleic acid construct in a suitable manner. In the example of expressing antibodies in mRNA, the mRNA encoding each antibody can be expressed separately on the same nucleic acid construct by ligating the mRNA encoding each antibody sequences through a linker. The joints include, but are not limited to: coding sequence for Furin cleavage site, coding sequence for 2A (e.g., F2A, T2A), IRES sequence, etc. Illustratively, the linker is Furin-GSG-T2A, and the coding sequence is shown in SEQ ID NO. 9 at positions 435-509. The coding sequence of an antibody typically also comprises a stop codon (e.g., TGATAA) and may comprise a cleavage site for ease of genetic engineering operations.

Furthermore, in embodiments in which tumor-associated antigens and binding molecules (e.g., binding molecules such as antibodies) are expressed, the coding sequences for the tumor-associated antigens and binding molecules may be introduced into the cell separately or simultaneously. Similarly, the coding sequences for the tumor-associated antigen and each binding molecule may be located on separate nucleic acid constructs or combined on the same nucleic acid construct in a suitable manner.

Specific procedures for preparing DC cells for example, antigen Presenting Cells (APCs) such as DCs are taken from a subject, such as a patient suffering from or at risk of developing cancer, using white blood cell apheresis. The purified dendritic cells are cultured in the presence of the maturation composition to obtain mature DC cells. Mature DC cells are loaded with antigen (e.g., MAGE-A3, survivin, CEA), for example, by electrotransformation of mRNA encoding the antigen, thereby obtaining mature DC and DC vaccines comprising the antigen loaded. The DC cells can express the PD-1 binding molecules and CTLA-4 binding molecules described herein, e.g., mRNA for electrotransduce anti-PD-1 antibodies and anti-CTLA-4 antibodies, prior to, simultaneously with, or after antigen loading. The antigen-loaded and activated DCs are then administered to a patient. An exemplary course of treatment includes administration of 3 DCs over a 4 week period.

Herein, the culture medium and culture conditions required in the preparation of DC cells may employ conditions for conventional culture of DC cells. Exemplary media and culture conditions are shown in the examples.

The antigen presenting cells (e.g., DC cells) described herein can be used to prepare pharmaceutical compositions, e.g., DC vaccines, for preventing or treating 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, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, mesothelioma, carcinoma of large intestine, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, uterine fibroids and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, cholangiocarcinoma, thyroid cancer, parathyroid cancer, adrenal carcinoma, soft tissue sarcoma, urethral cancer, urothelial cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia), childhood solid tumor, lymphocytic lymphoma, renal or carcinoma, carcinoma of the renal pelvis, carcinoma of the anal system (schlemma), primary tumor, tumor of the human nervous system (T-cell carcinoma), tumor of the human tumor, cancer of the human nervous system, cancer of the human system, cancer of the brain, cancer of the human nervous system, cancer of the human system, cancer of the brain, cancer of the human nervous system, cancer of the human nervous system, cancer of the human tumor, including asbestos-induced cancers and various leukemia and lymphoma and various precancerous lesions. In particular tumors that are sensitive to anti-PD-1 antibodies and anti-CTLA-4 antibodies.

The pharmaceutical composition of the present invention may be different antigen-presenting cells loaded with different antigens, respectively, or may be one antigen-presenting cell loaded with a plurality of antigens. In an exemplary embodiment, the pharmaceutical composition comprises three antigen presenting cells (e.g., DC cells) loaded with MAGE-A3, survivin, CEA expressing the PD 1-binding molecules and CTLA 4-binding molecules described herein, respectively. The concentration and ratio of the various antigen presenting cells in the pharmaceutical composition can be adjusted as desired by one skilled in the art. Illustratively, the three antigen presenting cells are contained in equal proportions in a pharmaceutical composition.

In addition to the DC cells described herein, the pharmaceutical compositions herein also contain pharmaceutically acceptable excipients including, but not limited to, diluents, carriers, solubilizers, emulsifiers and/or preservative adjuvants. The adjuvant is preferably non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

The auxiliary materials in the pharmaceutical composition also comprise vaccine adjuvants. The adjuvant may be a small molecule, a biological macromolecule, a composition, a complex or an extract of a compound known in the art to be capable of enhancing the effect of an immune response. In one or more embodiments of the present invention, the adjuvant includes a material selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, interferon alpha, corynebacterium parvum, lipopolysaccharide, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, proteoliposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticle resins, nanoliposome polymeric gels (nanonolipogel) synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA, polycaprolactone, silicon polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ di- (carboxyacetyl phenoxy) phosphazene (PCPP), poly- (methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (e.g. polyamino acids, poly- (gamma-glutamic acid), poly-arginine-HCl, poly-L-lysine), polypeptides, biopolymers), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactams, bellens, glycerides, phospholipids and spirochetes.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In the case of lyophilization of a composition, this method may be used to sterilize the composition either before or after lyophilization and reconstitution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the composition may be selected for inhalation or delivery through the digestive 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 antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles 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 formulation may be stored in a ready-to-use form or reconstituted (e.g., lyophilized) prior to administration. The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides a method of treating a patient, particularly a mesothelin-related disorder in a patient, by administering a binding molecule according to any one of the embodiments of the invention or a pharmaceutical composition thereof. The terms "patient," "subject," "individual," "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). "preventing" refers to the use of a therapeutic regimen described herein by a subject at risk to achieve at least one effect of preventing the occurrence of a disease or condition. The treatment regimen effective to treat or prevent a patient may vary depending on a variety of factors such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject.

