Multi-antigen loaded engineered antigen presenting cell and application thereof

Abstract

The invention relates to the field of immunotherapy, in particular to an engineered antigen presenting cell which contains a coding sequence of a multispecific nano antibody. And / or can express and / or secrete a multispecific nano antibody; a tumor-associated antigen is loaded on the carrier, and the tumor-associated antigen is selected from one or more of the following components: P53, Survivin, MUC1, hTERT or KRAS. The antigen presenting cell disclosed by the invention can activate immature T cells and effectively stimulate the effector function of the activated T cells on cancers.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, specifically to engineered antigen-presenting cells loaded with multiple antigens and their uses. Background Technology

[0002] Tumor immunotherapy, as a novel cancer treatment modality, primarily works by activating the body's immune system to generate anti-tumor immunity, thereby eliminating tumor cells. It not only induces long-lasting anti-tumor immunity but also plays a crucial role in preventing postoperative recurrence. Dendritic cells (DCs), as the primary link in generating a specific immune response, are an important target for tumor immunotherapy. DC vaccines utilize DCs derived from peripheral blood mononuclear cells sensitized with autologous or allogeneic tumor cell components to directly induce a specific immune response.

[0003] DC vaccines involve introducing cultured dendritic cells (DCs) loaded with tumor antigens into the body. These DCs regulate the proliferation and activation of tumor antigen-specific Th1 cells through antigen presentation and cytokine secretion, further promoting NK cell and CTL activation and mediating tumor killing. DCs that can specifically recognize tumors and are induced or constructed in vitro, when reinfused into cancer patients, can activate T cell immune responses against tumors and express high levels of PD-1 and CTLA-4.

[0004] Therapeutic tumor vaccines targeting dendritic cells typically consist of three components: 1) Tumor-associated antigens: These antigens can be small peptides, recombinant proteins, tumor antigens expressed via modified tumor cells, viral vectors / or modified bacterial vectors, and DNA or RNA. These antigens, expressed or prepared in different ways, may be absorbed and processed by dendritic cells in vivo and expressed on the cell surface as MHC2, where they can be recognized by T cells (CD8). Antigens can also be expressed directly on dendritic cells. 2) Dendritic cells themselves (in vivo, isolated and purified, or isolated from peripheral blood leukocytes). 3) Adjuvants or immunostimulants.

[0005] Although clinical studies have shown that DC vaccines induced by patients' own mononuclear cells are well tolerated and can generate anti-tumor immune responses, they are still not effective in treating tumors. This may be due to a lack of specific tumor antigens, low antigen loading efficiency, and subsequent activation of regulatory T cells, leading to immunosuppression. The tumor immune microenvironment and tumor immunosuppressive mechanisms are key factors limiting the use of DC vaccines. Summary of the Invention

[0006] The first aspect of the present invention provides engineered antigen-presenting cells, wherein the antigen-presenting cells:

[0007] (1) Contains a coding sequence for a multispecific nanobody; and / or

[0008] (2) Capable of expressing and / or secreting multispecific nanobodies;

[0009] and,

[0010] (3) Loaded with a tumor-associated antigen, wherein the tumor-associated antigen is selected from one or more of the following: P53, Survivin, MUC1, hTERT or KRAS.

[0011] In one or more embodiments, the tumor antigen includes: P53, Survivin, MUC1, hTERT, and KRAS.

[0012] The multispecific nanobody contains multiple functional regions that target multiple targets, and the multiple functional regions are heavy chain antibodies or their antigen-binding fragments.

[0013] In one or more embodiments, the antigen-presenting cells are derived from mammalian peripheral blood.

[0014] In one or more embodiments, the antigen-presenting cells are derived from mammalian PBMCs.

[0015] In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

[0016] In one or more embodiments, the antigen-presenting cells are mature or immature dendritic cells.

[0017] In one or more embodiments, the plurality of targets are selected from: immune checkpoint proteins, immune cell-associated antigens, tumor-associated antigens, immune co-stimulatory molecules or their receptors, for example selected from: PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, CD244, CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3, SLAM, CD226, CD80, CD86, EIIIB fibronectin, Siglecl5, VEGF(R), HER2, PSMA, AXL, MUC1, MUC16.

[0018] In one or more embodiments, the multispecific nanobody contains two functional regions that target two targets respectively (i.e., a bispecific antibody), or three functional regions that target three targets respectively (i.e., a trispecific antibody).

[0019] In one or more embodiments, the plurality of targets are selected from immune checkpoint proteins, such as PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, and CD244.

[0020] In one or more embodiments, the multispecific nanobody is an anti-PD1 / CTLA4 bispecific nanobody containing an anti-PD-1 nanobody and an anti-CTLA-4 nanobody.

[0021] In one or more embodiments, the complementarity-determining region of the anti-PD-1 nanobody comprises P-CDR1, P-CDR2, and P-CDR3, wherein P-CDR1 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:1 or 4; P-CDR2 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:2 or 5; and P-CDR3 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:4; and P-CDR3 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:5; and P-CDR3 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:4. The sequence shown in NO:3 or 6 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:7, and the complementarity-determining region of the anti-CTLA-4 heavy chain antibody includes C-CDR1, C-CDR2, and C-CDR3, wherein C-CDR1 includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:8, C-CDR2 includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:8, and C-CDR3 includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:7, and C-CDR3 ... The sequence shown in NO:9 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. In one or more embodiments, the anti-PD-1 nanobody has the sequence shown in SEQ ID NO:10, 11, 38, or 39, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and wherein each CDR sequence is identical to SEQ ID NO:10, 11, 38, or 39.

[0022] In one or more embodiments, the anti-CTLA-4 heavy chain antibody has the sequence shown in SEQ ID NO:12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and wherein each CDR sequence is identical to the sequence in SEQ ID NO:12.

[0023] In one or more implementations, the plurality of functional areas are independently one, two, or more.

[0024] In one or more embodiments, the plurality of functional areas are fused together by a connector. Preferably, the connector is (GGSGG)p or (G4S)mGn, where m, n, and p are each an independent positive integer from 1 to 10. Preferably, m, n, and p are each an independent positive integer from 1 to 6 or from 1 to 4.

[0025] In one or more embodiments, the connector has the sequence shown in SEQ ID NO:13-17.

[0026] In one or more embodiments, the anti-PD-1 nanobody is a chimeric antibody, preferably a humanized antibody.

[0027] In one or more embodiments, the anti-CTLA-4 heavy chain antibody is a chimeric antibody, preferably a humanized antibody.

[0028] In one or more embodiments, the multispecific nanobody further comprises: an Fc region; preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4.

[0029] In one or more embodiments, the Fc region further includes a mutation, and after the mutation, the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced compared to before the mutation.

[0030] In one or more embodiments, the Fc region is the Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more mutations selected from the following: L234A, L235A, G237A; or the Fc region is the Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more mutations selected from the following: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0031] In one or more embodiments, the antigen-presenting cells contain a coding sequence for a tumor-associated antigen; preferably, the coding sequence is RNA; more preferably, the RNA sequence encoding P53 is shown in SEQ ID NO:70, the RNA sequence encoding Survivin is shown in SEQ ID NO:71, the RNA sequence encoding MUC1 is shown in SEQ ID NO:72, the RNA sequence encoding hTERT is shown in SEQ ID NO:73, and the RNA sequence encoding KRAS is shown in SEQ ID NO:74.

[0032] A second aspect of the present invention provides a method for generating engineered antigen-presenting cells, comprising:

[0033] (1) Loading the antigen-presenting cell with the aforementioned tumor antigen or introducing the coding sequence of the tumor antigen; and introducing the coding sequence of the aforementioned multispecific nanobody into the antigen-presenting cell.

[0034] In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

[0035] In one or more embodiments, loading an antigen-presenting cell with a tumor antigen or introducing a coding sequence of a tumor antigen includes contacting the antigen-presenting cell with a tumor antigen peptide or with RNA encoding a tumor antigen.

[0036] In one or more embodiments, introducing the coding sequence of a multispecific nanobody includes: contacting an antigen-presenting cell with RNA encoding the multispecific nanobody; or contacting a plasmid vector containing a polynucleotide encoding the multispecific nanobody.

[0037] In one or more embodiments, the plasmid is a microcarrier with a backbone sequence length of less than 600 bp, reduced and / or free of CpG DNA motifs, and / or the plasmid is an antibiotic-free microplasmid.

[0038] In one or more embodiments, dendritic cells are derived from monocytes.

[0039] In one or more embodiments, the antigen-presenting cells are contacted with a maturation composition (e.g., maturation cocktails) before or after step (1). The maturation composition contains one or more selected from IFN-γ, PolyI:C, R848, and PGE2.

[0040] In one or more embodiments, the tumor includes respiratory system tumors, digestive system tumors, urinary system tumors, nervous system tumors, reproductive system tumors, and skin tumors; preferably, it includes one or more of the following: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulvar cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gallbladder cancer, kidney cancer, bladder cancer, and brain cancer.

[0041] A third aspect of the present invention also provides a method for in vitro activation of immune-active cells derived from a patient with a tumor, comprising:

[0042] Immune-active cells were obtained from the patient.

[0043] Engineered antigen-presenting cells are generated by the method described in the second aspect of the present invention, wherein the tumor antigen is at least one tumor antigen of the patient's tumor; and

[0044] The various immune-active cells are co-cultured with the engineered antigen-presenting cells for a sufficient period of time to activate the immune-active cells, thereby obtaining activated immune-active cells.

[0045] The fourth aspect of the present invention also provides activated immune-active cells, obtained by the method of the third aspect of the present invention.

[0046] In one or more embodiments, the immune-active cell is a T cell.

[0047] The fifth aspect of the present invention also provides a pharmaceutical composition comprising engineered antigen-presenting cells as described in the first aspect of the present invention or engineered antigen-presenting cells produced by the method described in the second aspect of the present invention, and / or activated immune-active cells as described in the fourth aspect of the present invention; and pharmaceutically acceptable excipients.

[0048] In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

[0049] In one or more embodiments, the pharmaceutical composition is used to treat or prevent tumors in a subject that express the tumor antigen.

[0050] In one or more embodiments, the pharmaceutical composition is a vaccine composition.

[0051] The present invention also provides the use of engineered antigen-presenting cells as described in the first aspect of the present invention, engineered antigen-presenting cells produced by the method described in the second aspect of the present invention, or activated immune-active cells as described in the fourth aspect of the present invention in the preparation 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, wherein the tumor expresses the tumor antigen.

[0052] In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

[0053] In one or more embodiments, the object is a mammal, such as a human, dog, cat, horse, cow, or pig.

[0054] In one or more embodiments, the subject has a tumor or is at risk of developing a tumor.

