TW202208415A - Il-12 armored immune cell therapy and uses thereof - Google Patents

Abstract

This disclosure relates to IL-12 armored immune cell therapies and the use of IL-12 in combination with immune cell therapies.

Description

IL-12 Armored Immune Cell Therapy and Its Uses

Field of Invention

The present disclosure relates to IL-12 armored immune cell therapy and the use of IL-12 in combination with immune cell therapy.

Background of the Invention

Cancer is caused by biological and environmental factors (such as age, gender, genetic mutation, environmental exposure (such as UV radiation), occupational risk factors, carcinogens, asbestos, radioactive substances and viral infections (eg, HPV, EBV, HBV, HCV, HTLV-1 and KSHV)) (Margaret E et al., "Viruses Associated With Human Cancer," Biochimica et Biophysica Acta. 1782:127-150 (2008)).

Recent clinical and commercial successes of CAR T-cell therapy have generated significant interest in immune cell therapy. Despite advances in cancer treatment, various treatments are relatively less effective for some cancers. Therefore, there is an unmet need for effective therapies against cancer.

Summary of Invention

The present disclosure provides modified (eg, membrane-tethered) or unmodified IL-12 that can be used in combination with immune cell therapy (eg, TCR-T, CAR-T, or TIL) to treat cancer (eg, solid tumors) ). The present disclosure also provides the use of IL-12 fused to a tumor-targeting antibody (eg, NHS76) in combination with immune cell therapy (eg, TCR-T, CAR-T, CAR-NK, or TIL) for the treatment of cancer. The use of modified IL-12 in combination with immune cell therapies can greatly increase the efficacy of these immune cell therapies; in addition, these approaches have an improved safety profile for clinical use.

In one aspect, provided herein is a cell expressing (a) a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); and (b) IL-12.

In some embodiments, the IL-12 is membrane-tethered IL-12.

In some embodiments, the membrane-tethered IL-12 comprises a CD4 transmembrane domain.

In some embodiments, the membrane-tethered IL-12 comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, and a CD4 transmembrane region.

In some embodiments, the membrane-tethered IL-12 comprises two or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a CD4 transmembrane region.

In some embodiments, the immunoglobulin CH2 domain is a wild-type immunoglobulin constant domain.

In some embodiments, the amino acid residue at position 235 (EU numbering) of the immunoglobulin CH2 domain is Glu, and the amino acid residue at position 297 (EU numbering) of the immunoglobulin CH2 domain is Gln .

In some embodiments, the membrane-tethered IL-12 further comprises an immunoglobulin hinge region.

In some embodiments, the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain, and the CD4 transmembrane region is a human CD4 transmembrane region.

In some embodiments, the IL-12 is soluble IL-12.

In some embodiments, IL-12 is linked to a tumor-targeting antibody or antigen-binding fragment thereof.

In some embodiments, the tumor-targeting antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

In some embodiments, the tumor-targeting antibody is NHS76.

In some embodiments, the IL-12 is human IL-12 or mouse IL-12.

In some embodiments, the TCR or CAR targets BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (IL-3R), CEA, IL13Ra2, ALPP, EBV-associated antigens (eg, LMP2), EGFR, EGFRvIII, GD2 , GPC3, HER2, HPV-associated antigens (eg, E6, E7), MAGE (eg, MAGE-A3), mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.

In some embodiments, the CAR comprises an extracellular domain,

In some embodiments, the extracellular domain is a single-chain variable fragment (scFv), a ligand (eg, a receptor binding ligand), or an antibody mimetic.

In some embodiments, the cells also express chemokines, such as CXCL10 or XCL1. In some embodiments, the cells also express Flt3L.

In one aspect, provided herein is a vector comprising: a) a first nucleic acid sequence encoding an IL-12α subunit and an IL-12β subunit; b) a second nucleic acid sequence encoding one or more immunoglobulins A CH2 domain, one or more immunoglobulin CH3 domains, and a transmembrane region. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a first linker sequence.

In some embodiments, the first linker sequence encodes an immunoglobulin hinge polypeptide sequence.

In some embodiments, the transmembrane domain is a CD4 transmembrane domain.

In some embodiments, the transmembrane region is an immunoglobulin transmembrane region.

In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence.

In some embodiments, the vector further comprises a sequence encoding a message peptide.

In some embodiments, the IL-12α subunit is a human IL-12α subunit, the IL-12β subunit is a human IL-12β subunit, the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, the immunoglobulin The CH3 domain is a human immunoglobulin CH3 domain.

In some embodiments, the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

In some embodiments, the third nucleic acid sequence is linked to the first nucleic acid through a second linker sequence. In some embodiments, the second linker sequence encodes a P2A sequence.

In some embodiments, the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter).

In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are under the control of a regulatory element (eg, a promoter).

In some embodiments, the vector further comprises a sequence encoding a chemokine such as CXCL10 or XCL1. In some embodiments, the vector further comprises a sequence encoding Flt3L.

In one aspect, provided herein is a vector comprising: a) a first nucleic acid sequence encoding the variable heavy (VH) and variable light (VL) regions of a tumor-targeting antibody; b) a second Nucleic acid sequences encoding the IL-12α subunit and the IL-12β subunit. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a first linker sequence.

In some embodiments, the tumor-targeting antibody targets a tumor-associated antigen. In some embodiments, the tumor-targeting antibody is NHS76.

In some embodiments, the vector further comprises a nucleic acid encoding a message peptide.

In some embodiments, the message peptide is a human message peptide, the IL-12α subunit is a human IL-12α subunit, and the IL-12β subunit is a human IL-12β subunit.

In some embodiments, the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

In some embodiments, the third nucleic acid sequence is linked 5' to the first nucleic acid through a second linker sequence. In some embodiments, the second linker sequence encodes a P2A sequence.

In some embodiments, the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter).

In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are under the control of a regulatory element (eg, a promoter).

In one aspect, provided herein is a vector comprising, in a 5' to 3' orientation: a) a first nucleic acid sequence encoding a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); b) A second nucleic acid sequence encoding the message peptide, the IL-12α subunit and the IL-12β subunit. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a linker sequence.

In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence.

In some embodiments, the linker sequence encodes a P2A sequence

In one aspect, provided herein is a fusion polypeptide comprising: a) a first region comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker peptide sequence. b) a second region comprising one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region.

In some embodiments, the first region and the second region are linked by an immunoglobulin hinge peptide.

In one aspect, provided herein is a fusion polypeptide comprising: a) a first region comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a first linker peptide sequence. b) A second region comprising the variable heavy (VH) and light chain (VL) regions of the tumor-targeting antibody. In some embodiments, VH and VL are linked by a second linker peptide sequence. In some embodiments, the first region and the second region are linked by a third linker peptide sequence.

In some embodiments, the third linker polypeptide sequence has a sequence that is at least 80% identical to SEQ ID NO: 17.

In one aspect, provided herein is a nucleic acid encoding a polypeptide as described herein.

In one aspect, provided herein is a vector comprising a nucleic acid as described herein.

In one aspect, provided herein are cells expressing a fusion polypeptide as described herein.

In one aspect, provided herein is a cell comprising a vector as described herein.

In some embodiments, the cell secretes a higher level of interferon than the same cell except that the cell does not comprise a vector as described herein. In some embodiments, the cell stimulates one or more cells in the vicinity of the cell to secrete interferons. In some embodiments, the interferon is IFNy.

In some embodiments, the cell exhibits a higher level of early TCR activation markers, and/or stimulates one or more cells in the vicinity of the cell, compared to the same cell except that the cell does not comprise a vector described herein Shows early TCR activation markers.

In some embodiments, the activation marker is CD69.

In some embodiments, the cell is a cell line.

In some embodiments, the cells are primary cells obtained from a subject (eg, a human subject).

In some embodiments, the cells are immune cells (eg, lymphocytes).

In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs) or NK cells (eg, CAR-NK cells).

In some embodiments, the cells are T cells. In some embodiments, the T cells are isolated from a human subject. In some embodiments, the T cells are CD8+. In some embodiments, the T cells are CD4+.

In some embodiments, the vector is an expression vector, a viral vector, a retroviral vector, or a lentiviral vector. In some embodiments, the retroviral vector is pMP71.

In one aspect, provided herein is a method for producing a cell, the method comprising introducing a vector as described herein into the cell in vitro or ex vivo. In some embodiments, the vector is introduced into the cell by transduction.

In one aspect, provided herein is a method of treating a subject having cancer, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.

In some embodiments, the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.

In some embodiments, the cells are isolated from peripheral blood mononuclear cells (PBMCs) of the subject.

In one aspect, provided herein is a method of treating a subject with cancer, the method comprising administering to a subject in need thereof: a) an effective amount of an expressed T cell receptor (TCR) or a chimeric antigen receptor ( CAR); and b) an effective amount of a protein comprising IL-12 and a tumor-targeting antibody or antigen-binding fragment thereof.

In some embodiments, the cells are isolated from peripheral blood mononuclear cells of the subject.

In one aspect, provided herein is a method of treating a human subject having cancer, the method comprising providing cells collected from the human subject or a different human subject; introducing a vector as described herein into the cells; culturing and expanding the cells; and administering to the subject an effective amount of a composition comprising the cells.

In some embodiments, the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.

In some embodiments, the cells are peripheral blood mononuclear cells (PBMCs).

In some embodiments, the cells are tumor-infiltrating lymphocytes and the vector comprises nucleic acid encoding IL-12. In some embodiments, the IL-12 is membrane-tethered IL-12.

In some embodiments, the cells are T cells and the vector comprises nucleic acid encoding a TCR or CAR. In some embodiments, the vector further comprises a nucleic acid encoding IL-12. In some embodiments, the IL-12 is membrane-tethered IL-12.

As used herein, the term "genetically engineered cell" or "genetically modified cell" refers to a cell having a modification of a nucleic acid sequence in the cell, including but not limited to having an insertion of one or more nucleotides in its genome, Deleted, substituted, or modified cells, and/or cells with exogenous nucleic acid sequences (eg, vectors) that are not necessarily integrated into the genome.

As used herein, the term "membrane-tethered IL-12" refers to a modified form of IL-12 that is tethered to the cell membrane.

As used herein, the term "peripheral blood cells" refers to cells commonly found in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, K cells, granulocytes, and B cells .

As used herein, the term "cancer" or "cancer cell" refers to a cell that divides in an uncontrolled manner. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growths (eg, tumors), oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. Cancer cells can form solid tumors or excess tumor cells in the blood (eg, blood cancers). Alternatively or additionally, it may include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasion. Examples of solid tumors include malignancies of various organ systems (eg, sarcomas), adenocarcinomas, and carcinomas, such as involving the liver, lung, breast, lymph, gastrointestinal tract (eg, colon), genitourinary tract (eg, kidney, urothelial cells) , prostate and pharynx. Adenocarcinomas include malignancies such as most colon, rectal, renal cell, liver, non-small cell lung, small bowel, and esophageal cancers. Examples of cancers that can be treated by the methods described herein include, for example, bone cancer, pancreatic cancer, skin cancer (eg, melanoma), head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia ), lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary gland Adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and/or T-cell lymphoma.

As used herein, the term "vector" refers to a vehicle by which a polynucleotide sequence (eg, a foreign gene) can be introduced into a host cell so as to obtain the desired gene expression of the introduced nucleotide sequence. Cloning vectors can include, for example, plastids, phages, viruses, and the like. The most popular type of vector is the "plastid," which refers to a closed circular double-stranded DNA loop into which additional DNA segments containing the gene of interest can be ligated. Another type of vector is a viral vector that links the nucleic acid construct to be delivered into the viral genome. Viral vectors are capable of replicating autonomously in the host cell into which they are introduced, or they can integrate themselves into the genome of the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In some embodiments, the vector is a viral vector (eg, replication-defective retrovirus, adenovirus, and adeno-associated virus).

As used herein, a "subject" is a mammal, such as a human or non-human animal. In some embodiments, the subject (eg, patient) to which the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. Subjects can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a dog, cat, horse, rodent, rat or mouse.

As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and encompasses ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified value The change.

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 to which this invention belongs. Methods and materials are described herein for use in the present invention, other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the present invention will be apparent from the following detailed description and drawings, and from the scope of the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The human immune system is able to recognize and eliminate cells that have been infected or damaged as well as those cells that have become cancerous. Immune cell therapy takes advantage of the human immune system and is revolutionizing cancer therapy. It involves the transfer of immune cells into a patient. The cells are most commonly derived from the immune system, and can be derived from a patient or another individual. In autologous cancer immunotherapy, immune cells are extracted from a patient, genetically modified and cultured in vitro, and returned to the same patient. In comparison, allogeneic therapy involves cells isolated and expanded from a donor subject.

Many different kinds of immune cells are used in immune cell therapy. These cell therapies include, for example, tumor infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, and natural killer (NK) cell therapy.

Cancer patients have naturally occurring T cells that are often able to target their cancer cells. Tumor-infiltrating lymphocyte (TIL) therapy involves harvesting these naturally occurring T cells from the patient and then activating and expanding them. These activated T cells are then reinfused into the patient. In contrast, engineered T cell receptor (TCR) therapy involves isolating T cells from patients, but instead of merely activating and expanding available antitumor T cells, T cells are also transfected with vectors encoding novel T cell receptors. cells that allow T cells to target specific cancer antigens presented by the major histocompatibility complex (MHC). Chimeric antigen receptor (CAR) T cell therapy is similar to TCR therapy. In CAR T cell therapy, the cells are transfected with a vector encoding a chimeric antigen receptor. Chimeric antigen receptors can bind cancer antigens and do not require the cancer antigen to be presented by the MHC. Some other immune cells can also be used in these cell therapies. For example, natural killer cells can also be transfected with vectors encoding chimeric antigen receptors.

The present disclosure shows that immune cell therapy can be synergistically combined with interleukin-12 (IL-12). However, IL-12 administration is often associated with severe toxicity, including, for example, hematological toxicity, anemia, lymphopenia, neutropenia, muscle and liver toxicity, and even death in some cases. The toxicity of IL-12 administration greatly limits its use in immune cell therapy.