The therapeutically effective amount of the pharmaceutical composition comprising the binding molecule of the invention to be employed will depend, for example, on the degree of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule 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. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect. Such as from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

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

The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional route injection; 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 intra-articular injection. Alternatively, depending on the target of the vaccine, the vaccine may be administered subcutaneously or intradermally to the extremities of a patient suffering from cancer that is being treated. Other routes of administration, such as intramuscular injection or blood injection, may also be employed.

Depending on the type of pharmaceutical composition (e.g. vaccine) prepared, the production scale of the pharmaceutical composition may be enlarged if necessary by culturing the cells in a bioreactor or fermenter or similar vessel and apparatus suitable for cell mass growth. In one or more embodiments, devices or compositions comprising the vaccine or antigen produced or recovered according to the present invention are suitable for sustained or intermittent release, and may be implanted in the body or administered locally at the corresponding location in the body, to achieve the effect of slowly and periodically releasing these materials into the body.

The invention also provides a method of treating and/or preventing cancer comprising administering to a subject an effective dose of one or more of the foregoing cells and pharmaceutical compositions. The method comprises the effect of at least one of treating and preventing. In one or more embodiments, the methods of the invention are for prophylactic purposes, and one or more of the cells and pharmaceutical compositions of the invention are administered to a subject individual prior to the occurrence of a cancer or precancerous condition. In certain instances, the pharmaceutical composition is administered to a subject individual after the onset of one or more of the cancers described above, in order to prevent further symptoms from occurring or further exacerbation of symptoms that have occurred. Prophylactic administration of one or more of the cells and pharmaceutical compositions of the invention is intended to prevent or alleviate any subsequent symptoms. In one or more embodiments, the methods of the invention are for therapeutic purposes, and one or more of the cells and pharmaceutical compositions of the invention are administered to a subject individual at or after the onset of cancer, with the aim of alleviating the symptoms of the cancer that has developed.

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

Examples

Experimental method

Antibody mRNA preparation

The 1194-NLA of the anti-PD 1 VHH and the Z12 of the anti-CTLA 4 VHH are connected through Furin-GSG-T2A, an Nco I enzyme cutting site and a human kappa chain signal peptide sequence are added in 5', a human immunoglobulin light chain signal peptide is added between the Z12 of the CTLA4 VHH and the Furin-GSG-T2A, two stop codons TGATAA and Sal I enzyme cutting sites are added in 3', and the sequence 1194-NLA-T2A-Z12 is synthesized by Jin Wei intelligent company. The self-constructed EGFP transcription template vector pT7-m5U-eGFP contains a T7 promoter, a 5'UTR sequence of HBBmRNA (NM_ 000518), an EGFP sequence, a3' UTR sequence of HBB mRNA and a polyA sequence (SEQ ID NO: 10), a 2122bp skeleton fragment is recovered by double digestion of Nco I and Sal I, and a 860bp target fragment is recovered by the same digestion with the synthetic sequence and is connected to obtain the transcription template pT7-m5U-1194-2VHH.

In vitro transcription reactions produce mRNA. The in vitro transcription system comprises: template cDNA 1.0. Mu.g, 10 Xtranscription buffer (400 mM Tris-HCl pH 8.0, 190mM MgCl2, 50mM DTT,10mM spermidine) 2.0. Mu.l, NTP (25 mM each) 7.2. Mu.l, RNase inhibitor 20U, T RNA polymerase 3000U, dH20 to 20.0. Mu.l.

Incubate at 37℃for 3 hours to 5 hours. The crude IVT mixture can be stored overnight at 4 ℃ for purification the next day. The original template was digested using 1U DNase without rnase at 37 ℃ for 15 min and mRNA was purified using the Ambion MEGACLEARTM kit (Austin, TX) according to the manufacturer's instructions. The kit can purify RNA of 500 mug. After purification, RNA was quantified using NanoDrop and analyzed by agarose gel electrophoresis to confirm that the RNA was of the appropriate size and that no degradation of the RNA occurred.

Antigen mRNA preparation

Antigen mRNA was prepared according to a similar method to that for antibody mRNA.

DC cell culture and induction

(1) Isolation of PBMC

1) The blood was collected from the blood bag by syringe in a 50ml centrifuge tube, the blood bag was washed with PBS (brand name HyClone) having the same volume as the blood collected by syringe, and the blood was mixed with the collected blood collected by syringe (1: 1 dilution);

2) A50 ml centrifuge tube (15 ml Ficoll lymphocyte separation liquid (brand name GE)) is taken, the mixed liquid of blood and PBS in the step 1 is slowly added along the tube wall, and then the centrifugation is carried out for 800g, 20min, 1 for rising speed and 0 for falling speed.

3) The white cell layer was carefully aspirated into another 50ml centrifuge tube, and washed with PBS for 10min at a speed of 9.

4) Pouring out the waste liquid, adding DPBS, cleaning again, and centrifuging (step 3).

5) The waste liquid was poured off, AIM-V (brand gibco) suspension cells were added to the culture flask and allowed to adhere overnight.

(2) DC cytogenesis

Monocyte-derived DCs were generated from Peripheral Blood Mononuclear Cells (PBMCs) by standard Ficoll density centrifugation to isolate PBMCs from patient leukapheresis samples. PBMC were inoculated 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 50ng/ml rhIL-4 and 100ng/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 100IU/ml IFN-. Gamma.30. Mu.g/ml poly (I: C), 5. Mu.g/ml R848 and 1. Mu.g/ml PGE2 was used to induce DC maturation for 24 hours.