[0055] The present invention also provides a method for preventing or treating the occurrence, growth, or metastasis of tumors in a subject, comprising administering a therapeutically effective amount of engineered antigen-presenting cells as described in the first aspect of the present invention or engineered antigen-presenting cells produced by the method described in the second aspect of the present invention, activated immune-active cells as described in the fourth aspect of the present invention, or a pharmaceutical composition as described in the fifth aspect of the present invention.

[0056] In one or more embodiments, the engineered antigen-presenting cells prevent the occurrence of the tumor or reduce the growth and metastasis of the tumor, and the engineered antigen-presenting cells are cultured in vitro with a mature composition prior to administration, the antigen-presenting cells carrying the tumor antigen.

[0057] In one or more embodiments, the antigen-presenting cells include one or more selected from macrophages, B cells, and dendritic cells.

[0058] In one or more embodiments, the mature composition comprises one or more selected from IFN-γ, PolyI:C, R848, and PGE2.

[0059] Following injection, dendritic cells (DCs) migrate to lymph nodes, where they encounter and activate naive T cells. The activation mechanisms are: 1. Interaction between MHC on the surface of DCs and the TCR of T cells; 2. Interaction between the B7 molecule of DCs and the CD28 of T cells. The secretion of anti-PD-1 / CTLA-4 bispecific nanobodies or DCs containing their encoding sequences in some embodiments of this invention has the following advantages:

[0060] The activation mechanism of naive T cells is as follows: In lymph nodes, Tregs express high levels of CTLA-4, which interacts with the B7 molecule in dendritic cells (DCs), reducing the ability of DCs to stimulate tumor-specific T cells. The DCs of this invention can secrete a PD-1 / CTLA-4 bispecific antibody, blocking CTLA-4 in Tregs and reversing the reduction in the ability of Tregs to stimulate tumor-specific T cells. Therefore, the DCs of this invention can effectively activate naive T cells.

[0061] Activated T cells express high levels of PD-1 and CTLA-4. At the tumor site, the function of tumor-specific T cells is inhibited by three factors, preventing them from killing cancer cells: A. Tumor cells, macrophages, and other immune cells express PD-L1; B. Tregs express high levels of CTLA-4; C. Tumor cells express B7 molecules that bind to CTLA-4 on T cells. The DCs of this invention can secrete a PD-1 / CTLA-4 bispecific antibody, thus blocking the expression of PD-1 and CTLA-4 on T cells. This means that CTLA-4 and PD-1 expression on T cells is inhibited before T cells migrate to the tumor site, thereby effectively stimulating the effector function of activated T cells against cancer.

[0062] Moreover, compared with secreting PD-1 nanobodies and CTLA-4 nanobodies separately, secreting PD-1 / CTLA-4 bispecific nanobodies can increase the amount of antibody secreted, especially much more than the amount of CTLA-4 nanobodies alone, thereby better blocking Treg's CTLA-4.

[0063] Some embodiments of the present invention also propose a method for producing DC vaccines by introducing exogenous RNA, which can trigger the body's innate and adaptive immune responses. In tumor treatment, this mechanism can be used to introduce exogenous RNA in the form of a vaccine, which can promote the release of inflammatory cytokines and interferon, activate NK cells, macrophages, and effector T cells, turn "cold tumors" without immune cell infiltration into "hot tumors", promote the killing effect of the immune system on cancer cells, and also synergistically improve the efficacy of immune checkpoint inhibitor antibodies, that is, "relaxing the brakes" and "stepping on the gas" for the immune system, thereby further enhancing the anti-tumor immune response. Compared with traditional vaccines, mRNA has a greater safety advantage, does not insert gene mutations, can be degraded by normal cells, and its half-life can be changed by regulating sequence modification and delivery vectors. Many pieces of evidence show that mRNA can not only mediate better transfection efficiency and longer protein expression time, but also has greater advantages than DNA. These advantages include: (1) mRNA can function without entering the cell nucleus. Once it reaches the cytoplasm, mRNA initiates protein translation. In contrast, DNA needs to enter the nucleus first and then be transcribed into mRNA. This process makes DNA less efficient than mRNA because its function depends on the disruption of the nuclear membrane during cell division. (2) Compared to DNA and viral vectors, mRNA does not insert into the genome but only transiently expresses the encoded protein. Therefore, due to its low insertion risk, it provides an excellent safe option for researchers and pharmaceutical companies. (3) mRNA is easily synthesized through in vitro transcription (IVT). This process is relatively inexpensive and can be rapidly applied to various therapies. Moreover, mRNA is theoretically capable of expressing any protein, so it can be used to treat almost all diseases. This solves the previous problems of expensive and cumbersome preparation processes for DC vaccines. Attached Figure Description

[0064] Figure 1 : The ability of the antibody to bind to the PD1 antigen.

[0065] Figure 2 : The ability of the antibody to bind to the CTLA4 antigen.

[0066] Figure 3 : The ability of the antibody to bind to CTLA4-positive cells.

[0067] Figure 4 : The ability of the antibody to bind to PDL1-positive cells.

[0068] Figure 5 : 1194nla bispecific antibody RGA results.

[0069] Figure 6 RGA results of 1194nla and C43z11 bispecific antibodies.

[0070] Figure 7 Results of the bispecific antibody CTLA4 ligand binding inhibition experiment.

[0071] Figure 8 Results of CTLA4 ligand binding inhibition assay for bispecific antibodies. Bi-KLH: Isotype control, a nonfunctional bispecific antibody of the same type.

[0072] Figure 9 : The spectral structure of the anti-antibiotic microplasmid (empty vector).

[0073] Figure 10 : The spectral structure of the anti-antibiotic microplasmid (empty vector).

[0074] Figure 11 : The spectral structure of the anti-PD-1 / CTLA-4 bispecific nanobody plasmid.

[0075] Figure 12 Detection of PD-1 antibody secretion levels after electroporation of five antigen mRNAs and anti-PD-1 / CTLA4 bispecific nanobody plasmids.

[0076] Figure 13 Cell viability detection after electroporation of five antigen mRNAs and anti-PD-1 / CTLA4 bispecific nanobody plasmids.

[0077] Figure 14 A and C show the antigen expression level detection, where A is the P53 expression level in the cell extract detected by ELISA, B is the survivin expression level in the cell extract detected by ELISA, and C is the MUC1 antigen expression in DC PLUS cells detected by Western blot.

[0078] Figure 15 A and D respectively show the activation capacity of DC PLUS for T cells. A is the CBA assay to detect the secretion levels of TNF-α and IFN-γ in the supernatant of co-cultured DC and T cells; B is the flow cytometry assay to detect the proportion of CD4 and CD8 and the positive rates of CD69 and CD25 in T cells after co-culture of DC and T cells; C is the ELISPOT assay to detect the ability of T cells co-cultured with DC and T cells to secrete IFN-γ; D is the T cell expansion capacity after co-culture of DC and T cells.

[0079] Figure 16 The effect of DC PLUS on the tumor-killing ability of T cells. Detailed Implementation

[0080] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0081] In this invention, the term "expression frame" refers to the complete elements required to express a gene, including the promoter and the gene coding sequence.

[0082] The term "coding sequence" is defined in this text as the portion of a nucleic acid sequence that directly identifies its protein product (e.g., CAR, single-chain antibody, hinge region, and transmembrane region). The boundaries of a coding sequence are typically defined by a ribosome-binding site (for prokaryotic cells) immediately upstream of the 5' open reading frame of the mRNA and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. Coding sequences can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0083] The term "Fc" refers to the crystallizable fragment of an antibody, which is a peptide segment located at the end of the stalk of the "Y" structure of the antibody molecule, containing the CH2 and CH3 domains of the antibody heavy chain constant region. It is the site where the antibody interacts with effector molecules or cells.

[0084] The term "co-stimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that binds to co-stimulatory molecule receptors on Th cells, generating a co-stimulatory signal. Lymphocyte proliferation requires not only antigen binding but also the reception of signals from co-stimulatory molecules. Co-stimulatory signals are primarily transmitted to T cells through the binding of co-stimulatory molecules CD80 and CD86, expressed on the surface of antigen-presenting cells, to CD28 molecules on the surface of T cells. B cells receive co-stimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on the surface of Th cells.

[0085] The term "connector" or hinge is a polypeptide fragment that links different proteins or peptides, with the purpose of maintaining the spatial conformation of the linked proteins or peptides to preserve their function or activity. Exemplary connectors include those containing G and / or S, and, for example, the Furin 2A peptide.

[0086] The term "specific binding" refers to the reaction between an antibody or antigen-binding fragment and the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds at a concentration of less than about 10... -5 M, for example, less than approximately 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen. "Specific recognition" has a similar meaning.

[0087] The term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0088] The term "effective dose" refers to a dose that can achieve therapeutic, preventive, alleviating and / or relieving disease or condition as described in this invention in a subject.

[0089] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0090] The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0091] The term "engineering" and its grammatical equivalents may refer to one or more human-designed alterations to nucleic acids (e.g., nucleic acids within an organism's genome). In another embodiment, engineering may refer to alterations, additions, and / or deletions of genes. "Engineered antigen-presenting cells" may refer to immune cells having added, deleted, and / or altered genes. As used herein, the terms "immune cells" or "engineered antigen-presenting cells" and their grammatical equivalents may refer to immune cells of human or non-human animal origin.

[0092] In this document, the term "antibody" includes monoclonal antibodies, antibody compositions and single-chain molecules with multi-epitope specificity, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0093] The "heavy chain antibody" described in this article refers to antibodies derived from camelids or cartilaginous fishes. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is connected to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH or single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0094] As used herein, the terms "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," and "VHH" are used interchangeably to refer to the VHH that specifically recognizes and binds to the antigen. The VHH is the variable region of a heavy chain antibody. Typically, a VHH contains three CDRs and four FRs.

[0095] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. A variable domain (or variable region) contains an antigen-binding site that mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all the amino acids spanned by the variable domain. For heavy chain antibodies or VHHs, the variable regions include CDR1, CDR2, and CDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy chains (and light chains) each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of the β-sheet structure. The CDRs in each chain are held together very closely by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antibody's antigen-binding site.

[0096] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of heavy chain antibody fragments include Fv fragments; biantibodies; linear antibodies; single-domain antibodies (VHH); single-chain antibody molecules; scFv-Fc fragments; and any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes.

[0097] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. For heavy chain antibodies, this fragment consists of heavy chain variable domains. "Single-chain Fv" can also be abbreviated as "sFv" or "scFv", and is an antibody fragment containing the VHH domain of the heavy chain antibody linked together to form a single polypeptide chain.

[0098] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by human-derived polynucleotides or is identical to that of the antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems.

[0099] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0100] This invention also includes the antibody derivatives and analogs described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a cmyc and / or 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art. For example, a polypeptide may be tandemly linked with cmyc and 6His to form a polypeptide-cmyc-his structure. Linking sequences may be present between the polypeptide and cmyc, and between cmyc and his; an exemplary cmyc-his containing two linking sequences is shown as residues 260-285 of SEQ ID NO:18.