The present disclosure provides an improved immune cell therapy in which engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) produce IL-12 in a controlled manner and deliver IL-12 directly to a target site points, thereby significantly improving efficacy and safety. In one aspect, IL-12 is membrane-tethered IL-12, which further limits the effects of IL-12 on localized target areas. In another aspect, IL-12 was conjugated to the scFv of NHS76 as an antibody targeting the tumor necrosis center.

The expression of IL-12 further enhances the efficacy of immune cell therapy. Antigen escape and downregulation (eg, antigen loss) have emerged as major issues affecting the durability of immune cell therapy. In particular, in solid tumors, most antigens are expressed at lower levels and/or in a more heterogeneous manner. Due to heterogeneity, certain tumor cells can escape immune cell therapy targeting a specific antigen by reducing the expression of that antigen. Immune stress from immune cell therapy can drive cancer cell evolution by modulating the expression of target antigens in cancer cells, either through the loss of detectable antigens or by reducing the expression of antigens to levels below the threshold required for immune cell activity. . Once most tumor cells are killed, those remaining resistant to immune cell therapy grow rapidly, and the entire relapsed solid tumor becomes resistant to immune cell therapy. Antigen loss or low antigen escape has become an even greater obstacle to the treatment of solid tumors. Details of the mechanism of antigen loss and related clinical data can be found, for example, in Majzner and Crystal, "Tumor antigen escape from CAR T-cell therapy." Cancer discovery 8.10 (2018): 1219-1226, which is hereby incorporated by reference in its entirety. The expression of IL-12 in engineered immune cells can further increase the immune response in the local tumor microenvironment (eg, stimulate peripheral immune cells), thereby killing peripheral cancer cells that do not express those antigens or express low-level antigens, preventing any Cancer cells escape immune cell therapy.

In one aspect, the present disclosure relates to IL-12 armored immune cell therapy (eg, TCR-T, CAR-T, CAR-NK, or TIL). In some embodiments, IL-12 is fused to one or more (eg, 1, 2, 3, 4, or 5) immunoglobulin constant domains and/or membrane tethered regions (eg, transmembrane regions) to be modified so that IL-12 is tethered to the cell membrane. In some embodiments, IL-12 is modified by linking it to variable regions (eg, VH and VL) of a tumor-targeting antibody. These IL-12 armored immune cell therapies may enhance anti-tumor immunity or efficacy, induce activation of the host immune system, induce epitope spread, and/or increase safety in humans. The present disclosure also provides vectors, cells comprising such vectors, and methods of making such vectors. Also provided are methods of immune cell therapy using the IL-12 armor of the present disclosure, including but not limited to the treatment of various cancers and some other disorders in humans. IL-12 and immune cell therapy

IL-12 is a heterodimeric molecule consisting of an alpha chain (p35 subunit) and a beta chain (p40 subunit) covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. Biologically, IL-12 is an inflammatory interleukin produced by various cells of the immune system, including phagocytes, B cells, and activated dendritic cells, in response to infection. IL-12 mediates the interaction of the innate and adaptive arms of the immune system, acts on T cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes, and produces other inflammatory interferons (especially It plays a key role in interferon-γ (IFN-γ). A detailed description of IL-12 can be found, for example, in Colombo et al., "Interleukin-12 in anti-tumor immunity and immunotherapy." Cytokine & growth factor reviews 13.2 (2002): 155-168; and Hamza et al., "Interleukin 12 a key immunoregulatory cytokine in infection applications." International journal of molecular sciences 11.3 (2010): 789-806; these documents are incorporated by reference in their entirety.

The IL-12 receptor is expressed in a constitutive or inducible manner on a variety of immune cells, including NK cells and T and B lymphocytes. Ligand-bound IL-12R becomes phosphorylated on tyrosine, which provides a carrier site for two kinases, JAK2 and TYK2. Among the STAT family of transcription factors, STAT4 is considered to be the most specific mediator of cellular responses elicited by IL-12. A remarkable function of IL-12 is its ability to induce IFNy release from natural killer (NK) cells as well as CD4 ⁺ and CD8 ⁺ T cells. Indeed, IL-12 signaling via STAT-4 is critical for Th1 differentiation and acquisition of cytolytic function of CD8 ⁺ T cells. IFNγ in turn strongly alters the tumor microenvironment. The best studied beneficial mechanisms of IFNγ are: 1) enhanced MHC I antigen presentation by tumor cells; 2) induction of CXCL9, 10 and 11 chemokine expression to attract NK, Th1 and CD8 ⁺ T cells; phagocytosis into activated anti-tumor M1 macrophages; and 4) acting on endothelial cells to mediate anti-angiogenesis in a CXCR3-dependent manner, while also enhancing the expression of homing receptors for T cell recruitment.

Although the potent antitumor effects of IL-12 are well established, IL-12 administration also induces severe side effects. These side effects associated with systemic administration of IL-12 in clinical studies, as well as the very narrow therapeutic index of this interferon, significantly limit the use of IL-12 as an anticancer therapy. A detailed description of IL-12 can be found, for example, in Lasek et al., "Interleukin 12: still a promising candidate for tumor immunotherapy?" Cancer Immunology, Immunotherapy 63.5 (2014): 419-435; Berraondo et al., "Revisiting interleukin-12 as a cancer immunotherapy agent." Clinical Cancer Research 24.12 (2018): 2716-2718; these references are incorporated by reference in their entirety.

To increase efficacy and reduce toxicity, the present disclosure provides modified IL-12 and/or improved means of delivering IL-12. In particular, IL-12 is expressed in immune cells that target cancer cells. These engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) can produce IL-12 in a controlled manner and deliver it directly to target sites. In one aspect, IL-12 is membrane-tethered IL-12, which further limits the effects of IL-12 on localized target areas.

In some embodiments, the modified IL-12 protein can be any of the fusion proteins described herein. In some embodiments, provided herein are fusion proteins comprising an IL-12α subunit and an IL-12β subunit. IL-12 protein can be derived from any species. In some embodiments, the IL-12α subunit is human IL-12α subunit. In some embodiments, the IL-12α subunit is mouse IL-12α subunit. In some embodiments, the IL-12beta subunit is human IL-12beta subunit. In some embodiments, the IL-12beta subunit is a mouse IL-12beta subunit. In some embodiments, the IL-12 alpha and beta subunits are linked by a polypeptide linker sequence.

In some embodiments, the amino acid sequence of an IL-12 subunit (eg, alpha or beta) can be modified. In some embodiments, the IL-12 subunit includes one or more amino acid variants, such as substitutions, deletions, insertions, and/or substitutions, as compared to the sequence of a wild-type molecule, such as any of the IL-12 subunits described herein. or mutation. Exemplary variants include those designed to increase binding affinity to the IL-12 receptor and/or other biological properties of the IL-12 subunit (eg, protein stability). Amino acid sequence variants of the IL-12 subunits can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the IL-12 alpha and/or beta subunits or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the IL-12 alpha and/or beta subunit amino acid sequence. Any combination of such deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as specific binding to the IL-12 receptor.

In some embodiments, the IL-12α subunit has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% identical to SEQ ID NO: 15 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the IL-12 beta subunit has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% identical to SEQ ID NO: 16 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the IL-12 alpha and beta subunits are linked by a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 17). In some embodiments, the IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

In some embodiments, the fusion proteins described herein comprise a membrane tethered region. In some embodiments, the membrane tethered region is the transmembrane region of a transmembrane protein (eg, CD4). In some embodiments, the transmembrane region is the transmembrane domain of 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, B7 (eg, B7-1), CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of the T cell receptor, or any combination thereof. In some embodiments, the fusion protein comprises the transmembrane domain of a membrane immunoglobulin (mIg). In some embodiments, the fusion protein comprises a CD28, CD3-zeta, CD3-alpha or CD3-beta transmembrane domain. In some embodiments, the fusion proteins described herein comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more constant domains of an immunoglobulin. In some embodiments, the constant domain is a heavy chain constant domain. In some embodiments, the constant domain is a light chain constant domain. In some embodiments, the protein comprises 2 constant domains. In some embodiments, the two constant domains are CH2 and CH3 domains. In some embodiments, the two constant domains are CH1 and CH3 domains. In some embodiments, the two constant domains are CH1 and CH2 domains. In some embodiments, the two constant domains are both CH1 domains. In some embodiments, the two constant domains are both CH2 domains. In some embodiments, the two constant domains are both CH3 domains. In some embodiments, the protein comprises the entire Fc region of an immunoglobulin. In some embodiments, the protein comprises 3 constant domains. In some embodiments, the three constant domains are two CH2 and one CH3. In some embodiments, the three constant domains are sequentially linked as CH2-CH2-CH3. In some embodiments, the three constant domains are linked sequentially as CH1-CH2-CH3. In some embodiments, the protein comprises 1, 2, 3, 4, 5 or more CH2 domains. In some embodiments, the protein comprises 1, 2, 3, 4, 5 or more CH3 domains. In some embodiments, the immunoglobulin is a human immunoglobulin. In some embodiments, the immunoglobulin is a mouse immunoglobulin. In some embodiments, the immunoglobulin is an immunoglobulin G (IgG), IgM, IgE, IgA, or IgD molecule. In some embodiments, the immunoglobulin is IgGl, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin is human IgG4. In some embodiments, the constant domains are from the same immunoglobulin class (eg, IgG, IgM, IgA). In some embodiments, the constant domains comprise CH2, CH3 and CH4 of IgM.

In some embodiments, the constant domains are wild-type constant domains from humans. In some embodiments, the constant domains are mutated human constant domains. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain. In some embodiments, the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids are mutated in the CH2 domain. In some embodiments, at least or about 1%, 2%, 3%, 4%, 5%, 6%, or 7% of all amino acid residues of the CH2 domain are mutated. In some embodiments, the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the mutated constant domains described herein (eg, hmCH2) have reduced binding affinity for soluble FcyRs compared to the corresponding wild-type constant domains. In some embodiments, a mutated constant domain (eg, hmCH2) described herein has enhanced T cell persistence in vivo as compared to a corresponding wild-type constant domain. In some embodiments, the mutations in the CH2 domain are L235E and/or N297Q according to EU numbering. In some embodiments, the mutation includes one or more of the following: N297A, N297Q, or N297G (EU numbering). In some embodiments, the mutation is L234A/L235A(LALA) (EU numbering). In some embodiments, the mutation is F234A and or L235A. In some embodiments, the mutation includes one or more of the following: H268Q, V309L, A330S and/or P331S (EU numbering). In some embodiments, the mutation includes one or more of the following: V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S. A detailed description can be found in, eg, Jonnalagadda et al., "Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy." Molecular Therapy 23.4 (2015): 757-768; and US Patent No. 9,914,909 B2, Each of these documents is incorporated herein by reference in its entirety.

In some embodiments, the CH2 domain described herein comprises, or has at least 80%, 85%, 90%, 91%, 92%, of the amino acid sequence set forth in any one of SEQ ID NO: 41 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CH2 domain described herein consists of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with SEQ ID NO: 42 , 98% or 99% identical amino acid sequence encoding. In some embodiments, the CH3 domain described herein comprises, or has at least 80%, 85%, 90%, 91%, 92%, of the amino acid sequence set forth in any one of SEQ ID NO: 43 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CH3 domain described herein consists of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with SEQ ID NO: 44 , 98% or 99% identical amino acid sequence encoding.

In some embodiments, the constant domain is linked to the IL-12 subunit through a hinge region. In some embodiments, the amino acid sequences of the constant domains described herein can be modified. In some embodiments, the constant domain (eg, CH2 or CH3) includes one or more amino acid variants, eg, substitutions, as compared to the sequence of the wild-type molecule (eg, any of the constant domains described herein). , deletions, insertions and/or mutations.

In some embodiments, the fusion protein is a membrane tethered protein. In some embodiments, the membrane tethered region is fused to a constant domain as described herein. In some embodiments, the membrane-tethered IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the membrane-tethered IL-12 fusion proteins described herein have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein comprises a message peptide (eg, a human message polypeptide). In some embodiments, the message peptide sequence is a secretion message peptide.

Another strategy to improve the safety of IL-12 is to direct its delivery to tumors by fusion to tumor-targeting antibodies. Such antibody-interleukin fusion proteins or "immune interleukins" have previously demonstrated the ability to enhance anti-tumor immunity in preclinical models. To maximize immune interleukin tolerance, antibodies chosen as vehicles typically bind specifically to tumor-associated antigens. For example, antibodies directed against necrosis-associated antigens that are abundant in tumors but not in normal tissues provide an attractive delivery method. In one aspect, the antibody-interleukin fusion proteins are expressed in tumor-targeted cells (eg, immune cells, T cells, NK cells, tumor-infiltrating cells); thus, the antibody-interleukin fusion proteins are in the target site-specific performance, which further enhances their safety.

In some embodiments, the fusion protein comprises a tumor-targeting antibody or antigen-binding fragment thereof. Tumor-targeted antibodies or antigen-binding fragments can deliver IL-12 to the target site, thereby further reducing the side effects of IL-12. In some embodiments, the fusion protein is not a membrane tethered protein and/or does not have a transmembrane protein. Tumor-targeting antibodies or antigen-binding fragments can target tumor-associated antigens. As used herein, the term "tumor-associated antigen" refers to an antigen that is or can be presented on the surface of a tumor cell and that is located on or within a tumor cell. In some other embodiments, tumor-associated antigens may be expressed exclusively on tumor cells, or may express tumor-specific mutations as compared to non-tumor cells. In some other embodiments, tumor-associated antigens may be found in both tumor cells and non-tumor cells, but are overexpressed on tumor cells when compared to non-tumor cells, or due to the presence of tumor tissue compared to non-tumor tissue Less compact structure and easier antibody binding in tumor cells. In some embodiments, the tumor-associated antigen is located on the vasculature of the tumor. Illustrative examples of tumor-associated surface antigens include CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135 ), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activation protein (FAP), CDCP1 , Derlin1, tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR/FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1-8 and Tie2, A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN/CA IX), de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, folate binding protein, G250, Fms Like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate) proteoglycan), Muc-1, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA) and TAG-72.

