Treatment involving car-engineered t cells and cytokines

Abstract

This disclosure relates to methods and substances for enhancing the effects of T cells engineered to express a chimeric antigen receptor (CAR). These methods and substances are particularly intended for treating diseases characterized by diseased cells expressing an antigen targeted by the CAR. Specifically, this disclosure relates to a method comprising providing a subject with genetically modified T cells to express a chimeric antigen receptor (CAR) and administering IL2 or a polynucleotide encoding IL2 to the subject. The method of this disclosure may include administering IL2 or a polynucleotide encoding IL2, as well as other cytokines or polynucleotides encoding other cytokines, wherein the other cytokines may be IL7 or IL21. The genetically modified T cells to express CAR can be provided to the subject by administering the genetically modified T cells to express the CAR or by generating genetically modified T cells to express the CAR in the subject. The method of this disclosure may further include administering an antigen or a variant thereof, or a polynucleotide encoding an antigen or a variant thereof, to the subject, wherein the genetically modified T cells to express the CAR target the antigen. In a preferred embodiment, the polynucleotide administered according to this disclosure is RNA.

Description

Technical Field

[0001] This disclosure relates to methods and substances for enhancing the effects of T cells engineered to express a chimeric antigen receptor (CAR). These methods and substances are particularly intended for treating diseases characterized by diseased cells expressing an antigen targeted by the CAR. Specifically, this disclosure relates to a method comprising providing a subject with genetically modified T cells to express a chimeric antigen receptor (CAR) and administering IL2 or a polynucleotide encoding IL2 to the subject. The method of this disclosure may include administering IL2 or a polynucleotide encoding IL2, as well as other cytokines or polynucleotides encoding other cytokines, wherein the other cytokines may be IL7 or IL21. The genetically modified T cells to express CAR may be provided to the subject by administering the genetically modified T cells to express the CAR or by generating genetically modified T cells to express the CAR in the subject. The method of this disclosure may further include administering an antigen or a variant thereof, or a polynucleotide encoding an antigen or a variant thereof, to the subject, wherein the T cells modified to express the CAR target the antigen. In a particularly preferred embodiment, the polynucleotide administered according to this disclosure is RNA. Background Technology

[0002] The immune system plays a crucial role in cancer, autoimmune diseases, allergies, and pathogen-associated diseases. T cells are central to cell-mediated immunity in humans and animals. The recognition and binding of specific antigens by T cells is mediated by the T cell receptor (TCR) expressed on the surface of T cells. The TCR of T cells can interact with immunogenic peptides (epitopes) that bind to the major histocompatibility complex (MHC) molecule and are presented on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells.

[0003] TCR diversity arises from the genetic rearrangement of discontinuous segments of genes encoding different structural regions of the TCR. A TCR comprises either an α-chain and a β-chain or a γ-chain and a δ-chain. The TCR α / β chains include a highly polymorphic variable region at the N-terminus involved in antigen recognition and an invariant region. At the genetic level, these chains are divided into several regions: variable (V) regions, diverse (D) regions (containing only β- and δ-chains), connecting (J) regions, and invariant (C) regions. During T cell differentiation, specific T cell receptor genes are generated by rearranging a V, a D (containing only β and δ-chains), a J, and a C region gene. TCR diversity is further amplified by imprecise V-(D)-J rearrangements, in which random nucleotides are introduced and / or deleted at recombination sites. Because TCR locus rearrangements occur within the genome during T cell maturation, each mature T cell expresses only one specific α / β TCR or γ / δ TCR. The TCR is part of a complex signal transduction mechanism, comprising a heterodimeric complex of TCRα- and β-chains, a common receptor CD4 or CD8, and a CD3 transduction module. The CD3 chain transmits activation signals intracellularly, while the TCRα / β heterodimer is solely responsible for antigen recognition.

[0004] Immunotherapy based on adoptive cell transfer (ACT) can be broadly defined as a form of passive immunization in which previously sensitized T cells are expanded in vitro from low precursor frequencies to clinically relevant cell numbers and then transferred to non-immune receptors or autologous hosts. The cell types used in ACT experiments include lymphokine-activated killer (LAK) cells (Mule, JJ et al. (1984) Science 225, 1487-1489; Rosenberg, SA et al. (1985) N. Engl. J. Med. 313, 1485-1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, SA et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166), hematopoietic stem cell transplant donor lymphocytes (HSCT), and tumor-specific T cell lines or clones (Dudley, ME et al. (2001) J. Immunother. 24, 363-373; Yee, C. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 16168-16173). Adoptive T-cell transfer has been shown to have therapeutic activity against human viral infections such as CMV. While CMV infection and reactivation of endogenous latent viruses are controlled by the immune system in healthy individuals, it can lead to significant morbidity and mortality in immunocompromised individuals, such as transplant recipients or AIDS patients. Riddell et al. demonstrated that immunosuppressed patients could reconstitute viral immunity with adoptive T-cell therapy after transfer of CD8+ CMV-specific T-cell clones from HLA-matched CMV seropositive transplant donors (Riddell, SR (1992) Science 257, 238-241). As an alternative, transfer of polyclonal donor-derived CMV or EBV-specific T-cell populations to transplant recipients has resulted in increased persistence of the transferred T cells (Rooney, CM et al. (1998) Blood 92, 1549-1555; Peggs, KSet et al. (2003) Lancet 362, 1375-1377). For adoptive immunotherapy of melanoma, Rosenberg and colleagues developed an ACT approach that relies on the infusion of in vitro expanded autologous tumor-infiltrating lymphocytes (TILs) isolated from resected tumors, combined with non-myeloablative lymphoablation chemotherapy and high-dose IL2. A recently published clinical study showed that the objective response rate in treated metastatic melanoma patients was approximately 50% (Dudley, ME et al. (2005) J. Clin. Oncol. 23:2346-2357).

[0005] An alternative approach involves adoptively transferring autologous T cells reprogrammed to express tumor-reactive immune receptors with defined specificity during a short period of in vitro culture, followed by infusion into a patient (Kershaw MH et al. (2013) Nature Reviews Cancer 13(8):525-41). This strategy makes ACT applicable to a wide range of common malignancies, even when tumor-reactive T cells are absent in the patient. Since the antigen specificity of T cells depends entirely on the heterodimeric complex of the TCR α- and β-chains, transferring a cloned TCR gene into T cells offers the potential to redirect them to antigens of interest. Therefore, TCR gene therapy provides an attractive strategy for developing antigen-specific immunotherapies using autologous lymphocytes as a treatment option. A major advantage of TCR gene transfer is the generation of therapeutic doses of antigen-specific T cells within days and the potential to introduce specificity not present in the patient's endogenous TCR repertoire.

[0006] Several groups have demonstrated that TCR gene transfer is an attractive strategy for redirecting the antigen specificity of primary T cells (Morgan, RA et al. (2003) J. Immunol. 171, 3287-3295; Cooper, LJ et al. (2000) J. Virol. 74, 8207-8212; Fujio, K. et al. (2000) J. Immunol. 165, 528-532; Kessels, HWe et al. (2001) Nat. Immunol. 2, 957-961; Dembic, Z. et al. (1986) Nature 320, 232-238). Rosenberg and his team were the first to demonstrate the feasibility of TCR gene therapy in humans during clinical trials for the treatment of malignant melanoma. Adoptive transfer of autologous lymphocytes transduced with melanoma / melanocyte antigen-specific TCR retroviruses resulted in cancer regression in up to 30% of treated melanoma patients (Morgan, RA et al. (2006) Science 314, 126-129; Johnson, LA et al. (2009) Blood 114, 535-546). Meanwhile, clinical trials of TCR gene therapy have expanded beyond melanoma to target many different tumor antigens (Park, TS et al. (2011) Trends Biotechnol. 29, 550-557).

[0007] The use of genetic engineering methods to insert specific antigen-targeting receptors into T cells has greatly expanded the potential of ACT. Chimeric antigen receptors (CARs) are antigen-targeting receptors that include an intracellular T-cell signaling domain fused to an extracellular antigen-binding portion, most commonly a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs directly recognize cell surface antigens, independently of MHC-mediated presentation, allowing the use of a single receptor construct specific to any given antigen in all patients. The earliest CARs fused the antigen-recognition domain to the CD3ζ activation chain of the T-cell receptor (TCR) complex. Subsequent CAR iterations included secondary co-stimulatory signals tandem with CD3ζ, including intracellular domains from CD28 or various TNF receptor family molecules such as 4-1BB (CD137) and OX40 (CD134). Furthermore, third-generation receptors contain two co-stimulatory signals in addition to CD3ζ, most commonly from CD28 and 4-1BB. Second- and third-generation CARs have significantly improved antitumor efficacy, inducing complete remission in some patients with advanced cancer in certain cases.

[0008] The number of transferred T cells is generally considered to be correlated with treatment response. However, the number of cells available for adoptive T cell transfer in patients is limited, and generating a large number of T cells for adoptive T cell transfer remains a challenge. Significant increases in cell persistence can be achieved when patients undergo lymphocyte removal preparation protocols prior to TIL or engineered recipient T cell infusion. However, transferring a large number of engineered T cells into an empty host carries the risk of serious adverse events if the target antigen is unexpectedly expressed in the relevant normal tissue. Therefore, it is desirable to transfer a limited number of engineered T cells that can be expanded in patients after their safety has been confirmed.

[0009] The inventors have discovered that CAR-T cells can be expanded in subjects by administering RNA encoding IL2, optionally in combination with RNA encoding other cytokines such as IL7 or IL21, and optionally using RNA vaccination to provide antigens for CAR-T cell stimulation. The method of this invention allows for the delivery of only a small number of CAR-engineered T cells to a patient, followed by in vivo expansion of the T cells. Summary of the Invention

[0010] This invention generally includes treating disease by targeting cells that express antigens on their cell surface, such as diseased cells expressing antigens on their cell surface, particularly cancer cells expressing tumor antigens on their cell surface. The method provides selective eradication of cells expressing antigens on their surface, thereby minimizing adverse effects on normal cells that do not express said antigens. For example, T cells are administered to a subject, which are genetically modified to express chimeric antigen receptors (CARs), targeting cells by binding to the antigen. IL2 or a nucleic acid encoding it is administered. In one embodiment, other cytokines such as IL7 or IL21 or nucleic acids encoding them are administered. In one embodiment, an antigen or a variant thereof or a nucleic acid encoding it is administered to provide (optionally after expression of the nucleic acid on suitable target cells) antigens for the stimulation, initiation, and / or expansion of T cells. The stimulated, initiated, and / or expanded T cells in the patient are able to recognize cells expressing antigens on their cell surface, such as diseased cells, thereby eliminating the diseased cells. The methods of this invention can be considered to involve passive and active immunity. Treatment involving the administration of genetically modified T cells to express CARs can be considered a form of passive immunity. Treatments involving the administration of antigens or variants thereof to stimulate T-cell-mediated immune responses against target cell populations or tissues can be considered a form of active immunization.

[0011] The immune response disclosed herein is directed against a target cell population or target tissue in mammals that expresses an antigen, and T cells genetically modified to express a chimeric antigen receptor (CAR) target said antigen. The method of the invention optionally further includes administration of said antigen or a variant thereof. In one embodiment, the immune response is a T cell-mediated immune response. In one embodiment, the immune response is an anti-tumor immune response, and the target cell population or target tissue is tumor cells or tumor tissue.

[0012] The methods and substances described herein are particularly effective when administered RNA encoding IL2 (hereinafter referred to as "IL2 for prolonged pharmacokinetics (PK)") linked to a pharmacokinetic modifying group, optionally in combination with RNA encoding other cytokines such as IL7 or IL21 (hereinafter referred to as "cytokines for prolonged pharmacokinetics (PK)") linked to a pharmacokinetic modifying group. The methods and substances described herein are particularly effective when the RNA encoding IL2 for prolonged PK and / or the RNA encoding cytokines for prolonged PK are targeted to the liver to achieve systemic availability. Hepatocytes can be efficiently transfected and are capable of producing large amounts of protein. RNA encoding antigens preferably targets secondary lymphoid organs.

[0013] In one aspect, the present invention provides a method for inducing an immune response in a subject, comprising:

[0014] a. Provide the subjects with genetically modified T cells that express chimeric antigen receptor (CAR), and

[0015] b. Administer IL2 or a polynucleotide encoding IL2 to the subject.

[0016] In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, as well as other cytokines or polynucleotides encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, and IL7 or a polynucleotide encoding IL7. In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, and IL21 or a polynucleotide encoding IL21.

[0017] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding other cytokines is RNA.

[0018] In one embodiment, the subject is provided with genetically modified T cells that express CAR by administering the genetically modified T cells to express CAR or by generating genetically modified T cells to express CAR in the subject.

[0019] In one embodiment, the method further includes administering an antigen or a variant thereof, or a polynucleotide encoding the antigen or variant, to the subject, wherein the genetic modification to T cells expressing a CAR targets the antigen, and the immune response is an immune response against a target cell population or target tissue expressing the antigen. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

[0020] In one aspect, the present invention provides a method for inducing an immune response in a subject, comprising:

[0021] a. Provide the subjects with genetically modified T cells that express chimeric antigen receptor (CAR), and

[0022] b. Administer RNA encoding IL2 to the subject.

[0023] In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding IL7. In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding IL21.

[0024] In one embodiment, the subject is provided with genetically modified T cells that express CAR by administering the genetically modified T cells to express CAR or by generating genetically modified T cells to express CAR in the subject.

[0025] In one embodiment, the method further includes administering RNA encoding an antigen or a variant thereof to the subject, wherein the genetic modification is such that T cells expressing CAR target the antigen, and the immune response is an immune response against a target cell population or target tissue expressing the antigen.

[0026] In one embodiment across all aspects, the immune response is a T-cell-mediated immune response.

[0027] In one aspect, the present invention provides a method for treating a subject suffering from a disease, symptom, or condition related to the expression or elevated expression of an antigen, the method comprising:

[0028] a. Providing the subject with genetically modified T cells that express chimeric antigen receptor (CAR) targeting the antigen, and

[0029] b. Administer IL2 or a polynucleotide encoding IL2 to the subject.

[0030] In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, as well as other cytokines or polynucleotides encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, and IL7 or a polynucleotide encoding IL7. In one embodiment, the method includes administering IL2 or a polynucleotide encoding IL2, and IL21 or a polynucleotide encoding IL21.

[0031] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding other cytokines is RNA.

[0032] In one embodiment, the subject is provided with genetically modified T cells that express CAR by administering the genetically modified T cells to express CAR or by generating genetically modified T cells to express CAR in the subject.

[0033] In one embodiment, the method further includes administering the antigen or a variant thereof, or a polynucleotide encoding the antigen or antibody, to the subject. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

[0034] In one aspect, the present invention provides a method for treating a subject suffering from a disease, symptom, or condition related to the expression or elevated expression of an antigen, the method comprising:

[0035] a. Providing the subject with genetically modified T cells that express chimeric antigen receptor (CAR) targeting the antigen, and

[0036] b. Administer RNA encoding IL2 to the subject.

[0037] In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding IL7. In one embodiment, the method includes administering RNA encoding IL2 and RNA encoding IL21.

[0038] In one embodiment, the subject is provided with genetically modified T cells that express CAR by administering the genetically modified T cells to express CAR or by generating genetically modified T cells to express CAR in the subject.

[0039] In one embodiment, the method further includes administering RNA encoding the antigen or a variant thereof to the subject.

[0040] In one embodiment across all aspects, the disease, symptom, or condition is cancer, and the antigen is a tumor-associated antigen.

[0041] In one embodiment across all aspects, the IL2 is an IL2 with prolonged pharmacokinetic (PK). In one embodiment, the PK-prolonged IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises an IL2 moiety and a portion selected from: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0042] In one embodiment across all aspects, the other cytokines, particularly IL7 or IL21, are cytokines that prolong pharmacokinetic (PK), particularly PK-prolonging IL7 or PK-prolonging IL21. In one embodiment, the PK-prolonging cytokines, particularly PK-prolonging IL7 or PK-prolonging IL21, comprise a fusion protein. In one embodiment, the fusion protein comprises portions of other cytokines, particularly the IL7 or IL21 portion, and portions selected from: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0043] In one embodiment, the serum albumin comprises mouse serum albumin or human serum albumin. In one embodiment, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

[0044] In one embodiment of all aspects, the method is a method for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0045] In one aspect, the present invention provides a pharmaceutical product comprising:

[0046] a. T cells genetically modified to express chimeric antigen receptor (CAR), and

[0047] b. IL2 or polynucleotides encoding IL2.