[0101] As used in this article, the term EC50 refers to the half-maximal effect concentration, which is the concentration that produces 50% of the maximum effect.

[0102] Engineered antigen-presenting cells

[0103] This article provides an engineered antigen-presenting cell containing a coding sequence for a multispecific nanobody; and / or capable of expressing and / or secreting multispecific nanobodies. The multispecific nanobodies contain multiple functional regions that target multiple targets, and each of these functional regions is a nanobody.

[0104] Antigen-presenting cells are cells that can transmit antigen information to lymphocytes (such as T cells) to trigger an immune response, including macrophages, B cells, and dendritic cells (DC cells or DCs).

[0105] In this article, "multiple" or "various types" refers to more than one or more kinds, that is, two or more types.

[0106] In one or more embodiments, the plurality of targets are selected from: immune checkpoint proteins, immune cell-associated antigens, tumor-associated antigens, immune co-stimulatory molecules, or their receptors. Immune checkpoint proteins include, but are not limited to: PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, and CD244. Immune cell-associated antigens include, but are not limited to: CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3, SLAM, CD226, CD80, and CD86. Tumor-associated antigens include, but are not limited to: EIIIB fibronectin, Siglecl5, VEGF(R), HER2, PSMA, AXL, MUC1, MUC16, P53, Survivin, hTERT, and KRAS.

[0107] In one or more embodiments, at least one of the plurality of functional regions is an immune checkpoint inhibitory nanobody.

[0108] In one or more embodiments, at least one of the plurality of functional regions is an activating antibody against an immune co-stimulatory molecule or its receptor.

[0109] In one or more embodiments, the plurality of functional regions are different immune checkpoint inhibitory nanobodies.

[0110] In one or more embodiments, the multispecific nanobody contains two or three functional regions that target two or three targets respectively.

[0111] In one or more preferred embodiments, the multispecific nanobody comprises: a first functional region targeting PD-1 and a second functional region targeting CTLA4; the first functional region is an anti-PD-1 nanobody and the second functional region is an anti-CTLA-4 nanobody.

[0112] As used herein, when referring to the amino acid sequence of the CTLA4 protein (Cytotoxic T-Lymphocyte Antigen 4), it includes the full-length CTLA4 protein, or an extracellular fragment of CTLA4 or a fusion protein thereof. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in this invention, the term "CTLA4 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the CTLA4 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0113] As used herein, when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NM_005018), it includes the full-length PD-1 protein, or an extracellular fragment of PD-1 or its fusion protein. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the PD-1 protein without affecting its biological function. Therefore, in this invention, the term "PD-1 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0114] Preferably, in this invention, the anti-PD-1 nanobody has P-CDR1 shown in SEQ ID NO:1 or 4, P-CDR2 shown in SEQ ID NO:2 or 5, and P-CDR3 shown in SEQ ID NO:3 or 6; the anti-CTLA-4 nanobody has C-CDR1 shown in SEQ ID NO:7, C-CDR2 shown in SEQ ID NO:8, and C-CDR3 shown in SEQ ID NO:9. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0115] In one or more embodiments, the nanobody of the present invention has FR1 as shown in SEQ ID NO:26, 30, 34, 40 or 44, FR2 as shown in SEQ ID NO:27, 31, 35, 41 or 45, FR3 as shown in SEQ ID NO:28, 32, 36, 42 or 46, and FR4 as shown in SEQ ID NO:29, 33, 37, 43 or 47. The sequences of the above-mentioned FRs can be arbitrarily combined for use in the anti-PD-1 nanobody or anti-CTLA-4 nanobody of the present invention. Exemplarily, the anti-PD-1 nanobody of the present invention has FR1 as shown in SEQ ID NO:26, 30, 40 or 44, FR2 as shown in SEQ ID NO:27, 31, 41 or 45, FR3 as shown in SEQ ID NO:28, 32, 42 or 46, and FR4 as shown in SEQ ID NO:29, 33, 43 or 47. The anti-CTLA-4 nanobody of the present invention has FR1 as shown in SEQ ID NO:34, FR2 as shown in SEQ ID NO:35, FR3 as shown in SEQ ID NO:36, and FR4 as shown in SEQ ID NO:37.

[0116] In some embodiments, the antigen-binding fragment of the anti-PD-1 nanobody has the sequence shown in SEQ ID NO:10, 11, 38, or 39, or a sequence having at least 80% sequence identity with it, wherein each CDR sequence is identical to SEQ ID NO:10, 11, 38, or 39. The antigen-binding fragment of the anti-CTLA-4 nanobody has the sequence shown in SEQ ID NO:12, or a sequence having at least 80% sequence identity with it, wherein each CDR sequence is identical to SEQ ID NO:12.

[0117] Multiple functional regions can be monovalent or multivalent single-domain antibodies, multispecific single-domain antibodies, heavy chain antibodies, or antigen-binding fragments thereof, comprising one, two, or more single-domain antibodies. For example, the first functional region targeting PD-1 described herein can be a monovalent or multivalent single-domain antibody, multispecific single-domain antibody, heavy chain antibody, or antigen-binding fragment thereof, comprising one, two, or more anti-PD-1 single-domain antibodies described herein. The first functional region targeting CTLA-4 described herein can be a monovalent or multivalent single-domain antibody, multispecific single-domain antibody, heavy chain antibody, or antigen-binding fragment thereof, comprising one, two, or more anti-CTLA-4 single-domain antibodies described herein.

[0118] Each of the multiple functional areas can be independently one, two, or more, with one of each being preferred.

[0119] The multiple functional regions can be directly connected or connected via adapters. The adapter sequence is not limited and can be any amino acid adapter sequence known in the art suitable for nanobodies. An exemplary adapter could be (GGSGG). p Or (G4S) m G n Where m, n, and p are each an independent positive integer from 1 to 10 (preferably 1 to 6, more preferably 1 to 4). For example, the connector is (G4S). m G4, where m is a positive integer from 1 to 4. Preferably, the connector has the sequence shown in SEQ ID NO:13-17.

[0120] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0121] The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention.

[0122] In some embodiments, the sequence of the variants described in this invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its source sequence. The sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. This invention also includes molecules having a variable region of the antibody heavy chain with a CDR, provided that its CDR has at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDR identified herein.

[0123] In some embodiments, the multispecific nanobody is a bispecific nanobody having the following structure from the N-terminus to the C-terminus: a first functional region targeting CTLA4, an optional linker, and a second functional region targeting PD-1. In some embodiments, the bispecific antibody has the following structure from the N-terminus to the C-terminus: a first functional region targeting PD-1, an optional linker, and a second functional region targeting CTLA4. In one or more embodiments, the bispecific nanobody has an anti-PD-1 single-domain antibody, a linker, and an anti-CTLA-4 single-domain antibody from the N-terminus to the C-terminus, wherein the complementarity-determining regions CDR1-CDR3 of the anti-PD-1 single-domain antibody are as shown in SEQ ID NO:4, 5, and 6, respectively, the complementarity-determining regions CDR1-CDR3 of the anti-CTLA-4 single-domain antibody are as shown in SEQ ID NO:7, 8, and 9, respectively, and the linker sequence is as shown in any of SEQ ID NO:13-17. In one or more embodiments, the bispecific nanobody comprises an anti-PD-1 single-domain antibody, a linker, and an anti-CTLA-4 single-domain antibody from the N-terminus to the C-terminus, wherein the complementarity-determining regions CDR1-CDR3 of the anti-PD-1 single-domain antibody are as shown in SEQ ID NO:1, 2, and 3, respectively; the complementarity-determining regions CDR1-CDR3 of the anti-CTLA-4 single-domain antibody are as shown in SEQ ID NO:7, 8, and 9, respectively; and the linker sequence is as shown in SEQ ID NO:14 or 15. In one or more embodiments, the bispecific nanobody comprises an anti-CTLA-4 single-domain antibody, a linker, and an anti-PD-1 single-domain antibody from the N-terminus to the C-terminus, wherein the complementarity-determining regions CDR1-CDR3 of the anti-CTLA-4 single-domain antibody are as shown in SEQ ID NO:7, 8, and 9, respectively; the complementarity-determining regions CDR1-CDR3 of the anti-PD-1 single-domain antibody are as shown in SEQ ID NO:4, 5, and 6, respectively; and the linker sequence is as shown in SEQ ID NO:15.

[0124] In one or more embodiments, the bispecific nanobody comprises an anti-PD-1 single-domain antibody, a linker, and an anti-CTLA-4 single-domain antibody from the N-terminus to the C-terminus, wherein the sequence of the anti-PD-1 single-domain antibody is selected from one of SEQ ID NO:10, 11, 38, or 39, the sequence of the anti-CTLA-4 single-domain antibody is as shown in SEQ ID NO:12, and the linker sequence is as shown in any one of SEQ ID NO:13-17, preferably as shown in SEQ ID NO:14. In one or more embodiments, the bispecific nanobody comprises an anti-CTLA-4 single-domain antibody, a linker, and an anti-PD-1 single-domain antibody from the N-terminus to the C-terminus, wherein the sequence of the anti-CTLA-4 single-domain antibody is as shown in SEQ ID NO:12, the sequence of the anti-PD-1 single-domain antibody is selected from one of SEQ ID NO:10, 11, 38, or 39, and the linker sequence is as shown in any one of SEQ ID NO:13-17, preferably as shown in SEQ ID NO:15.

[0125] In one or more embodiments, the bispecific nanobody comprises, from the N-terminus to the C-terminus: the anti-PD-1 single-domain antibody shown in SEQ ID NO:11, the adapter shown in SEQ ID NO:13, 14, or 15, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12; or the anti-PD-1 single-domain antibody shown in SEQ ID NO:38, the adapter shown in SEQ ID NO:14, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12; or the anti-PD-1 single-domain antibody shown in SEQ ID NO:39, the adapter shown in SEQ ID NO:14, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12; or the anti-PD-1 single-domain antibody shown in SEQ ID NO:10, the adapter shown in SEQ ID NO:15, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:10; or the anti-PD-1 single-domain antibody shown in SEQ ID NO:10, the adapter shown in SEQ ID NO:14, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12; or the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12, SEQ ID NO:10, the adapter shown in SEQ ID NO:14, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12; or the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12, SEQ ID NO:11, the adapter shown in SEQ ID NO:14, and the anti-CTLA-4 single-domain antibody shown in SEQ ID NO:12. The adapter shown in NO:15 and the anti-PD-1 single-domain antibody shown in SEQ ID NO:11.

[0126] In one or more embodiments, the bispecific nanobody has the sequences shown in SEQ ID NO:48-55. The bispecific nanobodies with the cmyc-his tag are shown in SEQ ID NO:18-25, respectively.

[0127] In one or more embodiments, the multispecific nanobody further comprises an Fc region; preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4; wherein the IgG1, IgG2, IgG3 or IgG4 is human-derived.