In some embodiments, provided herein are fusion proteins comprising an IL-12α subunit and an IL-12β subunit. In some embodiments, the protein further comprises the heavy and light chain variable regions of the tumor-targeted antibody or antigen-binding fragment thereof. In some embodiments, the tumor-targeting antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the antibody targeting tumor necrosis is a human antibody. In some embodiments, the antibody targeting tumor necrosis is human IgGl. In some embodiments, the antibody targeting tumor necrosis is NHS76. NHS76 is a fully human, phage-display-derived IgG1 antibody selected for its specific ability to bind necrotic regions and thus target tumors in vivo. Detailed descriptions can be found, for example, in Fallon et al., "The immunocytokine NHS-IL12 as a potential cancer therapeutic." Oncotarget 5.7 (2014): 1869; and Sharifi et al., "Characterization of a phage display-derived human monoclonal antibody counterpart (NHS76) to chimeric TNT-1 directed against necrotic regions of solid tumors." Hybridoma and hybridomics 20.5-6 (2001): 305-312; each of these references is incorporated herein by reference in its entirety. Thus, in some embodiments, the antibody-interferon fusion protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. T cell receptors and binding molecules

T cells are a type of lymphocyte that normally develops in the thymus and plays a major role in the adaptive immune response. What distinguishes T cells from other lymphocytes is the presence of T cell receptors on the cell surface. Differentiated T cells have an important role in controlling the immune response. CD8+ T cells (also known as "killer cells") are cytotoxic. Once they identify the target cell, they are able to directly kill the target cell (eg, virus-infected cells or cancer cells). CD8+ T cells also produce interferons and recruit other cells (eg, macrophages and natural killer (NK) cells) to initiate immune responses. CD4+ T cells (also known as "helper cells") can kill target cells indirectly, eg, by promoting the maturation of B cells into plasma cells and memory B cells and by activating cytotoxic T cells and macrophages. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist the immune response. Regulatory T cells are important for tolerance, preventing or suppressing autoimmune responses. The main role of regulatory T cells is to shut down T cell-mediated immunity at the end of the immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus.

T cells play an important role in cancer immunity, where antigens from cancer cells are taken up and presented on the cell surface of specialized immune cells called antigen-presenting cells (APCs), allowing other immune cells to recognize the antigen. In lymph nodes, APCs activate T cells and activate them to recognize tumor cells. The activated T cells can then travel through blood vessels to the tumor, where they infiltrate, recognize cancer cells and kill them.

Activation of T cells requires T cell receptors. "T cell receptors" or "TCRs" are those containing variable alpha (or alpha) chains and b (or beta) chains (referred to as TCRalpha and TCRbeta, respectively) or variable g (or gamma) chains and d (or delta) chains A molecule that chains (also referred to as TCRγ and TCRδ, respectively) or antigen-binding portions thereof and is capable of specifically binding an antigen (eg, a peptide antigen or peptide epitope bound to a major histocompatibility complex (MHC) molecule).

The present disclosure provides T cell receptors (TCRs) or antigen-binding fragments thereof, and binding molecules derived from TCRs. In some embodiments, the TCR is in the form of ab. TCRs in the αβ and γδ forms are often structurally similar, but the T cells that express them may have unique anatomical locations or functions. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are typically responsible for the recognition of antigens (such as peptides bound to major histocompatibility complex (MHC) molecules).

In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing an a-chain and a b-chain. In some embodiments, the TCR is an antigen-binding portion that is smaller than a full-length TCR but binds to a specific peptide bound in an MHC molecule, such as to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may contain only a portion of the structural domain of a full-length or intact TCR, but still be capable of binding a peptide epitope (such as an MHC-peptide complex) to which the intact TCR binds. In some cases, the antigen-binding portion contains a variable domain of a TCR (such as the variable a (Va or Vα) chain and variable b (Vb or Vβ) chain of a TCR, or is sufficient to form a TCR for interaction with a particular MHC -An antigen-binding fragment of the binding site to which the peptide complex binds.

The variable domains of TCRs contain complementarity determining regions (CDRs), which are generally the major contributors to antigen recognition and binding capacity and specificity of peptides, MHCs and/or MHC-peptide complexes. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen binding site of a given TCR molecule. The various CDRs within the variable regions of a TCR chain are typically separated by framework regions (FRs), which typically show less variability between TCR molecules than the CDRs. In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or the processed peptide among the three CDRs on the variable region of a given TCR for antigen recognition and/or for peptide-MHC complexes Some interactions are the most important. In some contexts, CDR1 of the alpha chain can interact with the N-terminal portion of certain antigenic peptides. In some cases, CDR1 of the beta chain can interact with the C-terminal portion of the peptide. In some contexts, CDR2 most strongly facilitates the interaction with or recognition of the MHC portion of the MHC-peptide complex, or is responsible for interacting with the MHC portion of the MHC-peptide complex Or the major CDRs that recognize the MHC portion of the MHC-peptide complex.

The a-chain and/or b-chain of the TCR may also contain constant domains, transmembrane regions and/or short cytoplasmic tails. In some aspects, each chain (eg, alpha or beta) of a TCR can possess an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction, eg, via a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3 and its subunits. For example, TCRs comprising constant domains with transmembrane regions can anchor proteins in the cell membrane and associate with the invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (eg, CD3γ, CD3δ, CD3e, and CD3z chains) contain one or more immunoreceptor tyrosine-based activation motifs, or ITAMs, and are typically involved in the signaling capacity of the TCR complex.

The exact location of a domain or region can vary depending on the particular structure or homology modeling or other features used to describe the particular domain. It should be understood that references to amino acids (including specific sequences listed as SEQ ID NOs used to describe the domain organization of the TCR) are for illustrative purposes and are not meant to limit the scope of the provided embodiments. In some cases, a particular domain (eg, variable or constant) may be several amino acids longer or shorter (such as one, two, three, or four). In some aspects, the residues of the TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see, eg, www.imgt.org; Lefranc et al., "IMGT unique numbering" for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77). The structures and variations of TCRs are known in the art and are described, for example, in WO 2019/195486, which is incorporated herein by reference in its entirety.

In some embodiments, the a and b chains of the TCR each further contain a constant domain. In some embodiments, the a-chain constant domain (Ca) and b-chain constant domain (Cb) are individually mammalian constant domains, such as human constant domains or non-human constant domains (eg, mouse constant domains) domain). In some embodiments, the constant domains are adjacent to the cell membrane. For example, in some cases, the extracellular portion of a TCR formed from two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains a CDR.

In some aspects, a TCR as described herein can contain human constant regions, such as an alpha chain containing a human Ca region and a beta chain containing a human Cb region. In some embodiments, the TCR is fully human. In some embodiments, the expression and/or activity of the TCR, such as when expressed in human cells (eg, human T cells, such as primary human T cells), is not or substantially unaffected by endogenous human TCRs .

In some embodiments, when engineered compared to the level of expression, functional activity and/or anti-tumor activity of the same TCR in similar human cells (but in which the expression of endogenous TCRs has been reduced or eliminated) The engineered TCRs are expressed at similar or improved levels on the cell surface and exhibit similar or greater functional activity when expressed by human cells (such as human T cells) that contain or express endogenous human TCRs (eg, cytolytic activity) and/or exhibit similar or greater antitumor activity. In some instances, when expressed in human T cells, an engineered TCR as described herein is expressed on the cell surface at levels that are comparable when expressed in similar human T cells (but where endogenous At least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or at least about the performance level of the same TCR at the time when the performance of the sexual TCR has been reduced or eliminated) 120%.

In some embodiments, each of the Ca domain and the Cb domain is human. In some embodiments, Ca is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, the variant of Ca contains at least one substitution of unnatural cysteine.

In some embodiments, the TCR may be a heterodimer such as two chains a and b linked by one or more disulfide bonds. In some embodiments, the constant domains of the TCR may contain short linking sequences in which the cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine residues in each of the a and b chains, such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, each of the constant and variable domains contain disulfide bonds formed by cysteine residues.

In some embodiments, the TCR comprises CDRs, Va and/or Vb and constant region sequences as described herein.

In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the dTCR comprises a first polypeptide in which the sequence corresponding to the provided TCR alpha chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR alpha chain constant region extracellular sequence, and in which the provided sequence corresponds to the TCR alpha chain constant region extracellular sequence. The sequence corresponding to the provided sequence of the variable region of the TCR b chain is fused to a second polypeptide at the N-terminus of the sequence corresponding to the extracellular sequence of the constant region of the TCR b chain, the first polypeptide and the second polypeptide passing through two Sulfur bond.

In some embodiments, the TCR may be cell-bound or in a soluble form. In some embodiments, the TCR is in a cell-bound form expressed on the cell surface.

In some embodiments, the TCR is a single-chain TCR (scTCR). The scTCR is a monoamino acid chain containing a and b chains capable of binding to the MHC-peptide complex. In general, scTCRs can be generated using methods known to those skilled in the art. These methods are described, for example, in WO 96/13593, WO 96/18105, WO 99/18129, WO 04/033685, WO2006/037960, WO2011/044186; WO 2019/195486; US Patent No. 7,569,664; each of these patents The entire contents are incorporated herein by reference.

TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules can bind or recognize peptide epitopes associated with an antigen of interest (eg, a tumor-associated antigen). In some embodiments, the antigen may be a peptide epitope expressed on the surface of cancer cells and/or virus-infected cells. In some embodiments, the antigen is presented in the context of an MHC molecule. Such binding molecules include, for example, T cell receptors (TCRs) and antigen-binding fragments thereof, antibodies and antigen-binding fragments thereof, and TCR-like CARs. They exhibit antigen specificity for binding or recognizing such peptide epitopes. In some aspects, engineered cells expressing a provided binding molecule (eg, TCR or antigen-binding fragment) express cellular Toxic activity.

In some aspects, TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules recognize or bind epitopes in the context of MHC molecules, such as MHC class I molecules or MHC class II molecules. Both MHC class I molecules or MHC class II molecules are human leukocyte antigens (HLA). They act as an important component of the adaptive immune system. HLA expression is controlled by genes located on chromosome 6. It encodes a cell surface molecule that specifically presents antigenic peptides to T cell receptors on T cells.

In some embodiments, TCRs, antigen-binding fragments thereof, and TCR-derived binding molecules recognize or bind epitopes in the context of MHC class I molecules. MHC class I molecules are human leukocyte antigen (HLA)-A2 molecules, including any one or more subtypes thereof, such as HLA-A *0201, *0202, *0203, *0206, or *0207. Human leukocyte antigen A2 (HLA-A2) is one of the most common human serotypes. In some cases, the frequency of subtypes may vary between different groups. For example, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency of HLA-A*0201 has been reported to be approximately 23% and the frequency of HLA-A*0207 to be 45% , the frequency of HLA-A*0206 was 8%, and the frequency of HLA-A*0203 was 23%. In some embodiments, the MHC molecule is HLA-A*0201.

In some embodiments, a binding molecule (eg, a TCR or antigen-binding fragment or TCR-derived binding molecule) is isolated or purified, or recombinant. In some aspects, the binding molecule (eg, TCR or antigen-binding fragment or TCR-derived binding molecule) is fully human. In some embodiments, the binding molecule is monoclonal. In some aspects, the binding molecule is single-stranded. In other embodiments, the binding molecule contains two chains. In some embodiments, the binding molecule (eg, TCR, antigen-binding fragment thereof, or TCR-derived binding molecule) is expressed on the cell surface.

In some embodiments, the TCR, antigen-binding fragment thereof, or TCR-derived binding molecule specifically binds a tumor-associated antigen, eg, BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R), CEA , IL13Ra2, ALPP, EBV-associated antigen (eg, LMP2), EGFR, EGFRvIII, GD2, GPC3, HER2, HPV-associated antigen (eg, E6 or E7), MAGE antigen, mesothelin, MUC-1, NY-ESO- 1. PSCA, PSMA, ROR1, WT1 or Claudin 18.2.

A TCR, antigen-binding fragment thereof, or TCR-derived binding molecule can have Va and Vb, or a region similar to Va and a region similar to Vb. In some embodiments, the TCR binds latent membrane protein 2 (LMP2) of Epstein Barr virus (EBV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the TCR binds the tumor antigen E6 of human papillomavirus (HPV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the TCR binds the tumor antigen E7 of human papillomavirus (HPV). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the TCR binds NY-ESO-1 (cancer/testis antigen 1, also known as LAGE2). In some embodiments, the Va region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the Vb region comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the Va region comprises one or more Va CDR sequences as described herein. In some embodiments, the Vb region comprises one or more Vb CDR sequences as described herein.

In some embodiments, the nucleic acid encoding the alpha chain and the nucleic acid encoding the beta chain can be linked by a linker, such as any of the linkers described elsewhere herein.

In some embodiments, the TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule can activate T cells by binding to the antigen of interest (eg, by activating the TCR signaling pathway). In some embodiments, activation can upregulate the immune response, increase the expression of cytokines (eg, IFNy) and/or CD107a, promote T cell proliferation and T cell mediated killing.

In some embodiments, a TCR or antigen-binding fragment thereof or TCR-derived binding molecule as described herein can increase the immune response, activity or number of T cells by at least 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, 100%, 2X, 3X, 5X, 10X or 20X. In some embodiments, the TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule can increase the serum concentration of IFN-γ in the presence of the antigen of interest. In some embodiments, activation can induce at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5x, 10x, 100x or 1000x. In some embodiments, activation can induce at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold increase in specific killing of target cells , 3 times, 4 times or 5 times.

In some aspects, provided recombinant TCRs include TCRs that are at least partially independent of CD8. In some aspects, provided recombinant TCRs include TCRs that are at least partially dependent on CD8.

In some embodiments, the TCR or antigen-binding fragment thereof or TCR-derived binding molecule has a relatively high expression efficiency. For example, a TCR, or antigen-binding fragment thereof, or TCR-derived binding molecule described herein can perform at least 10%, 20%, 30%, 40% higher than a reference molecule (eg, an endogenous TCR) under the same conditions %, 50% or 100%.