(3) DC cell transformation

mRNA encoding the anti-PD-1, anti-CTLA-4 nanobody sequence (hereinafter referred to as antibody mRNA) required for preparing an electrotransfer reagent was prepared. According to the LONZA electrotransfer kit, an electrotransfer reagent with an antibody mRNA of 60ug/2.5 x 10e6 cells was prepared. DC induced and matured from a mononuclear source were collected, electrotransformed according to LONZA electrotransformation kit instructions, transferred into 24 well plates and cultured with AIM-V medium to a cell density of 1X 10e6/ml and a volume of 1ml in the well plates. Culturing for 3 days, respectively collecting cell supernatants of 24h, 48h and 72h, and detecting the anti-PD-1 and anti-CTLA-4 nano antibody by ELISA method.

Antibody mRNAs required for preparing the electrotransfer reagent and 3 mRNAs encoding antigen sequences (hereinafter referred to as MAGEA-3mRNA, CEA mRNA, and survivin mRNA, wherein NCBI Reference Sequence of MAGEA-3 mRNA: NCBI Reference Sequence of NM-005362.4,CEA mRNA: NCBI Reference Sequence of NM-004363.6,Suvivin mRNA: NM-001168.3) were prepared. Preparing corresponding MAGEA-3mRNA according to LONZA electrotransformation kit; CEA mRNA; the survivin mRNA was 5ug/2.5 x 10e6 cells electrotransport reagent. Respectively preparing antibody mRNA and MAGEA-3mRNA; antibody mrna+cea mRNA; antibody mrna+survivin mRNA (antibody mRNA 60ug/2.5 x 10e6 cells, antigen mRNA 5ug/2.5 x 10e6 cells). Collecting DC induced and matured by a mononuclear source, dividing the DC into 6 parts, carrying out electrotransformation on the electrotransformation liquids according to LONZA electrotransformation kit instructions, and uniformly mixing cells respectively subjected to electrotransformation on 3 antigen mRNA; the cells respectively electrically transformed with 3 antigen+antibody mRNAs are uniformly mixed, transferred into a 24-well plate and cultured by adding AIM-V culture medium, so that the cell density in the well plate is 1 x 10e6/ml and the volume is 1ml, and immature DC and mature DC are set as experimental control. Culturing for 3 days, and collecting DC function after DC flow detection and electrotransformation for 24 hours. Respectively collecting cell supernatants of 24h, 48h and 72h, and detecting anti-PD-1, anti-CTLA-4 nano antibodies by an Elisa method; the Elisa method detects the cytokine IL-12; the CBA method detects cytokines IL-6, IL-10 and TNF-alpha.

Antibody mRNAs required for preparing an electrotransfer reagent and 3 mRNAs encoding antigen sequences (hereinafter referred to as MAGEA-3mRNA, CEA mRNA, and survivin mRNA) were prepared. Preparing corresponding MAGEA-3mRNA according to LONZA electrotransformation kit; CEA mRNA; the survivin mRNA was 5ug/2.5 x 10e6 cells electrotransport reagent. Respectively preparing antibody mRNA and MAGEA-3mRNA; antibody mrna+cea mRNA; antibody mrna+survivin mRNA (antibody mRNA 60ug/2.5 x 10e6 cells, antigen mRNA 5ug/2.5 x 10e6 cells). Collecting DC induced and matured by a mononuclear source, dividing the DC into 6 parts, carrying out electrotransformation on the electrotransformation liquids according to LONZA electrotransformation kit instructions, and uniformly mixing cells respectively subjected to electrotransformation on 3 antigen mRNA; the cells respectively electrically transformed with 3 antigen+antibody mRNAs are uniformly mixed, each group of cells is transferred into a 12-well plate and is added with a 1640 culture medium for culture, so that the number of DC cells in the well plate is 2 x 10e5, the volume is 1ml, and immature DC and mature DC are set as experimental control. After 2h, inactive T cells and active T cells (placed in a cd3\cd28 coated culture plate for 48 h) were added to the well-grouped DCs to make the number of cells in the well plate, t=1:10=2×10e5:2×10e6, 2ml in volume, and cultured for 3 days, and T cell flow assay T cell function was collected for 48 h. Respectively collecting cell supernatants of 24h, 48h and 72h, and detecting anti-PD-1, anti-CTLA-4 nano antibodies by an Elisa method; the CBA method detects cytokines IFN-gamma, TNF-alpha, IL-6 and IL-10.

Flow cytometry

The flow detection basically comprises the following steps:

(1) 1X 106 cells were added to each tube.

(2) Washing with 1ml PBS phosphate buffer for 2 times, centrifuging 400g for 5min, and discarding

A supernatant; adding 100 mu L of PBS phosphate buffer solution for resuspension;

(3) Adding a flow antibody to be detected, uniformly mixing, placing in a refrigerator at 2-8 ℃, and incubating for 30min in a dark place;

setting a group of blank control, and adding no test agent or corresponding isotype;

(4) Adding 1ml PBS phosphate buffer solution, centrifuging at 400g for 5min, cleaning for 2 times, discarding supernatant,

the cells were resuspended by pipetting 400. Mu.l of PBS phosphate buffer, examined by flow cytometry, and all fines were set up

Cells were collected 1×104. Data were analyzed using Kaluza Analysis software.