[0128] Because the Fc region can cause significant ADCC and CDC effects, potentially leading to immune cell damage and exhibiting negative pharmacological effects, Fc is often modified (referred to as variant Fc in this article). Through site mutation, the affinity constant of multispecific nanobodies for FcγRIIIa and / or C1q is reduced compared to the original value, thereby improving the efficacy of antibody drugs. Currently disclosed mutation sites, according to the EU numbering system, include: IgG1 Fc region mutation sites include L234A, L235A, L235E, L235G, G236A, G237A, N297A, G318A, L320A, and L322A; IgG3 Fc region mutation sites include V234A, G237A, P238S, H28A, and V309L. The mutation sites in the Fc region of IgG3 include Leu281, Leu282, Gly283, Gly284, Asn344, and Pro378; the mutation sites in the Fc region of IgG4 include S228P, E233P, F234V, L235A, F243L, D254A, R292P, Y300L, L309V, and R409K. The variant Fc in this article includes, but is not limited to, the Fc of each IgG having the above-mentioned mutation sites.

[0129] In one or more embodiments, the Fc region is the Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more of the following mutations: L234A, L235A, G237A. In one or more embodiments, the Fc region is the Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more of the following mutations: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0130] The heavy chain antibodies of the present invention can be prepared using methods conventional in the art, such as phage display technology well known in the art. Alternatively, the antibodies or heavy chain antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequence encoding the antibody of the present invention. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc.

[0131] The engineered antigen-presenting cells described in this article are also loaded with tumor antigens and / or contain coding sequences for tumor antigens.

[0132] The term "antigen" has its conventional meaning and refers to a molecule capable of inducing an immune response. In the context of this invention, an antigen can be a protein or a fragment thereof, such as a (poly)peptide that presents an epitope of said protein. However, the antigen used may also be an artificial peptide or a peptide mimic. The antigens used in this invention are preferably proteins or portions thereof obtained from or derived from tumor cells.

[0133] In this article, tumor antigens can be tumor-associated antigens, tumor-specific antigens, or neotumor antigens.

[0134] In some embodiments, the tumor antigen is selected from one or more of the following: P53, Survivin, MUC1, hTERT, or KRAS.

[0135] The coding sequence of tumor antigens can be DNA or RNA, such as mRNA.

[0136] In one or more embodiments, the RNA sequence encoding P53 is shown in SEQ ID NO:70, the RNA sequence encoding Survivin is shown in SEQ ID NO:71, the RNA sequence encoding MUC1 is shown in SEQ ID NO:72, the RNA sequence encoding hTERT is shown in SEQ ID NO:73, and the RNA sequence encoding KRAS is shown in SEQ ID NO:74.

[0137] Preparation method of engineered antigen-presenting cells

[0138] In this study, DCs can be derived from DC precursor cells isolated from the subject's own blood, such as CD34+ hematopoietic precursor cells derived from umbilical cord blood, or differentiated from CD14+ monocytes derived from peripheral blood. DCs were obtained after isolation, culture, expansion, and differentiation from the subject.

[0139] The method for culturing DC precursor cells to differentiate into DCs can be any method known in the art or any other method capable of differentiating DC precursor cells into DCs, such as adding cytokines GM-CSF and IL-4 to the culture medium for differentiation culture. In other embodiments, the DCs can be cells obtained by in vitro expansion and culture followed by differentiation culture of an immortalized DC precursor cell line. The immortalized DC precursor cell line can 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 CN201810368646.3. The immortalized DC precursor cell line can be expanded in large quantities in vitro and then differentiated into DCs by the aforementioned method.

[0140] In some implementations, engineering antigen-presenting cells includes: loading antigen-presenting cells with tumor antigens or introducing coding sequences for tumor antigens; and introducing coding sequences for multispecific nanobodies into antigen-presenting cells.

[0141] The term "load" as used herein refers to enabling antigen-presenting cells to contain (capture) tumor antigens in a certain way, thereby processing the antigens and presenting them to other immune cells. Taking dendritic cells (DCs) as an example, loading can be achieved through various methods of contacting the antigen or its coding sequence, such as incubation with recombinant, synthetic, or purified tumor antigen peptides or proteins, incubation with tumor cell lysates, incubation with apoptotic or necrotic tumor cells, or enabling the cells to express the antigen. Enabling cells to express antigens can be achieved by contacting the cells with nucleic acids (DNA or RNA) encoding the tumor antigen (e.g., co-incubation) or by introducing the nucleic acids into the cells (e.g., via electroporation). Introducing a DNA-coding sequence into cells typically involves a nucleic acid (DNA) construct, such as an expression vector and an integration vector, containing the DNA sequence along with a suitable promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. Alternatively, the RNA-coding sequence of the antigen (e.g., mRNA) can be directly introduced into the cells to express the antigen. Depending on the tumor to be targeted, the antigen can be contacted with and loaded with the corresponding tumor antigen or its encoding nucleic acid (e.g., mRNA). Methods of loading antigens are known in the art, such as incubation, cell transformation (e.g., electroporation of DNA or mRNA), etc. In this document, the antigen or its coding sequence is in a soluble form or the antigen or its coding sequence is attached to a solid support. The solid support may include polystyrene beads. The solid support is biodegradable.

[0142] Antigen-presenting cells (e.g., dendritic cells) are induced to mature by contact with a maturation composition (maturation cocktail). The maturation composition used exemplary herein comprises one or more selected from IFN-γ, PolyI:C, R848, and PGE2. The dendritic cells are contacted with the maturation composition for at least 10 hours, at least 20 hours, at least 30 hours, or at least 40 hours.

[0143] There are generally no particular restrictions on the order of engineering and maturation of antigen-presenting cells; that is, cells can be loaded with antigens before being exposed to the cytokine composition, or antigens can be exposed to the cytokine composition before being loaded with antigens. This is within the knowledge of those skilled in the art.

[0144] Multispecific nanobodies or their encoding sequences can be introduced into antigen-presenting cells in the form of proteins, RNA, or DNA. For example, DNA vectors expressing antibodies can be constructed and transformed into antigen-presenting cells. Therefore, the present invention also includes polynucleotides (in DNA or RNA form) encoding the antigens or antibodies described herein or fragments thereof, and nucleic acid constructs containing these polynucleotides (e.g., expression vectors and integration vectors). The vectors described herein typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to in some embodiments as “flanking sequences”) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence encoding a polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-linker region for inserting a nucleic acid encoding the antibody to be expressed, and optional labeling elements.

[0145] The polynucleotides introduced into antigen-presenting cells according to the present invention can be in the form of DNA or RNA (e.g., mRNA or saRNA). The inventors have found that mRNA offers advantages in safety compared to traditional vaccines, such as not introducing gene mutations, being degraded by normal cells, and having its half-life altered by regulating sequence modifications and delivery vectors. Methods for introducing mRNA into antigen-presenting cells (e.g., dendritic cells) are well known in the art, for example, by electroporation.

[0146] The coding sequence of the antibody may also have a signal peptide to guide antibody secretion, and those skilled in the art will know of signal peptides that can be used in this invention. Exemplary signal peptides include: human κ chain signal peptide coding sequences and human immunoglobulin light chain signal peptides.

[0147] This invention provides a method for preparing the DC cells, comprising enabling dendritic cells to secrete multispecific nanobodies and loading them with a tumor antigen. The method may include the steps of: (1) loading the multispecific nanobodies-capable dendritic cells with a tumor antigen or its coding sequence; or (2) enabling the tumor antigen-loaded dendritic cells to secrete multispecific nanobodies; or (3) contacting the dendritic cells with the tumor antigen or its coding sequence and the coding sequence of the multispecific nanobodies. The method of loading the antigen is as described herein, for example by contacting the cells with the antigen or its coding nucleic acid (DNA or RNA) (e.g., co-incubation) or introducing the antigen or its coding nucleic acid into the cells (e.g., by electroporation, or by electroporation of RNA).

[0148] Common methods for enabling cells to secrete antibodies or possess the ability to secrete antibodies include expressing the antibody. Processes for expressing antibodies in cells are known in the art, such as introducing a nucleic acid (e.g., DNA or RNA) that encodes the antibody into the cell. Introducing a DNA-coding sequence into a cell typically involves a nucleic acid (DNA) construct, such as an expression vector and an integration vector, containing that DNA sequence along with a suitable promoter or control sequence. These vectors can be used to transform suitable host cells to enable them to express proteins. Exemplary vectors are found in CN105154473A and CN111206043A, which are incorporated herein by reference in their entirety. Alternatively, the RNA-coding sequence of the antibody (e.g., mRNA or saRNA) can be directly introduced into the cell to express the antibody.

[0149] Specifically, any DNA vector in the art that facilitates the introduction of antibody-encoding nucleic acid (DNA) into cells can be used, including but not limited to conventional circular DNA plasmids, linear DNA plasmids, microcircular plasmids, nanoparticles, doggybone, and other DNA forms that do not contain antibiotic or / and replicon DNA sequences. In some embodiments, the DNA vector is a DNA microcarrier whose DNA backbone sequence does not contain an antibiotic expression cassette and is preferably limited to a length of less than 600 bp, and / or does not contain a CpG DNA motif. In some embodiments, the DNA vector is an antibiotic-free microplasmid, i.e., a microplasmid without an antibiotic resistance gene (antibiotic-free expression cassette microplasmid), also known as a tiny or tiniplasmid. For examples of antibiotic-free microplasmids applicable to this invention, please refer to Chinese Patent Application No. 202310072956.1, filed January 13, 2023, entitled "An Antibiotic-Free Microplasmid and Its Preparation Method and Application," submitted by Shanghai Jiliang Pharmaceutical Engineering Co., Ltd., etc., the entire contents of which are incorporated herein by reference. In some embodiments, the antitoxin-free microplasmid comprises a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein contains the following sequences: (1) the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having one or more mutations of E24D, I35V, V43I compared to SEQ ID NO:56; or (2) the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared to SEQ ID NO:57; the replicon is ≤800bp in length, preferably ≤600bp or ≤300bp. In some embodiments, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NO:56-62. In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; preferably R6K or pUC57. In some embodiments, the length of the plasmid backbone of the antitoxin-free plasmid is ≤1000 bp, preferably ≤900 bp, ≤800 bp, or ≤600 bp. In some embodiments, the nucleotide sequence encoding the antitoxin protein does not contain a CpG motif; preferably, the nucleotide sequence encoding the antitoxin protein is as shown in any one of SEQ ID NO:63-65. In some embodiments, the nucleotide sequence of the replicon does not contain a CpG motif. In a preferred embodiment, the length of the backbone sequence of the antitoxin-free plasmid is ≤600 bp, and the replicon is an R6K replicon without a CpG motif. The nucleotide sequence of an R6K replicon without a CpG motif is, for example, shown in SEQ ID NO:66.In some implementations, the nucleotide sequence of the antimicroplasmid (empty vector) is as shown in SEQ ID NO:67 or 68; the spectral structure is as shown. Figure 9 Or as shown in 10.