In some embodiments, the binding molecules (eg, TCRs) do not exhibit cross-reactivity or off-target binding, such as undesired off-target binding, eg, off-target binding to antigens present in healthy or normal tissues or cells. Chimeric Antigen Receptors and Binding Molecules

Chimeric antigen receptors (CARs) combine many aspects of normal T cell activation into a single protein. They link extracellular antigen recognition domains to intracellular signaling domains that activate T cells when bound to antigen. CAR is usually composed of four regions: antigen recognition domain, extracellular hinge region, transmembrane domain and intracellular T cell signaling domain.

The antigen recognition domain is exposed outside the cell in the extracellular domain portion of the receptor. It interacts with potential target molecules and is responsible for targeting CAR-T cells to any cell that expresses the matching molecule. Antigen recognition domains are typically derived from the variable regions of monoclonal antibodies linked together as single-chain variable fragments (scFvs). scFvs are chimeric proteins consisting of the light (VL) and heavy (VH) chains of immunoglobulins linked with a short linker peptide. The linker between the two chains consists of hydrophilic residues, with a stretch of glycine and serine for flexibility and a stretch of glutamic and lysine for solubility. In some embodiments, the antigen binding domain specifically binds a tumor-associated antigen, eg, BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R), CEA, EBV-associated antigen (eg, LMP2) , EGFR, GD2, GPC3, HER2, HPV-associated antigen (eg, E6), MAGE antigen, mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.

A hinge, also known as a spacer, is a domain of a small structure located between the antigen-knot recognition region and the outer cell membrane. An ideal hinge enhances the flexibility of the scFv receptor head, thereby reducing the steric confinement between the CAR and its target antigen. This promotes antigen binding and synapse formation between CAR-T cells and target cells. Hinge sequences are often based on membrane-proximal regions from other immune molecules, including IgG, CD8, and CD28.

The transmembrane domain is a structural component consisting of hydrophobic alpha helices that span the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen recognition domain with the intracellular signaling region. This domain is critical for the stability of the entire receptor. Typically, the transmembrane domain from the closest membrane component of the intracellular domain is used, but different transmembrane domains result in different receptor stability. The CD28 transmembrane domain is known to result in a highly expressed stable receptor. In some embodiments, the transmembrane domain is the transmembrane domain of 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of the T cell receptor, or any combination thereof.

The intracellular T cell signaling domain is located in the receptor intracellular domain inside the cell. After antigen binding to the external antigen recognition domain, CAR receptors come together and transmit activation messages. The inner cytoplasmic end of the receptor then perpetuates the signaling inside the T cell. Normal T cell activation relies on phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) present in the cytoplasmic domain of CD3-ζ. To mimic this process, the cytoplasmic domain of CD3-ζ is typically used as the main CAR intracellular domain component. Other ITAM-containing domains were also tried, but not as effective. T cells require co-stimulatory molecules in addition to CD3 signaling in order to persist after activation. For this reason, the intracellular domains of CAR receptors often also include one or more chimeric domains from co-stimulatory proteins. Messaging domains from a variety of costimulatory molecules have been successfully tested, including CD28, CD27, CD134 (OX40) and CD137 (4-1BB).

A variety of CAR molecules and vectors expressing these CAR molecules can be used in the methods described herein. In some embodiments, the CAR molecule specifically binds a tumor-associated antigen, such as BCMA. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, Amino acid sequences of 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the CAR molecule specifically binds CD19. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, of the amino acid sequence set forth in any one of SEQ ID NO: 36 %, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the CAR molecule specifically targets IL13Ra2. In some embodiments, the CAR comprises, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, of the amino acid sequence set forth in any one of SEQ ID NO: 40 %, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. Some of these CAR molecules or target binding domains thereof are described, for example, in WO2018200496A1, WO2019241686A1, WO2018085690A1, WO2018028647A1, and WO2018052828A1, each of which is incorporated herein by reference in its entirety.

Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells include those described, for example, in Chandran et al., "T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.” Immunological reviews 290.1 (2019): 127-147; Cartellieri, Marc et al., “Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.” BioMed Research International 2010 (2010); and PCT Publication No. WO2017173256A1; US2002 /131960, US2013/287748, US2013/0149337, U.S. 6,451,995, U.S. 7,446,190, U.S. 8,252,592; each of these documents are incorporated herein by reference in their entirety.

In some embodiments, the CARs described herein comprise antibody mimetics. Antibody mimetics are compounds that behave in a similar manner to antibodies and bind a specific antigen but are unrelated to antibodies (eg, antibody fragments). In some embodiments, the antibody mimetic is a peptide, nucleic acid, small molecule, or a combination thereof. In some embodiments, the antibody mimetic is an avidin molecule (eg, the Z domain of protein A), adnectin, a monomer (eg, the class 10 III domain of fibronectin), a peptide aptamer, a cohesin ( affimer) (eg, cystatin), affilin (eg, gamma-B crystallin or ubiquitin), affitin (eg, Sac7d), alpha body (eg, triple helix coiled-coil), anticalin (eg, lipocalin) protein), avimer (eg, A domain of various membrane receptors), fynomer (eg, SH3 domain of Fyn), armadillo repeat protein, DARPin (eg, ankyrin repeat motif), Kunitz Domain peptides (eg, Kunitz domains of various protease inhibitors), designed ankyrin repeat proteins, nanoCLAMPs (eg, carbohydrate binding module 32-2), cysteine knot peptides (knottins), or combinations thereof . Detailed descriptions can be found, for example, in Yu et al ., "Beyond antibodies as binding partners: the role of mimetics in bioanalysis." Annual Review of Analytical Chemistry 10 (2017): 293-320; and Ta and Brian, "Antibody and antibody mimetic antibody immunotherapeutics ." (2017): 1301-1304; each of these documents is incorporated by reference in its entirety. engineered cells

The present disclosure provides engineered cells (eg, immune cells, T cells, NK cells, tumor infiltrating cells) expressing IL-12 (eg, membrane-tethered IL-12), TCR, CAR, and/or various proteins as described herein lymphocytes). These engineered cells can be used to treat various disorders or diseases as described herein (eg, viral infections, cancer, virus-induced disorders).

In various embodiments, the engineered cells can be obtained from, for example, humans and non-human animals. In various embodiments, the engineered cells can be obtained from bacteria, fungi, human, rat, mouse, rabbit, monkey, pig, or any other species. Preferably, the cells are from human, rat or mouse. In some embodiments, the cells are mouse lymphocytes and are engineered (eg, transduced) to express TCRs, CARs, or antigen-binding fragments thereof. In some embodiments, the cells are obtained from a human. In various embodiments, the engineered cells are blood cells. Preferably, the cells are leukocytes (eg T cells), lymphocytes or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are T cells, B cells, or NK cells. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs). In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells.

In some embodiments, the cells are T cells. In some embodiments, T cells may express cell surface receptors that recognize specific antigenic moieties on the surface of target cells. Cell surface receptors can be wild-type or recombinant T cell receptors (TCRs), chimeric antigen receptors (CARs), or any other surface receptors capable of recognizing antigenic moieties associated with target cells. T cells can be obtained by various methods known in the art, eg, by in vitro culture of T cells (eg, tumor-infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (eg, isolated from the peripheral blood of a patient) with a viral vector. In some embodiments, the T cells are TCR gene-modified or CAR-modified T cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are type 1 T helper cells and type 2 T helper cells. In some embodiments, the T cells expressing this receptor are αβ-T cells. In an alternative embodiment, the T cells expressing this receptor are γδ-T cells. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are naive T cells.

In some embodiments, the cells are NK cells. In some embodiments, the preparation of engineered cells includes one or more culturing and/or preparation steps. The cells used to introduce the binding molecule (eg, TCR or CAR) can be isolated from a sample, such as a biological sample, eg, obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of, or to which cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

In some embodiments, the cells are stem cells, such as pluripotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells may be primary cells, such as those cells isolated directly from the subject and/or isolated from the subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain the desired cell type (eg, T cells).

Different cell types can be obtained from appropriate isolation methods. The methods of separation include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, eg, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is an affinity or immunoaffinity-based separation. For example, in some aspects, the isolating comprises, for example, the expression or level of expression of cells based on, for example, one or more markers (usually cell surface markers), by incubating with antibodies or binding partners that specifically bind such markers, followed by usually It is the washing steps and separation of cells and cell populations that separate cells that have bound the antibody or binding partner from cells that have not bound the antibody or binding partner.

Such isolation steps may be based on positive selection, in which cells that have bound the reagent are retained for further use, and/or based on negative selection, in which cells not bound to the antibody or binding partner are retained. In some instances, both fractions are reserved for further use. In some aspects, negative selection may be particularly useful where no antibodies are available that specifically identify cell types in a heterologous population such that separation is best performed based on markers expressed by cells other than the desired population.

Also provided are methods, nucleic acids, compositions and kits for expressing binding molecules and for producing genetically engineered cells expressing such binding molecules. Genetic engineering typically involves transduction, transfection, or transformation, such as by retroviral transduction, encoding a therapeutic molecule (eg, TCR, CAR (eg, TCR-like CAR), IL-12 (eg, membrane-tethered IL-12), polypeptide, fusion protein) nucleic acid into cells. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with an induction response (such as proliferation, survival, and/or activation, eg, as measured by the expression of interleukins or activation markers) The combination of stimuli, activated cells are then transduced and expanded in culture to a sufficient number for clinical use.

In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant lentiviral or retroviral vectors (such as gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells. In some embodiments, the retroviral vector has long terminal repeats (LTR), eg, derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Retroviral vector for stem cell virus (MSCV) or spleen foci forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses are generally amphiphilic, which means that they are capable of infecting host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, recombinant nucleic acids are transferred into T cells by electroporation. In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment and strontium phosphate DNA coprecipitation. Many of these methods are described, for example, in WO2019195486, which is incorporated herein by reference in its entirety.

In some aspects, the development of humanized and/or fully human recombinant TCRs presents technical challenges. For example, in some aspects, humanized and/or fully human recombinant TCR receptors, when engineered into human T cells, can compete with endogenous TCR complexes and/or can compete with endogenous TCRα chains and/or The TCRb chains form mispairings, which in some ways may reduce recombinant TCR signaling, activity, and/or expression, and ultimately lead to reduced activity in the engineered cells. The engineered cells can be genetically modified. In some embodiments, the engineered cells may comprise genetic disruption of the T cell receptor alpha constant (TRAC) gene and/or the T cell receptor beta constant (TRBC) gene. In some embodiments, the TRBC gene is one or both of the T cell receptor beta constant 1 (TRBCJ) or T cell receptor beta constant 2 (TRBC2) genes. In some embodiments, the engineered cells do not express endogenous TCR alpha chains and/or TRC beta chains. In some other aspects, non-human constant domains are used, eg, rodent (eg, mouse) constant domains. The use of non-human constant domains can effectively reduce the possibility of pairing errors.

Also provided are populations of engineered cells, containing such cells and/or enriched for such cells (such as in which cells expressing the binding molecule constitute at least 15%, 20%, 25%, 30%, 35%, 40%, 50%) , 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentages of the total in the composition A composition of cells or cells of a certain type, such as T cells, CD8+ or CD4+ cells.

In some embodiments, engineered cells (eg, TCR-T cells) are co-cultured with target cells (eg, antigen-presenting cells) for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours , 12 hours, 18 hours, 1 day, 2 days, 3 days, or longer, so that engineered cells (eg, TCR-T cells) can be activated. In some embodiments, the target cells are Jurkat cells. In some embodiments, the target cells are LLC-HLA-A2-Peplinker (LLW) melanoma cells. In some embodiments, the target cells are antigen presenting cells. In some embodiments, the target cell expresses a major histocompatibility complex (MHC) (eg, class I, class II, and/or class III MHC). In some embodiments, the target cells comprise the human leukocyte antigen (HLA) system.

In some embodiments, the engineered cells can express IL-12 and modified IL-12. For example, a fusion protein comprising modified IL-12 described herein can be expressed on the cell surface of an engineered cell, eg, when the fusion protein is a membrane-tethered protein. In some cases, such as when the fusion protein is a soluble protein, a fusion protein comprising the modified IL-12 described herein can be expressed and secreted.

The expression of IL-12 in engineered cells provides several additional benefits. For example, it can increase the production of IFN-γ (which is the strongest mediator of IL-12 action) from NK and T cells, stimulate the growth and cytotoxicity of activated NK cells, CD8+ and CD4+ T cells, reduce the production of CD4+ Th0 cells Differentiation changes towards a Th1 phenotype, increases antibody-dependent cytotoxicity (ADCC) against tumor cells, and induces IgG and inhibition of IgE production from B cells, eg, at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold times, 10 times or 20 times.

In some embodiments, co-cultivation with target cells can increase the secretion of interleukins (eg, IFNγ) by the engineered cells by at least or about 1 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times or more.

In some embodiments, the expression of modified IL-12 may allow engineered cells (eg, TCR-T cells) to compare to the level of interferon expression or secretion of engineered cells that do not express modified IL-12 ) at least or about a 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000 times, 10000 times or more. In some embodiments, the modified IL in an engineered cell (eg, a TCR-T cell) is compared to the level of cytokine expression or secretion in cells adjacent to the engineered cell that does not express the modified IL-12 -12 expression can stimulate at least or about a 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold increase in interferon (eg, IFNγ) expression or secretion by immune cells adjacent to the engineered cells times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times or more. In some embodiments, one or more early TCR activation markers (eg, CD69) of immune cells in the vicinity of engineered cells that do not express modified IL-12 or engineered cells that do not express modified IL-12 Modified IL-12 expression may increase the expression of one or more early TCR activation markers by engineered cells (e.g., TCR-T cells) or immune cells adjacent to the engineered cells by at least or about the expression level of 1x, 2x, 5x, 10x, 20x, 50x, 100x, 200x, 500x, 1000x or more.