[0048] In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2, as well as other cytokines or polynucleotides encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL7. In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL21.

[0049] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding other cytokines is RNA.

[0050] In one embodiment, the pharmaceutical product further comprises an antigen or a variant thereof, or a polynucleotide encoding said antigen or variant, wherein the genetic modification to express a CAR by T cells targets said antigen. In one embodiment, the polynucleotide encoding said antigen or variant is RNA.

[0051] In one embodiment, the pharmaceutical product is a reagent kit.

[0052] In one embodiment, the pharmaceutical product comprises genetically modified T cells to express CAR, IL2 or a polynucleotide encoding IL2, optionally other cytokines or polynucleotides encoding other cytokines, and optionally an antigen or a variant thereof or a polynucleotide encoding said antigen or variant, in separate containers.

[0053] In one embodiment, the pharmaceutical product further includes instructions for use of the pharmaceutical product for the treatment or prevention of cancer, wherein the antigen is a tumor-associated antigen.

[0054] In one embodiment, the pharmaceutical product is a pharmaceutical composition.

[0055] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0056] In one aspect, the present invention provides a pharmaceutical product comprising:

[0057] a. T cells genetically modified to express chimeric antigen receptor (CAR), and

[0058] b. RNA encoding IL2.

[0059] In one embodiment, the pharmaceutical product comprises RNA encoding IL2 and RNA encoding other cytokines. In one embodiment, the other cytokines are selected from IL7 and IL21. In one embodiment, the pharmaceutical product comprises RNA encoding IL2 and RNA encoding IL7. In one embodiment, the pharmaceutical product comprises RNA encoding IL2 and RNA encoding IL21.

[0060] In one embodiment, the pharmaceutical product further comprises RNA encoding an antigen or a variant thereof, wherein the genetic modification to express CAR T cells targets the antigen.

[0061] In one embodiment, the pharmaceutical product is a reagent kit.

[0062] In one embodiment, the pharmaceutical product comprises genetically modified T cells to express CAR, RNA encoding IL2, optionally present RNA encoding other cytokines, and optionally present RNA encoding an antigen or a variant thereof, in separate containers.

[0063] In one embodiment, the pharmaceutical product further includes instructions for use of the pharmaceutical product for the treatment or prevention of cancer, wherein the antigen is a tumor-associated antigen.

[0064] In one embodiment, the pharmaceutical product is a pharmaceutical composition.

[0065] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0066] In one embodiment across all aspects, the IL2 is an IL2 with prolonged pharmacokinetic (PK). In one embodiment, the PK-prolonged IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises an IL2 moiety and a portion selected from: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0067] In one embodiment across all aspects, the other cytokines are cytokines that prolong pharmacokinetic (PK). In one embodiment, the PK-prolonging cytokines comprise fusion proteins. In one embodiment, the fusion protein comprises a cytokine portion and a portion selected from: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0068] In one embodiment, the serum albumin comprises mouse serum albumin or human serum albumin.

[0069] In one embodiment, the immunoglobulin fragment includes an immunoglobulin Fc domain.

[0070] In one aspect, the present invention provides the pharmaceutical products described herein for pharmaceutical use. In one embodiment, the pharmaceutical use includes therapeutic or preventative treatment of a disease or condition.

[0071] In one aspect, the present invention provides the pharmaceutical product described herein, and a method for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0072] In one aspect, the present invention provides substances and compositions described herein for use in the methods described herein.

[0073] In one aspect, the present invention provides genetically modified T cells that express chimeric antigen receptors (CARs) and target antigens for use in the methods described herein.

[0074] In one aspect, the present invention provides IL2 or a polynucleotide encoding IL2 for use in the methods described herein.

[0075] In one aspect, the present invention provides cytokines other than IL2, such as IL7 or IL21, or polynucleotides encoding them, for use in the methods described herein.

[0076] In one aspect, the present invention provides an antigen or a variant thereof, or a nucleic acid encoding said antigen or a variant thereof, for use in the methods described herein.

[0077] In one embodiment of the pharmaceutical product, the RNA is present in a form selected from liquid, solid, or combinations thereof. In one embodiment, the solid form is a frozen or dehydrated form. In one embodiment, the dehydrated form is a freeze-dried or spray-dried form.

[0078] In one implementation scheme, the cancers described herein are selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer. Attached Figure Description

[0079] Figure 1: mAlb-mIL-2 and mIL-7-mAlb enhance in situ repeat antigen-specific amplification of genetically engineered CAR T cells in pre-conditioned mice. (A) C57BL / 6BrdCrHsd-Tyr cells irradiated with 2.5 Gy (XRAD320) c Mice (n=2-3 / group) IV transplanted 5x10 6 One CLDN6-CAR-BBz-Luc-GFP transduced C57Bl / 6-Thy1.1 + T cells. One day later, mice were treated with an inoculation (20 μg, iv) of mRNA lipoplex encoding hCLDN6 or OvaI (control RNA), followed by intraperitoneal administration of nucleoside-modified formulated RNA (1 μg / mRNA / mouse) encoding mAlb-mIL-2 and mIL-7-mAlb. Buffer was used as a dummy control. Treatment was repeated after 7 days. Bioluminescent imaging (BLI) was performed to monitor expansion and persistence from day 1 (baseline) to day 15 post-ACT. (B) Transgenic expression in adoptively transferred mouse CAR-transduced T cells. Cells were stained with antibodies conjugated to fluorescent dyes against CD8 and CD4, as well as a unique type-specific antibody against the scFv portion of CLDN6-CAR (anti-IMAB206), and analyzed by flow cytometry. Left: Cells gated on a single lymphocyte; Right: Cells gated on CD8. + Gates are set on T cells. (C) In ACT and with antigen RNA (LIP) Bioluminescence imaging of lateral mice at different time points following treatment with mRNA encoding albumin-cytokines. Non-color images represent light intensity superimposed on a grayscale reference image (black, least intense; white to dark gray, most intense). (D) Expansion index of total flux calculated at 4 and 11 days after ACT (mean ± sd) compared to baseline on day 1; ACT: adoptive T cell transfer; TBI: whole-body irradiation; BLI: bioluminescence imaging; Luc: effective firefly luciferase; mAlb: mouse serum albumin; mIL-2: mouse interleukin-2; mIL-7: mouse interleukin-7.

[0080] Figure 2: Even in immune-active mice, mAlb-mIL-2 and mIL-7-mAlb prolong the persistence of antigen-specifically expanded CAR T cells in vivo. (A) Unirradiated C57BL / 6BrdCrHsd-Tyr c Mice (n = 2-3 / group) received the same dose of CAR-transduced T cells and were treated as described in Figures 1A-B. (B) In ACT with antigen RNA (LIP)Bioluminescence imaging of lateral mice at different time points following treatment with mRNA encoding albumin-cytokines. Non-color images represent light intensity superimposed on grayscale reference images (black, least intense; white to dark gray, most intense). (C) Expansion index of total flux at 4 and 11 days after ACT compared to baseline on day 1 (mean ± sd); ACT: adoptive T cell transfer; BLI: bioluminescence imaging; Luc: effective firefly luciferase; mAlb: mouse serum albumin; mIL-2: mouse interleukin-2; mIL-7: mouse interleukin-7.

[0081] Figure 3: The presence of mAlb-mIL-2 combined with mIL-7-mAlb or mIL-21-mAlb leads to the accumulation of in situ repeat antigen-specific amplification and prolonged persistence of genetically engineered CAR T cells in vivo. (A) C57BL / 6BrdCrHsd-Tyr cells irradiated with 2.5 Gy c Mice (n = 2-3 / group) received the same dose of CAR-transduced T cells and an mRNA lipoplex vaccine encoding hCLDN6 or OvaI (control RNA), in combination with formulated cytokines with different nucleoside modifications (1 μg of each single mRNA used per animal), such as Figure 1A (B) During the three rounds of vaccination (imaging typically on labeled RNA) (LIP) The relative increase in bioluminescence in mice treated with cytokine RNA was quantified and calculated 2–3 days after each round of treatment. The amplification index was calculated as follows: total throughput of each round of amplification [p / s] / total throughput at baseline on day 1 after ACT [p / s] (mean ± sem). (CD) In ​​the presence of labeled nucleoside-modified formulated cytokine RNA (mean + / - sem), CLDN6-RNA was used. (LIP) Quantification of bioluminescence during and after the amplification round. Arrows indicate hCLDN6 RNA. (LIP) Vaccination and nucleoside-modified formulations of cytokines (ribocytokines). ACT: adoptive T-cell transfer; TBI: whole-body irradiation; BLI: bioluminescent imaging; Luc: effective firefly luciferase; mAlb: mouse serum albumin; mIL-2: mouse interleukin-2; mIL-7: mouse interleukin-7. Detailed Implementation

[0082] Although this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which is defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0083] Preferably, the terminology used herein is defined as “A Multilingual Glossary of Biotechnology Terms: (IUPAC Recommendation)”, H.G. Leuenberger, B. Nagel, and H. Those described in Eds., Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

[0084] Unless otherwise stated, the implementation of this disclosure employs conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, as explained in the literature in the field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0085] The elements of this disclosure will be described below. These elements are listed with particular embodiments; however, it should be understood that they can be combined in any way and in any number to produce additional embodiments. The various examples and embodiments described should not be construed as limiting this disclosure to the explicitly described embodiments. The description of this disclosure should be understood as disclosing and covering embodiments that combine the explicitly described embodiments with any number of disclosed elements. Furthermore, unless the context otherwise requires, any permutation and combination of all described elements is considered to be disclosed in this specification.

[0086] The term “about” means approximately or almost, and in the context of a numerical value or range set forth in one embodiment herein, it means ±20%, ±10%, ±5%, or ±3% of the cited or claimed numerical value or range.

[0087] Unless otherwise stated herein, or obviously contradicted by the context, the terms “an,” “a,” and “the,” and similar references used in the context of describing this disclosure (especially in the context of the claims) are interpreted to cover both singular and plural forms. References to numerical ranges herein are intended only as a shorthand method for individually referring to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually referenced herein. Unless otherwise stated herein, or obviously contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate this disclosure and does not constitute a limitation on the scope of the claims. No language in the specification should be construed as indicating any unclaimed element essential to the implementation of this disclosure.

[0088] Unless otherwise expressly stated, the term “comprising” as used in the context of this document means that additional members may optionally exist in addition to the list of members described by “comprising”. However, as contemplated as a particular embodiment of this disclosure, the term “comprising” is intended to cover the possibility that no other members exist; that is, for the purposes of this embodiment, “comprising” should be understood to mean: “consisting of…”.

[0089] Several documents are referenced throughout this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether mentioned above or below, is incorporated herein by reference. Nothing herein should be construed as an admission that this disclosure is not earlier than such disclosure.

[0090] Definitions applicable to all aspects of this disclosure will be provided below. Unless otherwise stated, the following terms have the following meanings. Any undefined term has its generally accepted meaning in the art.

[0091] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150 consecutive amino acids linked together by peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0092] When administered to a subject in a therapeutically effective amount, a "therapeutic protein" has a positive or beneficial effect on the subject's disease condition or disease status. In one embodiment, the therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of one or more symptoms of a disease or condition, or reduce its severity. Therapeutic proteins may have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides, and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, cytokines.

[0093] A “fragment” of an amino acid sequence (peptide or protein) refers to a portion of an amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) is available, for example, by translating a truncated open reading frame (OPF) lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) is also available, for example, by translating a truncated OPF lacking the 5' end, provided the truncated OPF contains a start codon for initiating translation. The amino acid sequence fragment comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. Preferably, the amino acid sequence fragment comprises at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0094] For the purposes of this disclosure, “variants” of an amino acid sequence (peptide or protein) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” specifically includes fragments of an amino acid sequence.

[0095] Amino acid insertion variants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants contain the fusion of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids, at their N-terminus and / or C-terminus. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any position in the protein. Amino acid deletion variants containing N-terminal and / or C-terminal deletions of a protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue into its position. Modification of non-conserved amino acid sequence positions between homologous proteins or peptides and / or substitution of amino acids with other amino acids having similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conserved amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of the related amino acid families in its side chain. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

[0096] Preferably, the degree of similarity between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is preferably at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Preferably, the degree of similarity or identity is given for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, it is preferred that the degree of similarity or identity be given for consecutive amino acids of at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignments used to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0097] "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0098] The term "percentage similarity" is intended to represent the percentage of identical amino acid residues between two sequences to be compared after optimal alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their full length. Sequence comparisons between two amino acid sequences are typically performed by comparing these sequences after optimal alignment, using segments or "comparison windows" to identify and compare local regions of sequence similarity. Besides manual methods, the best alignments for comparison can be generated by: using the local homology algorithm of Smith and Waterman, 1981, AdsApp.Math.2, 482; using the local homology algorithm of Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; using the similarity retrieval method of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85, 2444; or by using computer programs of these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package (Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0099] The percentage similarity is calculated by determining the number of identical positions between the two sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100.

[0100] According to this disclosure, the homologous amino acid sequences exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% amino acid residue similarity.

[0101] Those skilled in the art can readily prepare the amino acid sequence variants described herein, for example, through recombinant DNA manipulation. For instance, Sambrook et al. (1989) described in detail the procedures for preparing DNA sequences of peptides or proteins with substitutions, additions, insertions, or deletions. Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[0102] In one embodiment, the fragment or variant of the amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant exhibiting one or more functional properties that are the same as or similar to those properties of the amino acid sequence from which it originates, i.e., they are functionally equivalent. Regarding cytokines, a specific function is one or more immunomodulatory activities exhibited by the amino acid sequence from which the fragment or variant originates and / or binding to a receptor of the amino acid sequence from which the fragment or variant originates.

[0103] The phrase "derived from" a specific amino acid sequence (peptide or protein) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from a particular amino acid sequence has the same, substantially the same, or homologous amino acid sequence as that particular sequence or a fragment thereof. The amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, those skilled in the art will understand that antigens and cytokines (e.g., IL2, IL7, or IL21) suitable for use herein can be modified such that their sequences differ from naturally occurring sequences or the natural sequences from which they are derived, while retaining the desired activity of the natural sequence.

[0104] T cells

[0105] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by special receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ responsible for T cell maturation. Several distinct T cell subsets have been identified, each with different functions.

[0106] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor consists of two separate polypeptide chains, produced by independent T cell receptor α and β (TCRα and TCRβ) genes, and are called α- and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells with a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is less numerous than αβ T cells (comprising 2% of the total number of T cells).

[0107] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells, derived from these stem cells, reside in the thymus and proliferate through cell division to produce a large number of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as double-negative (CD4-CD8-) cells. As they develop, they become double-positive thymocytes (CD4+CD8+), and eventually mature into single-positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

[0108] The terms “T cell” and “T lymphocyte” are used interchangeably herein and include both helper T cells (CD4+ T cells) and cytotoxic T cells, including cytolytic T cells (CTLs, CD8+ T cells). The term “antigen-specific T cell” or similar term refers to a T cell that recognizes an antigen targeted by a T cell, particularly when presented on the surface of an antigen-presenting cell or a diseased cell (e.g., a cancer cell), and preferably performs an effector function. A T cell is considered antigen-specific if it kills a target cell expressing an antigen. T cell specificity can be assessed using any of a variety of standard techniques, such as in a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0109] Helper T cells assist other white blood cells in the immune process, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, among other functions. These cells are also called CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when MHC class II molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens. Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist in active immune responses.

[0110] Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. These cells are also called CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to MHC class I-associated antigens, which are present on the surface of almost every cell in the body.

[0111] T cell-mediated effector functions include those of helper T cells (CD4+). + In the presence of T cells, cytokines are released and / or CD8 is activated. + Lymphocytes (CTLs) and / or B cells, and in the case of CTLs, the elimination of cells, i.e., cells characterized by antigen expression, for example, through apoptosis or perforin-mediated cell lysis, the production of cytokines (such as IFN-γ and TNF-α), and specific cytolytic killing of target cells expressing antigens.

[0112] According to the present invention, the term "T cell" also includes cells that can mature into T cells under appropriate stimulation.

[0113] T cells can typically be prepared in vitro or ex vivo using standard procedures. For example, commercially available cell isolation systems can be used to isolate T cells from the bone marrow, peripheral blood, or portions of bone marrow or peripheral blood of mammals (e.g., patients). Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures. Samples containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0114] The T cells used in this invention may express endogenous T cell receptors or may lack the expression of endogenous T cell receptors.

[0115] CAR

[0116] Nucleic acids, such as RNA encoding CAR, can be introduced into T cells or other cells with lytic potential, especially lymphoid cells.