[0150] The RNA coding sequence of the antibody can be synthesized by a gene company or obtained through in vitro transcription. Those skilled in the art are familiar with methods for preparing RNA sequences through in vitro transcription. Exemplary in vitro transcription methods include the steps of constructing a transcription template DNA vector and incubating it in a transcription system. Transcription systems and incubation conditions are well known in the art; transcription systems include, for example, transcription buffers (containing, but not limited to, Tris-HCl, MgCl2, DTT, spermidine), NTPs, RNase inhibitors, RNA polymerase, etc.; incubation conditions include, for example, 37°C for at least 2 hours.

[0151] The coding sequences of each antibody can be located on separate nucleic acid constructs or combined in a suitable manner on the same nucleic acid construct. The coding sequences of antibodies usually also contain stop codons (e.g., TGATAA) and may contain restriction enzyme sites to facilitate genetic engineering.

[0152] Furthermore, in embodiments expressing tumor antigens and multispecific nanobodies, the coding sequences for the tumor antigens and multispecific nanobodies can be introduced into cells separately or simultaneously. Similarly, the coding sequences for the tumor antigens and multispecific nanobodies can be located on separate nucleic acid constructs or combined in a suitable manner on the same nucleic acid construct.

[0153] Specific steps for preparing dendritic cells (DCs) include, for example, antigen-presenting cells (APCs) such as DCs being obtained from subjects, such as patients with cancer or at risk of developing cancer, via apheresis. Purified dendritic cells are cultured in the presence of a maturation composition to obtain mature DCs. Mature DCs are loaded with antigens (e.g., P53, Survivin, MUC1, hTERT, and KRAS), for example, by electroporation of the mRNA encoding the antigen, thereby obtaining mature DCs containing the antigen and a DC vaccine. Before, during, or after antigen loading, the DCs may be introduced with the multispecific nanobody encoding sequences described herein, for example, by electroporation of the mRNA, saRNA, or DNA of the multispecific nanobody. The resulting DCs are then given to the patient. An exemplary treatment procedure involves administering DCs three times over a 4-week period.

[0154] The DC cells used in this invention are either freshly prepared or obtained by cryopreservation followed by thawing, such as DC cells obtained by thawing and culturing after one month of cryopreservation. The reagents, conditions, etc., required for cryopreservation and thawing can be obtained using conventional methods in the art.

[0155] In this article, the culture medium and culture conditions required for preparing DC cells can be the same as those for conventional DC cell culture. Exemplary culture media and culture conditions are shown in the examples.

[0156] The antigen-presenting cells (e.g., dendritic cells) described herein can be used to prepare pharmaceutical compositions for the prevention or treatment of the various conditions and diseases described herein, such as DC vaccines. The conditions and diseases primarily refer to the occurrence, growth, and / or metastasis of tumors (cancers), including but not limited to: lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, mesothelioma, colorectal cancer, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial tumors and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, malignant melanoma of the skin or eye, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestinal cancer, endocrine system cancers, bile duct cancer, and thyroid cancer. Cancer, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, urothelial carcinoma, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, Hodgkin's lymphoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and various types of leukemia and lymphoma, as well as various precancerous lesions.

[0157] The pharmaceutical composition of the present invention can be different antigen-presenting cells loaded with different antigens, or it can be a single antigen-presenting cell loaded with multiple antigens. In an exemplary embodiment, the pharmaceutical composition comprises five antigen-presenting cells (e.g., dendritic cells) expressing the multispecific nanobodies described herein, respectively loaded with P53, Survivin, MUC1, hTERT, and KRAS. The concentration and proportion of the various antigen-presenting cells in the pharmaceutical composition can be adjusted by those skilled in the art as needed. Exemplarily, the above five antigen-presenting cells are included in the pharmaceutical composition in equal proportions.

[0158] 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 preservatives and adjuvants. These excipients are preferably non-toxic to the recipient at the doses and concentrations used. Such excipients include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0159] The excipients in the pharmaceutical composition also include vaccine adjuvants. The adjuvants can be small molecules, biomolecules, compositions, complexes, or extracts of compounds known in the art that can enhance immune responses. In one or more embodiments, the adjuvants include those selected from aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), prostaglandin E2, alpha-interferon, Corynebacterium breviculae, lipopolysaccharides, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, protein liposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro / nanoparticles, polymeric sheet substrate particles (PLSP), microparticle resins, nanoliposome polymeric gels, synthetic / biodegradable and biocompatible semi-synthetic or natural polymers or dendritic polymers (e.g., PLG, PL...). GA, PLA, polycaprolactone, silicone polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyltrimethylammonium chloride, polystyrene divinylbenzene resin, polyphosphazene, poly-[di-(carboxyacetylphenoxy)phosphazene (PCPP), poly-(methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and its derivatives, chitosan and its derivatives, polysaccharides, δ-inulin polysaccharide, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (such as polyamino acids, poly-(γ-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides, biopolymers), cationic dimethyl di(octadecyl)ammonium (DDA), α-galactoside ceramide and its derivatives, archaeal lipids and their derivatives, lactams, gallons, glycerides, phospholipids, and spirochetes.

[0160] Pharmaceutical compositions for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization can be performed using this method before or after lyophilization and rehydration. The pharmaceutical compositions of the present invention may be used for parenteral delivery. Compositions for parenteral administration may be lyophilized or stored in solution. They are prepared, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution bands or vials with stoppers puncturable by a hypodermic needle. Alternatively, the compositions may be used for inhalation or delivery via the digestive tract (e.g., orally). The preparation of the pharmaceutically acceptable compositions is within the scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled-release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioeasily perishable microparticles or porous beads, and accumulation injection) are also known to those skilled in the art.

[0161] Once formulated, the pharmaceutical composition is stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. The formulation may be stored in a ready-to-use form or rehydrated before administration (e.g., lyophilized). The present invention also provides kits for generating single-dose administration units. The kits of the present invention may each contain a first container with dried protein and a second container with an aqueous formulation. In some embodiments of the invention, kits containing single-lumen and multi-lumen pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0162] This invention also provides a method for treating patients (especially patients with mesothelin-related diseases) by applying engineered antigen-presenting cells or pharmaceutical compositions thereof as described in any embodiment of the invention. In this document, the terms “patient,” “subject,” “individual,” and “object” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. “Treatment” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). “Prevention” refers to a subject at risk receiving the treatment regimen described herein to achieve at least one effect of preventing the occurrence of disease or symptoms. Effective treatment or prevention regimens for patients can vary depending on various factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject).

[0163] The therapeutically effective amount of the pharmaceutical composition containing the engineered antigen-presenting cells of this invention will depend, for example, on the degree of treatment and the target. Those skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the delivered molecules, indication, route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health status). In some embodiments, clinicians may titrate the dose and vary the route of administration to obtain optimal therapeutic effect. For example, approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight per day.

[0164] The frequency of administration will depend on the pharmacokinetic parameters of the bound molecules in the formulation used. Clinicians typically administer the composition until a dose is reached to achieve the desired effect. The composition can therefore be administered as a single dose, or over time as two or more doses (with or without the same amount of the desired molecule), or via implanted device or catheter as a continuous infusion.

[0165] The drug composition can be administered via known methods, such as oral, intravenous, intraperitoneal, intracerebral (within brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional injection; via a sustained-release system; or via an implantable device. In one or more embodiments, the drug composition as a vaccine can be administered to the inguinal region via intrasegmental injection. Optionally, depending on the target of the vaccine, the vaccine can be administered subcutaneously or intradermally to the hands and feet of a cancer patient receiving treatment. Other routes of administration, such as intramuscular or blood injection, may also be used.

[0166] Depending on the type of pharmaceutical composition (e.g., a vaccine) being prepared, the production scale of the pharmaceutical composition can be scaled up, if necessary, by culturing cells in a bioreactor or fermenter or similar containers and devices suitable for mass cell growth. In one or more embodiments, according to the invention, a device or composition comprising the produced or recycled vaccine or antigen is adapted for continuous or intermittent release and can be implanted in the body or administered locally at a corresponding location in the body to achieve the effect of slow and timed release of these materials into the body.

[0167] The present invention also provides methods for treating and / or preventing cancer, the methods comprising administering an effective dose of one or more of the aforementioned cell and pharmaceutical compositions to a subject. The methods include effects of at least one of treatment and prevention. In one or more embodiments, the methods of the present invention are for preventative purposes, wherein one or more of the cell and pharmaceutical compositions of the present invention are administered to the subject before the occurrence of cancer or precancerous lesions. In some cases, the pharmaceutical compositions are administered to the subject after the onset of one or more of the aforementioned cancers, with the aim of preventing the occurrence of further symptoms or further deterioration of existing symptoms. Prophylactic administration of one or more of the cell and pharmaceutical compositions of the present invention is intended to prevent or alleviate any subsequent symptoms. In one or more embodiments, the methods of the present invention are for therapeutic purposes, wherein one or more of the cell and pharmaceutical compositions of the present invention are administered to the subject at the onset of cancer or after the onset of cancer, with the aim of alleviating symptoms of existing cancer.

[0168] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0169] Experimental methods

[0170] Example

[0171] The bispecific nanobody provided in this embodiment

[0172] The amino acid sequence of the epitope that binds to PD-1 is selected from:

[0173] C43-z11 sequence:

[0174] ELQLVESGGGLVQPGGSLRLSCAASGHSFSIYDMGWFRQAADKERESVAVI

[0175] NFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPPLADHWGQGSQVTVSS(SEQ ID NO:10)

[0176] C43-z14 sequence:

[0177] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPGKERESVAVI

[0178] NFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPPLADHWGQGSQVTVSS(SEQ ID NO:38)

[0179] C43-z15 sequence:

[0180] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPDKERESVAVI

[0181] NFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPPLADHWGQGTQVTVSS(SEQ ID NO:39)

[0182] 1194nla (abbreviated as 1194) sequence:

[0183] EVQLVESGGGLVQPGGSLRLSCAASGRPFSIYDMGWFRQAPDKERESVAVI

[0184] NLARGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGVDRRQYGLGIPPLADHWGQGTQVTVSS(SEQ ID NO:11)

[0185] The amino acid sequence of the epitope that binds to CTLA-4 is as follows:

[0186] 12-z1 sequence:

[0187] EVQLVESGGGLVQPGGSLRLSCAASGFSSDYYDIGWFRQAPGKEREMVSCI

[0188] RSSGGSTKYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCGLAPISPVHAVCNQHYFGYWGQGTRVTVSS(SEQ ID NO:12)

[0189] The CDR and FR regions of the above VHH are shown in the table below.