The procedure described above was tested and the different antibodies added are shown in the following table:

DC detection

Detection item antibody	Luminous signal
		CD80(Biolegend)	FITC
CD83(Biolegend)	APC
		CD86(Biolegend)	APC
CD40(Biolegend)	AF700
		HLA-ABC(Biolegend)	PE
HLA-DR(Biolegend)	PE-Cy7
		CD197(CCR7)(Biolegend)	PE-Cy7

T detection

Detection item antibody	Luminous signal
		CD3(Biolegend)	FITC
CD4(Biolegend)	FITC
		CD8(Biolegend)	PE
CD279(PD-1)(BD)	BV421
		CD152(CTLA-4)(BD)	PE
CD62L(BD)	PE
		CD25(BD)	APC
CD69(Biolegend)	BV421
		HLA-DR(Biolegend)	PE-Cy7

Multiplex cytokine CBA method detection

According to the operation steps of the instruction, firstly, the standard substance is subjected to gradient dilution, after 2mL of the standard substance is resuspended, 9 flow-type loading pipes are taken out, and the multiples of gradient dilution of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256 are marked respectively. In order to ensure that each experimental tube contains 6 microspheres, 10 mu l of each capturing microsphere is needed, the volume of the mixed microsphere is 60 mu l, the amount of the added microspheres in each tube of detection sample or standard product in the specific experimental process is 50, and the microspheres are configured according to the proportion. For example, the number of samples to be tested is 8, 9 standards, 1 negative control, and 18 total tubes. Therefore, it was necessary to add 50. Mu.l of diluted sample/standard (50. Mu.l) per tube after 180. Mu.l of each microsphere was mixed and add 50. Mu.l of PE detection reagent thereto and incubate at room temperature for 3 hours in the absence of light.

The method comprises the steps of calibrating an instrument and adjusting voltage by combining magnetic beads before the machine, compensating and adjusting by using control magnetic beads, and calculating the concentration of a detection sample by combining CBA analysis software FCAP with standard curve analysis after the machine is acquired after incubation.

ELISA detection

ELISA detection of anti-PD-1 antibody, anti-CTLA-4 antibody secretion essentially follows:

(1) Antigen coated plate: the coated antigen is prepared. The antigen was diluted with coating solution, and the enzyme-labeled reaction plate was coated at 100 ul/well, and at 4℃overnight. After overnight, the wells were washed 5 times with 200 ul/well for 3 minutes each with blotting paper.

(2) Closing: each well was incubated with 300ul of blocking solution in a biochemical incubator at 37℃for 2 hours. The wells were washed 5 times with PBST, 200 ul/well for 3 minutes each, and then blotted dry with absorbent paper.

(3) Sample adding: and diluting the sample and the standard by using a diluent, diluting the standard in a gradient manner, setting 7 gradients and 0ng/ml, and diluting the sample according to actual conditions. Samples and standards were added, 100 ul/well, and duplicate wells and control wells were set. The biochemical incubator was incubated at 37℃for 1 hour. The wells were washed 5 times with PBST, 200 ul/well for 3 minutes each, and then blotted dry with absorbent paper.

(4) Adding a secondary antibody: the secondary antibody was diluted in proportion with blocking solution, 100 ul/well and incubated in a 37℃biochemical incubator. The wells were washed 5 times with PBST, 200 ul/well for 3 minutes each, and then blotted dry with absorbent paper.

(5) Color development: adding color development solution TMB (brand Abcam), and developing at room temperature in dark place for 5-15min at 100 ul/hole.

(6) And (3) terminating: the reaction was terminated by adding 50 ul/well of a stop solution. The on-board readings were immediately taken.

The steps described above were tested and the different parameters are shown in the following table:

parameters (parameters)	PD-1	CTLA-4
			Coating antigen concentration	1ug/mL (Arco brand)	2ug/mL (self-produced)
Maximum gradient of standard	12.5ng/mL (self-produced)	20ng/mL (self-producing)
			Standard substance and sample incubation time	1h	1.5h
Dilution ratio of secondary antibody	1:20000 (brand Abcam)	1:10000 (GenScript brand)
			Incubation time of secondary antibody	30min	1h

Example 1

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. anti-PD-1/anti-CTLA-4 antibody mRNA was electroporated to electroporate the mature DC. The electric conversion amounts are 40ug, 60ug, 80ug, 100 ug/2.5X10 respectively ⁶ And (3) cells. Cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24h and 48h, collecting the supernatant, and detecting 1X 10 by ELISA ⁶ Cells secrete a-PD-1, a-CTLA4 content. The detection results are as follows: electrotransport of different concentrations of a-PD-1, a-CTLA-4 affects the secretion of a-PD-1, a-CTLA-4. Secretion amount of the a-PD-1 and the a-CTLA-4 after electrotransformation is 24 hours, and secretion amount is higher than 48 hours; 100ug>80ug>60ug>40ug group.

The results are shown in FIG. 1:

A.100ug group 24h a-PD-1 secretion amount was 145.3ng, 48h secretion amount was 72.2ng,80ug group 24h a-PD-1 secretion amount was 133.7ng, 48h secretion amount was 61.4ng,60ug group 24h a-PD-1 secretion amount was 122.2ng, 48h secretion amount was 56.6ng,40ug group 24h a-PD-1 secretion amount was 93.8ng, 48h secretion amount was 38.9ng.