[0117] According to this disclosure, as described above, when present on T cells, CARs recognize antigens on the surface of cells such as antigen-presenting cells or diseased cells (e.g., cancer cells), thereby stimulating, initiating, and / or expanding the T cells or enabling them to perform effector functions.

[0118] According to the present invention, the term "chimeric antigen receptor (CAR)" is synonymous with the terms "chimeric T-cell receptor" and "artificial T-cell receptor".

[0119] Preferably, the CAR is expressed on the cell surface.

[0120] According to the present invention, the term "CAR" (or "chimeric antigen receptor") refers to an artificial receptor comprising a single molecule or molecular complex that recognizes, i.e., binds to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding to an antigen expressed on the surface of the target cell via an antigen-binding domain), and can confer specificity to immune effector cells (e.g., T cells expressing the CAR on their cell surface). Such cells do not necessarily need to process and present antigens to recognize target cells, but can preferably specifically recognize any antigen present on the target cell. Preferably, the recognition of the target structure by the CAR leads to activation of the immune effector cell expressing the CAR. The CAR may comprise one or more protein units containing one or more of the domains described herein. The term "CAR" does not include T cell receptors.

[0121] According to the present invention, a CAR may typically include several domains. In one embodiment of all aspects of the present invention, the CAR includes an antigen-binding domain, a transmembrane domain, and a T-cell signaling domain.

[0122] The binding domain recognizes and binds to antigens. In one embodiment, a single-chain variable fragment (scFv) derived from a monoclonal antibody is used as the binding domain. Other antigen-recognition domains that can be used include T-cell receptor (TCR) α and β single chains. In fact, almost any substance that binds to a given target with high affinity can be used as an antigen-recognition domain. In one embodiment of all aspects of the invention, the CAR comprises an antigen-binding domain. In one embodiment, the exodomain of the CAR comprises an antigen-binding domain. In one embodiment, the antigen-binding domain comprises a single-chain variable fragment (scFv) of an antibody against the antigen. In one embodiment, the antigen-binding domain comprises a heavy chain variable region (VH) of an immunoglobulin specific to the antigen (VH(antigen)) and a light chain variable region (VL) of an immunoglobulin specific to the antigen (VL(antigen)). In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker, preferably a peptide linker comprising the amino acid sequence (GGGGS)3.

[0123] In one embodiment of all aspects of the invention, the CAR includes a transmembrane domain. In one embodiment, the transmembrane domain is a transmembrane hydrophobic α-helix. In one embodiment, the transmembrane domain includes a CD28 transmembrane domain or a fragment thereof.

[0124] Activation signaling domains (or T-cell signaling domains) are used to activate cytotoxic lymphocytes after CAR binding to an antigen. The characteristic of the activation signaling domain is limited to its ability to induce activation of selected cytotoxic lymphocytes upon CAR binding to an antigen. Suitable activation signaling domains include the T-cell CD3[ζ] chain and the Fc receptor[γ]. Those skilled in the art will understand that sequence variants of these mentioned activation signaling domains can be used without adversely affecting the invention, wherein said variants have the same or similar activity as the domain they model. Such variants will have at least about 80% sequence identity with the amino acid sequence of their source domain.

[0125] In one embodiment, the T cell signaling domain is located intracellularly. In one embodiment, the T cell signaling domain includes CD3-ζ (zeta), preferably the inner domain of CD3-ζ, optionally combined with CD28.

[0126] Another possible domain is a costimulatory domain. Costimulatory domains are used to enhance the proliferation and survival of cytotoxic lymphocytes after the CAR binds to the target moiety. The property of a costimulatory domain is limited to its ability to enhance cell proliferation and survival after the CAR binds to the target moiety. Suitable costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family, CD134 (OX40), a member of the TNFR receptor superfamily, and CD278 (ICOS), a costimulatory molecule of the CD28 superfamily expressed on activated T cells. Those skilled in the art will understand that sequence variants of these mentioned costimulatory domains can be used without adversely affecting the invention, wherein said variants have the same or similar activity as the domain they model. Such variants will have at least about 80% sequence identity with the amino acid sequence of their source domain. In some embodiments of the invention, the CAR construct comprises two costimulatory domains. While specific combinations include all possible variations of the four mentioned domains, concrete examples include CD28+CD137(4-1BB) and CD28+CD134(OX40).

[0127] The CAR of the present invention can comprise the aforementioned domains together in the form of a fusion protein. Such a fusion protein typically comprises a binding domain, one or more co-stimulatory domains, and an activation signaling domain linked from the N-terminus to the C-terminus. However, the CAR of the present invention is not limited to this arrangement, and other arrangements are also acceptable, and include a binding domain, an activation signaling domain, and one or more co-stimulatory domains. It should be understood that because the binding domain must be able to freely bind to the antigen, the placement of the binding domain in the fusion protein typically results in the display of that region outside the cell. Similarly, because the co-stimulatory and activation signaling domains are used to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein typically displays these two domains inside the cell. The CAR may include other elements, such as a signal peptide that ensures the proper export of the fusion protein to the cell surface, a transmembrane domain that ensures the fusion protein remains an integrated membrane protein, and a hinge domain (or spacer region) that imparts flexibility to the binding domain and allows for strong binding to the antigen.

[0128] In one embodiment of all aspects of the invention, the CAR includes a signal peptide that guides nascent proteins into the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen-binding domain.

[0129] In one embodiment of all aspects of the invention, the CAR includes a spacer region connecting the antigen-binding domain to the transmembrane domain. In one embodiment, the spacer region allows the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. In one embodiment, the spacer region includes a hinge region derived from IgG1.

[0130] In one embodiment of all aspects of the present invention, the CAR includes the following structure:

[0131] NH2-signal peptide-antigen binding domain-spacer region-transmembrane domain-T cell signaling domain-COOH.

[0132] In one embodiment of all aspects of the invention, the CAR preferably has specificity for the antigen it targets, particularly when it is present on the surface of cells (e.g., diseased cells or antigen-presenting cells).

[0133] In one embodiment of all aspects of the invention, the CAR may be expressed and / or present on the surface of T cells, preferably cytotoxic T cells. In one embodiment, the T cells are reactive to the antigen targeted by the CAR.

[0134] The cells used in conjunction with the CAR system of the present invention are preferably T cells, particularly cytotoxic lymphocytes, preferably selected from T cells, especially cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes triggers the destruction of the target cell. For example, cytotoxic T cells trigger the destruction of the target cell through any one or two of the following methods: First, upon activation, the T cell releases cytotoxins, such as perforin, granzyme, and granzyme. Perforin and granzyme form pores in the target cell, and granzyme enters the cell and triggers a caspase cascade reaction in the cytoplasm, which induces apoptosis (programmed cell death). Second, apoptosis can be induced through Fas-Fas ligand interactions between the T cell and the target cell. Although heterologous or allogeneic cells can be used, the cytotoxic lymphocytes are preferably autologous cells.

[0135] Adoptive cell transfer therapy using T cells expressing chimeric antigen receptors is a promising anticancer therapy because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (genetically modified) to express CARs that specifically target antigens on the patient's tumor cells and then infused back into the patient.

[0136] According to the present invention, CAR can replace the function of T cell receptors and, in particular, can confer responsiveness, such as cytolytic activity, to cells (e.g., T cells). However, compared to T cell receptor-antigen peptide-MHC complexes, CAR can bind to antigens, especially when expressed on the cell surface.

[0137] Various methods can be used to introduce CAR constructs into T cells, including non-viral DNA transfection, transposon-based systems, and virus-based systems. Non-viral DNA transfection carries a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids without integrative elements. Virus-based systems include those using gamma retroviruses and lentiviral vectors. Gamma retroviruses are relatively easy to produce, efficiently and permanently transduce T cells, and have shown initial safety in integrating primary human T cells. Lentiviral vectors can also efficiently and permanently transduce T cells, but are more expensive to manufacture. They may also be safer than retrovirus-based systems.

[0138] In one embodiment of all aspects of the invention, the method further includes transfecting T cells or T cell progenitor cells in vitro or in vivo with a nucleic acid encoding CAR to provide T cells genetically modified to express CAR.

[0139] CAR T cells can be generated in vivo, making the use of T cell-targeting nanoparticles virtually instantaneous. For example, poly(β-amino ester)-based nanoparticles can be coupled with an anti-CD3e f(ab) fragment to bind to CD3 on T cells. For this purpose, the anti-CD3e f(ab) fragment can be covalently linked to polyglutamate (PGA). The PGA surrounds and is linked to a particle core containing nucleic acid and an excess of poly(β-amino ester) (PBAE) polymer via charge interactions. Upon binding to T cells, these nanoparticles are internalized. Their contents, such as plasmid DNA encoding the anti-tumor antigen CAR, can be directed to the T cell nucleus due to the presence of peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) covalently linked to the PBAE polymer. Separate plasmids containing transposons flanking the CAR gene expression cassette and encoding highly active transposases can allow for efficient integration of CAR vectors into chromosomes. Such a system that allows the generation of CAR T cells in vivo after nanoparticle infusion is described in Smith et al. (2017) Nat. Nanotechnol. 12:813-820.

[0140] Another possibility is to intentionally place the CAR coding sequence at a specific locus using the CRISPR / Cas9 method. For example, the existing T cell receptor (TCR) can be knocked out, and a CAR can be knocked in and placed under the dynamic regulatory control of an endogenous promoter, which would otherwise weaken TCR expression; see, for example, Eyquem et al. (2017) Nature 543:113-117.

[0141] In one embodiment of all aspects of the invention, T cells genetically modified to express CAR are stably or transiently transfected with nucleic acids encoding CAR. Thus, the nucleic acids encoding CAR are integrated into or not integrated into the genome of the T cells.

[0142] In one embodiment of all aspects of the invention, the T cells or T cell progenitor cells are derived from the subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell progenitor cells are derived from a subject different from the subject to be treated.

[0143] In one embodiment of all aspects of the invention, the T cells for the subject to be treated may be autologous, allogeneic, or syngeneic. The T cells may be genetically modified in vitro to express a chimeric antigen receptor (CAR) targeting the antigen.

[0144] In one embodiment of all aspects of the invention, the expression of endogenous T cell receptors and / or endogenous HLA on T cells genetically modified to express CAR is inactivated.

[0145] The term "autologous" is used to describe any substance derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organ from the same subject. Such procedures are advantageous because they overcome the immune barrier that can lead to rejection.

[0146] The term "alien" is used to describe any substance that comes from different individuals of the same species. Two or more individuals are said to be alliens of each other when the genes at one or more loci are different.

[0147] The term "homogeneous" is used to describe any substance derived from an individual or tissue having the same genotype, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0148] The term "heterologous" is used to describe substances composed of multiple different components. For example, transplanting bone marrow from one individual to another constitutes a heterologous transplant. Heterologous genes are genes from sources other than the subject.

[0149] RNA

[0150] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, the polynucleotides are preferably isolated.

[0151] Nucleic acids can be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage (e.g., λ phage) vectors, viral vectors (e.g., adenovirus or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and typically contain the desired coding sequence and appropriate DNA sequence required to express an operable linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify specific desired DNA fragments and may lack the functional sequences required to express the desired DNA fragment.

[0152] In one embodiment of all aspects of the invention, a nucleic acid encoding a cytokine or an antigen or a variant thereof is expressed in the cells of a treated subject to provide the cytokine or antigen or a variant thereof. In one embodiment of all aspects of the invention, the nucleic acid is transiently expressed in mammalian cells. Therefore, in one embodiment, the nucleic acid is not integrated into the cell's genome. In one embodiment of all aspects of the invention, the nucleic acid is RNA, preferably in vitro transcribed RNA. In one embodiment of all aspects of the invention, the antigen or a variant thereof is expressed on the cell surface.

[0153] In one embodiment of all aspects of the invention, a nucleic acid encoding an antigen or a variant thereof is expressed in mammalian cells to provide an antigen or a variant thereof to be bound by T cells genetically modified to express a CAR, said binding resulting in stimulation, initiation, and / or amplification of the T cells genetically modified to express a CAR.

[0154] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or peptides or proteins, for example, through transcription and / or translation. Expression can be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "aberrant expression".

[0155] According to the present invention, the term "nucleic acid encoding" refers to nucleic acids, which, if present in a suitable environment, such as inside a cell, can be expressed to produce the proteins or peptides they encode.

[0156] The nucleic acids described in this article can be recombinant and / or isolated molecules.

[0157] As used in this article, "isolated molecules" refers to molecules that are essentially free of other molecules, such as other cellular material.

[0158] In the context of this invention, the term "recombinant" means "prepared by genetic engineering." Preferably, the "recombinant object," such as recombinant cells in the context of this invention, is not naturally occurring.

[0159] As used in this article, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources without being intentionally modified by humans in a laboratory are naturally occurring.

[0160] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of this invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, wherein said cells may be present in a subject, such as a patient. Thus, according to the invention, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, cells that may form part of a patient's organ, tissue, and / or organism. According to the invention, transfection can be transient or stable. For some transfection applications, transient expression of the transfected genetic material is sufficient. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the exogenous nucleic acids are diluted or degraded by mitosis. Cells that allow free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cell and its daughter cells, then stable transfection must be performed. RNA can be transfected into cells to transiently express the protein it encodes.

[0161] In one embodiment of all aspects of the invention, a nucleic acid encoding a cytokine or an antigen or a variant thereof is formulated in a delivery carrier (e.g., particles). In one embodiment, the delivery carrier comprises at least one lipid. In one embodiment, the at least one lipid comprises at least one cationic lipid. In one embodiment, the lipid forms a complex with and / or encapsulates the nucleic acid. In one embodiment, the lipid is contained in vesicles encapsulating the nucleic acid. In one embodiment of all aspects of the invention, the nucleic acid is formulated in liposomes.

[0162] In this disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. These alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, such altered RNA is considered an analogue of naturally occurring RNA.

[0163] In some embodiments of this disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNA typically comprises a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, wherein the DNA refers to a nucleic acid containing deoxyribonucleotides.

[0164] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter controlling transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0165] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine, and / or 1-methyl-pseudouridine.

[0166] In some embodiments, the RNA of this disclosure comprises a 5'-cap. In one embodiment, the RNA of this disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5'-cap analog. The term "5'-cap" refers to a structure found at the 5' end of an mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA via a 5'-to-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. RNA providing a 5'-cap or a 5'-cap analog can be provided by in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA strand, or it can be ligated to the RNA post-transcriptionally using a capping enzyme.

[0167] In some embodiments, the RNA disclosed herein comprises a 5'-UTR and / or a 3'-UTR. The terms "untranslated region" or "UTR" refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as mRNA. An untranslated region (UTR) may be present at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR). The 5'-UTR, if present, is located at the 5' end upstream of the start codon in a protein-coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. The 3'-UTR, if present, is located at the 3' end downstream of the stop codon in a protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) tail. Thus, the 3'-UTR is located upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0168] In some embodiments, the RNA of this disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" refers to a sequence of adenosine (A) residues, which are typically located at the 3' end of an RNA molecule. According to this disclosure, in one embodiment, the poly(A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and up to about 500, about 400, about 300, about 200, or about 150 A nucleotides, particularly about 120 A nucleotides.

[0169] In the context of this disclosure, the term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA. The RNA can then be translated into peptides or proteins.

[0170] Regarding RNA, the terms "expression" or "translation" refer to a process in the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to create peptides or proteins.

[0171] According to this disclosure, the term "RNA-encoded" refers to RNA, if present in a suitable environment, such as within the cells of a target tissue, which can direct the assembly of amino acids to produce its encoded peptides or proteins during translation. In one embodiment, RNA is capable of interacting with cellular translation mechanisms, allowing the translation of peptides or proteins. Cells may produce encoded peptides or proteins intracellularly (e.g., in the cytoplasm and / or nucleus), secrete encoded peptides or proteins, or produce encoded peptides or proteins on their surface.

[0172] The terms “connected,” “integrated,” or “integrated” used in this document are used interchangeably. These terms refer to connecting two or more elements, components, or domains together.

[0173] As used herein, "half-life" refers to the time required for a 50% reduction in serum or plasma concentration of a peptide or protein in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Cytokines with extended PK suitable for this purpose, such as interleukins (ILs) with extended PK, are stable in vivo, and their half-life is increased, for example, by fusion with serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, typically include the following steps: appropriately administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood samples or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood sample; and calculating from the data thus obtained (plotting) the time until the level or concentration of the amino acid sequence or compound decreases by 50% compared to the initial level at the time of dose administration. Further details are provided, for example, in standard manuals such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0174] Cytokines

[0175] Cytokines are a class of small proteins (~5-20 kDa) that play a crucial role in cell signaling. Their release influences the behavior of surrounding cells. Cytokines act as immunomodulators in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in terminology). Cytokines are produced by a variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one type of cell. Cytokines function through receptors and are particularly important in the immune system; they regulate the balance between humoral and cellular immune responses, and they modulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the effects of other cytokines in complex ways.