[0190] Table 1. FR and CDR regions of VHH

[0191]

[0192]

[0193] The bispecific nanobody 1194-NLA-L1-12-Z1 used in this embodiment

[0194] The amino acid sequences of 1194-NLA-L2-12-Z1, 1194-NLA-L3-12-Z1, C43-Z14-L2-12-Z1 (abbreviated as C43-Z14-12-Z1), C43-Z15-L2-12-Z1 (abbreviated as C43-Z15-12-Z1), 12-Z1-L3-1194, C43-z11-L3-12-Z1, and C43-z11-L2-12-Z1 are shown in SEQ ID NO:48-55, and the corresponding amino acid sequences of the bispecific nanobodies with the cmyc-his tag are shown in Table 2.

[0195] Table 2. Names and sequences of tagged bispecific nanobodies (names are not case-sensitive)

[0196]

[0197]

[0198]

[0199] Preparation in Example 1

[0200] The bispecific antibody was prepared by Baiying Biotechnology, expressed using HEK293, purified using Ni column, and tagged with his. The storage buffer was PBS. Preparation data are shown in Table 3.

[0201] Table 3. Antibody Preparation Data (Provided by Bio-Tech)

[0202]

[0203] Example 2 Affinity Test

[0204] (1) SPR detection binding dynamics

[0205] Protein-level affinity assays: The binding kinetics and affinity of bispecific nanobodies or PD1 nanobodies (1194nla, C43z11) or CTLA4 nanobodies (12-z1) for human PD-1.IgG and CTLA4.IgG antigens were determined using surface plasmon resonance (SPR) technology.

[0206] Purified PD-1 IgG or CTLA4 IgG antigens were passed through a CM5 chip pre-coated with IgG (GE, cat#BR-1008-39). The antigens were captured by the IgG on the chip. Different concentrations of bispecific nanobodies or PD-1 nanobodies and CTLA4 nanobodies were then used as the mobile phase to detect binding kinetics and affinity. Biacore Evaluation Software 2.0 (GE) was used to analyze the binding rate (kon), dissociation rate (koff), and equilibrium constant (KD). Simultaneously, the marketed PD-1 antibody drugs Keytruda and Opdivo were selected as controls.

[0207] As shown in the table below, the ability of bispecific antibodies to bind to PD1 is comparable to that of PD1 nanobodies, and the ability of bispecific antibodies to bind to CTLA4 is comparable to that of CTLA4 nanobodies.

[0208] Table 4. Antibody binding kinetics and affinity

[0209]

[0210] (2) ELISA detection of antigen binding ability

[0211] Coat the ELISA plate with 1 μg / mL PD.fc or CTLA4.fc protein. Add 50 μL of serially diluted antibody to the ELISA plate and incubate. After washing, add HRP Streptavidin (Biolegend) and incubate at room temperature for 1 hour. After washing, add TMB chromogenic solution and then terminate the reaction with 2M HCl. Read the OD450 value using a microplate reader.

[0212] ELISA assays showed that the binding ability of different dual-characteristic antibodies to PD1 antigen or CTLA4 antigen was not significantly different. Figure 1 , Figure 2 ).

[0213] (3) FACS detection of cell binding ability

[0214] HEK293T cells expressing CTLA4 or CHO / K1 cells expressing PD1 were seeded into 96-well plates, 3 × 10⁶ cells per well. 5 Cells were then incubated with serially diluted nanobodies on ice for half an hour, followed by incubation with secondary antibody for detection. Detection was then performed using a CytoFLEX flow cytometer. The EC50 of the antibody was calculated using a fitted curve. Isotype was used as a negative control.

[0215] The binding ability of different nanobodies to PD1-positive or CTLA4-positive cells was detected by flow cytometry, and the results are as follows: Figure 3 As shown: Difference in EC50 between penicillin antibodies on 293T-CTLA4 cells:

[0216] C43-Z11-(G4S)-3G4-12-Z1 EC50 is slightly better than other dual antibodies.

[0217] On CHO / K1-PD1 cells, the difference in EC50 among bispecific antibodies was as follows: C43-Z11-(G4S)-3G4-12-Z1 had a slightly better EC50 compared to other bispecific antibodies. Results are as follows... Figure 4 As shown.

[0218] Based on the above data, at the ELISA level, the EC50 values ​​of different linker-linked bispecific antibodies for binding to PD1 and CTLA4 antigens were not significantly different; however, in cell lines overexpressing CTLA4 or PD1, different bispecific antibodies showed certain differences in EC50 values, with C43z11-L3-12-Z1 exhibiting better binding ability to stable cell lines than other bispecific antibodies (Table 5).

[0219] Table 5. Summary of antibody ELISA and FACS binding data

[0220]

[0221] Example 3: Ligand Binding Inhibition

[0222] (1) PD1 / PDL1 reporter gene experiment

[0223] First, 10,000 GS / C2-PDL1 (GenScript) target cells were resuspended in 20 μL of antibiotic-free medium and cultured in 384-well plates for 16-20 h. The nanobody was serially diluted using RMPI1640 + 10% FBS buffer, and 40,000 GS-J2 / PD1 (GenScript) effector cells were dissolved in 20 μL of antibiotic-free medium. The old medium was removed from the 384-well plates, and 20 μL of antibody and 20 μL of GS-J2 / PD1 effector cells were added to each well, and the plates were co-cultured for 6 h. Finally, 40 μL of One-Glo substrate was added to each well, and the reaction was carried out for 10 minutes before detection using a microplate reader.

[0224] The results of the Reporter Gene Assay experiment are as follows Figure 5 or Figure 6As shown, the IC50 order of the inhibitory effects of different linker bispecific antibodies against PD1 / PDL1 binding on 1194nla is: 1194NLA-L2 > 1194NLA-L1 > 1194NLA-L3, all of which are not significantly different from the IC50 values ​​of the 1194nla antibody. Under the same linker conditions, the inhibitory effect of the bispecific antibody with 1194nla first (1194nla-L3) on PD1 / PDL1 binding is greater than that of the bispecific antibody with 12-z1 first (12-z1-L3).

[0225] (2) CTLA4 ligand binding inhibition experiment

[0226] Adjust the GS-J1 / CD28 cell density to 2 × 10⁻⁶ using Assay buffer (RMPI 1640 + 10% FBS buffer). 6 Cells / mL; GS-C1 / CD80 target cells (GenScript) were adjusted to a density of 1×10⁶ cells / mL using antibiotic-free medium. 6 Cells / mL. The test antibody was serially diluted with Assay buffer, and CTLA-4 protein was diluted to 100 μg / mL with Assay buffer. In each well of a 384-well plate, 20 μL of GS-J1 / CD28 cells, 20 μL of GS-C1 / CD80 cells, 20 μL of CTLA-4 protein, and 20 μL of the test antibody or Assay buffer were added sequentially to the adjusted density. The 384-well plate was then incubated for 24 hours. After 24 hours, the cell supernatant was collected by centrifugation, and the IL-2 concentration was quantified using an IL-2 quantification kit (Cisbio). The EC50 of the antibody was calculated by curve fitting using Graphprism software.

[0227] CTLA4 ligand binding inhibition assay Reporter Gene Assay results are as follows Figure 7 and Figure 8 As shown ( Figure 7 and Figure 8 (These are experimental results from different batches). The IC50 order of the inhibitory effects of different linker bispecific antibodies against CTLA4 / CD80 binding on 1194nla is as follows:

[0228] 1194NLA-L2>1194NLA-L3>1194NLA-L1; Under the same linker conditions, for the inhibitory effect on CTLA4 / CD80 binding, the effect of 1194nla first (1194nla-L3) is less than that of the bispecific antibody with 12-z1 first (12-z1-L3).

[0229] Based on the combined results of the PD1 / PDL1 reporter gene assay and the CTLA4 ligand competition assay, 1194nla-L2-12-z1 and C43z11-L2-12-z1 showed better performance.

[0230] Example 4: Tissue Cross-Reactivity (TCR)

[0231] Thirty-four tissue samples were selected for frozen sectioning. After air-drying at room temperature, the sections were soaked in PBST for 5-10 min. Blocking was performed using reagents A and B of the endogenous biotin blocking kit (Sangon Biotech, E674001). After washing, imported sheep serum working solution was added and incubated for 10-15 min. Primary antibody was added and incubated at room temperature for 10-15 min. After washing, horseradish peroxidase-labeled streptavidin (Abcam, ab7403) was added and incubated for 15 min. DAB staining and hematoxylin counterstaining were performed. After dehydration with alcohol, the sections were mounted on neutral plastic and air-dried for microscopic examination. The positive control was Anti-PD-1 antibody (abcam), and the negative control was IgG4 isotype control. All antibody concentrations in this experiment were 10 μg / ml.

[0232] The experimental results are shown in Table 6. The TCR results of the four bispecific antibodies 1194nla-L2-12-z1, C43-z14-12-z1, C43-z15-12-z1 and C43z11-L3-12-z1 were all relatively clean and had good specificity.

[0233] Table 6. Results of Tissue Cross-Reactivity of Bispecific Antibodies

[0234]

[0235]

[0236]

[0237] The anti-PD-1 / CTLA-4 bispecific nanobody used in the following examples is C43-Z15-(G4S)2G4-12-Z1 (i.e., C43-Z15-L2-12-Z1, abbreviated as Z15-1).

[0238] 1. Preparation of mRNA

[0239] According to NEB The T7 High Yield RNA Synthesis Kit was used for in vitro transcription and purification of mRNA. After thawing the reagents, they were kept on ice. A 20 mM GTP solution was prepared by mixing 2 μl of 100 mM GTP and 8 μl of nuclease-free water to prepare a 40 mM cap analog. During RNA synthesis, the four ribonucleoside triphosphates, cap analog, template DNA, DTT, and T7 RNA Polymerase Mix were thoroughly mixed and incubated at 37°C for 2 hours. The purity of the mRNA was controlled using an Agilent 5200 capillary electrophoresis system.

[0240] The sequences of each segment are shown below:

[0241] Excerpt SEQ ID NO P53 mRNA sequence 70 Survivin mRNA sequence 71 MUC1 mRNA sequence 72 hTERT mRNA sequence 73 KRAS mRNA sequence 74

[0242] 2. DC cell culture and induction

[0243] 2.1 Separation of PBMC

[0244] (1) Use a syringe to draw apheresis blood from the blood bag into a 50ml centrifuge tube, wash the blood bag with an equal volume of PBS (HyClone brand) and mix it with the extracted apheresis blood (1:1 dilution);

[0245] (2) Take a 50ml centrifuge tube (containing 15ml Ficoll lymphocyte separation solution (brand name: GE)) and slowly add the blood and PBS mixture from step 1 along the tube wall. Then centrifuge at 800g for 20min with an acceleration of 1 and a deceleration of 0.

[0246] (3) Carefully aspirate the white cell layer into another 50ml centrifuge tube, add PBS, centrifuge and wash at 400g / min for 10min, with acceleration and deceleration at 9.

[0247] (4) Discard the waste liquid, but do not discard it completely. Add DPBS, wash again, and centrifuge (same as step 3).