B.100ug group 24h a-CTLA-4 secretion amount is 60.9ng, 48h secretion amount is 24.3ng,80ug group 24h a-CTLA-4 secretion amount is 56.5ng, 48h secretion amount is 20.9ng,60ug group 24h a-CTLA-4 secretion amount is 52.1ng, 48h secretion amount is 19.2ng,100ug group 24h a-CTLA-4 secretion amount is 40.9ng, 48h secretion amount is 15.1ng.

Example 2

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. The anti-PD-1/anti-CTLA-4 antibody mRNA and MAGE-A3 antigen mRNA were electroporated to form mature DCs. The electrotransformation amounts were respectively (antigen mRNA:5 ug/2.5X10) ⁶ Cells) and (antibody mRNA:40ug, 60ug, 80 ug/2.5X10 ⁶ Cells), collecting supernatant after 24h, 48h and 72h, and detecting 1×10 by ELISA ⁶ Cells secrete a-PD-1, a-CTLA4 content. The detection results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electrotransformation tends to decrease with time. Higher levels of a-PD-1, a-CTLA4 can be secreted at 24 h.

The results are shown in FIG. 2:

80ug group 24h a-PD-1 secretion amount is 120.5ng, 48h secretion amount is 41.2ng, 72h secretion amount is 16.9ng;60ug group 24h a-PD-1 secretion amount is 89.1ng, 48h secretion amount is 24.0ng, 72h secretion amount is 12.8ng;40ug group 24h a-PD-1 secretion amount is 72.3ng, 48h secretion amount is 21.1ng, 72h secretion amount is 13.0ng;

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

Example 3

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. The anti-PD-1/anti-CTLA-4 antibody mRNA and survivin antigen mRNA were electroporated to form mature DCs. The electrotransformation amounts were respectively (antigen mRNA:5 ug/2.5X10) ⁶ Cells) and (antibody mRNA:40ug, 60ug, 80 ug/2.5X10 ⁶ Cells). Cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24 hours, 48 hours and 72 hours, the supernatant is collected and detected to be 1 multiplied by 10 by ELISA ⁶ Cell secretion of a-PD-1, a-CTLA4 content. The detection results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electrotransformation tends to decrease with time. Higher levels of a-PD-1, a-CTLA4 can be secreted at 24 h.

The results are shown in FIG. 3:

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

80ug group 24h a-CTLA-4 secretion amount is 69.1ng, 48h secretion amount is 28.5ng, and 72h secretion amount is 16.9ng;60ug group 24h a-CTLA-4 secretion amount is 57.3ng, 48h secretion amount is 23.4ng, 72h secretion amount is 12.5ng;40ug group 24h a-CTLA-4 secretion amount is 45.6ng, 48h secretion amount is 18.1ng, 72h secretion amount is 10.1ng.

Example 4

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. Electroporation was used to electroporate the anti-PD-1/anti-CTLA-4 antibody mRNA and CEA antigen mRNA into mature DCs. The electrotransformation amounts were respectively (antigen mRNA:5 ug/2.5X10) ⁶ Cells) and (antibody mRNA:40ug, 60ug, 80 ug/2.5X10 ⁶ Cells). Cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24 hours, 48 hours and 72 hours, the supernatant is collected and detected to be 1 multiplied by 10 by ELISA ⁶ Cells secrete a-PD-1, a-CTLA4 content. The detection results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electrotransformation tends to decrease with time. Higher levels of a-PD-1, a-CTLA4 can be secreted at 24 h.

The results are shown in FIG. 4:

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

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

Example 5

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. The mature DC cells were divided into three groups for electrotransformation. A first group: anti-PD-1/anti-CTLA-4 antibody mRNA and MAGE-A3 antigen mRNA; second group: anti-PD-1/anti-CTLA-4 antibody mRNA and survivin antigen mRNA; the third group is anti-PD-1/anti-CTLA-4 antibodies and CEA antigen mRNA). The electrotransformation amounts were respectively (antigen mRNA:5 ug/2.5X10) ⁶ Cells) and (antibody mRNA:60 ug/2.5X10 ⁶ Cells). After electrotransformation, three groups of cells were mixed together and the cells were cultured in 24 well plates (1X 10) ⁶ Cells/ml). After 24 hours, 48 hours and 72 hours, the supernatant is collected and detected to be 1 multiplied by 10 by ELISA ⁶ Cells secrete a-PD-1, a-CTLA4 content. The detection results are as follows: the secretion of a-PD-1 and a-CTLA-4 after electrotransformation tends to decrease with time. Higher levels of a-PD-1, a-CTLA4 can be secreted at 24 h.

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

Example 6

On day 5, the DC cells were either untreated (first group: immature DC-iDC-) or matured with a maturation cocktail (IFN-. Gamma., polyI: C, R848, PGE 2) for 24 hours (mature DC-mDC-). On day 6, mature DC cells were divided into three groups of electrotransformation. Second group: no electric rotation; third group: three antigen mRNAs (MAGE-A3, survivin and CEA); fourth group (superdc): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNAs. Cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24 hours of incubation, the expression of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CCR7 in the four groups was compared by flow-through assays.