[0176] IL2

[0177] Interleukin-2 (IL2) is a cytokine that induces the proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL2 is mediated by the multi-subunit IL2 receptor complex (IL2R) consisting of three transmembrane polypeptide subunits: p55 (IL2Rα, α subunit, also known as CD25 in humans), p75 (IL2Rβ, β subunit, also known as CD122 in humans), and p64 ​​(IL2Rγ, γ subunit, also known as CD132 in humans). The T cell response to IL2 depends on various factors, including: (1) the concentration of IL2; (2) the amount of IL2R molecules on the cell surface; and (3) the amount of IL2R occupied by IL2 (i.e., the affinity of the binding interaction between IL2 and IL2R (Smith, "Cell Growth Signal Transduction is Quantal" In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)). The IL2:IL2R complex is internalized after ligand binding, and different components are differentially sorted. When administered intravenously (iv) bolus, IL2 exhibits rapid systemic clearance (with an initial clearance phase half-life of 12.9 min, followed by a slower clearance phase with a half-life of 85 min) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0178] The outcomes of systemic IL2 administration in cancer patients are far from ideal. While 15-20% of patients objectively respond to high doses of IL2, the majority do not, and many experience severe, life-threatening side effects, including nausea, confusion, hypotension, and septic shock. The severe toxicities associated with high-dose IL2 treatment are primarily attributed to the activity of natural killer (NK) cells. Attempts have been made to reduce serum concentrations by decreasing the dose and adjusting the dosing regimen, with reduced toxicity, but these treatments have not been very effective.

[0179] According to this disclosure, in some embodiments, IL2 is linked to a pharmacokinetic modification group. The resulting molecule, hereinafter referred to as "prolonged pharmacokinetic (PK) IL2," has a prolonged circulating half-life relative to free IL2. The prolonged circulating half-life of prolonged PK IL2 allows serum IL2 concentrations in vivo to be maintained within the therapeutic range, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, prolonged PK IL2 can be administered at a lower frequency and for a longer duration compared to unmodified IL2.

[0180] According to this disclosure, IL2 (optionally as part of an extended PK IL2) can be naturally occurring IL2 or a fragment or variant thereof. IL2 can be human IL2 and can be derived from any vertebrate, especially any mammal. In one embodiment, IL2 comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. In one embodiment, IL2 or an IL2 fragment or variant binds to an IL2 receptor or a subunit of the IL2 receptor, such as the α subunit and / or β / γ subunit.

[0181] In some embodiments, the IL2 portion of the extended PK is human IL2. In other embodiments, the IL2 portion of the extended PK is a fragment or variant of human IL2.

[0182] In some embodiments described herein, IL2 is fused with a heterologous peptide (i.e., a peptide that is not IL2). The heterologous peptide can increase the circulating half-life of IL2. As discussed in further detail below, the peptide that increases the circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

[0183] IL7

[0184] IL-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. It is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but not by normal lymphocytes. IL-7 is an important cytokine for the development of B cells and T cells. IL-7 cytokines form heterodimers with hepatocyte growth factor, which function as a pre-progenitor B cell growth stimulator. Mouse gene knockout studies have shown that IL-7 plays a crucial role in the survival of lymphoid cells.

[0185] IL7 binds to the IL7 receptor, a heterodimer composed of the IL7 receptor α and a common γ chain receptor. Binding leads to a signaling cascade important for T cell development within the thymus and for peripheral survival. Knockout mice genetically lacking the IL7 receptor exhibit thymic atrophy, T cell development arrest at the double-positive stage, and severe lymphopenia. Administration of IL7 to mice results in an increase in recently emigrating thymic cells, an increase in B cells and T cells, and an increased recovery of T cells following cyclophosphamide administration or bone marrow transplantation.

[0186] According to this disclosure, in some embodiments, IL7 is linked to a pharmacokinetic modification group. The resulting molecule, hereinafter referred to as "prolonged pharmacokinetic (PK) IL7," has a prolonged circulating half-life relative to free IL7. The prolonged circulating half-life of PK IL7 allows serum IL7 concentrations in vivo to be maintained within the therapeutic range, potentially leading to enhanced survival of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, PK IL7 can be administered at a lower frequency and for a longer duration compared to unmodified IL7.

[0187] According to this disclosure, IL7 (optionally as part of an extended PK IL7) can be naturally occurring IL7 or a fragment or variant thereof. IL7 can be human IL7 and can be derived from any vertebrate, especially any mammal. In one embodiment, IL7 comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In one embodiment, IL7 or an IL7 fragment or variant binds to an IL7 receptor.

[0188] In some embodiments, the IL7 portion of the extended PK is human IL7. In other embodiments, the IL7 portion of the extended PK is a fragment or variant of human IL7.

[0189] In some embodiments described herein, IL7 is fused with a heterologous peptide (i.e., a peptide that is not IL7). The heterologous peptide can increase the circulating half-life of IL7. As discussed in further detail below, the peptide that increases the circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

[0190] IL21

[0191] Interleukin-21 (IL21) is a cytokine that effectively regulates cells of the immune system, including natural killer (NK) cells and cytotoxic T cells. This cytokine induces cell division / proliferation in its target cells. IL21 is expressed in activated human CD4+ T cells but not in most other tissues. Furthermore, IL21 expression is upregulated in the Th2 and Th17 subsets of T helper cells and in T follicular cells. Additionally, IL21 is expressed in NK T cells, regulating the function of these cells. Interleukin-21 is also produced by Hodgkin's lymphoma (HL) cancer cells.

[0192] The IL21 receptor (IL21R) is expressed on the surface of T, B, and NK cells. Structurally, IL21R is similar to the receptors of other type I cytokines (such as IL2 or IL-15) and requires dimerization with a common γ chain (γc) to bind IL-21. When bound to IL21, the IL21 receptor functions via the Jak / STAT pathway, utilizing Jak1 and Jak3, as well as the STAT3 homodimer, to activate its target genes.

[0193] According to this disclosure, in some embodiments, IL21 is linked to a pharmacokinetic modification group. The resulting molecule, hereinafter referred to as "extended pharmacokinetic (PK) IL21," has an extended circulating half-life relative to free IL21. The extended circulating half-life of PK IL21 allows serum IL21 concentrations to be maintained within the therapeutic range in vivo, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, PK IL21 can be administered at a lower frequency and for a longer duration compared to unmodified IL21.

[0194] According to this disclosure, IL21 (optionally as part of an extended PK IL21) can be naturally occurring IL21 or a fragment or variant thereof. IL21 can be human IL21 and can be derived from any vertebrate, especially any mammal. In one embodiment, IL21 comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In one embodiment, IL21 or an IL21 fragment or variant binds to an IL21 receptor.

[0195] In some embodiments, the IL21 portion of the extended PK is human IL21. In other embodiments, the IL21 portion of the extended PK is a fragment or variant of human IL21.

[0196] In some embodiments described herein, IL21 is fused with a heterologous peptide (i.e., a peptide that is not IL21). The heterologous peptide can increase the circulating half-life of IL21. As discussed in further detail below, the peptide that increases the circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

[0197] Extended PK group

[0198] Cytokines, such as interleukins described herein, such as IL2, IL7, or IL21, can be fused with a group that extends the PK, thereby increasing the circulating half-life. Non-limiting examples of groups that extend the PK are described below. It should be understood that other PK groups that increase the circulating half-life of cytokines or variants thereof are also applicable to this disclosure. In some embodiments, the group that extends the PK is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0199] As used herein, the term “PK” is an acronym for “pharmacokinetics” and encompasses properties of a compound, including, for example, absorption, distribution, metabolism, and elimination by a subject. As used herein, a “PK-prolonging group” refers to a protein, peptide, or moiety that, when fused with or administered co-with a biologically active molecule, increases the circulating half-life of that molecule. Examples of PK-prolonging groups include serum albumin (e.g., HSA), Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publications 2005 / 0287153 and 2007 / 0003549). Other exemplary PK-prolonging groups are disclosed in Kontermann et al., Current Opinion in Biotechnology 2011; 22:868-876, the entirety of which is incorporated herein by reference. As used herein, a “PK-prolonging cytokine” refers to a cytokine moiety combined with a PK-prolonging group. In one embodiment, the cytokine of extended PK is a fusion protein, wherein the cytokine portion is linked to or fused to a group of extended PK. As used herein, "IL of extended PK" refers to the interleukin (IL) portion combined with a group of extended PK. In one embodiment, the IL of extended PK is a fusion protein, wherein the IL portion is linked to or fused to a group of extended PK. Exemplary fusion proteins include the HSA / IL2 fusion protein, wherein the IL2 portion is fused to HSA. Another exemplary fusion protein is the HSA / IL7 fusion protein, wherein the IL7 portion is fused to HSA. Another exemplary fusion protein is the HSA / IL21 fusion protein, wherein the IL21 portion is fused to HSA.

[0200] In some embodiments, the serum half-life of the cytokine in the extended PK is increased relative to the cytokine alone (i.e., the cytokine is not fused with the group of the extended PK). In some embodiments, the serum half-life of the cytokine in the extended PK is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the cytokine alone. In some embodiments, the serum half-life of the cytokine in the extended PK is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40, or 50 times that of the cytokine alone. In some implementations, the serum half-life of the PK cytokines is extended by at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0201] In some embodiments, the groups extending the PK include serum albumin or fragments thereof, or variants of serum albumin or fragments thereof (all of which are included in the term "albumin" for the purposes of this disclosure). The polypeptides described herein may be fused with albumin (or fragments or variants thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

[0202] As used herein, "albumin fusion protein" refers to a protein formed by fusing at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of protein, such as a therapeutic protein, particularly IL2, IL7, or IL21 (or a fragment or variant thereof). Albumin fusion proteins can be produced by the translation of nucleic acids, wherein a polynucleotide encoding a therapeutic protein is in-frame linked to a polynucleotide encoding albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a "part," "region," or "fraction" of the albumin fusion protein (e.g., "therapeutic protein fraction" or "albumin fraction"). In a particularly preferred embodiment, the albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to, a mature form of a therapeutic protein) and at least one molecule of albumin (including, but not limited to, a mature form of albumin). In one embodiment, the albumin fusion protein is processed by host cells, such as cells of a target organ receiving RNA (e.g., hepatocytes), and secreted into circulation. Processing of newly formed albumin fusion proteins occurring in the secretory pathway of host cells used to express RNA can include, but is not limited to, signal peptide cleavage; disulfide bond formation; proper folding; addition and processing of carbohydrates (e.g., N- and O-linked glycosylation); specific proteolytic cleavage; and / or assembly into polyproteins. Albumin fusion proteins are preferably encoded by unprocessed RNA, particularly having a signal peptide at its N-terminus, and are preferably present in a processed form after cell secretion, wherein the signal peptide has been cleaved. In the most preferred embodiment, "processed form of albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as "mature albumin fusion protein."

[0203] In a preferred embodiment, the albumin fusion protein containing the therapeutic protein exhibits higher plasma stability compared to the plasma stability of the same therapeutic protein not fused with albumin. Plasma stability generally refers to the time interval between the administration of a therapeutic protein into the bloodstream and its degradation and clearance from the bloodstream to an organ (e.g., kidney or liver), ultimately being cleared from the body. Plasma stability is calculated based on the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined using commonly used assays known in the art.

[0204] As used herein, “albumin” generally refers to an albumin protein or amino acid sequence, or a fragment or variant of albumin, that possesses one or more functional activities (e.g., biological activities) of albumin. Specifically, “albumin” refers to human albumin or fragments or variants thereof, particularly the mature form of human albumin, or albumin or fragments thereof from other vertebrates, or variants of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, cattle, sheep, or pig. Non-mammalian albumins include, but are not limited to, chicken and salmon. The albumin portion of an albumin fusion protein may originate from a different animal than the therapeutic protein portion.

[0205] In some embodiments, albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed below: US 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO2011 / 0514789.

[0206] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably in this document. The terms “albumin” and “serum albumin” are broader and encompass human serum albumin (and its fragments and variants) as well as albumins (and their fragments and variants) from other species.

[0207] As used herein, an albumin fragment sufficient to prolong the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment that is long or structurally sufficient to stabilize or prolong the therapeutic activity or plasma stability of the protein, thereby prolonging or extending the plasma stability of the therapeutic protein portion of the albumin fusion protein compared to the plasma stability of the non-fusion state.

[0208] The albumin portion of an albumin fusion protein may comprise the full length of the albumin sequence, or it may comprise one or more fragments capable of stabilizing or prolonging therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids long, or may comprise about 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise specific domains of albumin, partially or entirely. For example, one or more HSA fragments may be used, spanning the first two immunoglobulin-like domains. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0209] Generally, albumin fragments or variants are at least 100 amino acids in length, preferably at least 150 amino acids.

[0210] According to this disclosure, albumin can be naturally occurring albumin or fragments or variants thereof. Albumin can be human albumin and can be derived from any vertebrate, especially any mammal. In one embodiment, albumin comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4.

[0211] Preferably, the albumin fusion protein comprises albumin as the N-terminal portion and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N-terminus and C-terminus are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminus and C-terminus are different therapeutic proteins. In one embodiment, the different therapeutic proteins may be used to treat or prevent the same or related diseases, symptoms, or disease conditions. In one embodiment, the different therapeutic proteins are all cytokines.

[0212] In one embodiment, the therapeutic protein is linked to albumin via a peptide linker. The linker peptide between the fusion moieties can provide greater physical separation between the moieties, thereby maximizing the accessibility of the therapeutic protein moieties, for example, for binding to their homologous receptors. The linker peptide can be composed of amino acids, making it flexible or more rigid. The linker sequence can be cleaved by proteases or chemicals.

[0213] As used herein, the term "Fc region" refers to a portion of a native immunoglobulin formed by the corresponding Fc domains (or Fc portions) of its two heavy chains. The term "Fc domain" as used herein refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not contain an Fv domain. In some embodiments, the Fc domain begins in a hinge region immediately upstream of the papain cleavage site and terminates at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the Fc domain comprises at least one of the following: a hinge (e.g., an upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In some embodiments, the Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc domain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In some embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). The term Fc domain herein generally refers to a polypeptide containing all or part of the Fc domain of the immunoglobulin heavy chain. This includes, but is not limited to, polypeptides containing the entire CH1, hinge, CH2, and / or CH3 domains, and fragments of such peptides containing only, for example, the hinge, CH2, and CH3 domains. The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses both native Fc and Fc variant molecules. As described herein, those skilled in the art will understand that any Fc domain can be modified such that its amino acid sequence differs from the native Fc domain of a naturally occurring immunoglobulin molecule. In some embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0214] The Fc domain of the peptide described herein can be derived from different immunoglobulin molecules. For example, the Fc domain of the peptide may include a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In yet another example, the Fc domain may include a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0215] In some embodiments, the group extending the PK includes an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are included in the term "Fc domain" for the purposes of this disclosure). The Fc domain does not contain a variable region that binds to the antigen. The Fc domains suitable for use in this disclosure can be obtained from several different sources. In some embodiments, the Fc domain is derived from human immunoglobulins. In some embodiments, the Fc domain is derived from the constant region of human IgG1. However, it should be understood that the Fc domain may be derived from immunoglobulins of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques).

[0216] Furthermore, the Fc domain (or fragments or variants thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0217] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in publicly available deposits. Constant region domains containing Fc domain sequences can be selected that lack specific effector functions and / or have specific modifications that reduce immunogenicity. Sequences of many antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be obtained from these sequences using techniques recognized in the art.

[0218] In some embodiments, the group extending the PK is a serum albumin-binding protein, such as those disclosed in US2005 / 0287153, US2007 / 0003549, US2007 / 0178082, US2007 / 0269422, US2010 / 0113339, WO2009 / 083804, and WO2009 / 133208, all of which are incorporated herein by reference. In some embodiments, the group extending the PK is transferrin, such as those disclosed in US 7,176,278 and US 8,158,579 (all of which are incorporated herein by reference). In some embodiments, the group extending the PK is a serum immunoglobulin-binding protein, such as those disclosed in US2007 / 0178082 (all of which are incorporated herein by reference). In some embodiments, the group extending the PK is a fibronectin (Fn) scaffold domain protein that binds serum albumin, such as those disclosed in US2012 / 0094909 (which are incorporated herein by reference in their entirety). US2012 / 0094909 also discloses methods for preparing fibronectin-based scaffold domain proteins. A non-limiting example of a PK extension group based on Fn3 is Fn3(HSA), i.e., the Fn3 protein that binds human serum albumin.