[0248] (5) Drain the waste liquid, add AIM-V culture medium (gibco brand) to suspend the cells, and add them to the culture flask. Let them adhere to the wall overnight.

[0249] 2.2 DC cell generation

[0250] Monocyte-derived dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMCs) via standard Ficoll density centrifugation to isolate PBMCs from patient leukocyte removal samples. PBMCs were seeded in serum-free AIM-V medium and allowed to adhere to culture flasks with 0.22 μm filter caps. After 2 hours, non-adherent cells were removed, and adherent monocytes were subsequently cultured for 6 days in AIM-V medium containing 50 ng / ml rhIL-4 and 100 ng / ml rhGM-CSF. On day 3, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. A maturation mixture consisting of 100 IU / ml IFN-γ, 30 μg / ml poly(I:C), 5 μg / ml R848, and 1 μg / ml PGE2 was used to induce DC maturation for 24 hours.

[0251] 2.3 DC cell transformation

[0252] Observe cell morphology under a microscope, collect DCs into centrifuge tubes, and centrifuge at 300g for 10 min at room temperature; discard the supernatant, add an appropriate amount of DPBS to resuspend, and count the cells; take the number of cells for electroporation, and centrifuge at 300g for 10 min at room temperature. Take the electroporation kit (from Lonza), add 100 μl of electroporation reagent according to the 4D electroporation kit instructions, and then add the electroporation plasmid or mRNA; resuspend the centrifuged cell pellet, gently mix, transfer the mixture to an electroporation cuvette, and select the desired program for electroporation; use the micropipettes in the kit to transfer the electroporated cell suspension to a 6-well or 12-well plate (containing DC induction medium), mix well, and incubate at 37℃ in a 5% CO2 incubator.

[0253] 3. Flow cytometry detection

[0254] (1) Add 1×10 to each tube 6 One cell;

[0255] (2) Add 1 ml of PBS phosphate buffer and wash twice, centrifuge at 400 g for 5 min, discard the supernatant, and resuspend in 100 μL of PBS phosphate buffer;

[0256] (3) Add the flow cytometry antibody to be detected, mix well, place in a refrigerator at 2-8℃, incubate in the dark for 30 minutes, and set up a blank control, without adding reagents or adding the corresponding isotype;

[0257] (4) Add 1 ml of PBS phosphate buffer, centrifuge at 400 g for 5 min, wash twice, and discard the supernatant;

[0258] (5) Resuspend the cells in 400 μl of PBS phosphate buffer and analyze them using flow cytometry. Set the total cell collection to 1×10⁶ cells / cells. 41. Data was analyzed using Kaluza Analysis software.

[0259] The detection process is as described above, and the different antibodies added are shown in the table below:

[0260] DC detection

[0261] Antibody testing items Light emission 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

[0262] T-detection

[0263] Antibody testing items Light emission signal CD3 (Biolegend) FITC CD4 (Biolegend) FITC CD8 (Biolegend) PE CD25 (BD) APC CD69 (Biolegend) BV421

[0264] 4. Detection of multiple cytokines using the CBA method

[0265] Follow the instructions. First, serially dilute the standards. After resuspending the standards in 2 mL, take out 9 flow cytometry tubes and label them with serial dilution factors of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. To ensure that each experimental tube contains 6 types of microspheres, take 10 μl of each type of capture microsphere, and the total volume of the mixed microspheres should be 60 μl. In the actual experiment, the amount of microspheres added to each tube of sample or standard is 50 μl. Prepare the microspheres according to this ratio. For example, if there are 8 samples, 9 standards, and 1 negative control, for a total of 18 tubes, then the amount of each type of microsphere needs to be 180 μl. After mixing 6 × 180 μl, add 50 μl of the diluted sample / standard (50 μl) to each tube, and then add 50 μl of PE detection reagent. Incubate at room temperature in the dark for 3 hours.

[0266] Before running the instrument, it is necessary to calibrate the instrument and adjust the voltage using magnetic beads, and use control magnetic beads for compensation adjustment. After incubation, the concentration of the test sample is calculated by analyzing the CBA analysis software FCAP in conjunction with the standard curve.

[0267] 5. ELISA testing

[0268] 5.1 The basic steps for ELISA detection of anti-PD-1 antibody secretion are as follows:

[0269] (1) Antigen coating: Prepare the coating antigen. Dilute the antigen with coating buffer and coat the enzyme-labeled reaction plate with 100 μl / well. Incubate overnight at 4°C. After incubation, wash 5 times with PBST, 200 μl / well, for 3 minutes each time, and pat dry with absorbent paper.

[0270] (2) Blocking: Add 300 μL of blocking solution to each well and incubate at 37°C for 2 hours. Wash 5 times with PBST, 200 μL / well, for 3 minutes each time, and pat dry with absorbent paper.

[0271] (3) Sample addition: Dilute the samples and standards with diluent. Perform serial dilutions of the standards, setting up 7 gradients and 0 ng / ml. Dilute the samples according to the actual situation. Add 100 μl of sample and standard to each well, including replicates and controls. Incubate at 37℃ in a biochemical incubator for 1 hour. Wash 5 times with PBST, 200 μl per well, for 3 minutes each time, and pat dry with absorbent paper.

[0272] (4) Add secondary antibody: Dilute the secondary antibody with blocking buffer according to the ratio, 100 μl / well, and incubate in a biochemical incubator at 37°C. Wash 5 times with PBST, 200 μl / well, 3 minutes each time, and pat dry with absorbent paper.

[0273] (5) Color development: Add 100 μl of color development solution TMB (brand name Abcam) to each well and develop at room temperature in the dark for 5-15 min.

[0274] (6) Termination: Add 50 μL of stop solution per well to terminate the reaction. Immediately take instrument readings.

[0275] The detection process follows the steps outlined above, with different parameters shown in the table below:

[0276] parameter PD-1 Coating antigen concentration 1ug / mL (brand: Arco) highest gradient of standard products 12.5 ng / mL (self-produced) Incubation time for standards and samples 1h Secondary antibody dilution ratio 1:20000 (brand: Abcam) Secondary antibody incubation time 30min

[0277] 5.2 The basic steps for ELISA detection of p53 and survivin antigen expression levels are as follows:

[0278] (1) Prepare reagents: Prepare sample dilution solution and antibody reaction solution in advance;

[0279] (2) Sample addition: Dilute the samples and standards with diluent. Perform serial dilutions of the standards, setting up 7 gradients and 0 ng / ml. Dilute the samples according to the actual situation. Add the samples and standards to the pre-coated plate (provided with the kit), 50 μl / well, with replicates and control wells. Add the detection antibody, 50 μl / well, and incubate at 37°C in a biochemical incubator for 1 hour. Wash 3 times with PBST, 300 μl / well, 3 minutes each time, and pat dry with absorbent paper.

[0280] (3) Color development: Add 100 μl of color development solution TMB (brand name Abcam) to each well and develop at room temperature in the dark for 5-10 min.

[0281] (4) Termination: Add 50 μl of stop solution per well to terminate the reaction. Immediately take instrument readings.

[0282] Example 5: Detection of PD-1 antibody secretion levels after electroporation of five antigen mRNAs and anti-PD-1 / CTLA4 bispecific nanobody plasmids into DC (DCPLUS)

[0283] Immature dendritic cells (DCs) were stimulated with IFN-γ, PolyI:C, R848, and PGE2 for 24 hours to obtain mature DCs. Five antigen mRNAs and an anti-PD-1 / CTLA-4 bispecific nanobody plasmid (the anti-PD-1 / CTLA-4 bispecific nanobody is C43-Z15-12-Z1) were then electroporated. The plasmid backbone includes the nucleotide sequences of the R6K replicon and antitoxin protein 0637. The nucleotide sequence of the R6K replicon is shown in SEQ ID NO: 66, the nucleotide sequence of the antitoxin protein 0637 is shown in SEQ ID NO: 65, and the nucleotide sequence of the anti-PD-1 / CTLA-4 bispecific nanobody plasmid is shown in SEQ ID NO: 69. The complete plasmid map is shown below. Figure 11 Mature DCs were electroporated. The electroporation volume was 2E6 cells. Cells were cultured in 24-well plates (1×10⁶ cells / well). 6 Cells / ml). After 24 hours, the supernatant was collected and analyzed by ELISA at a concentration of 1×10⁻⁶. 6 The level of PD1 antibody secreted by cells.

[0284] The results are as follows Figure 12 As shown, the secretion level of PD-1 antibody was significantly increased after electroporation of the five antigen mRNAs and the anti-PD-1 / CTLA4 bispecific nanobody plasmid compared with the non-electroporated group.

[0285] Example 6: Cell viability detection after electroporation of DCs with five antigen mRNAs and anti-PD-1 / CTLA4 bispecific nanobody plasmids (DCPLUS)

[0286] Mature dendritic cells (DCs) were divided into two groups and electroporated using electroporation: Group 1: no electroporation; Group 2: DC PLUS group. The electroporation concentrations of the five antigen mRNAs were 10 pmol per 2 × 10⁻⁶ cells. 6 Cells, the electroporation concentration of the nanoparticle anti-antibody plasmid is 3 μg per 2 × 10⁻⁶ cells. 6 cell.

[0287] The instrument used was a Lonza 4D-Nucleofector electroporator (LONZA, Switzerland). Mature DCs were collected for cell counting, and the cell number was adjusted to 2 × 10⁻⁶ cells / year according to experimental requirements. 6 Cells were prepared using 100 μL / component of electroporation buffer (LONZA, Switzerland). Cells were transferred from the electroporation cuvette to a 24-well plate, resulting in a final cell count of 1.0 × 10⁶ cells / well. 6 / well, place the 24-well plate in a 37℃, 5% CO2 incubator and continue culturing. After 24 hours, collect cells and measure cell viability. The number of cells electroporated was 2.0 × 10⁶. 6 .

[0288] The results are as follows Figure 13As shown, the cell viability after electroporation of the five antigen mRNAs and anti-PD-1 / CTLA4 bispecific nanobody plasmid for 24 hours was 84.75%, which was less than 10% different from the DC viability of the control group.

[0289] Example 7: Detection of antigen expression levels

[0290] The protein expression of tumor-associated antigens (P53, survivin, MUC1, hTERT, KRAS) was detected by Western blot. Anti-PD-1 / CTLA-4 bispecific nanobody plasmids and DCs (DC Plus) containing the mRNAs of the five antigens were electroporated. The electroporation concentrations of the five antigen mRNAs were 10 pmol per 2 × 10⁻⁶ mcg. 6 Cells, nanobody plasmid electroporation concentration was 3 μg per 2 × 10⁻⁶ cells. 6 cell.