The results are shown in FIG. 6:

HLA-ABC positive cell MFI for the first set of DCs was 4611, for the second set of DCs 12746MFI, for the third set of DCs 13186MFI and for the fourth set of DCs 15112MFI. The first group of DCs had an HLA-DR positive cell MFI of 1761, the second group had a DC of 2786MFI, the third group had a DC of 2898MFI, and the fourth group had a DC of 2999MFI. The first set of DCs had a CD80 positive cell MFI of 2809, the second set had a DC of 8666MFI, the third set had a DC of 9210MFI, and the fourth set had a DC of 12015MFI. The first set of DCs had a CD86 positive cell MFI of 3722, the second set of DCs had a MFI of 6357, the third set of DCs had a MFI of 9771 and the fourth set of DCs had a MFI of 10634. E, CD40 positive cell MFI of the first set of DCs is 2674, DC of the second set is 4221MFI, DC of the third set is 6084MFI, and DC of the fourth set is 6884MFI. F, the first group of DCs had a CCR7 positive cell MFI of 790, the second group had a DC of 1799MFI, the third group had a DC of 1998MFI, and the fourth group had a DC of 2205MFI. From the above results, it can be seen that mRNA transfection has a positive effect on maturation and activation of DCs.

Example 7

On day 5, the DC cells were either untreated (first group: immature DC-iDC-) or matured with a maturation cocktail (IFN-. Gamma., polyI: C, R848, PGE 2) for 24 hours (mature DC-mDC-). On day 6, mature DC cells were divided into three groups of electrotransformation. Second group: no electric rotation; third group: three antigen mRNAs (MAGE-A3, survivin and CEA); fourth group (superdc): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNAs. Cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24 hours of incubation, supernatants were collected and assayed for secretion of IL-6, TNF- α and IL-10 by CBA flow-through methods. The secreted amount of IL-12 was detected by ELISA.

The results are shown in FIG. 7:

the IL-6 secretion amount of the DCs of the first group was 34.4pg/ml, the DCs of the second group was 64.2pg/ml, the DCs of the third group was 86.4pg/ml, and the DCs of the fourth group was 214.5pg/ml. The IL-12 secretion amount of the DCs of the first group was 45.0pg/ml, the DCs of the second group was 101.7pg/ml, the DCs of the third group was 121.1pg/ml, and the DCs of the fourth group was 154.7pg/ml. The amount of TNF-. Alpha.secretion was 21.6pg/ml in the first DC, 96.9pg/ml in the second DC, 118.4pg/ml in the third DC, and 169.3pg/ml in the fourth DC. The IL-10 secretion amount of the DCs of the first group was 31.2pg/ml, the DCs of the second group was 34.0pg/ml, the DCs of the third group was 31.5pg/ml, and the DCs of the fourth group was 32.2pg/ml. From the above results, it can be seen that superdcs secrete the highest levels of pro-inflammatory cytokines, which are important in T cell responses.

Example 8

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. anti-PD-1/anti-CTLA-4 antibody mRNA and three antigen mRNAs (MAGE-A3, survivin and CEA) were electroporated to form mature DCs. After 4h of electrotransformation, the cells were incubated with T cells according to 1:10 ratio (DC: 2X 10) ⁵ cells, T cells: 2X 10 ⁶ ) Co-culturing, respectively taking supernatants of 24h, 48h and 96h for ELISA detection of a-PD-1 and a-CTLA-4, and collecting cells for flow detection at 96 h. The detection results are shown in fig. 8: a is ELISA detection result, and secretion of 24h, 48h, 96h a-PD-1 is 29.2ng,14.6ng and 5.2ng; secretion of 24h, 48h, 96h a-CTLA-4 was 17.5ng, 10.44ng, 3.8ng. B is 96h flow detection T cell surface marker CTLA-4 and PD-1. The analysis modes are CD3+ (CTLA-4) and CD3+ (PD-1). From the upper panel of FIG. 8, B, it can be seen that T cell surface CTLA-4 is 35.05%, super DC: t cell surface CTLA-4 of T group 23.83%; from the lower graph of FIG. 8, B, it can be seen that the T cell surface PD-1 is 16.07%, super DC: the surface PD-1 of T-group T cells was 5.05%. It can be seen that superDCs secrete a-CTLA-4 and a-PD1, resulting in reduced expression of CTLA-4 and PD-1 on the surface of T cells.

Example 9

On day 5, the DC cells were either untreated (first group: immature DC-iDC-) or matured with a maturation cocktail (IFN-. Gamma., polyI: C, R848, PGE 2) for 24 hours (mature DC-mDC-). On day 6, mature DC cells were divided into three groups of electrotransformation. Second group: no electric rotation; third group: three antigen mRNAs (MAGE-A3, survivin and CEA); fourth group (superdc): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNAs. After 4h of electrotransformation, the cells were incubated with T cells according to 1:10 ratio (DC: 2X 10) ⁵ cells, T cells: 2X 10 ⁶ ) Co-culturing. After 96 hours of incubation, T cells were collected to detect surface markers (CD 3 ⁺ CD8 ⁺ 、CD3 ⁺ CD4 ⁺ 、CD3 ⁺ CD62L ⁺ 、CD3 ⁺ CD25 ⁺ 、CD3 ⁺ CDHLA-DR ⁺ 、CD3 ⁺ CD69 ⁺ )。