[0219] In some aspects, cytokines suitable for use according to this disclosure, such as extended PK ILs, may utilize one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that links two or more domains (e.g., the extended PK portion and the IL portion, such as IL2, IL7, or IL21) within a linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to link the IL2 portion to the HSA domain. In another embodiment, a peptide linker may be used to link the IL7 portion to the HSA domain. In yet another embodiment, a peptide linker may be used to link the IL21 portion to the HSA domain.

[0220] It is well known in the art that suitable linkers for fusing groups that extend the PK to, for example, IL2, IL7, or IL21. Exemplary linkers include glycine-serine peptide linkers, glycine-proline peptide linkers, and proline-alanine peptide linkers. In some embodiments, the linker is a glycine-serine peptide linker, i.e., a peptide composed of glycine and serine residues.

[0221] antigen

[0222] The peptide and protein antigens applicable to this disclosure, i.e., antigens or variants thereof, generally comprise peptides or proteins containing epitopes for inducing an immune response. The peptide or protein or epitope may be derived from a target antigen, i.e., an antigen against which an immune response is to be elicited. For example, the peptide or protein antigen or the epitope contained in the peptide or protein antigen may be a target antigen or a fragment or variant of a target antigen.

[0223] The administered or nucleic acid-encoded peptide and protein antigens, particularly the administered RNA, i.e., vaccine antigens, preferably cause stimulation, initiation, and / or expansion of T cells genetically modified to express CAR in the subject receiving the administered peptide or protein antigen or nucleic acid. The stimulated, initiated, and / or expanded T cells are preferably targeted at a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., disease-related antigens. Therefore, vaccine antigens may contain disease-related antigens, or fragments or variants thereof. In one embodiment, such fragments or variants are immunologically equivalent to disease-related antigens. In the context of this disclosure, the terms "fragment of antigen" or "variant of antigen" refer to substances that cause stimulation, initiation, and / or expansion of CAR-engineered T cells, and the stimulated, initiated, and / or expanded T cells target antigens, i.e., disease-related antigens, particularly when presented by diseased cells, tissues, and / or organs. Therefore, vaccine antigens may correspond to or may contain disease-related antigens, may correspond to or may contain fragments of disease-related antigens, or may correspond to or may contain antigens homologous to disease-related antigens or fragments thereof. If a vaccine antigen comprises a fragment of a disease-related antigen or an amino acid sequence homologous to a fragment of a disease-related antigen, then said fragment or amino acid sequence may contain an epitope of the disease-related antigen (the epitope targeted by the CAR-engineered T cell), or contain a sequence homologous to the epitope of the disease-related antigen. Therefore, according to this disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-related antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-related antigen. The “immunogenic fragment of antigen” in this disclosure preferably relates to an antigen fragment capable of stimulating, initiating, and / or amplifying T cells carrying a CAR that binds to the antigen or a cell expressing the antigen. Preferably, the vaccine antigen (similar to a disease-related antigen) may be expressed on the surface of cells (e.g., antigen-presenting cells) to provide an associated epitope that binds to the CAR-engineered T cell. The vaccine antigen may be a recombinant antigen.

[0224] The term "immunologic equivalence" refers to immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, that exhibit the same or substantially the same immunological properties and / or perform the same or substantially the same immunological effects, for example, in terms of the type of immunological effect. In the context of this disclosure, the term "immunologic equivalence" is preferably used for the immunological effects or properties of antigens or antigen variants used for immunization. For example, if an amino acid sequence induces an immune response with specificity to a reference amino acid sequence upon exposure to the immune system of a subject, such as T cells that bind to a reference amino acid sequence or cells expressing a reference amino acid sequence, then said amino acid sequence is immunologically equivalent to the reference amino acid sequence. Therefore, molecules that are immunologically equivalent to antigens exhibit the same or substantially the same properties and / or perform the same or substantially the same effects as the antigens targeted by T cells, in terms of T cell stimulation, initiation, and / or amplification.

[0225] The term "initiation" refers to the process by which a T cell first comes into contact with its specific antigen, leading to its differentiation into an effector T cell.

[0226] The term "clonal expansion" or "expansion" refers to the process in which a specific entity multiplies. In the context of this disclosure, the term is preferably used in the context of an immune response, in which lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes that recognize said antigen are expanded. Preferably, clonal expansion leads to lymphocyte differentiation.

[0227] The term "antigen" refers to a substance containing an epitope against which an immune response can be elicited. The term "antigen" particularly includes proteins and peptides. In one embodiment, the antigen is present on the surface of immune system cells, such as antigen-presenting cells, like dendritic cells or macrophages. In one embodiment, the antigen or a processed product thereof (such as a T-cell epitope) is bound to a CAR molecule. Therefore, the antigen or its processed product can specifically react with T lymphocytes (T cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, and the epitope is derived from such an antigen.

[0228] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a disease-specific cellular antigen-based immune response and / or a humoral antibody response. Therefore, disease-associated antigens or their epitopes can be used for therapeutic purposes. Disease-associated antigens may be related to microbial infections, typically microbial antigens, or to cancer, typically tumors.

[0229] The term "tumor antigen" refers to components of cancer cells, which can originate from the cytoplasm, cell surface, and nucleus. Specifically, it refers to antigens produced intracellularly or acting as tumor cell surface antigens. Tumor antigens are typically preferentially expressed by cancer cells (e.g., their expression levels are higher in cancer cells than in non-cancer cells), and in some cases, they are expressed only by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, and Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the tight junction protein family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, and MAGE-A1. 1 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 small BCR-abL, Pml / RARa, PRAME, Protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT and WT-1.

[0230] The term "viral antigen" refers to any viral component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0231] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Bacterial antigens can originate from the bacterial cell wall or cytoplasmic membrane.

[0232] The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be about 5 to about 100 amino acids in length, for example, about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids. For example, the length of an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T-cell epitopes.

[0233] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the case of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and refer to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present T cells with self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms). In the case of class I MHC / peptide complexes, the length of the bound peptide is typically about 8 to about 10 amino acids, but longer or shorter peptides can be effective. In the case of class II MHC / peptide complexes, the length of the bound peptide is typically about 10 to about 25 amino acids, particularly about 13 to about 18 amino acids, but longer and shorter peptides can be effective.

[0234] In one embodiment, the target antigen is a tumor antigen, and the vaccine antigen or a fragment thereof (e.g., an epitope) is derived from the tumor antigen. The tumor antigen can be a "standard" antigen, which is generally known to be expressed in various cancers. The tumor antigen can also be a "neoantigen," which is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may be caused by one or more cancer-specific mutations in the genome of a cancer cell, resulting in amino acid changes. If the tumor antigen is a neoantigen, the vaccine antigen preferably contains an epitope or fragment of the neoantigen, which contains one or more amino acid changes.

[0235] The length of peptides and protein antigens can range from 2 to 100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptide can be longer than 50 amino acids. In some embodiments, the peptide can be longer than 100 amino acids.

[0236] According to the present invention, the antigen or a variant thereof should be recognizable by a CAR. Preferably, if recognized by a CAR, the antigen or a variant thereof is capable of inducing stimulation, priming, and / or expansion of T cells carrying a CAR that recognizes the antigen or a variant thereof in the presence of a suitable co-stimulatory signal. In the context of embodiments of the present invention, the antigen or a variant thereof is preferably present on the cell surface, preferably on the surface of antigen-presenting cells. Recognition of an antigen on the surface of diseased cells can lead to an immune response against the antigen (or cells expressing the antigen).

[0237] According to various aspects of the invention, the preferred objective is to provide an immune response against cancer cells expressing tumor antigens (e.g., CLDN6 or CLDN18.2) and to treat cancerous diseases involving cells expressing tumor antigens (e.g., CLDN6 or CLDN18). Preferably, the invention relates to the administration of CAR-engineered T cells that target cancer cells expressing tumor antigens (e.g., CLDN6 or CLDN18.2).

[0238] "Cell surface" is used according to its common meaning in the art and therefore includes the extracellular space that is readily bound by proteins and other molecules. An antigen is expressed on the cell surface if it is located on the cell surface and can be readily bound by, for example, an antigen-specific antibody added to the cell. In one embodiment, the antigen expressed on the cell surface is an integrated membrane protein having an extracellular portion that is recognized by the CAR.

[0239] In the context of this invention, the term "extracellular portion" or "extracellular domain" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of the cell and is preferably accessible from the outside of the cell, for example, by a binding molecule located outside the cell, such as an antibody. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0240] In one embodiment of all aspects of the invention, the antigen is expressed in diseased cells (e.g., cancer cells). In one embodiment, the antigen is expressed on the surface of diseased cells (e.g., cancer cells). In one embodiment, the CAR binds to the extracellular domain of the antigen or a variant thereof, or an epitope within the extracellular domain. In one embodiment, the CAR binds to a natural epitope of the antigen or a variant thereof present on the surface of a living cell. In some embodiments, the antigen is a tight junction protein, particularly tight junction protein 6 or tight junction protein 18.2, and the CAR binds to a first extracellular loop of the tight junction protein. In one embodiment, when expressed by and / or present on T cells, the binding of the CAR to the antigen or a variant thereof present on cells, such as antigen-presenting cells, leads to stimulation, initiation, and / or expansion of the T cells. In one embodiment, when expressed by and / or present on T cells, the binding of the CAR to the antigen present on diseased cells (e.g., cancer cells) leads to cell lysis and / or apoptosis of the diseased cells, wherein the T cells preferably release cytotoxic factors, such as perforin and granzymes.

[0241] Immune checkpoint inhibitors

[0242] In some implementations, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein.

[0243] As used herein, an "immune checkpoint" refers to a co-stimulatory and inhibitory signal that regulates the amplitude and quality of T-cell receptor recognition of antigens. In some embodiments, the immune checkpoint is an inhibitory signal. In some embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In some embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 instead of CD28 binding. In some embodiments, the inhibitory signal is the interaction between LAG3 and MHC class II molecules. In some embodiments, the inhibitory signal is the interaction between TIM3 and galactagoguein 9.

[0244] As used herein, an "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. In some embodiments, an immune checkpoint inhibitor blocks inhibitory signals associated with an immune checkpoint. In some embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that disrupts inhibitory signaling associated with an immune checkpoint. In some embodiments, an immune checkpoint inhibitor is a small molecule that disrupts inhibitory signaling. In some embodiments, an immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic that prevents interactions between checkpoint blocking proteins, such as an antibody, fragment thereof, or antibody mimic that prevents the interaction between PD-1 and PD-L1. In some embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that prevents the interaction between CTLA-4 and CD80 or CD86. In some embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that prevents the interaction between LAG3 and its ligand or TIM-3 and its ligand. Checkpoint inhibitors may also be in a soluble form of the molecule (or a variant thereof), such as soluble PD-L1 or a PD-L1 fusion protein.

[0245] The programmed death-1 (PD-1) receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isotypes, and species homologs of hPD-1, as well as analogs that share at least one common epitope with hPD-1.

[0246] Programmed death-ligand 1 (PD-L1) is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2). Upon binding to PD-1, PD-L1 downregulates T cell activation and cytokine secretion. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, as well as analogs sharing at least one common epitope with hPD-L1.

[0247] "Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)" is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 and CD86. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one common epitope with hCTLA-4.

[0248] Lymphocyte activation gene-3 (LAG3) is an inhibitory receptor that is associated with the suppression of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits the function of CD8+ effector T cells. As used herein, the term "LAG3" includes human LAG3 (hLAG3), variants, subtypes, and species homologs of hLAG3, as well as analogs sharing at least one common epitope.

[0249] T-cell membrane protein-3 (TIM3) is an inhibitory receptor that participates in suppressing lymphocyte activity by inhibiting the TH1 cell response. Its ligand is galactagogue 9, which is upregulated in various types of cancer. As used herein, the term "TIM3" includes human TIM3 (hTIM3), variants, subtypes, and species homologs of hTIM3, as well as analogs sharing at least one common epitope.

[0250] The "B7 family" refers to inhibitory ligands with undefined receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells.

[0251] In some embodiments, the immune checkpoint inhibitors suitable for the methods disclosed herein are antagonists of the inhibitory signal, such as antibodies targeting PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM3. Pardoll, D., Nature. 12:252-264, 2012 reviewed these ligands and receptors.

[0252] In some embodiments, the immune checkpoint inhibitor is an antibody or its antigen-binding moiety that disrupts or inhibits signal transduction from an inhibitory immunomodulator. In some embodiments, the immune checkpoint inhibitor is a small molecule that disrupts or inhibits signal transduction from an inhibitory immunomodulator.

[0253] In some embodiments, the inhibitory immunomodulator is a component of the PD-1 / PD-L1 signaling pathway. Therefore, certain embodiments of this disclosure provide administration to a subject of an antibody or its antigen-binding portion that disrupts the interaction between the PD-1 receptor and its ligand PD-L1. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1 are known in the art. In some embodiments, the antibody or its antigen-binding portion specifically binds to PD-1. In some embodiments, the antibody or its antigen-binding portion specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity.

[0254] In some embodiments, an immunomodulatory agent is a component of the CTLA4 signaling pathway. Therefore, certain embodiments of this disclosure provide administration to a subject of an antibody or its antigen-binding moiety that targets CTLA4 and disrupts its interaction with CD80 and CD86.

[0255] In some embodiments, the immunomodulatory agent is a component of the LAG3 (lymphocyte activation gene 3) signaling pathway. Therefore, certain embodiments of this disclosure provide administration to a subject of an antibody or its antigen-binding moiety that targets LAG3 and disrupts its interaction with MHC class II molecules.

[0256] In some embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In some embodiments, the B7 family members are B7-H3 and B7-H4. Therefore, some embodiments of this disclosure provide administration to a subject of an antibody or antigen-binding moiety targeting B7-H3 or H4. The B7 family does not have any defined receptors, but these ligands are upregulated on tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity.

[0257] In some embodiments, the immunomodulatory agent is a component of the TIM3 (T cell membrane protein 3) signaling pathway. Therefore, certain embodiments of this disclosure provide administration to a subject of an antibody or its antigen-binding moiety that targets TIM3 and disrupts its interaction with galactagogue 9.

[0258] Those skilled in the art will understand that other immune checkpoint targets can also be targeted by antagonists or antibodies, as long as the target stimulates an immune response, such as an anti-tumor immune response, as reflected in, for example, increased T cell proliferation, enhanced T cell activation and / or increased production of cytokines (e.g., IFN-γ, IL2).

[0259] According to this disclosure, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain comprises a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.

[0260] Antibodies can be derived from different species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0261] The antibodies described herein include IgA (e.g., IgA1 or IgA2), IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more specifically, an IgG1,κ, or IgG1,λ isotype (i.e., IgG1,κ,λ), an IgG2a antibody (e.g., IgG2a,κ,λ), an IgG2b antibody (e.g., IgG2b,κ,λ), an IgG3 antibody (e.g., IgG3,κ,λ), or an IgG4 antibody (e.g., IgG4,κ,λ).

[0262] The terms "antigen-binding portion" (or simply "binding portion") or "antigen-binding fragment" (or simply "binding fragment") of an antibody, or similar terms, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments covered in the term "antigen-binding moiety" of antibodies include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds through hinge regions; (iii) Fd fragments, consisting of VH and CH domains; (iv) Fv fragments, consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), consisting of VH domains; (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via synthetic linkers using recombination methods, allowing them to be prepared as single protein chains in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be covered within the term "antigen-binding fragment" in the antibody terminology. Further examples include binding domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a variable region of the heavy chain or a variable region of the light chain. Domain-binding immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments were obtained using conventional techniques known to those skilled in the art, and the utility of the fragments was screened in the same manner as for intact antibodies.

[0263] RNA targeting

[0264] According to this disclosure, following administration of the RNA described herein, at least a portion of the RNA is delivered to target cells. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is translated by the target cells to produce an encoded peptide or protein.

[0265] Some aspects of this disclosure relate to the targeted delivery of RNAs disclosed herein (e.g., RNA encoding cytokines and RNA encoding antigens or variants thereof).

[0266] In one embodiment, this disclosure relates to targeting the lymphatic system, particularly secondary lymphatic organs, and more particularly the spleen. Targeting the lymphatic system, particularly secondary lymphatic organs, and more particularly the spleen, is especially preferred if the administered RNA is an RNA encoding an antigen or a variant thereof.