[0291] After electroporation overnight, the cell pellet was collected. Lysis buffer (RIPA:PMSF mixed at a ratio of 100:1) was added to the centrifuge tube containing the cell pellet, and the cell suspension was pipetted to lyse the cells. The supernatant was collected by centrifugation. The protein concentration was determined and quantified using a BCA (BCA protein assay kit, Beijing Dingguo, China). The protein suspension was boiled before Western blot experiments. Five antibodies and secondary antibodies were purchased from Abcam and diluted according to the manufacturer's instructions.

[0292] The results are as follows Figure 14 As shown in (AC), ELISA was used to detect the expression levels of p53 and survivin in the cell extract, and the expression level of DCPLUS antigen was significantly increased. Western blot was used to detect the expression of MUC1 antigen in DCPLUS cells.

[0293] Example 8: Detection of the activation ability of DC PLUS on T cells

[0294] Mature dendritic cells (DCs) were divided into three groups and electroporated using electroporation: empty DCs (no electroporation); DC(Ags) group (containing 5 antigen mRNAs (P53, survivin, MUC1, hTERT, KRAS); and DC PLUS group (containing 5 antigen mRNAs (P53, survivin, MUC1, hTERT, KRAS) and anti-PD-1 / CTLA-4 bispecific nanobody plasmid). The electroporation concentrations of the 5 antigen mRNAs were 10 pmol per 2 × 10⁻⁶ cells. 6 Cells, the electroporation concentration of nanobody mRNA is 3 μg per 2 × 10⁻⁶ cells. 6 cell.

[0295] The ability of DC cells to activate T lymphocytes was tested, and the experiment was conducted in four groups: Group 1: control T cell group, T cells alone; Group 2: empty DC / T cell group; Group 3: DC(Ags) / T cell group; Group 4: DC(PLUS) / T cell group.

[0296] 1) After 4 hours of electroporation, mix with T cells at a ratio of 1:10 (DC: 2×10⁻⁶). 5 T cells: 2 × 10 6 Co-culture. After 48 hours of culture, the cell supernatant was collected and the levels of TNF-α and IFN-γ were detected by CBA.

[0297] 2) Mix DC cells and T cells at a ratio of 1:10 (DC: 2 × 10⁻⁶ T cells). 5 T cells: 2 × 10 6 The cells were co-cultured at a ratio of 1:1. After three days, the supernatant and cells were collected as needed for the experiment. T cell activation markers (CD69, CD25) were detected using flow cytometry.

[0298] 3) After 4 hours of electroporation, mix with T cells at a ratio of 1:10 (DC: 2×10⁻⁶). 5 T cells: 2 × 10 6 Co-culture. The level of IFN-γ expression in T cells was detected using ELISpot.

[0299] 4) After electroporation for 4 hours, mix with T cells at a ratio of 1:10 (DC: 2×10⁻⁶). 5 T cells: 2 × 10 6 Co-culture. The fold increase of T cells was calculated by cell counting.

[0300] The results are as follows Figure 15 As shown in (AD), the secretion levels of TNF-α and IFN-γ in the supernatant of co-cultured DC and T cells were detected by the CBA assay. The secretion level of T cells in the DC PLUS co-culture group was significantly higher than that in the DC (Ags) co-culture group, the DC co-culture group, and the T cell-only group. Figure 15 -A). Flow cytometry analysis revealed that the proportions of CD4 and CD8 in T cells after co-culturing DCs and T cells, as well as the positivity rates of CD69 and CD25, were significantly higher in the DC PLUS co-culture group compared to the DC (Ags) co-culture group and the DC co-culture group. These proportions were also significantly higher than those in the T cell-only group. Figure 15 -B). ELISPOT results showed that the IFN-γ secretion capacity of T cells in the DCPLUS co-culture group was significantly higher than that of T cells in the DC(Ags) co-culture group and the DC co-culture group. Figure 15-C). Comparing the T cell proliferation capacity of different groups, the T cell proliferation capacity of the DC PLUS co-culture group was significantly higher than that of the DC co-culture group and the T cell-only group. Figure 15 -D).

[0301] Example 9: Effect of DC PLUS on T-cell tumor-killing ability

[0302] Mature dendritic cells (DCs) were divided into three groups and electroporated using electroporation: empty DCs (no electroporation); DC(Ags) group: containing 5 antigen mRNAs (P53, survivin, MUC1, hTERT, KRAS); DC(PLUS) group: containing 5 antigen mRNAs (P53, survivin, MUC1, hTERT, KRAS) and anti-PD-1 / CTLA-4 bispecific nanobody plasmid. The electroporation concentrations of the 5 antigen mRNAs were 10 pmol per 2 × 10⁻⁶ cells. 6 Cells, nanobody plasmid electroporation concentration was 3 μg per 2 × 10⁻⁶ cells. 6 cell.

[0303] The ability of DC-activated T cells to kill was tested. The experiment was conducted in four groups: Group 1: control T cell group, T cells alone; Group 2: empty DC / T cell group; Group 3: DC(Ags) / T cell group; Group 4: DC(PLUS) / T cell group.

[0304] After 24 hours of electroporation, the cells were mixed with T cells at a ratio of 1:5 (DC: 5 × 10⁻⁶). 5 T cells: 2.5 × 10 6 T cells were co-cultured for 7 days. T cell cytotoxicity was assessed using a lactate dehydrogenase cytotoxicity assay kit (Roche). HepG2 cells (ATCC) were used as the target cells at an effector-to-target ratio of 10:1, and co-cultured for 4 hours. The cytotoxicity difference between the MHCI-blocked and unblocked groups was calculated.

[0305] The results are as follows Figure 16 As shown, the T cell killing ability was detected using a lactate dehydrogenase cytotoxicity assay kit. HepG2 cells were used as the target cells, with an effector-to-target ratio of 10:1. Co-culture time was 4 hours. The difference in tumor killing ability between the MHCI blocking group and the non-blocking group was calculated. The tumor killing ability of T cells in the DC PLUS co-culture group was significantly higher than that in the DC (Ags) co-culture group and the DC co-culture group.

Claims

1. An engineered antigen presenting cell, characterized in that, the antigen presenting cell (1) a coding sequence of the multispecific nanobody; and / or (2) is capable of expressing and / or secreting the multispecific nanobody; and, (3) is loaded with a tumor associated antigen and / or a coding sequence comprising a tumor associated antigen selected from one or more of: P53, Survivin, MUC1, hTERT or KRAS; Preferably, the tumor antigens comprise: P53, Survivin, MUC1, hTERT and KRAS; Preferably, the coding sequence is RNA; more preferably, the RNA sequence encoding P53 is set forth in SEQ ID NO: 70, the RNA sequence encoding Survivin is set forth in SEQ ID NO: 71, the RNA sequence encoding MUC1 is set forth in SEQ ID NO: 72, the RNA sequence encoding hTERT is set forth in SEQ ID NO: 73, and the RNA sequence encoding KRAS is set forth in SEQ ID NO:

74.

2. The engineered antigen presenting cell of claim 1, wherein the multispecific nanobody comprises an anti-PD-1 nanobody and an anti-CTLA-4 nanobody, preferably the complementarity determining regions of the anti-PD-1 nanobody comprise a P-CDR1 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1 or 4, a P-CDR2 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2 or 5, and a P-CDR3 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 3 or 6, and the complementarity determining regions of the anti-CTLA-4 heavy chain antibody comprise a C-CDR1 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 7, a C-CDR2 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 8, and a C-CDR3 comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 9, Preferably, the anti-PD-1 Nanobody has a sequence as set forth in SEQ ID NO: 10, 11, 38 or 39, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and wherein each CDR sequence is identical to SEQ ID NO: 10, 11, 38 or 39, and / or the anti-CTLA-4 heavy chain antibody has a sequence as set forth in SEQ ID NO: 12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and wherein each CDR sequence is identical to SEQ ID NO:

12.

3. The engineered antigen presenting cell of claim 1 or 2, wherein has one or more of the following features: (A) the plurality of functional regions of the multispecific Nanobody are fused by a linker; preferably, the linker is (GGSGG)p or (G4S)mGn, each of m, n and p is independently a positive integer from 1 to 10; more preferably, the linker has a sequence as set forth in SEQ ID NO: 13-17. (B) the multispecific Nanobody further comprises: an Fc region; preferably, the Fc region is an Fc region of IgGl, IgG2, IgG3 or IgG4; preferably, the Fc region further comprises a mutation that reduces the affinity constant of the multispecific Nanobody to FcyRIIIa and / or Clq; more preferably, the Fc region is an Fc region of IgGl, and the Fc region has one or more mutations selected from the following: L234A, L235A, G237A according to the EU numbering system; or the Fc region is an Fc region of IgG4, and the Fc region has one or more mutations selected from the following: S228P, E233P, F234V, L235A, D254A, L309V, R409K according to the EU numbering system.

4. The engineered antigen presenting cell of any of claims 1-3, wherein, the antigen presenting cell is selected from a macrophage, a B cell or a dendritic cell.

5. A method of producing an engineered antigen presenting cell, comprising: (1) loading the antigen presenting cell with a tumor associated antigen as recited in claim 1 or introducing a coding sequence of a tumor associated antigen as recited in claim 1; and introducing a coding sequence of the multispecific Nanobody as recited in claim 1 into the antigen presenting cell. preferably, the multispecific Nanobody comprises an anti-PD-1 Nanobody and an anti- CTLA-4 Nanobody.

6. The method of claim 5, wherein, loading the antigen presenting cell with a tumor associated antigen or introducing a coding sequence of a tumor associated antigen comprises: contacting the antigen presenting cell with a tumor associated antigen peptide, or with a RNA encoding a tumor associated antigen; and / or introducing a coding sequence of the multispecific Nanobody comprises: contacting the antigen presenting cell with a RNA encoding the multispecific Nanobody; or with a plasmid vector containing a polynucleotide encoding the multispecific Nanobody, preferably, the plasmid is a microcarrier, the backbone sequence of which is within 600 bp in length, reduced and / or free of CpG DNA motifs, and / or the plasmid is an anti-microplasmid; Preferably, the antigen presenting cells are contacted with a maturation composition prior to or after step (1).

7. A method of activating immune competent cells ex vivo from a patient having a tumor, comprising: obtaining immune competent cells from the patient; producing engineered antigen presenting cells by the method of claim 5 or 6; and co-culturing the plurality of immune competent cells with the engineered antigen presenting cells for a time sufficient to activate the immune competent cells, resulting in activated immune competent cells.

8. An activated immune competent cell, obtained by the method of claim 7.

9. A pharmaceutical composition comprising the engineered antigen presenting cell of any one of claims 1-4 or produced by the method of claim 5 or 6 or the activated immune competent cell of claim 8, and a pharmaceutically acceptable excipient.

10. Use of the engineered antigen presenting cell of any one of claims 1-4 or produced by the method of claim 5 or 6 or the activated immune competent cell of claim 8 for the manufacture of a medicament; preferably, the medicament is for preventing the occurrence or metastasis of a tumor in a subject, or inhibiting the growth or metastasis of a tumor in a subject, the tumor expressing the tumor-associated antigen.

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