In some embodiments, the cells are human PBMCs and are engineered (eg, transduced) to express IL-12, TCR, CAR, or antigen-binding fragments thereof. In some embodiments, the engineered cells may also express modified IL-12 as described herein. In some embodiments, the modified IL-12 is tethered to the membrane of the engineered cell. In some embodiments, when engineered cells (eg, PBMCs, TCRb+PBMCs, or TCRb-PBMCs) are co-cultured with target cells (eg, antigen-presenting cells), engineered cells that do not express membrane-tethered IL-12 Membrane-tethered IL-12 can increase the expression or secretion of interferons (e.g., IFNγ) by at least or about 10%, 20%, 30%, 40% in engineered cells compared to the level of interleukin expression or secretion , 50%, 60%, 70%, 80%, 90%, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times times, 80 times, 90 times, 100 times or more. In some embodiments, when engineered cells (eg, PBMCs or TCRb-PBMCs) are co-cultured with target cells (eg, antigen-presenting cells), activated T cells in engineered cells that do not express membrane-tethered IL-12 Membrane-tethered IL-12 can increase activated T cell population by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold compared to cell population , 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or more. In some embodiments, T cell activation status can be measured by the level of CD69 expression.

In some embodiments, membrane-tethered IL-12 enables a population of CD4+ T cells in engineered cells (eg, PBMCs) as compared to a population of CD4+ T cells in engineered cells that do not express membrane-tethered IL-12 Increase at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20 times, 50 times, 100 times or more. In some embodiments, membrane-tethered IL-12 enables a population of CD8+ T cells in engineered cells (eg, PBMCs) as compared to a population of CD8+ T cells in engineered cells that do not express membrane-tethered IL-12 Reduce to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less.

In some embodiments, membrane-tethered IL-12 can increase IL-12 in engineered cells (eg, CD4+ PBMC or CD8+ PBMC) as compared to a population of effector memory cells in engineered cells that do not express membrane-tethered IL-12 Increase in effector memory cell population by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6x, 7x, 8x, 9x, 10x, 20x, 50x, 100x or more. In some embodiments, membrane-tethered IL-12 can increase the number of cells in an engineered cell (eg, CD4+ PBMC or CD8+ PBMC) compared to a population of central memory cells in engineered cells that do not express membrane-tethered IL-12 The central memory cell population is reduced to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less.

In some embodiments, cells (eg, PBMCs) are engineered to express a membrane-tethered IL-12 fusion protein comprising a soluble moiety. In some embodiments, the soluble fraction comprises IL-12A and/or IL-12B. In some embodiments, the soluble portion comprises a sequence at least or about 50%, 60%, 70%, 80%, 90%, or 100% identical to SEQ ID NO: 8. In some embodiments, the membrane-tethered IL-12 fusion protein cannot be released from the cell. In some embodiments, the level of IL-12 from cells expressing a membrane-tethered IL-12 fusion protein is less than 10%, 5%, 4% in culture medium compared to cells engineered to express soluble IL-12 , 3%, 2%, 1% or less. In some embodiments, the concentration of IL-12 in the culture medium is less than 300 pg/ml, 200 pg/ml or 100 pg/ml as detected by ELISA.

In some embodiments, the engineered cells can express chemokines such as CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, XCL1 or XCL2. In some embodiments, the chemokine is CXCL10 or XCL1. In some embodiments, the engineered cells express FLT3L. In some embodiments, expression is under the control of an exogenous regulatory element (eg, a promoter) as described herein. Chemokines and FLT3L increase the activity of antigen-presenting cells. Antigen-presenting cells can present many tumor antigens to host immune cells, such that the host immune cells will recognize these tumor antigens and kill these tumor cells, even if these tumor cells do not express the antigen recognized by CAR or TCR. In some embodiments, chemokines (eg, CXCL10) and/or FLT3L can enhance memory T cell function and thus provide long-term protection against tumors. In some embodiments, the host immune cells can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, at least 11, or at least 11 after treatment of the subject with the engineered cells. Effectively kills tumor cells after at least 12 months.

Accordingly, in one aspect, the present disclosure provides a method of improving immune cell therapy (eg, TCR-T, CAR-T). In some embodiments, immune cells are engineered to express IL-12 (eg, membrane-tethered IL12) in addition to the TCR or CAR. In some embodiments, the cells are further engineered to express chemokines (eg, CXCL10) and/or FLT3L. recombinant vector

The present disclosure also provides recombinant vectors (eg, expression vectors) comprising an isolated polynucleotide disclosed herein (eg, a polynucleotide encoding a polypeptide disclosed herein), into which the recombinant vector is introduced (ie, such that a host cell contains the polynucleotide) and/or a polynucleotide-containing vector), and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

A vector is a construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering one or more polynucleotides of interest and expressing it as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, by operably linking at or near or flanking the integration site of the polynucleotide of interest with regulatory elements such as promoters, enhancers and/or poly A tails within the vector or in the genome of the host cell such that the polynucleotide of interest will Translation in the host cell introduced with the expression vector locates the polynucleotide of interest in the vector for expression.

Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (eg, DEAE-dextran), transformation, transfection, and infection and/or transduction (eg, with recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plastids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

The present disclosure provides recombinant vectors comprising nucleic acid constructs suitable for genetic modification of cells, which can be used to treat pathological diseases or disorders.

Any vector or type of vector can be used to deliver genetic material to cells. These vectors include, but are not limited to, plastid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors can include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy viral vectors, recombinant adeno-associated virus (AAV) vectors, hybrid vectors, and plastid transposons (eg, Sleeping Beauty). (sleeping beauty) transposon system and PiggyBac transposon system) or integrase-based vector system. Other vectors known in the art can also be used in conjunction with the methods described herein.

In some embodiments, the vector is a viral vector. Viral vectors can be grown in media specific for viral vector production. Any suitable growth media and/or supplements for growing viral vectors may be used in accordance with the embodiments described herein. In some embodiments, the MP71 vector is used.

In some embodiments, the vector used is a recombinant retroviral vector. Retroviral vectors are capable of directing the expression of nucleic acid molecules of interest. Retroviruses exist in their viral membranes as RNA and form double-stranded DNA intermediates when replicated in host cells. Similarly, retroviral vectors exist in both RNA and double-stranded DNA forms. Retroviral vectors also include DNA forms containing recombinant DNA fragments and RNA forms containing recombinant RNA fragments. The vector may include at least one transcriptional promoter/enhancer, or other elements that control gene expression. Such vectors may also include packaging information, long terminal repeats (LTRs) or portions thereof, and binding sites for plus-strand and minus-strand primers appropriate to the retrovirus used. Long terminal repeats (LTRs) are identical DNA sequences found at the ends of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA that are repeated many times (eg, hundreds or thousands of times). Viruses use them to insert their genetic material into the host genome. Optionally, the vector may also include a message directing polyadenylation, a selectable marker (such as ampicillin resistance, neomycin resistance, TK, hygromycin resistance, phleomycin resistance, histamine alcohol resistance, or DHFR), and one or more restriction sites and translation termination sequences. For example, such a vector can include a 5' LTR, a leader sequence, a tRNA binding site, packaging information, an origin for second strand DNA synthesis, and a 3' LTR or a portion thereof. In addition, the retroviral vector used herein may also refer to a recombinant vector produced by removing the retroviral gag, pol and env genes and replacing them with a gene of interest.

In some embodiments, a vector or construct may contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters may be polycistronic (bicistronic or tricistronic). For example, in some embodiments, the transcription unit can be a bicistronic unit engineered to contain an IRES (internal ribosome entry site) that allows for gene products (eg, the alpha chain encoding TCR and modified IL-12) and/or beta strands) are co-expressed by messages from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (eg, encoding the alpha and/or beta chains of the TCR) in a single open reading frame (ORF) ), the two or three genes are separated from each other by a sequence encoding a self-cleaving peptide (eg, P2A or T2A) or a protease recognition site (eg, furin). Thus, the ORF encodes a single polyprotein that is cleaved into individual proteins either during translation (in the case of 2A (eg T2A)) or after. In some cases, a peptide (such as T2A) can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in separation between the end of the 2A sequence and the downstream adjacent peptide.

Various cell lines can be used in conjunction with the vectors as described herein. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells and FUT8 CHO cells; PER.C6™ cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modification of the binding molecule. For example, in some embodiments, a polypeptide produced by CHO cells has a higher level of sialylation than the same polypeptide produced in 293 cells. In one aspect, the present disclosure relates to cells comprising a vector or vector pair as described herein. In some embodiments, the cells are T cells.

In some cases, certain TCRs may exhibit poor performance or activity, in part due to mispairing and/or competition with endogenous TCR chains and/or other factors. One approach to address these challenges is to design recombinant TCRs with mouse constant domains to prevent mispairing with endogenous human TCR a- or b-chains. However, the use of recombinant TCRs with mouse sequences may pose a risk of immune responses. In some embodiments, genetic disruption is introduced at an endogenous gene encoding one or more TCR chains, eg, by gene editing.

As shown in Figures 1A-1B, provided herein are mouse and human IL-12 expression vectors comprising nucleic acid sequences encoding IL-12B (p40) and IL-12A (p35). In some embodiments, the nucleic acid sequences encoding IL-12B and IL-12A are linked by a linker sequence. In some embodiments, the vector further comprises an immunoglobulin-encoding hinge region, two or three constant domains (eg, one or both CH2 and CH3), and a transmembrane region (eg, CD4) linked after IL-12A transmembrane region).

In some embodiments, the vector encoding IL-12 further comprises variable heavy (vH) and variable light (vL) regions encoding a tumor-targeting antibody (eg, NHS-76) prior to IL-12B the sequence of. In some embodiments, the nucleic acid sequences encoding vH and vL are joined by a linker sequence. In some embodiments, the nucleic acid sequences encoding vL and IL-12B are joined by a linker sequence.

In some embodiments, the vectors described herein comprise a sequence encoding a message peptide sequence. In some embodiments, the sequence encoding the message peptide sequence is linked to the sequence encoding IL-12B. In some embodiments, the sequence encoding the message peptide sequence is linked to the sequence encoding the vH of the tumor-targeting antibody. In some embodiments, the vector further comprises a sequence encoding a linker peptide sequence (eg, P2A) preceding the message peptide sequence.

1C-1D, in some embodiments, the vectors described herein further comprise a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the sequence encoding the TCR or CAR is ligated before the sequence encoding the message peptide sequence.

In some embodiments, the vector further comprises a sequence encoding a chemokine such as CXCL-8, CCL2, CCL3, CCL4, CCL11, CXCL10, XCL1 or XCL2. In some embodiments, the chemokine is CXCL10 or XCL1. In some embodiments, the vector further comprises a sequence encoding an FMS-like tyrosine kinase 3 ligand (FLT3L). In some embodiments, the XCL1 amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 94%, 95%, 96%, 97% identical to SEQ ID NO: 46 or 47. 98% or 99% the same. In some embodiments, the CXCL10 amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% identical to SEQ ID NO: 48 or 49, 98% or 99% the same. In some embodiments, the Flt3L amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% identical to SEQ ID NO: 50 or 51, 98% or 99% the same. In some embodiments, these sequences are separated from other sequences by the sequence encoding the 2A self-cleaving peptide.

The present disclosure also provides nucleic acids encoding human IL-12 fusion proteins. In some embodiments, the nucleic acid encoding the IL-12 fusion protein described herein is set forth in SEQ ID NO: 7, or has at least 80%, 85%, 90%, 91%, 92%, 93% therewith , 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 9, or has at least 80%, 85%, 90%, 91%, 92% therewith , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 11, or has at least 80%, 85%, 90%, 91%, 92% therewith , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences. In some embodiments, the nucleic acid encoding a soluble IL-12 fusion protein described herein is set forth in SEQ ID NO: 13, or is at least 80%, 85%, 90%, 91%, 92%, 93% therewith %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequences.

As used herein, the term "linker" (L) or "linker domain" or "linker region" refers to an oligomeric or polypeptide region of about 1 to 100 amino acids in length that joins any domain/region together . Linkers can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used when it is desired to ensure that two adjacent domains do not sterically interfere with each other. Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg, P2A, T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker; the CHYSEL sequence of porcine Teshin virus (P2A), Thosea asigna virus (T2A); or a combination thereof, Variants and functional equivalents. Other linkers will be apparent to those skilled in the art and can be used in the methods described herein.

In some embodiments, the linker peptide sequence comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker sequence comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues base. In some embodiments, the linker sequence comprises or consists of both glycine and serine residues. In some embodiments, the linker sequence comprises or consists of a sequence that is at least or about 70% identical to SEQ ID NO: 17, GGGGSGGGGS (SEQ ID NO: 18), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19), At least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100% identical. In some embodiments, the linker sequence comprises at least 1, 2, 3, 4, 5, or 6 repeats of GGGGS (SEQ ID NO: 20).

The disclosure also provides nucleic acid sequences comprising encoding IL-12, modified IL-12, CAR, TCR, antigen-binding fragments thereof, and/or TCR-derived binding molecules (including, for example, those described herein) The nucleotide sequence of any of the functional parts and functional variants, polypeptides or proteins). As used herein, "nucleic acid" may include "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which may be single- or double-stranded, synthetic Or obtained from natural sources, which may contain natural, non-natural or altered nucleotides. Furthermore, nucleic acids comprise complementary DNA (cDNA). It is generally preferred that the nucleic acid does not contain any insertions, deletions, inversions and/or substitutions. However, as discussed herein, in some cases it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions and/or substitutions.

Nucleic acids as described herein can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, naturally occurring nucleotides or various modified nucleotides can be used to chemically synthesize nucleic acids. In some of any such embodiments, the nucleotide sequence is codon-optimized.

The present disclosure also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleoside that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein acid sequence.

In some embodiments, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome hopping. In some embodiments, the peptide that causes ribosome skipping is a P2A or T2A peptide. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.

In some embodiments, the vector may additionally include a nucleic acid sequence encoding a checkpoint inhibitor (CPI) (eg, an arrestin). In some embodiments, the checkpoint inhibitor is, for example, any antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7- H2, B7-H3, B7-H4, B7-H6, BTLA, Butyrophilin, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG -3, OX-40, PIR-B, SIRPα (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, or VISTA. In some embodiments, the inhibitory protein is an scFv (eg, an anti-PD-1 scFv). In some embodiments, the vector may additionally include a nucleic acid sequence encoding a bifunctional trap fusion protein. In some embodiments, the bifunctional trap protein targets both PD-1 and TGF-beta. In some embodiments, the bifunctional trap protein targets both PD-L1 and TGF-beta. In some embodiments, the bifunctional fusion protein is designed to block PD-L1 and sequester TGF-beta. Human IgG1 based monoclonal antibody (mAb) avelumab, M7824 (MSB0011395C) contains the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-L1 scFv . In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-1 scFv.