[0267] In one embodiment, the target cells are spleen cells. In another embodiment, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In yet another embodiment, the target cells are dendritic cells in the spleen.

[0268] The lymphatic system is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a lymphatic conduction network, and circulating lymph. Primary or central lymphatic organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphatic organs. Secondary or peripheral lymphatic organs, including lymph nodes and the spleen, maintain mature immature lymphocytes and initiate adaptive immune responses.

[0269] RNA can be delivered to the spleen via a so-called lipoplex formulation, wherein RNA is bound to a liposome comprising cationic lipids and optionally additional or auxiliary lipids to form an injectable nanoparticle formulation. The liposomes can be obtained by injecting an ethanolic solution of lipids into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeting RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Therefore, RNA accumulation and / or RNA expression occur in the spleen following administration of the RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver following administration of the RNA lipoplex particles. In one embodiment, following administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occur in antigen-presenting cells, such as specialized antigen-presenting cells in the spleen. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0270] In the context of this disclosure, the term "RNA lipoplex particle" refers to a particle containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to the recombination and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can typically be synthesized using cationic lipids (e.g., DOTMA) and additional lipids (e.g., DOPE). In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0271] As used herein, “cationic lipid” refers to lipids with a net positive charge. Cationic lipids bind negatively charged RNA to a lipid matrix through electrostatic interactions. Typically, cationic lipids have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and the lipid head is usually positively charged. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethylbis(octadecylammonium) (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane, bis(octadecyldimethylammonium chloride) (DODAC), and 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylazonium (2,3-di( The following lipids are preferred: tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propane (DOSPA). DOTMA, DOTAP, DODAC, and DOSPA are preferred. In specific embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0272] Additional lipids can be added to adjust the overall charge-to-weight ratio and physical stability of the RNA lipoplex particles. In some embodiments, the additional lipids are neutral lipids. As used herein, "neutral lipid" refers to a lipid with a net charge of zero. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, and cerebrosides. In specific embodiments, the additional lipids are DOPE, cholesterol, and / or DOPC.

[0273] In some embodiments, the RNA lipoplex particles comprise cationic lipids and additional lipids. In an exemplary embodiment, the cationic lipid is DOTMA, and the additional lipid is DOPE.

[0274] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0275] In one embodiment, the average diameter of the RNA lipoplex particles described herein is about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific implementations, the average diameter of the RNA lipoplex particles is approximately 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, 900 nm, 925 nm, 950 nm, 975 nm, or 1000 nm. In one implementation, the average diameter of the RNA lipoplex particles is approximately 250 nm to approximately 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0276] The charge of the RNA lipoplex particles disclosed herein is the sum of the charge present in at least one cationic lipid and the charge present in RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA is calculated by the following equation: Charge ratio = [(Cationic lipid concentration (mol)) * (Total positive charge in cationic lipid)] / [(RNA concentration (mol)) * (Total negative charge in RNA)].

[0277] At physiological pH, the spleen-targeting RNA lipoplex particles described herein preferably have a net negative charge, for example, a positive to negative charge ratio of about 1.9:2 to about 1:2. In specific embodiments, at physiological pH, the positive to negative charge ratio in the RNA lipoplex particles is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0278] Cytokines, such as cytokines that prolong PK, particularly interleukins that prolong PK, such as those described herein, can be delivered to a subject's target organ or tissue, including administering RNA encoding the cytokine to the subject in a formulation for preferential delivery of the RNA to said target organ or tissue.

[0279] In one embodiment, the target organ is the lymphatic system, particularly secondary lymphatic organs, more specifically the spleen, and the target tissue is tissue of the lymphatic system, particularly tissue of secondary lymphatic organs, more specifically spleen tissue. Delivery of cytokines to such target tissues is preferred, especially if the presence of cytokines in the organ or tissue is desired (e.g., for inducing an immune response, particularly during T cell initiation or for activating resident immune cells), while systemic presence of the cytokines, especially in large quantities, is not desired (e.g., because cytokines have systemic toxicity). Particularly preferred examples of suitable cytokines include those involved in T cell initiation.

[0280] In another embodiment of delivering cytokines to a subject's target organ or target tissue, the target organ is the liver and the target tissue is liver tissue. Delivery of cytokines to such a target tissue is preferred, particularly if the presence of cytokines in this organ or tissue is desired, and / or if the expression of a large amount of cytokines is desired, and / or if the systemic presence of cytokines, especially a large amount, is desired or required.

[0281] In one embodiment, the RNA encoding the cytokine is administered in a formulation for targeting the liver. Such formulations are described herein. Examples of suitable cytokines include IL2, IL7, or IL21, fragments and variants thereof, and fusion proteins of these cytokines, fragments, and variants, such as cytokines that prolong PK, as described herein. Particularly preferred examples of suitable cytokines are those involved in T cell proliferation and / or maintenance.

[0282] RNA delivery systems have an inherent preference for the liver. This involves lipid-based particles, cationic and neutral nanoparticles, particularly lipid nanoparticles such as liposomes, nanomicelles, and lipophilic ligands in bioconjugates. Hepatic accumulation is caused by the discontinuous nature of the hepatic vascular system or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0283] To deliver RNA to the liver in vivo, drug delivery systems can be used to transport it there by preventing RNA degradation. For example, polyplex nanomicelles, consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core, are a useful system because nanomicelles provide excellent in vivo stability for RNA under physiological conditions. Furthermore, the cloaking properties provided by the surface of polyplex nanomicelles containing dense PEG palisades effectively evade host immune defenses.

[0284] Pharmaceutical Composition

[0285] The substances described herein can be administered as pharmaceutical compositions or drugs, and can be administered in any suitable pharmaceutical composition.

[0286] In one embodiment of all aspects of the invention, the components described herein, such as genetically modified T cells to express CARs, nucleic acids encoding cytokines, or nucleic acids encoding antigens or variants thereof, may be administered together or separately in a pharmaceutical composition, said pharmaceutical composition may contain a pharmaceutically acceptable carrier and may optionally contain one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is used for therapeutic or prophylactic treatment, for example, for treating or preventing diseases involving antigens, such as cancer, as those described herein.

[0287] The term "pharmaceutical composition" refers to a formulation comprising a therapeutically effective substance, preferably comprising a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is intended to treat, prevent, or reduce the severity of a disease or condition by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also referred to in the art as pharmaceutical preparations.

[0288] The pharmaceutical compositions disclosed herein preferably comprise one or more adjuvants, or may be administered in combination with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. Chemokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, and LT-α. Other known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils, such as... ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides, such as Pam3Cys.

[0289] The pharmaceutical compositions disclosed herein are generally used in "pharmaceuticalally effective amounts" and "pharmaceutically acceptable formulations".

[0290] The term "pharmaceutical acceptable" refers to the non-toxicity of materials that do not interact with the active ingredient in a pharmaceutical composition.

[0291] The term "pharmaceuticalally effective amount" or "therapeutically effective amount" refers to the amount, alone or in combination with other doses, that achieves the desired response or desired effect. In the case of treating a specific disease, the desired response preferably involves the inhibition of disease progression. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay of the onset of the disease or disease condition or the prevention of the onset of the disease or disease condition. The effective amount of the composition described herein will depend on the disease condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physical condition, size and weight, duration of treatment, type of concurrent treatment (if present), specific route of administration, and similar factors. Therefore, the dosage of the composition described herein may depend on a variety of such parameters. In cases where the initial dose is insufficient to elicit a response in the patient, a higher dose may be used (or a higher dose that is effective can be achieved through a different, more localized route of administration).

[0292] The pharmaceutical compositions disclosed herein may comprise salts, buffers, preservatives, and other optional therapeutic agents. In one embodiment, the pharmaceutical compositions disclosed herein comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0293] Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, p-hydroxybenzoate and thimerosal.

[0294] As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0295] The term "diluting agent" refers to a diluting agent and / or a thinning agent. Furthermore, the term "diluting agent" includes any one or more fluid, liquid, or solid suspensions and / or mixtures. Examples of suitable diluting agents include ethanol, glycerol, and water.

[0296] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, in which an active component is bound to promote, enhance, or enable the administration of a pharmaceutical composition. The carriers used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalene, and especially biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of this disclosure comprises isotonic saline.

[0297] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. Rgennaro edit. 1985).

[0298] Drug carriers, excipients, or diluents can be selected based on the intended route of administration and standard pharmaceutical practices.

[0299] In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local or systemic administration. Systemic administration may include enteral administration, which involves absorption via the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” means administration by any means other than via the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is performed via intravenous administration.

[0300] In one embodiment of all aspects of the invention, a nucleic acid encoding a cytokine or an antigen or a variant thereof is administered systemically. In one embodiment of all aspects of the invention, following systemic administration of a nucleic acid encoding an antigen or a variant thereof, the antigen or variant thereof is expressed in the spleen. In one embodiment of all aspects of the invention, following systemic administration of a nucleic acid encoding an antigen or a variant thereof, the antigen or variant thereof is expressed in antigen-presenting cells, preferably specialized antigen-presenting cells. In one embodiment, the antigen-presenting cells are selected from dendritic cells, macrophages, and B cells. In one embodiment of all aspects of the invention, following systemic administration of a nucleic acid encoding an antigen or a variant thereof, there is no or substantially no expression of the antigen or variant thereof in the lungs and / or liver. In one embodiment of all aspects of the invention, following systemic administration of a nucleic acid encoding an antigen or a variant thereof, the expression level of the antigen or variant thereof in the spleen is at least 5 times that in the lungs.

[0301] As used herein, the term "co-administration" refers to the process of administering different compounds or compositions (e.g., RNA encoding interleukins and RNA encoding antigens or variants thereof) simultaneously, substantially simultaneously, or sequentially to the same patient. If co-administered, the different compounds or compositions do not need to be administered in the same composition.

[0302] treat

[0303] The substances, compositions, and methods described herein can be used to treat subjects suffering from diseases, such as those characterized by the presence of diseased cells expressing antigens. Particularly preferred diseases include cancer. For example, if the antigen is derived from a virus, the substances, compositions, and methods can be used to treat viral diseases caused by said virus. If the antigen is a tumor antigen, the substances, compositions, and methods can be used to treat cancerous diseases in which cancer cells express said tumor antigens.

[0304] In one embodiment, this disclosure relates to a method for inducing an immune response in a subject. In an exemplary embodiment, the immune response is directed against cancer.

[0305] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is generally interpreted as a medical condition associated with specific symptoms and signs. Disease may originate from exogenous factors, such as infectious diseases, or from internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in a distressed individual, or to any condition that causes similar problems in individuals in contact with that individual. In this broader sense, it sometimes includes injury, disability, impairment, syndrome, infection, isolation symptoms, abnormal behavior, and atypical changes in structure and function, while in other cases and for other purposes, these can be considered distinct categories. Disease often affects not only an individual's body but also their emotions, as infection and living with many diseases can alter a person's perspective on life and their personality.

[0306] In this context, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject for the purpose of combating a disease condition such as a disease or symptom. The term is intended to include a full spectrum of treatment for a given condition suffered by the subject, such as administering a therapeutically effective compound to reduce symptoms or complications, delay the progression of the disease, symptom, or condition, reduce or alleviate symptoms and complications, and / or cure or eliminate the disease, symptom, or condition, as well as prevention of the disease condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, disease condition, or symptom, and includes administering an active compound to prevent the onset of symptoms or complications.

[0307] The term "therapeutic treatment" refers to any treatment that improves health status and / or prolongs (increases) an individual's lifespan. Such treatment may eliminate an individual's disease, stop or slow the progression of an individual's disease, inhibit or slow the progression of an individual's disease, reduce the frequency or severity of an individual's symptoms, and / or reduce recurrence in individuals who currently or previously had the disease.

[0308] The term "preventive treatment" or "preventive therapy" refers to any treatment designed to prevent an individual from developing a disease. The terms "preventive treatment" or "preventive therapy" are used interchangeably in this document.

[0309] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (such as a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) who may have or is susceptible to a disease or condition (e.g., cancer), but may or may not have the disease or condition. In many embodiments, the individual is a person. Unless otherwise stated, the terms “individual” and “subject” do not indicate a specific age and therefore cover adults, the elderly, children, and newborns. In embodiments of this disclosure, “individual” or “subject” refers to a “patient.”

[0310] The term "patient" refers to an individual or subject undergoing treatment, particularly an individual or subject who is ill.

[0311] In one embodiment of this disclosure, the aim is to provide an immune response against diseased cells expressing antigens (such as cancer cells expressing tumor antigens) and to treat diseases involving cells expressing antigens (such as cancer).

[0312] It can elicit an immune response against the antigen, which can be therapeutic or partially or completely protective. The pharmaceutical compositions described herein are suitable for inducing or enhancing an immune response. Therefore, the pharmaceutical compositions described herein can be used for the prophylactic and / or therapeutic treatment of diseases involving antigens.

[0313] As used herein, "immune response" refers to the body's overall response to an antigen or cells expressing that antigen, and includes cellular and / or humoral immune responses. Cellular immune responses include, but are not limited to, cellular responses against cells expressing antigens. Such cells may be characterized by expressing antigens on their cell surface or by presenting antigens via class I or II MHC molecules. Cellular responses are associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+ T cells), and play a central role by modulating immune responses or killing cells (also known as cytotoxic T cells, CD8+ T cells, or CTLs) to induce apoptosis of infected cells or cancer cells. In one embodiment, administration of the pharmaceutical composition disclosed herein involves stimulating an antitumor CD8+ T cell response against cancer cells expressing one or more tumor antigens.

[0314] This disclosure is intended to consider protective, prophylactic, and / or therapeutic immune responses. As used herein, “inducing an immune response” can mean either the absence of an immune response against a specific antigen prior to induction, or the presence of a basal level of immune response against a specific antigen prior to induction, which is enhanced after induction. Therefore, “inducing an immune response” includes “enhancing an immune response.”

[0315] The term "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response.

[0316] The terms “vaccination” or “immunization” describe the process of administering an antigen to an individual with the aim of inducing an immune response, for example, for therapeutic or preventative purposes.

[0317] In one embodiment, this disclosure envisions an implementation in which an RNA preparation, such as the RNA particles described herein, is administered.

[0318] Therefore, this disclosure relates to RNA as described herein, which is used for preventive and / or therapeutic treatment of diseases involving antigens, preferably cancerous diseases.

[0319] The term "macrophage" refers to a subset of phagocytes differentiated from monocytes. Macrophages, activated by inflammation, immune cytokines, or microbial products, nonspecifically engulf and kill foreign pathogens within their cells through hydrolytic and oxidative attacks, leading to pathogen degradation. The peptides of the degraded proteins are displayed on the macrophage surface, where they can be recognized by T cells and can interact directly with antibodies on the surface of B cells, resulting in T and B cell activation and further stimulating the immune response. Macrophages belong to the antigen-presenting cell category. In one embodiment, the macrophages are splenic macrophages.

[0320] The term "dendritic cell" (DC) refers to another subtype of phagocytic cell belonging to the antigen-presenting cell category. In one embodiment, dendritic cells originate from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells continuously sample the surrounding environment, searching for pathogens such as viruses and bacteria. Once they come into contact with presentable antigens, they are activated into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small fragments, presenting these fragments on their cell surface using MHC molecules after maturation. Simultaneously, they upregulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemokine receptor that induces dendritic cells to reach the spleen via the bloodstream or to reach lymph nodes via the lymphatic system. Here, they act as antigen-presenting cells, activating helper T cells, cytotoxic T cells, and B cells by presenting antigens and non-antigen-specific co-stimulatory signals. Therefore, dendritic cells can actively induce immune responses associated with T cells or B cells. In one embodiment, the dendritic cells are splenic dendritic cells.

[0321] The term "antigen-presenting cell" (APC) refers to a cell of various types capable of displaying, acquiring, and / or presenting at least one antigen or antigen fragment on its cell surface (or at the cell surface). Antigen-presenting cells can be classified into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0322] The term "professional antigen-presenting cell" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules required for interaction with naive T cells. When a T cell interacts with an MHC class II molecule complex on its membrane, the antigen-presenting cell produces a co-stimulatory molecule that induces T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0323] The term "nonprofessional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do express them upon stimulation by certain cytokines (such as interferon-γ). Exemplary nonprofessional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells, or vascular endothelial cells.

[0324] "Antigen processing" refers to the degradation of an antigen into processed products, which are fragments of the antigen (e.g., protein degradation into peptides) and one or more of these fragments (e.g., through binding) interacting with the MHC for presentation by cells, such as cells that present antigens to specific T cells.