The results are shown in FIG. 9:

a is the firstCD3 of T cells of a group ⁺ CD8 ⁺ The percentage of positive cells expressed was 38.8%, the second group of T cells 38.8%, the third group of T cells 38.3%, and the fourth group of T cells 52.6%. CD3 of T cells of the first group ⁺ CD4 ⁺ The percentage of positive cells expressed was 45.1%, the second group of T cells 45.9%, the third group of T cells 50.3%, and the fourth group of T cells 40.9%. CD3 of T cells of the first group ⁺ HLA-DR ⁺ The percentage of positive cells expressed was 6.8%, the second group of T cells was 8.9%, the third group of T cells was 9.4%, and the fourth group of T cells was 15.6%. CD3 of T cells of the first group ⁺ CD62L ⁺ The percentage of positive cells expressed was 69.9%, the second group of T cells 65.3%, the third group of T cells 64.6%, and the fourth group of T cells 40.2%. CD3 of T cells of the first group ⁺ CD25 ⁺ The percentage of positive cells expressed was 5.2%, the second group of T cells was 6.1%, the third group of T cells was 8.7%, and the fourth group of T cells was 65.4%. F CD3 of T cells of the first group ⁺ CD69 ⁺ The percentage of positive cells expressed was 8.8%, the second group of T cells was 8.0%, the third group of T cells was 10.4%, and the fourth group of T cells was 39.2%. From the above results, it can be seen that superdcs can effectively activate T cells.

Example 10

On day 5, the DC cells were either untreated (first group: immature DC-iDC-) or matured with a maturation cocktail (IFN-. Gamma., polyI: C, R848, PGE 2) for 24 hours (mature DC-mDC-). On day 6, mature DC cells were divided into three groups of electrotransformation. Second group: no electric rotation; third group: three antigen mRNAs (MAGE-A3, survivin and CEA); fourth group (superdc): three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNAs. After 4h of electrotransformation, the cells were incubated with T cells according to 1:10 ratio (DC: 2X 10) ⁵ cells, T cells: 2X 10 ⁶ ) Co-culturing. After 48 hours of culture, the cell supernatants were assayed for secretion of IFN-gamma, TNF-alpha, IL-6 and IL-10 by CBA.

The results are shown in FIG. 10:

a, IFN-. Gamma.secretion amounts of 448pg/ml in the first group, 601pg/ml in the second group, 801pg/ml in the third group and 1092pg/ml in the fourth group. The secretion amount of TNF-alpha in the first group is 155pg/ml, the secretion amount of TNF-alpha in the second group is 252pg/ml, the secretion amount of TNF-alpha in the third group is 307pg/ml, and the secretion amount of TNF-alpha in the fourth group is 506pg/ml. The secretion amount of IL-6 in the first group was 100pg/ml, 178pg/ml in the second group, 226pg/ml in the third group and 422pg/ml in the fourth group. The secretion amount of IL-10 in the first group was 26pg/ml, the secretion amount in the second group was 28pg/ml, the secretion amount in the third group was 27pg/ml, and the secretion amount in the fourth group was 25pg/ml. From the above results, it can be seen that superdcs are effective in promoting T cell activity and enhancing the secretion of type 1 and type 2 cytokines.

Example 11

Mature DCs were obtained by IFN-. Gamma., polyI C, R848, PGE2 stimulation for 24 h. Three antigen mRNAs (MAGE-A3, survivin and CEA) and anti-PD-1/anti-CTLA-4 antibody mRNAs were electroporated to form mature DCs (superDCs). The electrotransport concentration was 60 ug/2.5X10 ⁶ And (3) cells. Cells were divided into two parts after electrotransformation: 1) Cells were cultured [ 24 well plate (1X 10) ⁶ Cell/ml) 24h,48h,72h, and then collecting the supernatant; 2) Cells were cultured [ 24 well plate (1X 10) ⁶ Cells/ml) and after 4h, the cells were collected for cryopreservation. Resuscitates after 4 weeks of cryopreservation and cells were cultured in 24-well plates (1X 10) ⁶ Cells/ml). After 24h,48h,72h, the supernatants were collected. Detection of the supernatant by ELISA 1X 10 ⁶ Cells secrete a-PD-1, a-CTLA4 content.

The results are shown in FIG. 11:

after 24h, the secretion of fresh cell a-PD-1 was 70.3ng, and the secretion of frozen cell a-PD-1 was 65.2ng. After 48h the secretion of fresh cell a-PD-1 was 25.0ng and that of cryopreserved cell a-PD-1 was 20.7ng. After 72h the secretion of fresh cell a-PD-1 was 11.9ng and that of cryopreserved cell a-PD-1 was 10.2ng.

The secretion of fresh cells a-CTLA-4 after 24h is 48.9ng, and the secretion of frozen cells a-CTLA-4 is 45.1ng. After 48h the secretion of fresh cells a-CTLA-4 was 22.8ng and that of frozen cells a-CTLA-4 was 21.0ng. After 72h the secretion of fresh cells a-CTLA-4 was 11.0ng and that of frozen cells a-CTLA-4 was 10.1ng.

ELISA experiment results show that the capacity of secreting high levels of a-PD-1 and a-CTLA-4 can be maintained after the cryopreserved DC cells after electrotransformation are recovered.