In some of any such embodiments, the IL-12 (eg, membrane-tethered IL-12), CAR, TCR, or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized. In certain embodiments, the polypeptide comprises a message peptide. In some embodiments of any such embodiments, the polypeptide and/or fusion protein is recombinant.

The present disclosure also provides that any nucleotide sequence described herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleic acid sequences, and at least 1%, 2%, 3%, 4% with any amino acid sequence described herein , 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein, or any amino acid sequence encoded by any of the nucleotide sequences described herein.

In some embodiments, the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (eg, one of the first and second amino acid or nucleic acid sequences may be or both to introduce gaps for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. A molecule is identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. Taking into account the number of gaps and the length of each gap (which needs to be introduced to achieve optimal alignment of the two sequences), the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Methods for making engineered cells

The present disclosure provides methods or processes for preparing, manufacturing, and/or using the engineered cells to treat a pathological disease or disorder.

Cells used to introduce the proteins described herein (eg, IL-12, TCR and/or CAR) can be isolated from a sample, such as a biological sample, eg, obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of, or to which cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

Thus, in some embodiments, the cells are primary cells, eg, primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples from one or more processing steps such as isolation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation . A biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, Liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs and/or cells derived therefrom. In the context of cell therapy (eg, adoptive cell therapy), samples include samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, eg, from a mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.

In some embodiments, blood cells collected from the subject are washed, eg, to remove plasma fractions and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is devoid of calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished by semi-automated "flow-through" centrifugation. In some aspects, the washing step is accomplished by tangential flow filtration (TFF). In some embodiments, cells are resuspended in various biocompatible buffers (such as, for example, PBS without Ca ²⁺ /Mg ²⁺ ) after washing. In certain embodiments, components of the blood cell sample are removed and the cells are directly resuspended in culture medium. In some embodiments, the method includes a density-based cell separation method, such as the preparation of leukocytes from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

In some embodiments, the method comprises one or more of the following steps: eg, isolating T cells from a patient's blood; transducing a population of T cells with a viral vector comprising encoding a genetically engineered antigen receptor expand the transduced cells in vitro; and/or infuse the expanded cells into a patient, where the engineered T cells will seek out and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further includes a sequence encoding an arrestin. In some embodiments, these engineered T cells can block PD-1/PD-L1 immunosuppression and enhance anti-tumor immune responses. The method also includes transfecting the T cells with the viral vector containing the nucleic acid construct.

In some embodiments, the methods involve introducing any of the vectors described herein into cells in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introduction is by transduction. In some embodiments, the cells are transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or more. In some embodiments, the method further involves introducing into the cell one or more agents, wherein each of the one or more agents is independently capable of inducing a T cell receptor alpha chain constant region (TRAC) gene and/or Genetic disruption of the T cell receptor beta chain constant region (TRBC) gene. In some embodiments, the one or more agents are inhibitory nucleic acids (eg, siRNA). In some embodiments, the one or more agents are fusion proteins comprising a DNA targeting protein and a nuclease or RNA-guided nuclease (eg, Clustered Regularly Interspaced Short Palindromic Nucleic Acids (CRISPR)-related nucleases) .

In some embodiments, the cells are tumor-infiltrating lymphocytes, and the cells are transfected with a vector encoding IL-12 or modified IL-12. In some embodiments, the cells are T cells and the cells are treated with a vector encoding IL-12 (eg, modified IL-12) and a TCR or a vector encoding IL-12 (eg, modified IL-12) and a CAR vector transfection.

Transfection of T cells can be accomplished by the skilled artisan using any standard method such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, bioelastic particle delivery systems, or any other known method. In some embodiments, transfection of T cells is performed using the calcium phosphate method.

The present disclosure provides methods for creating personalized anti-tumor immunotherapies. Genetically engineered T cells can be generated from a patient's blood cells. These engineered T cells are then re-infused into the patient as a cell therapy product.

Methods of preparing engineered cells and administering these engineered cells to a subject are known in the art and are described, for example, in US Pat. No. 10,174,098 and Draper et al., "Targeting of HPV-16+ Epithelial Cancer Cells By Tcr Gene Engineered t Cells Directed Against e6." Clinical Cancer Research 21.19 (2015): 4431-4439, both of which are incorporated by reference in their entirety. treatment method

The methods disclosed herein can be used for various therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, methods of reducing the rate of increase in tumor volume over time in a subject, methods of reducing the risk of developing metastases, or reducing the occurrence of extraneous lesions in a subject method of transferring risk. In some embodiments, the treatment can stop, slow, delay or inhibit the progression of the cancer. In some embodiments, the treatment can result in a reduction in the number, severity and/or duration of one or more symptoms of cancer in the subject.

In one aspect, the disclosure features methods comprising engineering a therapeutically effective amount of expressing IL-12 (eg, modified IL-12), TCR, CAR, antigen-binding fragments thereof, and TCR-derived binding molecules The cells are administered to a subject in need (eg, a subject suffering from or identified as or diagnosed with cancer).

In some embodiments, the subject has a solid tumor (eg, a heterogeneous solid tumor or a homogeneous solid tumor). In some embodiments, the subject has breast cancer (eg, triple negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma tumor, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is cervical cancer, head and neck cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

In some embodiments, the subject has a heterogeneous cancer. In some embodiments, the subject has a homogeneous cancer. In some embodiments, the heterogeneous cancer has at least 1% of the antigen or antigens expressed by the majority (eg, at least 50%, 60%, 70%, 80%, or 90%) of cancer cells in the heterogeneous cancer , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of cancer cells expressing different antigens. In some embodiments, TCRs, CARs, antigen-binding fragments thereof, and/or TCR-derived binding molecules can bind to a portion (eg, at least 50%, 60%, 70%, 80%, 90%, 95% or more) or one or more antigens expressed by all cells. In some embodiments, the one or more antigens are not expressed by a fraction (eg, less than 50%, 40%, 30%, 20%, 10%, or 5%) of cells in a heterogeneous cancer. In some embodiments, the one or more antigens are deactivated (eg, cleavage or a mechanism such as immune escape) such that the TCR, CAR, antigen-binding fragments thereof and/or TCR-derived binding molecules are no longer recognized. In some embodiments, a fraction (eg, less than 50%, 40%, 30%, 20%, 10%, or 5%) of cells in a heterogeneous cancer can express an immune checkpoint molecule (eg, PD-L1) to inhibit immune response. A detailed description of tumor antigen heterogeneity and antigen escape can be found in O'Rourke et al., "A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma." Science translational medicine 9.399 ( 2017): eaaa0984; Majzner and Crystal, "Tumor antigen escape from CAR T-cell therapy." Cancer discovery 8.10 (2018): 1219-1226; each of these documents are incorporated herein by reference in their entirety.

In some embodiments, the IL-12 (eg, modified IL-12) expressed by the engineered cells described herein can be therapeutically specific compared to similarly engineered TCR or CAR cells that do not express IL-12. At least or about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more.

In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Cancer patients can be identified by various methods known in the art.

Furthermore, the present disclosure provides methods for treating an infection or an infection-related disorder in a subject. In some embodiments, the treatment can stop, slow, delay or inhibit the progression of the disease. These methods generally involve administering to a subject in need thereof a therapeutically effective amount of a genetically engineered cell disclosed herein. In some embodiments, the disease or disorder to be treated is an infectious disease or disorder, such as, but not limited to, viral, retrovirus, bacterial and protozoal infections, immunodeficiency, human papilloma virus (HPV), cytomegalovirus (CMV), Epstein Barr virus (EBV), adenovirus, BK polyoma virus.

As used herein, an "effective amount" means an amount or dose sufficient to achieve beneficial or desired results, including halting, slowing, delaying or inhibiting the progression of a disease (eg, cancer). The effective amount will vary depending, for example, on the age and weight of the subject to which the therapeutic agent and/or therapeutic composition will be administered, the severity of symptoms and the route of administration, and thus administration can be determined on an individual basis.

As used herein, the term "delaying the development of a disease" refers to delaying, retarding, slowing, delaying, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer). This delay can be of varying lengths, depending on the medical history and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, since the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

An effective amount can be administered in one or more administrations. For example, an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay the progression of cancer in a patient, or to ameliorate, stop, stabilize, reverse, slow and/or in vitro or an amount that delays the proliferation of cells (eg, biopsied cells, any of the cancer cells described herein, or cell lines (eg, cancer cell lines)). As understood in the art, the effective amount may vary depending on, inter alia, the patient's medical history, as well as other factors such as the type (and/or dosage) of the composition used.

Effective amounts and schedules of administration can be determined empirically, and it is within the skill in the art to make such determinations. Those skilled in the art will appreciate that the dosage that must be administered will vary depending on, for example, the mammal to be treated, the route of administration, the particular type of therapeutic and other drugs administered to the mammal. Guidelines for choosing an appropriate dose can be found in the literature. Furthermore, treatment does not necessarily result in 100% or complete treatment or prevention of the disease or disorder. There are a variety of therapeutic/preventive methods available with varying degrees of therapeutic effect, which are considered potentially beneficial treatments by those of ordinary skill in the art.

In some aspects, the present disclosure also provides methods of diagnosing a disease/disorder in a mammal, wherein the TCR, CAR, antigen-binding fragment, TCR-derived binding molecule interacts with one or more samples obtained from a subject Act to form a complex, wherein the sample may comprise one or more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen-binding moieties; blood, whole cells, lysates thereof, or fractions of whole cell lysates, such as nuclei or a cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction thereof, wherein detection of the complex is indicative of the presence of a disorder in the mammal, wherein the disorder is cancer or infection. Furthermore, detection of complexes can be by any number of means known in the art, but not limited to ELISA, flow cytometry, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), microarray, Southern Spotting, electrophoresis, phage analysis, chromatography, etc. Thus, methods of treatment can further include determining whether a subject can benefit from the treatments disclosed herein, eg, by determining whether the subject has an infection or cancer.

In any of the methods described herein, the engineered cells and/or at least one additional therapeutic agent can be at least once a week (eg, once a week, twice a week, three times a week, four times a week, daily once, twice daily, or three times daily) to the subject. In some embodiments, at least two different engineered cells (eg, cells expressing different binding molecules) are administered in the same composition (eg, a liquid composition). In some embodiments, the engineered cells and the at least one additional therapeutic agent are administered in the same composition (eg, a liquid composition). In some embodiments, the engineered cells and at least one additional therapeutic agent are administered in two different compositions. In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

In some embodiments, one or more additional therapeutic agents can be administered to a subject before, concurrently with, or after administration of the engineered cells to the subject.

In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent may be a checkpoint inhibitor (CPI). In some embodiments, the checkpoint inhibitor is an inhibitory protein, such as an antibody or antigen-binding fragment thereof. Checkpoint inhibitors can inhibit or block one or more immune checkpoints including, for example, PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, Butteroprotein, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors , LAG-3, OX-40, PIR-B, SIRPα (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, and combinations thereof. In some embodiments, the arrestin blocks PD-1 or PD-L1. In various embodiments, the arrestin comprises an anti-PD-1 scFv. Arrestins can result in decreased expression of PD-1 or PD-L1 in a population of T cells and/or inhibit the upregulation of PD-1 or PD-L1, and/or physically block the PD-1/PD-L1 complex formation and subsequent message transduction. In some embodiments, arrestin blocks PD-1. In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody Antibody. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (eg, ipilimumab), an anti-CD20 antibody (eg, rituximab), an anti-EGFR antibody (eg, cetuximab), an anti-CD319 antibody (eg elotuzumab) or anti-PD1 antibody (eg nivolumab).

In some embodiments, the additional therapeutic agent is a bifunctional trap fusion protein. Bifunctional trap proteins can target both immune checkpoints and TGF-beta negative regulatory pathways. In addition to expressing immune checkpoints, the tumor microenvironment also contains other immunosuppressive molecules. Of particular interest is the interleukin TGF-beta (TGFB), which has multiple functions in cancer. TGF-β prevents tumor cell proliferation and promotes differentiation and apoptosis in the early stages of tumor development. However, during tumor progression, tumor TGF-β insensitivity occurs due to loss of TGF-β receptor expression or mutation of downstream signaling elements. TGF-β then promotes tumor progression through its effects on angiogenesis, induction of epithelial-mesenchymal transition (EMT), and immunosuppression. High TGF-beta serum levels and loss of TGF-beta receptor (TGFbetaR) expression on tumors are associated with poor prognosis. TGFβ-targeted therapy has demonstrated limited clinical activity. In some embodiments, the bifunctional trap protein targets both PD-1 and TGF-beta. In some embodiments, the bifunctional trap protein targets both PD-L1 and TGF-beta. In some embodiments, the bifunctional fusion protein is designed to block PD-L1 and sequester TGF-beta. Human IgG1 based monoclonal antibody (mAb) avelumab, M7824 (MSB0011395C) contains the extracellular domain of human TGF-β receptor II (TGFβRII) linked to the C-terminus of a human anti-PD-L1 scFv . In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-beta receptor II (TGFbetaRII) linked to the C-terminus of a human anti-PD-1 scFv. These bifunctional trap fusion proteins are described, for example, in Knudson et al., "M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine. "Oncoimmunology 7.5 (2018): e1426519; the entirety of which is incorporated herein by reference." In some embodiments, a subject is treated with a cell expressing a TCR or antigen binding molecule described herein and one or more bifunctional trap fusion proteins.