[0325] The terms "disease involving an antigen," "disease involving cells expressing an antigen," or similar terms refer to any disease involving an antigen, for example, a disease characterized by the presence of an antigen. The disease can be an infectious disease or a cancerous disease, or simply cancer. As mentioned above, the antigen can be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. Preferably, the disease involving the antigen is a disease involving cells expressing the antigen, preferably on the cell surface.

[0326] The term "infectious disease" refers to any illness (e.g., the common cold) that can be transmitted between individuals or between organisms and is caused by microbial material. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / Acquired Immunodeficiency Syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory disease syndrome (SARS), avian influenza, and influenza.

[0327] The term "cancer disease" or "cancer" refers to or describes an individual's physiological condition, typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, blood cancers, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancers of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal carcinoma, spinal cord axis tumors, gliomas, meningiomas, and pituitary adenomas. The term "cancer" according to this disclosure also includes cancer metastasis.

[0328] Due to the resulting synergistic effects, combination strategies in cancer treatment can be desirable, potentially having a stronger impact than single-treatment approaches. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any substance that can participate in activating a specific immune response and / or the function of immune effectors. This disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through various mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting tumor cell proliferation. In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or bind to complement proteins, resulting in direct cytotoxicity, termed complement-dependent cytotoxicity (CDC). Non-limiting examples of anticancer antibodies and potential antibody targets (in parentheses) that may be used in conjunction with this disclosure include: abagovomab (CA-125), aciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), atumomab pentetate (CEA), amatuximab (MORAb-009), and anatumomab... mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), beectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab mertansin (mucin CanAg), rapantuzumab ravtansine (MUC1), Capromab (carosumab)pendetide (prostate cancer cells), Carlumab (CNT0888), Catuximab (EpCAM, CD3), Cetuximab (EGFR), Citatuzumab bogatox (EpCAM), Cixutumumab (IGF-1 receptor), Claudiximab (tight junction protein), Clivatuzumab (Titan-Clivatuzumab) tetraxetan (MUC1), Conatumumab (TRAIL-R2), Dacetuzumab (CD40), Dalotuzumab (insulin-like growth factor I receptor), Denosumab (RANKL), Detumomab (B-lymphoma cells), Drozitumab (DR5), Ecromeximab (GD3 ganglioside), Edrecolomab (EpCAM), Erotuzumab (McCl2), and others. Anti-Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensituximab (NPC-1C), Epratuzumab (CD22), Ertumaxomab (HER2 / neu, CD3), Etaracizumab (integrin ανβ3), Farletuzumab (folate receptor 1), FBTA05 (CD20), Ficlatuzumab (SCH) 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (IL-1β), Girentuximab (carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomabtiuxetan (CD20), Icrucumab (VEGFR-1), Igovoma (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD152), Iratumumab (CD30), Labetuzumab (CEA), Lexatumumab (TRAIL-R2), Libivirumab (Hepatitis B surface antigen), Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Lucarumumab (CD40), Lumiliximab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR), Mepolizumab (IL5), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumomab pasudotox (CD22), Nacolomab tafenatox (C242 antigen), Naptumomab estafenatox (5T4), Namatumab (RON), Necitumumab (EGFR), Nimotuzumab (EGFR), Nivolumab (IgG4), Ofatumumab (CD20), Olaratumab (PDGF-RA), Onartuzumab (human dispersive factor receptor kinase), OportuzumabMonatox (EpCAM), Oregovomab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumoma (MUC1), Pertuzuma (HER2 / neu), Pintumomab (adenocarcinoma antigen), Pritumumab (vimentin), Racotumomab (N-hydroxyacetylneuraminic acid), Radretumab (Fibronectin extra domain-B), Rafivirumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrotuzumab (FAP), Siltuximab (IL6), Tabalumab (BAFF), Tacatuzumab Tetraxetan (alpha-fetoprotein), Tapipramomab paptox (CD 19), Tenatumomab (tenosynovitis C), Teprotumumab (CD221), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL13), Tositumomab (CD20), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Tucotuzumab celmoleukin (EpCAM), Ublituximab (MS4A1), Urelumab (4-1BB), Volociximab (integrin α5β1), Votumumab (tumor antigen CTAA) 16.88), zalumumab (EGFR) and zanolimumab (CD4).

[0329] The documents and studies cited herein are not intended to acknowledge any of them as relevant prior art. All statements regarding the content of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of these documents.

[0330] The following description is provided to enable those skilled in the art to make and use various embodiments. The descriptions of specific devices, techniques, and applications are provided by way of example only. Various modifications to the examples described herein will be apparent to those skilled in the art, and the general principles defined herein can be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but are consistent with the scope of the claims.

[0331] Example

[0332] method

[0333] animal

[0334] C57BL / 6BrdCrHsd-Tyr c Mice were purchased from Envigo Labs. Age- (8–10 weeks) and sex-matched (male or female) animals were used throughout the experiments. C57Bl / 6-Thy1.1 mice of the same line were bred at the animal facility of BioNTech AG, Germany.

[0335] CAR constructs / CAR T cells

[0336] The γ-retroviral self-inactivating (SIN) vector pES.12-6 is used to stably overexpress CLDN6-CAR-BBz-T2A-Luc-T2A-GFP in mouse T cells under the control of an internal eukaryotic promoter (a short, intronless form of the human elongation factor 1-α promoter (EFS–213 / +31)). The vector backbone contains wild-type MLV sequences in the R- and U5- regions at the 5' and 3'-LTRs and the packaging regions (psi and psi+). Enhancer elements (including the CAAT-Box) in the U3 region of the 3'-LTR are eliminated, and the TATA-Box sequence is mutated to prevent transcription initiation. A truncated form of the post-transcriptional regulatory element (PRE) of marmot hepatitis virus (WHV) is used to prevent the expression of unwanted viral proteins. CLDN6-CAR-BBz contains the signal peptide of human IgG (SEQ ID NO:12), a single-chain Fv fragment of the Claudin6-specific antibody IMAB206 (Ganymed Pharmaceuticals), and a heavy chain (V) H (SEQ ID NO:13) and light chain (V L(SEQ ID NO:15) has a (G4S)3 connector (SEQ ID NO:14) between them, and in V L The ScFv fragment has a cysteine-to-serine substitution at position 46. The fragment is fused with the human CD8α hinge and transmembrane region (SEQ ID NO:16), followed by the human 4-1BB (SEQ ID NO:17) and human CD3ζ(Q14K) (SEQ ID NO:18) signaling regions. The CAR is linked to an efficient firefly luciferase (SEQ ID NO:20) and eGFP (SEQ ID NO:21) via a T2A ribosomal jumping element (SEQ ID NO:19), enabling equimolar production of the specified protein in transduced T cells.

[0337] Retroviral gene manipulation and preparation of CAR T cells for adoptive T cell transfer

[0338] Using Dynabeads TM Mouse T-Activator CD3 / CD28, at a 1:1 bead-to-T-cell ratio (Invitrogen), isolated and preactivated naïve T cells in the presence of 5 ng / mL recombinant human (rh) IL-7 and 5 ng / mL rh IL-15 (Miltenyi Biotec). C57Bl / 6-Thy1.1 + Spleen cells. For transduction of mouse cells, MLV-E pseudotyped retrovirus supernatant was loaded into RetroNectin (2 μg / cm³) according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan). 2 Virus was loaded into non-tissue culture-treated plates and centrifuged (1,300 x g, 15 °C, 15 min) three times to increase binding. 24 h after pre-activation, cells were loaded at 0.5–0.6 x 10^6 cells / cm². 2 Centrifuge (300xg, 37℃, 1h) into virus-coated wells. After overnight incubation, repeat spin-down transduction with fresh virus-coated plates. Remove Dynabeads from the culture 72h after pre-activation. TMMouse T-Activator CD3 / CD28 was used to amplify cells in the presence of 5 ng / mL rh IL-7 and 5 ng / mL rh IL-15. After Ficoll washing, cells were washed twice with PBS to remove serum proteins and then prepared for adoptive cell transfer (ACT). The pES12.6-based retroviral vector contained OT1-TCR or CLDN6-CAR encoding an enhanced firefly luciferase (eff Luc; Rabinovich et al. (2008) PNAS 105(38):14342-6) and an eGFP (enhanced green fluorescent protein) reporter gene, which were expressed using 2A splicing elements (Szymczak et al. (2004) Nat Biotechnol. 22(5):589-94) for transduction.

[0339] Production of in vitro transcribed (IVT) mRNA

[0340] In vitro transcription of the mRNA encoding the cytokine-albumin fusion protein was based on the pST4-T7-GG-TEV-MCS-FI-A30LA70 plasmid backbone and derived DNA constructs. These plasmid constructs contained the 5' leader sequence of tobacco etch virus (TEV), a 3'Fl element (where F is an N-terminal cleavage enhancer of a 136-nucleotide-long 3'-UTR fragment, and I is a 142-nucleotide-long fragment of mitochondrial-encoded 12S RNA, both identified in humans; WO 2017 / 060314), and a 100-nucleotide poly(A) tail followed by a linker at 70 nucleotides. The cytokine and albumin-encoding sequences were derived from mice (Musmusculus), and no changes were introduced in the resulting amino acid sequences (mouse (m)IL-2, SEQ ID NO:5; mIL-7, SEQ ID NO:6; and mIL-21, SEQ ID NO:7). The encoded protein is equipped with an N-terminal signal peptide (SP), which is the native SP of the N-terminal portion. Only the N-terminal SP is retained; for the other portions, only the mature portion (the protein without the SP) is encoded. The stop codon is retained only at the C-terminus. The albumin and cytokine portions of the construct are separated by 30-nucleotide linker sequences encoding glycine and serine residues. The orientations of the albumin-cytokine fusion proteins used are as follows: albumin-linker-mIL2 (SEQ ID NOs: 8, 9, and 10 consecutively from N to C-terminus), mIL7-linker-albumin (SEQ ID NOs: 6, 9, and 11 consecutively from N to C-terminus), and mIL21-linker-albumin (SEQ ID NOs: 7, 9, and 11 consecutively from N to C-terminus). In vitro transcription of the mRNA encoding the antigen was based on the pST1-T7-GG-hAg-MCS-2hBg-A30LA70 plasmid backbone and the derived DNA construct. These plasmid constructs, in addition to the full-length human CLDN6 or chicken ovalbumin epitope SIINFEKL (OvaI; additionally flanked by 3'Sec and 5'TM1 – ​​as described by Kreiter et al. (2008) J Immunol. 180(1): 309-18), contain a 5' human α-globin, two consecutive 3' human β-globin UTRs, and a 100-nucleotide poly(A) tail followed by a linker at 70 nucleotides. As described in Holtkamp S. et al. (2006) Blood 108(13): 4009-17, mRNAs encoding antigens and cytokines were produced via in vitro transcription. The latter was further modified by replacing the normal nucleoside uridine with 1-methyl-pseudouridine. The resulting cytokine mRNAs were equipped with a Cap1 structure, and the double-stranded (dsRNA) molecules were removed by cellulose purification. The purified mRNAs were eluted in H2O and stored at -80°C until further use.In vitro transcription of all described mRNA constructs was performed at BioNTech RNA Pharmaceuticals GmbH.

[0341] IVT RNA (RNA) encoding antigens formulated by liposomes (LIP) The generation of )

[0342] The complexation of IVTRNA encoding an antigen with liposomes was previously described in Kranz et al. (2016) Nature 534(7607):396-401. The charge ratio of the cationic DOTMA to RNA used was 1.3:2. In addition to DOTMA, the lipid portion did indeed contain DOPE, an accessory lipid of DOTMA / DOPE in a molar ratio of 2:1.

[0343] Mouse experiment

[0344] Thy1.1, a CAR or TCR homolog transduced with 5 x 10^6 gamma retroviruses. + T cells were transferred intravenously (iv) at a dose of 200 μL to C57BL / 6BrdCrHsd-Tyr cells that were either immunocompetent or moderately irradiated (2.5 Gy–XRAD320). c In donor mice. Subsequently, at different time points after ACT, RNA encoding the antigen was used with an F12:RNA ratio of 1.3:2. (LIP) Mice were vaccinated intravenously (iv). At specified time points, mice were repeatedly treated with 1 μg of nucleoside-modified mRNA encoding a mouse albumin-cytokine fusion protein prepared with TransIT (Mirrus) or with buffer alone. Peripheral blood donation and whole-body bioluminescence imaging were performed at specified time points.

[0345] In vivo luciferase imaging (BLI)

[0346] The expansion and distribution of CAR- or TCR-effLuc-GFP transduced T cells were assessed using in vivo bioluminescence imaging with the IVIS Lumina imaging system (Caliper Life Sciences). In short, at designated time points following adoptive transfer of transduced T cells, D-luciferin aqueous solution (80 mg / kg body weight; Perkin Elmer) was injected intraperitoneally. Five minutes later, the emitted photons were quantified (integration time 1 min, pixel binning 8). In vivo bioluminescence in the region of interest (ROI) was quantified as total flux (photons / second) using IVIS Living Image 4.0 software. Transmitted light intensity from luciferase-expressing cells in animals was represented as grayscale images, with black representing the lowest intensity and white to dark gray representing the strongest bioluminescent signal. Grayscale reference images of mice were acquired under low-light LED illumination. Images were overlaid using Living Image 4.0 software.

[0347] Example 1: Selected pharmacokinetic-prolonging γ-chain cytokines (IL-2 / 7) lead to in vivo CAR T cell proliferation upon antigen contact.

[0348] Typically, a certain cytokine environment is required to maintain the persistence of T cells upon antigen contact. It has been demonstrated that gamma-chain cytokines (e.g., IL-2 and IL-7) enhance T cell proliferation and survival (e.g., Blattman et al. (2003) Nat. Med. 9(5): 540-7, Fry et al. (2001) Trends Immunol. 22(10): 564-71, Bradley et al. (2005) Trends Immunol. 26(3): 172-6, Jiang et al. (2005) Cytokine Growth Factor Rev. 16(4-5): 513-33). However, the use of recombinant cytokines (e.g., IL-2) is limited by their short half-life and dose-dependent toxicity (Vial et al. (1992) Drug Saf. 7(6): 417-33). To overcome the limited cytokine support for adoptive T cells, mRNA constructs encoding cytokine-albumin fusion proteins were developed, and these constructs indeed significantly increased the serum half-life of the encoded cytokines in vivo after systemic administration. When the cytokine-albumin constructs are encoded on nucleoside-modified mRNAs, their systemic availability is prolonged.

[0349] Therefore, we focus on combinations of liposome-formulated TAAs (e.g., RNA encoding CLDN6). (LIP)(It selectively targets APCs in secondary lymphoid organs) and whether the support of selected cytokines can lead to adequate replication and persistence of CAR-T cells in vivo.

[0350] To test this concept, γ-retroviral-transferred CLDN6-CAR T cells were adoptively transferred to moderately irradiated (2.5 Gy) or immunocompetent mice (Figures 1 and 2, respectively). To visualize the expansion and fate of those mouse CLDN6-CAR T cells in vivo, we utilized the co-expression of a luciferase and a GFP reporter gene on the same retroviral vector encoding CLDN6 CAR, but separated by the viral T2A sequence. Figure 1A Notably, co-expression of luciferase and GFP in CAR-transduced mouse T cells did not significantly affect the surface expression and antigen specificity of CLDN6-CAR (data not shown).

[0351] Moderately irradiated (2.5 Gy, XRAD320) albino C57Bl / 6 mice were transplanted with 5 x 10⁻⁶ cells. 6 A large number of syngeneic Thy1.1 cells transduced by the CLDN6-CAR-reporter gene. + Mouse T cells (approximately 2.5 x 10⁻⁶) 8 (cells / kg body weight). 20 μg of RNA encoding CLDN6 or a control was formulated into spleen-targeting liposomes and intravenously injected into mice one day after adoptive CAR-T transfer. Accompanying CLDN6 RNA (LIP)- Mice were vaccinated and intraperitoneally administered either albumin-conjugated mouse IL-2 and IL-7 mRNA or a mimic control (buffer) in TransIT (1 μg / cytokine RNA). Treatment was repeated 7 days later. At designated time points, CAR-T cell amplification and biodistribution were tracked in vivo by intraperitoneal administration of 1.66 mg of D-fluorescein solution per mouse. 24 hours post-ACT, most CAR-T cells were found in the spleen. Bioluminescence was detected on day 4 post-ACT using only CLDN6-RNA in the absence of cytokines (mimicry). (LIP) Treatment induced an approximately 21-fold increase in CAR-T cells (compared to day 1). CLDN6-RNA luminescence was also observed compared to baseline luminescence measured on day 1. (LIP) The second enhancement still produced a 15-fold increase in luminescence intensity on day 11. When TransIT-formulated mRNA encoding albumin fused with IL-2 and IL-7 was co-administered, the proliferative capacity of CAR T cells was significantly increased. CAR-T cells showed improved luminescence intensity in the first RNA... (LIP) The treatment resulted in a 75-fold amplification, and a second CLDN6 RNA was obtained. (LIP) The processing capacity was increased by up to 114 times. Figure 1C&D). In mice that received CLDN6-CAR T cells, RNA encoding CLDN6 was used separately. (LIP) This effect was observed after treatment with RNA encoding the cytokine albumin, but not after treatment with control RNA encoding OvaI. (LIP) This effect was not observed in the respective control groups, regardless of the presence or absence of RNA encoding the cytokine albumin. These data suggest that CAR-T cells can be successfully expanded in situ in a highly antigen-specific manner in moderately irradiated mice.