Sequence listing

<110> Shanghai cell therapy group Co., ltd

<120> super DC expressing immune checkpoint inhibitor and uses thereof

<130> 217890 1CNCN

<160> 10

<170> PatentIn version 3.5

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<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

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Gly Arg Pro Phe Ser Ile Tyr Asp

1 5

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<212> PRT

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<220>

<223> CDR2

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Ile Asn Leu Ala Arg Gly Asn Thr

1 5

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

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Gly Val Asp Arg Arg Gln Tyr Gly Leu Gly Ile Pro Pro Leu Ala Asp

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His

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<223> VHH

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Ile Tyr

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Asp Met Gly Trp Phe Arg Gln Ala Pro Asp Lys Glu Arg Glu Ser Val

35 40 45

Ala Val Ile Asn Leu Ala Arg Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys

85 90 95

Gly Val Asp Arg Arg Gln Tyr Gly Leu Gly Ile Pro Pro Leu Ala Asp

100 105 110

His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 5

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 5

Gly Phe Ser Ser Asp Tyr Tyr Asp

1 5

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 6

Ile Arg Ser Ser Gly Gly Ser Thr

1 5

<210> 7

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 7

Gly Leu Ala Pro Ile Ser Pro Val His Ala Val Cys Asn Gln His Tyr

1 5 10 15

Phe Gly Tyr

<210> 8

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Ser Asp Tyr Tyr

20 25 30

Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ser Cys Ile Arg Ser Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Leu Ala Pro Ile Ser Pro Val His Ala Val Cys Asn Gln His Tyr

100 105 110

Phe Gly Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 959

<212> DNA

<213> Artificial Sequence

<220>

<223> a-PD1-CTLA4

<400> 9

ccatggaggc cccagctcag ctgctgttcc tgctgctgct gtggctgcca gacaccacag 60

gagaggtgca gctggtggag agcggcggcg gcctggtgca gcccggcggc agcctgagac 120

tgagctgcgc cgccagcggc agacccttca gcatctacga catgggctgg ttcagacagg 180

cccccgacaa ggagagagag agcgtggccg tgatcaacct ggccagaggc aacacctact 240

acgccgacag cgtgaagggc agattcacca tcagcagaga caacgccaag aacaccgtgt 300

acctccagat gaacagcctg agagccgagg acaccgccgt gtacagctgc ggcgtggaca 360

gaagacagta cggcctgggc atcccccccc tggccgacca ctggggccag ggcacccagg 420

tgaccgtgag cagccgcgcc aagcgcggca gcggcgaggg ccgcggcagc ctgctgacct 480

gcggcgacgt ggaggagaac cccggcccca tggacatgcg cgtgcccgcc cagctgctgg 540

gcctgctgct gctgtggctg agcggcgccc gctgcgaggt gcagctggtg gagagcggcg 600

gcggcctggt gcagcccggc ggcagcctgc gcctgagctg cgccgccagc ggcttcagca 660

gcgactacta cgacatcggc tggttccgcc aggcccccgg caaggagcgc gagatggtga 720

gctgcatccg cagcagcggc ggcagcacca agtacgccga cagcgtgaag ggccgcttca 780

ccatcagccg cgacaacagc aagaacaccg tgtacctcca gatgaacagc ctgcgcgccg 840

aggacaccgc cgtgtactac tgcggcctgg cccccatcag ccccgtgcac gccgtgtgca 900

accagcacta cttcggctac tggggccagg gcacccgcgt gaccgtgagc agctgataa 959

<210> 10

<211> 1055

<212> DNA

<213> Artificial Sequence

<220>

<223> vector

<400> 10

taatacgact cactatagga catttgcttc tgacacaact gtgttcacta gcaacctcaa 60

acagacacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 120

gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 180

gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 240

tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 300

cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 360

accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 420

gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 480

ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 540

cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 600

cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 660

gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 720

cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 780

tacaagtaag tcgacgctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc 840

cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 900

ctaataaaaa acatttattt tcattgcaag cttaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaggaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1055

Claims

1. Antigen presenting cells secreting PD-1 binding molecules and CTLA-4 binding molecules,

preferably, the PD-1 binding molecule is an anti-PD-1 antibody or antigen-binding fragment thereof, and/or the CTLA-4 binding molecule is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

2. The antigen presenting cell of claim 1, wherein the antigen presenting cell is selected from the group consisting of a macrophage, a B cell, and a dendritic cell.

3. The antigen presenting cell of claim 1 or 2, wherein the antigen presenting cell comprises a coding sequence for a PD-1 binding molecule and a coding sequence for 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.

4. The antigen presenting cell of claim 1 or 2, wherein the antigen presenting cell is loaded with a tumor-associated antigen; preferably, the antigen presenting cells express the tumor-associated antigen.

5. A method of producing a dendritic cell loaded with a tumor-associated antigen comprising:

(1) Loading the antigen presenting cell of any one of claims 1 to 4 with a tumor-associated antigen;

(3) Contacting the antigen presenting cells with a tumor associated antigen or a coding sequence thereof and a coding sequence for a PD-1 binding molecule and a CTLA-4 binding molecule,

6. The method of claim 5, wherein (2) comprises introducing into the cell the coding sequences for the PD-1 binding molecule and the CTLA-4 binding molecule;

7. The method of claim 5 or 6, wherein loading the tumor-associated antigen comprises: contacting or expressing a tumor-associated antigen, and/or contacting or introducing a coding sequence for a tumor-associated antigen,

preferably, the coding sequence for the tumor-associated antigen is RNA.

8. The method of claim 5 or 6, wherein the dendritic cells are contacted with a maturation composition before or after the antigen is loaded.

9. A pharmaceutical composition comprising the antigen presenting cell of any one of claims 1 to 4 or produced by the method of any one of claims 5 to 8, and a pharmaceutically acceptable adjuvant,

preferably, the pharmaceutical composition is for use in the treatment or prevention of a tumor in a subject expressing the tumor-associated antigen.

10. Use of an antigen presenting cell of any one of claims 1 to 4 or produced by the method of any one of claims 5 to 8 in the manufacture of a medicament for preventing the occurrence or metastasis of a tumor in a subject or inhibiting the growth or metastasis of a tumor in a subject, said tumor expressing said tumor-associated antigen.