In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, MEK inhibitors, ERK inhibitors Inhibitor, K-Ras inhibitor, c-Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol 3 kinase (PI3K) inhibitor, Akt inhibitor, mTOR inhibitor, PI3K/mTOR dual Inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and inhibitors of isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (eg, epacadostat). In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, Trebananib, pazopanib, cediranib, pembocoxib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsimo Division, Axitinib, Everolimus, Sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, Cabozantinib, Vinflunine, Hsp90 Inhibitor, Ad-GM-CSF , temozolomide (Temazolomide), IL-2, IFNa, vinblastine, thalidomide (Thalomid), dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib , amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of adjuvant, TLR agonist, tumor necrosis factor (TNF) alpha, IL -1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, IL-17 antagonist, HVEM antagonist, ICOS agonist, therapy targeting CX3CL1, therapy targeting CXCL9, target CXCL10-targeted therapy, CCL5-targeted therapy, LFA-1 agonists, ICAM1 agonists, and Selectin agonists. In some embodiments, the additional therapeutic agent may be CXCL10, Flt3L, or XCL1.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, carbamazepine urea, paclitaxel, rituximab, vinblastine, vincristine, and/or combinations thereof. Compositions and Formulations

The present disclosure provides compositions (including pharmaceutical compositions and therapeutic compositions) containing engineered cells and populations thereof produced by the methods disclosed herein. Also provided are methods (eg, therapeutic methods) for administering the engineered cells and compositions thereof to a subject (eg, a patient).

Compositions comprising engineered cells for administration are provided, including pharmaceutical compositions and formulations, such as unit dosage compositions comprising the number of cells for administration in a given dose or fraction thereof. Pharmaceutical compositions and formulations may include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

A pharmaceutically acceptable carrier refers to ingredients other than the active ingredient in a pharmaceutical composition. A pharmaceutically acceptable carrier does not interfere with the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers or preservatives. Pharmaceutical formulation refers to the process of combining different substances and/or pharmaceutical agents to produce a final drug product. Formulation research involves the development of pharmaceutical formulations that are acceptable to patients. Additionally, are formulations that are in a form that renders the biological activity of the active ingredient contained therein effective and that are free of additional components that would have unacceptable toxicity to the subject to whom the formulation is administered.

In some embodiments, the choice of vector is determined in part by the particular cell (eg, T cells or NK cells) and/or by the method of administration. A variety of suitable formulations are available. For example, pharmaceutical compositions may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservatives or mixtures thereof are generally present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine ; preservatives (eg octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides ; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartamine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as Sodium; metal complexes (eg, zinc-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG).

Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. The buffer or mixtures thereof are generally present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Ed. (May 1, 2005).

Formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated with the engineered cells, preferably those ingredients having activities complementary to the cells, wherein the respective activities will not adversely affect each other. Such active ingredients are present in suitable combinations in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition may also include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, eg, asparaginase, busulfan, carboplatin, cisplatin , Daunorubicin, Doxorubicin, Fluorouracil, Gemcitabine, Hydroxyurea, Ammethopterin, Paclitaxel, Rituximab, Vinblastine and/or Vincristine.

In some embodiments, the pharmaceutical composition contains the cells in an amount effective to treat or prevent a disease or disorder, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodically evaluating the treated subject. The desired dose can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

Cells and compositions can be administered using standard administration techniques, formulations and/or devices. Administration of the cells can be autologous or allogeneic. For example, immune reactive T cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject after genetic modification thereof according to various embodiments described herein By. Peripheral blood-derived immunoreactive T cells or progeny thereof (eg, in vivo, ex vivo or in vitro derived) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. Typically, when a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immune-responsive cells) is administered, it is usually formulated in a unit dose injectable form (solution, suspension, emulsion).

The formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the population of cells is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

In some embodiments, the compositions are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. In another aspect, the viscous composition can be formulated in an appropriate viscosity range to provide longer contact times with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) alcohol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, with an appropriate carrier, diluent, or excipient (eg, sterile water, physiological saline, dextrose, dextrose, and the like). The compositions may contain auxiliary substances such as wetting agents, dispersing agents or emulsifying agents (eg methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents and/or pigments, depending on the on the route of administration and the desired formulation. In some aspects, reference can be made to standard texts for the preparation of suitable formulations.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile membranes.

The compositions or pharmaceutical compositions described herein can be included in a container, pack or dispenser with instructions for administration. Application method

Also provided are methods of administering cells, populations, and compositions, as well as the use of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, the methods described herein can reduce the risk of developing the diseases, conditions, and disorders described herein.

In some embodiments, the cells, populations, and compositions described herein are administered to a subject or patient suffering from a specific disease to be treated, eg, by adoptive cell therapy, such as adoptive T cell therapy disease or condition. In some embodiments, cells and compositions prepared by the provided methods (such as engineered compositions and final production compositions after incubation and/or other processing steps) are administered to a subject, such as suffering from a disease or A disorder or a subject at risk of developing a disease or disorder. In some aspects, the methods thereby treat a disease or disorder (eg, ameliorate one or more symptoms of the disease or disorder), such as by reducing tumor burden in cancers that express antigens recognized by engineered T cells.

Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in U.S. 2003/0170238; U.S. Patent No. 4,690,915; Rosenberg, "Cell transfer immunotherapy for metastatic solid cancer—what clinicians need to know." Nature reviews Clinical oncology 8.10 (2011): 577; Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Nature biotechnology 31.10 (2013): 928; Tsukahara et al., “CD19 target-engineered T cells accumulate at tumor lesions in human B -cell lymphoma xenograft mouse models.” Biochemical and biophysical research communications 438.1 (2013): 84-89; Davila et al., “CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia." PloS one 8.4 (2013); each of these documents is incorporated herein by reference in its entirety.

In some embodiments, cell therapy (eg, adoptive T cell therapy) is performed by autologous transfer, wherein isolation and/or other way to prepare T cells. Thus, in some aspects, the cells are derived from a subject (eg, a patient) in need of treatment, and the cells are administered to the same subject after isolation and treatment.

In some embodiments, cell therapy (eg, adoptive T cell therapy) is performed by allogeneic transfer, wherein the isolation and /or otherwise prepare T cells. In such embodiments, the cells are subsequently administered to a different subject (eg, a second subject) of the same species. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject exhibits the same HLA class or supertype as the first subject.

In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or disorder (eg, tumor) prior to administration of the cells or cell-containing composition. In some aspects, the subject is refractory or unresponsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease, eg, following treatment with another therapeutic intervention including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) (eg, allogeneic allogeneic HSCT). In some embodiments, the administration is effective to treat the subject despite the subject having developed resistance to another therapy.

In some embodiments, the subject is responsive to other therapeutic agents, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject initially responds to the therapeutic agent, but exhibits recurrence of the disease or disorder over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, eg, to reduce the likelihood of relapse or to prevent relapse. In some embodiments, the subject has not received prior treatment with another therapeutic agent.

In some embodiments, the cells are administered in a desired dose, which in some aspects includes a desired dose or number of cells or one or more cell types and/or a desired ratio of cell types. Thus, in some embodiments, the cell dosage is based on the total number of cells (or the number of cells per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dose of cells is based on the desired total number of cells or individual cell types in a single population (or number of cells per kg body weight). In some embodiments, the dosage is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in a single population.

In some embodiments, populations or subtypes of cells (such as CD8+ and CD4+ T cells) are administered with a desired dose of total cells (such as a desired dose of T cells) for a tolerance differential or within the tolerance differential . In some embodiments, the desired dose is the desired number of cells or the desired number of cells per unit body weight of the subject to which the cells are administered (eg, cells/kg). In some embodiments, the desired dose is equal to or higher than the minimum number of cells or the minimum number of cells per unit body weight. In some embodiments, the individual populations or subtypes are at or near the desired output ratio (such as the ratio of CD4+ to CD8+) in total cells administered at the desired dose (eg, some tolerated difference in this ratio or error) exists.

In some embodiments, the difference in tolerance to or between a desired dose of one or more individual cell populations or subtypes (such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells) intracellular administration of cells. In some embodiments, the desired dose is the desired number of cells of the subtype or population or the desired number of such cells per unit body weight of the subject to which the cells are administered (eg, cells/kg) . In some embodiments, the desired dose is equal to or higher than the minimum number of cells of said population or subtype or the minimum number of cells of said population or subtype per unit body weight.

Thus, in some embodiments, the dose is based on the desired fixed dose of total cells and the desired ratio, and/or based on one or more (eg, each) individual subtypes or subpopulations of the desired fixed dose. Thus, in some embodiments, the dosage is based on the desired fixed or minimum dose of T cells and the desired ratio of CD4+ to CD8+ cells, and/or based on the desired fixed or minimum dose of CD4+ and/or CD8+ cells.

In certain embodiments, cells, or a single population or subtype of cells, are divided into about 1 million to about 100 billion cells (such as, for example, 1 million to about 50 billion cells (eg, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or any two of the foregoing values defined range), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or any two of the foregoing values defined range), and in some cases from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or any value in between these ranges) are administered to the subject.

In some embodiments, the dose of total cells and/or the dose of individual cell subsets is in the range between at or about ¹⁰ and at or about ¹⁰ cells per kilogram (kg) body weight, such as at 10 Between ⁵ and 10 ⁶ cells/kg body weight, eg, at least or at least about or at or about 1 x 10 ⁵ cells/kg body weight, 1.5 x 10 ⁵ cells/kg body weight, 2 x 10 ⁵ cells/kg Body weight, or 1×10 ⁶ cells/kg body weight. For example, in some embodiments, at between at or about ¹⁰ and at or about ¹⁰ T cells/kilogram (kg) body weight, such as between ¹⁰ and ¹⁰ T cells/kg body weight, eg , at least or at least about or at or about 1 x 10 ⁵ T cells/kg body weight, 1.5 x 10 ⁵ T cells/kg body weight, 2 x 10 ⁵ T cells/kg body weight, or 1 x 10 ⁶ T cells/kg body weight /kg body weight, or administer cells within a certain margin of error.

In some embodiments, at between at or about ¹⁰ and at or about ¹⁰ CD4+ and/or CD8+ cells/kilogram (kg) body weight, such as between ¹⁰ and ¹⁰ CD4+ and/or CD8+ cells/ Between kg body weight, eg, at least or at least about or at or about 1 x 10 ⁵ CD4+ and/or CD8+ cells/kg body weight, 1.5 x 10 ⁵ CD4+ and/or CD8+ cells/kg body weight, 2 x 10 ⁵ cells CD4+ and/or CD8+ cells/kg body weight, or ¹ x 106 CD4+ and/or CD8+ cells/kg body weight, or cells were administered within a certain margin of error.

In some embodiments, with greater than, and/or at least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ CD4+ cells, and/or At least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ CD8+ cells, and/or at least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ T cells, or within a certain margin of the administered cells. In some embodiments, with between about 10 ⁸ and 10 ¹² , or between about 10 ¹⁰ and 10 ¹¹ T cells, between about 10 ⁸ and 10 ¹² , or between about 10 ¹⁰ and 10 ¹¹ between about 10 8 and 10 12 or between about 10 10 and 10 ¹¹ CD4+ cells, and/or between about 10 ⁸ and 10 ¹² , or between about 10 ¹⁰ and 10 11 , or administer the cells within a margin of error.

In some embodiments, the cells are administered at or within the tolerable range of the desired output ratio of the various cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. For example, in some embodiments, the desired ratio (eg, ratio of CD4+ to CD8+ cells) is between at or about 1:5 and at or about 5:1 (or greater than about 1:5 and less than about 5:1 ), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1), such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9: 1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1: 5. In some aspects, the tolerance difference is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30% of the desired ratio %, about 35%, about 40%, about 45%, about 50%, including any value between these ranges. In some aspects, the TCRs described herein provide improved performance and activity, thereby providing therapeutic effects even at low effector-to-target (E:T) ratios.

The optimal response to therapy may depend on the ability of engineered recombinant receptors, such as TCRs, to consistently and reliably express and/or bind target antigens on the cell surface. For example, in some cases, properties of certain recombinant receptors (eg, TCRs) may affect the expression and/or properties of certain recombinant receptors (eg, TCRs) when expressed in cells (such as human T cells) in some cases for use in cell therapy active. In some contexts, the level of expression of particular recombinant receptors (eg, TCRs) may be low, and the activity of engineered cells (such as human T cells) expressing such recombinant receptors may be due to poor expression or poor signaling activity and restricted. In some cases, the consistency and/or efficiency of expression of recombinant receptors and the activity of the receptors are limited in certain cells or certain populations of cells for available therapeutic methods. In some cases, large numbers of engineered T cells (high effector-to-target (E:T) ratios) are required to exhibit functional activity. In some embodiments, the desired ratio (E:T ratio) is between at or about 1:10 and at or about 10:1 (or greater than about 1:10 and less than about 10:1), or at or Between about 1:1 and at or about 10:1 (or greater than about 1:1 and less than about 5:1), such as between at or about 2:1 and at or about 10:1. In some embodiments, the E:T ratio is greater than or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

For prophylactic or therapeutic purposes, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are being administered for prophylactic or therapeutic purposes, previous therapy, the subject clinical history and response to cells and the discretion of the attending physician. In some embodiments, the compositions and cells are suitably administered to a subject at one time or over a series of treatments.

The cells described herein can be administered by any suitable means, eg, by bolus infusion, by injection, eg, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, scleral injection Sub-, intra-choroidal, anterior chamber, subconjectval, subconjuntival, sub-Tenon, retrobulbar, peribulbar, or posterior juxtascleral) delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus injection of cells. In some embodiments, a given dose is administered by multiple bolus administration of cells, eg, over a period of not more than 3 days, or by continuous infusion of cells.

In some embodiments, the cells are administered as part of a combination therapy, such as concurrently or sequentially in any order with another therapeutic intervention such as an antibody or engineered cell or receptor or agent (eg, a cytotoxic or therapeutic agent). apply. In some embodiments, the cells are co-administered with one or more additional therapeutic agents or in combination with another therapeutic intervention (simultaneously or sequentially in any order). In some cases, the cells are co-administered with another therapy sufficiently close in time that the population of cells enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include an interferon (eg, IL-2), eg, to enhance persistence. In some embodiments, the method includes administering a chemotherapeutic agent.