[0352] Having demonstrated the presence of RNA encoding cytokines, it is possible to use RNA encoding their respective antigens. (LIP) Following in situ replication of CAR T cells in moderately irradiated mice, we investigated whether this effect could also be achieved in immune-active hosts. However, lymphocyte removal has several drawbacks, including well-known side effects and chemotherapy-related risks such as potential infection and sepsis (Brentjens et al. (2010) Mol Ther. 18(4):666-8 & Robbinset et al. (2015) Clin Cancer Res. 21(5):1019-27). Furthermore, the rapid expansion of adoptively transferred CAR-T cells can be lethal in cases of on-target and / or off-target toxicity (Morgan et al. (2010) Mol Ther. 18(4):843-51). For this purpose, CLDN6-CAR-transduced mice were used to transform Thy1.1 cells. + Unirradiated albino C57Bl / 6 mice transplanted with T cells were treated as described above. Figure 2A Compared to the control group that received buffer (simulated) rather than TransIT-formulated RNA encoding the cytokine albumin, systemic IL-2 and IL-7 were present against CLDN6-RNA during the first round of stimulation. (LIP) Following vaccination, CLDN6-CAR T cell expansion was not significantly affected (Day 4: expansion index: simulated 192-fold and IL-2 / 7: 223-fold). However, in the absence of IL-2 / 7 cytokines, the CAR T cell population showed increased activity upon first CLDN6 RNA administration. (LIP) The mediated expansion was followed by strong contraction and could not be re-expanded. Only in the presence of RNA encoding IL-2 / IL-7 albumin could CLDN6-CAR T cells repeatedly expand and persist for several days in immune-active mice (Day 11: expansion index: simulated: 0.5-fold and IL-2 / 7: 79-fold). Figure 2B +C).

[0353] These data strongly support the use of RNA (LIP) The idea of ​​directly controlling CAR-T cell expansion in patients is feasible, but for persistence, cells need a favorable cytokine environment, such as IL-2 and IL-7, which can be achieved by administering RNA that encodes prolonged pharmacokinetic γ-chain cytokines.

[0354] Example 2: Optimal combination of cytokine-albumin fusion during CAR T cell replication expansion

[0355] Since several gamma-chain cytokines actively support T cell survival and support T cell therapeutic efficacy in an antigen-specific manner (e.g., Markley et al. (2010) Blood 115(17):3508-19, He et al. (2006) J Transl Med. 4:24.), we compared the effects of nucleoside-modified RNAs encoding mIL-2, mIL-7, mIL-21, and combinations of IL-2 / 7 and IL-2 / 21 on promoting CAR-modified T cell response to repetitive RNA in vivo. (LIP) The treatment supports proliferation and persistence.

[0356] Similar to that described in Example 1, moderately irradiated albino C57Bl / 6 mice transplanted with CLDN6-CAR-reporter gene-transduced T cells were vaccinated with liposome-formulated RNA encoding hCLDN6 or a control, and simultaneously treated with RNA encoding mouse albumin-conjugated mIL-2, mIL-7, mIL-21, or mouse albumin (Alb control) formulated in TransIT (1 μg / cytokine RNA). The antigen / cytokine mixture (cocktail) was administered at weekly intervals. Figure 3A The bioluminescence intensity was analyzed when CAR T cell proliferation reached its peak in vivo (typically 2-3 days after RNA-based treatment). Figure 3B Compared to albumin controls, the systemic presence of IL-7 and IL-21 alone resulted in repetitive RNA. (LIP)Antigen-specific CAR-T amplification capacity decreased after treatment. Compared with baseline, IL-2 co-treatment resulted in up to 164-fold CAR T cell amplification. However, in vivo accumulation of CAR T cells was only achieved when IL-2 RNA was co-administered with IL-7 (up to 214-fold after the third round of amplification) or IL-21 (up to 141-fold after the third round of amplification), respectively. In addition to the ability of CAR T cells to accumulate in vivo, the clinical success of adoptive metastasis tumor-reactive T cell therapy is also positively correlated with the persistence of those cells in vivo (Robbins et al. (2004) J Immunol. 173(12):7125-30, Huang et al. (2005) 28(3):258-67). Therefore, in the presence of IL-7 ( Figure 3C ) or IL-21 exists Figure 3D In the presence of albumin, IL-2 alone or in combination with IL-2, bioluminescence analysis was used to assess the contraction of CAR T cells after three rounds of antigen-specific amplification. However, in the presence of only albumin, IL-2, or IL-7, the CAR T cell population showed increased contraction after the third CLDN6 RNA assay. (LIP) Shortly after, they contract. However, only the combination of IL-2 and IL-7 can increase the decelerating contraction of CLDN6 CAR T cells after antigen withdrawal. Figure 3C This effect was even more pronounced in mice co-treated with IL-2 and IL-21-RNA. Figure 3D ).

[0357] Overall, these results indicate that systemic administration of nucleoside-modified RNA encoding IL-2, which binds to IL-7 and IL-21, can increase the highly antigen-dependent accumulation of CAR T cells in vivo in response to antigen-specific stimulation and prolong the duration of CAR T cell proliferation. sequence list <110> Biotechnology cell and gene therapy companies <120> Therapies involving CAR-engineered T cells and cytokines <130> 674-244 PCT2 <150> PCT / EP2019 / 053144 <151> 2019-02-08 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 2 <211> 152 <212> PRT <213> Homo sapiens <400> 2 Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu 1 5 10 15 Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser 20 25 30 Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp 35 40 45 Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg 50 55 60 Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu 65 70 75 80 Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val 85 90 95 Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser 100 105 110 Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu 115 120 125 Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys 130 135 140 Ile Leu Met Gly Thr Lys Glu His 145 150 <210> 3 <211> 133 <212> PRT <213> Homo sapiens <400> 3 Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser 65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser 130 <210> 4 <211> 585 <212> PRT <213> Homo sapiens <400> 4 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290,295,300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 405 410 415 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Only Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 <210> 5 <211> 169 <212> PRT <213> Mus musculus <400> 5 Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu 1 5 10 15 Leo Val Asn Ser Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala 20 25 30 Glu Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu 35 40 45 Glu Gln Leu Leu Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn 50 55 60 Tyr Arg Asn Leu Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu 65 70 75 80 Pro Lys Gln Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu 85 90 95 Leu Gly Pro Leu Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe 100 105 110 Gln Leu Glu Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val 115 120 125 Val Lys Leu Lys Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp 130 135 140 Glu Ser Ala Thr Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys 145 150 155 160 Gln Ser Ile Ile Ser Thr Ser Pro Gln 165 <210> 6 <211> 154 <212> PRT <213> House mouse (Mus musculus) <400> 6 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Ile Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Thr Ser Ser Glu Cys His Ile Lys Asp Lys 20 25 30 Glu Gly Lys Ala Tyr Glu Ser Val Leu Met Ile Ser Ile Asp Glu Leu 35 40 45 Asp Lys Met Thr Gly Thr Asp Ser Asn Cys Pro Asn Asn Glu Pro Asn 50 55 60 Phe Phe Arg Lys His Val Cys Asp Asp Thr Lys Glu Ala Ala Phe Leu 65 70 75 80 Asn Arg Ala Ala Arg Lys Leu Lys Gln Phe Leu Lys Met Asn Ile Ser 85 90 95 Glu Glu Phe Asn Val His Leu Leu Thr Val Ser Gln Gly Thr Gln Thr 100 105 110 Leu Val Asn Cys Thr Ser Lys Glu Glu Lys Asn Val Lys Glu Gln Lys 115 120 125 Lys Asn Asp Ala Cys Phe Leu Lys Arg Leu Leu Arg Glu Ile Lys Thr 130 135 140 Cys Trp Asn Lys Ile Leu Lys Gly Ser Ile 145 150 <210> 7 <211> 146 <212> PRT <213> Mus musculus <400> 7 Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val 1 5 10 15 Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu 20 25 30 Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp 35 40 45 Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys 50 55 60 Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser 65 70 75 80 Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu 85 90 95 Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile 100 105 110 Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu 115 120 125 Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His 130 135 140 Leu Ser 145 <210> 8 <211> 608 <212> PRT <213> Mus musculus <400> 8 Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala 1 5 10 15 Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala 50 55 60 Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala 130 135 140 Glu Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly 145 150 155 160 His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys 180 185 190 Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly 195 200 205 Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys 210 215 220 Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr 245 250 255 Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp 290 295 300 Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp 305 310 315 320 Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp 325 330 335 Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu 385 390 395 400 Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys 405 410 415 Asp Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460 Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile 465 470 475 480 Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His 485 490 495 Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525 Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu 530 535 540 Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala 565 570 575 Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe 580 585 590 Ser Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala 595 600 605 <210> 9 <211> 10 <212> PRT <213> artificial sequence <220> <223> GS connector <400> 9 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 <210> 10 <211> 149 <212> PRT <213> Mus musculus <400> 10 Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln 1 5 10 15 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu 20 25 30 Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn Leu 35 40 45 Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala 50 55 60 Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu 65 70 75 80 Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp 85 90 95 Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys 100 105 110 Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr 115 120 125 Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile 130 135 140 Ser Thr Ser Pro Gln 145 <210> 11 <211> 584 <212> PRT <213> Mus musculus <400> 11 Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu 1 5 10 15 Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln 20 25 30 Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu 65 70 75 80 Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu 100 105 110 Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys 115 120 125 Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Glu Gln 145 150 155 160 Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp Lys Glu Ser 165 170 175 Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys Ala Leu Val Ser 180 185 190 Ser Val Arg Gln Arg Met Lys Cys Ser Ser Met Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Thr Phe Pro 210 215 220 Asn Ala Asp Phe Ala Glu Ile Thr Lys Leu Ala Thr Asp Leu Thr Lys 225 230 235 240 Val Asn Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Glu Leu Ala Lys Tyr Met Cys Glu Asn Gln Ala Thr Ile Ser 260 265 270 Ser Lys Leu Gln Thr Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His 275 280 285 Cys Leu Ser Glu Val Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala 290 295 300 Ile Ala Ala Asp Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys 340 345 350 Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala 355 360 365 Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu Pro 370 375 380 Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu Gly Glu 385 390 395 400 Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln Lys Ala Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg Asn Leu Gly Arg 420 425 430 Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp Gln Arg Leu Pro Cys 435 440 445 Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg Val Cys Leu Leu His 450 455 460 Glu Lys Thr Pro Val Ser Glu His Val Thr Lys Cys Cys Ser Gly Ser 465 470 475 480 Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu Thr Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe Thr Phe His Ser Asp 500 505 510 Ile Cys Thr Leu Pro Glu Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu 530 535 540 Lys Thr Val Met Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys 545 550 555 560 Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val 565 570 575 Thr Arg Cys Lys Asp Ala Leu Ala 580 <210> 12 <211> 19 <212> PRT <213> Artificial sequence <220> <223> signal peptide <400> 12 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser <210> 13 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 13 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GS Linker <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 15 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region <400> 15 Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Asp Pro Ala 100 105 110 <210> 16 <211> 69 <212> PRT <213> Artificial sequence <220> <223> Human CD8 hinge and transmembrane domain <400> 16 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys 65 <210> 17 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Human 4-1BB signal transduction domain <400> 17 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 18 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Human CD3zeta signal transduction domain <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 19 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> T2A component <400> 19 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 20 <211> 550 <212> PRT <213> Artificial sequence <220> <223> Effective firefly luciferase <400> 20 Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 40 45 Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ile Ala Val 545 550 <210> 21 <211> 239 <212> PRT <213> Artificial sequence <220> <223> eGFP <400> 21 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> twenty two <211> 8 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> twenty two Ser Ile Ile Asn Phe Glu Lys Leu 1 5

Claims

1. Use of a pharmaceutical product in the preparation of a medicament for treating a subject with a disease, symptom, or condition related to the expression or elevated expression of an antigen, wherein the pharmaceutical product comprises: a. T cells genetically modified to express chimeric antigen receptors (CARs) that target said antigens. b. RNA encoding IL2 c. RNA encoding other cytokines, said other cytokines being selected from IL7 and IL21, and d. The RNA encoding the antigen, The drug is used to treat the subject's cancer, and the antigen is a tumor-associated antigen.

2. The use of claim 1, wherein the pharmaceutical product comprises RNA encoding IL2 and RNA encoding IL7.

3. The use of claim 1, wherein the pharmaceutical product comprises RNA encoding IL2 and RNA encoding IL21.

4. Use according to any one of claims 1-3, wherein the subject is provided with genetically modified T cells expressing CAR by administering genetically modified T cells to express CAR or by generating genetically modified T cells to express CAR in the subject.

5. The use of any one of claims 1-3, wherein the IL2 is the IL2 that prolongs pharmacokinetic (PK).

6. The use of claim 5, wherein the extended PK IL2 comprises a fusion protein.

7. The use of claim 6, wherein the fusion protein comprises an IL2 moiety and a moiety selected from: serum albumin, immunoglobulin fragments, transferrin and Fn3.

8. The use of claim 7, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

9. The use of claim 7, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

10. Use according to any one of claims 1-3, wherein the other cytokines are cytokines that prolong pharmacokinetic (PK).

11. The use of claim 10, wherein the cytokine that prolongs pharmacokinetic (PK) is IL7 or IL21 that prolongs PK.

12. The use of claim 10, wherein the cytokine that prolongs PK comprises a fusion protein.

13. The use of claim 12, wherein the fusion protein comprises portions of other cytokines, and portions selected from: serum albumin, immunoglobulin fragments, transferrin, and Fn3.

14. The use of claim 13, wherein a portion of said other cytokine is the IL7 portion or the IL21 portion.

15. The use of claim 13, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

16. The use of claim 13, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

17. A pharmaceutical product comprising: a. T cells genetically modified to express chimeric antigen receptor (CAR), b. RNA encoding IL2 c. RNA encoding other cytokines, said other cytokines being selected from IL7 and IL21, and d. RNA encoding an antigen, wherein the genetic modification thereof enables T cells expressing CAR to target the antigen.

18. The pharmaceutical product of claim 17, comprising RNA encoding IL2 and RNA encoding IL7.

19. The pharmaceutical product of claim 17, comprising RNA encoding IL2 and RNA encoding IL21.

20. The pharmaceutical product of any one of claims 17-19, wherein it is a reagent kit.

21. The pharmaceutical product of claim 20, comprising, in different containers, the genetically modified T cells expressing CAR, the RNA encoding IL2, the RNA encoding other cytokines, and the RNA encoding antigens.

22. The pharmaceutical product of claim 20, further comprising a description of the pharmaceutical product for treating cancer, wherein the antigen is a tumor-associated antigen.

23. The pharmaceutical product according to any one of claims 17-19, wherein it is a pharmaceutical composition.

24. The pharmaceutical product of claim 23, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

25. The pharmaceutical product of any one of claims 17-19, wherein IL2 is IL2 with prolonged pharmacokinetic (PK) activity.

26. The pharmaceutical product of claim 25, wherein the IL2 of the extended PK comprises a fusion protein.

27. The pharmaceutical product of claim 26, wherein the fusion protein comprises an IL2 portion and a portion selected from: serum albumin, immunoglobulin fragments, transferrin, and Fn3.

28. The pharmaceutical product of claim 27, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

29. The pharmaceutical product of claim 27, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

30. The pharmaceutical product of any one of claims 17-19, wherein the other cytokines are cytokines that prolong pharmacokinetic (PK).

31. The pharmaceutical product of claim 30, wherein the cytokine that prolongs PK comprises a fusion protein.

32. The pharmaceutical product of claim 31, wherein the fusion protein comprises portions of other cytokines and portions selected from: serum albumin, immunoglobulin fragments, transferrin, and Fn3.

33. The pharmaceutical product of claim 32, wherein the portion of said other cytokine is the IL7 portion or the IL21 portion.

34. The pharmaceutical product of claim 32, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

35. The pharmaceutical product of claim 32, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

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