In some embodiments, the biological activity of the engineered cell population is measured following administration of the cells, eg, by any of a number of known methods. Parameters to be assessed include the specific binding of engineered T cells to antigen, assessed in vivo, eg, by imaging, or ex vivo, eg, by ELISA or flow cytometry. In certain embodiments, the ability of an engineered cell to destroy a target cell can be measured using any suitable method known in the art, such as described in, eg, Kochenderfer et al., "Construction and pre-clinical evaluation of an anti- CD19 chimeric antigen receptor.” Journal of immunotherapy (Hagerstown, Md.: 1997) 32.7 (2009): 689 and Hermans et al., “The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets in in vitro and in vivo .” Journal of immunological methods 285.1 (2004): 25-40. In certain embodiments, the biological activity of the cells is measured by assaying the expression and/or secretion of one or more cytokines, such as CD107a, IFNy, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or burden.

Repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, the timing and size of multiple doses of cells are typically designed to increase the efficacy and/or activity and/or function of engineered cells as described herein. The method involves administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.

In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells in a single composition and/or in a single uninterrupted administration (eg, in a single injection or continuous infusion). , and also encompass the administration of a given amount or number of cells in divided doses provided in multiple separate compositions or multiple separate infusions over a specified period of time (eg, no more than 3 days). Thus, in some contexts, a first or continuous dose is a single or continuous administration of a specified number of cells administered or initiated at a single time point. However, in some situations, the first or consecutive doses are administered as multiple injections or infusions over a limited period of time (eg, no more than three days), such as once daily for three or two days or by Administered by multiple infusions over a period of one day.

The first dose of cells is administered in a single pharmaceutical composition. In some embodiments, consecutive doses of cells are administered in a single pharmaceutical composition.

In some embodiments, the first dose of cells is administered in multiple compositions that together contain the first dose of cells. In some embodiments, consecutive doses of cells are administered in multiple compositions that together contain consecutive doses of cells. In some aspects, additional consecutive doses of the various compositions may be administered over a period of not more than 3 days.

With respect to a previous dose, such as a first dose, the term "successive dose" refers to a dose administered to the same subject without any intervening dose being administered to the subject temporarily following the previous (eg, first) dose. However, the term does not cover the second, third and/or etc. injections or infusions in a series of infusions or injections contained within a single divided dose. Thus, unless otherwise stated, a second infusion over a one, two or three day period is not considered a "continuous" dose as used herein. Likewise, the second, third, etc. of a series of multiple doses within a divided dose are not considered "interventional" doses in the context of a "continuous" dose. Therefore, unless otherwise stated, as long as the second or subsequent injection or infusion occurs within a three-day period after the start of the first or previous dose, even if the subject received the first dose of cells after the start of the first dose Second or subsequent injections or infusions, doses administered over a period of time greater than three days after initiation of the first or previous dose, are considered "continuous" doses.

Therefore, unless otherwise stated, multiple administrations of the same cells over a period of up to 3 days are considered a single dose, and administration of cells within 3 days of the initial administration is not considered a consecutive dose and is not considered The intervention dose used for the purpose of determining whether the second dose is "consecutive" with the first dose.

In some embodiments, in some aspects, the same timing guidelines as for timing between the first dose and the first consecutive dose are used, eg, by administering the first and multiple consecutive doses, multiple consecutive doses are administered .

In some embodiments , the timing between the first dose and the first consecutive dose, or the first and multiple consecutive doses, is such that the dose is less than about 5 days, 6 days, 7 days, 8 days, 9 days , 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days Each successive dose is administered over a period of 27 days, 28 days, or longer. In some embodiments, consecutive doses are administered for a period of less than about 28 days following administration of the first dose or immediately preceding dose. Additional multiples of one or more consecutive doses are also referred to as subsequent doses or subsequent consecutive doses.

The first dose of cells and/or one or more consecutive doses are typically sized to provide improved efficacy and/or reduced risk of toxicity. In some aspects, the dose amount or size of the first dose or any successive doses is any dose or amount as described above. In some embodiments, the number of cells in the first dose or any successive doses is between about 0.5 x 10 ⁶ cells/kg subject body weight and 5 x 10 ⁶ cells/kg, between about 0.75 x 10 ⁶ cells/kg Between cells/kg and 3×10 ⁶ cells/kg, or between about 1×10 ⁶ cells/kg and 2×10 ⁶ cells/kg.

As used herein, "first dose" is used to describe the timing of a given dose prior to administration of successive or subsequent doses. The term does not necessarily imply that the subject has never received a dose of cell therapy before, or even that the subject has not previously received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.

In some embodiments, over an extended period of time (eg, at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) to the subject dose. A skilled medical professional can determine the length of a treatment period using any of the methods described herein for diagnosing or tracking treatment effectiveness (eg, observing at least one cancer symptom). Example

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Construct Design

Mouse and human IL-12 expression vectors (FIGS. 1A-1B) were designed to contain IL-12B (p40) and IL-12A (p35) separated by a peptide linker. For membrane-tethered IL-12, add two (membrane-tethered IL-12; mt-IL-12; or smt) or three (long membrane-tethered IL-12; long mt-IL) after IL-12A -12; or lmt) the immunoglobulin hinge region of the constant domains (CH2 and CH3) and the CD4 transmembrane region. A vector expressing soluble IL-12 was also designed to contain both the heavy chain variable region (VH) and light chain variable region (VL) of a human IgG1 targeting tumor necrosis, NHS-76 (NHS76-IL-12) .

To generate lymphocytes expressing IL-12 in combination with TCR or CAR, additional nucleic acid sequences encoding TCR (FIG. 1C) or CAR (FIG. 1D) were added to the expression vector. This additional sequence and the sequence encoding IL-12B or NHS-76 vH are separated by the P2A peptide sequence.

The mouse IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The mouse membrane-tethered IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The mouse long membrane tethered IL-12 construct is shown in Figure 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The human IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The human membrane-tethered IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The human long membrane tethered IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The human NHS76-IL-12 construct is shown in Figure IB, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Example 2. Lymphocytes engineered to express IL-12 secrete IFNγ

Purified primary mouse lymphocytes were engineered to express L202 TCR alone (L202) (anti-LMP2 TCR), L202 plus mouse membrane-tethered IL-12 (sample name: mt12), or L202 plus mouse NHS-76 - IL-12 (sample name: NHS76). Then, two days after transduction, the indicated lymphocyte populations were co-cultured overnight with LLC-HLA-A2-Peplinker (LLW) target cells. The secretion of mouse IL-12 (mIL12) and mouse IFNγ (mIFNγ) into the culture medium was quantified using BioLegend MAX ELISA kits for mIL12 p40 (catalog #430707) and mIFNγ (catalog #430801). The results in Figures 2A-2B demonstrate that mouse IL-12 and IFNγ are expressed at higher levels in both mt12 and NHS76 cells, and the interleukin concentration is further increased after co-culture with target cells. The amino acid sequences of mouse membrane-tethered IL-12 and NHS76-IL-12 are shown in SEQ ID NO: 4 and SEQ ID NO: 14, respectively. Example 3. IL-12 expression increases IFNγ production in naive lymphocytes

Naive mouse lymphocytes isolated from lymph nodes were either not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 together with membrane-tethered IL-12 (sample name: L202) mt12) or L202 together with mouse NHS76-IL-12 (sample name: NHS76). The indicated lymphocyte populations were co-cultured with Jurkat target cells overnight. Mouse IFNy was then measured in the culture medium using the Mouse IFNy ELISA MAX kit (BioLegend). As shown in Figure 3, the results show that IL-12 appears to stimulate IFNy production by lymphocytes when incubated with target cells, and that IL-12 appears to transactivate naive lymphocytes. Example 4 : IL-12 expression on the surface of armored TCR-T in mouse lymphocytes

Primary mouse lymphocytes were isolated from lymph nodes and transduced with L202 TCR alone or L202 together with mt-IL-12 or mouse NHS76-IL-12 for 5 days. The indicated cell types were then stained with 3 uL of the anti-mIL12 antibody PE-Cy7 to assess the surface expression of IL-12. As shown in Figures 4A to 4D, approximately 52% of mt-IL-12 PBMCs exhibited cell surface expression of human IL-12, while IL-12 was expressed in untransduced, L202 or L202-NHS76-IL-12 cells It doesn't appear on the surface. The amino acid sequences of human membrane-tethered IL-12 and NHS76-IL-12 are shown in SEQ ID NO: 10 and SEQ ID NO: 14, respectively. Example 5 : Intracellular IL-12 expression in NHS-76-IL-12 mouse lymphocytes

Mouse lymphocytes were isolated from lymph nodes and then transduced with NHS76-IL-12 for 2 days. Lymphocytes transduced with NHS76-IL-12 exhibited intracellular IL-12 expression as measured by staining with human Fab (FIG. 5A) or anti-IL12 antibody PE-Cy7 (FIG. 5B). Example 6 : TCRb and human IL-12 surface staining of human PBMC

Human PBMCs were transduced with L202 TCR alone or in combination with human mt-IL-12 or human long mt-IL-12 as indicated. 12 days after infection, TCR and IL-12 surface expression was measured by staining with anti-TCRb antibody (TCRb-PE) and anti-IL-12 antibody (IL-12-PE-Cy7), respectively. The results in Figures 6A-6H demonstrate that cells transduced with L202 have TCR surface expression for each cell type, whereas surface IL-12 was detected on PBMCs transduced with a membrane-tethered form of IL-12. The amino acid sequences of human membrane tethered IL-12 (mt-IL-12) and human long membrane tethered IL-12 (human long mt-IL-12) are shown in SEQ ID NO: 10 and SEQ ID NO: , respectively 12. Example 7 : CD69 expression in mt - IL-12 expressing PBMC co-cultured with target cells

On day 12 post-infection, as indicated, untransduced human PBMCs were either engineered to express L202 TCR, L202 plus human membrane-tethered IL-12 (smt), or L202 plus human long membrane-tethered IL-12 (lmt) PBMCs were co-cultured overnight with A375-HLA-A2-peplinker (LLW) melanoma target cells. Then, T cell activation was assessed by staining with APC anti-CD69 antibody.

Figure 7A is a graph showing CD69 expression in human PBMC co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells. Figure 7B is a graph showing CD69 expression in human PBMC without co-culture. As shown in Figures 7A-7B, IL-12-expressing PBMCs had more IL-12 than L202 or untransduced T cells when they were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells Strong CD69 expression. Example 8 : IFNγ expression in IL-12 armored PBMCs

Untransduced human PBMCs or PBMCs transduced with L202 TCR-T, L202 TCR-T plus human membrane tethered IL-12 (smt) or L202 TCR-T plus human long membrane tethered IL-12 (lmt) Co-culture with A375-HLA-A2-peplinker (LLW) target cells overnight followed by staining with hIFNγ-FITC to measure T cell activation. As shown in Figures 8A to 8H, the results show that about 20% IFNγ positivity in L202 TCR-T cells relative to greater than 40% IFNγ in L202 TCR-T cells expressing membrane-tethered IL-12 (smt or lmt) expression, indicating that the cell surface expression of IL-12 enhances TCR-T cell activation. Example 9 : CD69 expression in IL-12 armored mTCRb+ and mTCRb- cells

Experiments were conducted to determine whether IL12-armoured cells increased the activation of two types of cells: cells with IL12 armour and nearby unarmoured immune cells. To observe the effect of IL-12 armored cells on nearby unarmored cells, a mixed population of 50% L202 TCR-transduced and 50% untransduced human PBMCs was co-cultured with A375-LLW melanoma target cells. L202 TCR-transduced human PBMCs were transduced with L202 alone, L202 plus human membrane tethered IL-12 (smt) or L202 plus human long membrane tethered IL-12 (lmt). The L202 TCR has mouse TCRb.

Activation of CD3, CD8 double positive T cells in TCRb+ and TCRb- populations was measured by staining for CD69. In Figure 9A, the mTCRb+ population indicates cells with IL12 armor. CD69 is a marker of activation. Activation of TCRb+ cells was comparable for L202 TCR-T and armored L202 TCR-T cells (Figure 9A). In Figure 9B, the expression of CD69 on the mTCRb-population indicates activation of surrounding cells (unarmored cells). Unarmored cells were also activated. Addition of membrane-tethered IL-12 further increased activation of PBMCs that do not express mTCRb (Figure 9B). Example 10 : IFNγ activation in TCRb+ cells is increased by addition of membrane-tethered IL-12

Untransduced human PBMC cells, engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12), or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) ) were mixed with untransduced human PBMC cells at a ratio of 1:1.

These cells were then co-cultured with A375-LLW target cells and subsequently subjected to intracellular IFNy FITC staining using BD Perm/Wash buffer. Cells were gated on lymphocytes, followed by single cells, CD3+ cells, CD8+ cells and TCRb+ cells. As shown in Figures 10A-10H, the data demonstrate that expression of membrane-tethered IL-12 enhances T cell activation by TCR+ PBMCs. Example 11 : IFNγ activation in TCRb- cells is increased by addition of membrane-tethered IL-12

Untransduced human PBMC cells, engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12), or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) ) were mixed with untransduced human PBMC cells at a ratio of 1:1.

These cells were then co-cultured with A375-LLW target cells and subsequently subjected to intracellular IFNy FITC staining using BD Perm/Wash buffer. Cells were gated on lymphocytes, followed by single cells, CD3+ cells, CD8+ cells and TCRb- cells. As shown in Figures 11A-11H, the data demonstrate that expression of membrane-tethered IL-12 enhances T-cell activation of TCR-PBMCs. Example 12 : Phenotypic Analysis of IL-12 Armored TCR-T Cells

On day 12 post-infection, treatment with untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane-tethered IL-12 (mt-IL12), or human long membrane-tethered IL- 12 (lmt-IL12) PBMCs of L202 TCR-T were stained for CD4 (hCD4-Pacific Blue) and CD8 (hCD8-PerCP-Cy5.5). The results are shown in Figures 12A to 12D. IL-12-armoured L202 TCR-T cells exhibited significantly higher CD4 expression compared to untransduced and L202 TCR-T cells, indicating an IL-12-enriched CD4+ T cell population. Example 13 : Flow cytometry staining for quantification of central and effector memory T cells

Untransduced CD4+ PBMCs in the above experiments were stained for the effector memory marker CD45RO (hCD45RO-FITC antibody) and the central memory marker CCR7 (CCR7-R-PE antibody). As shown in Figure 13, flow cytometry analysis revealed that approximately 41% of PBMCs were effector memory cells, while 58% were central memory cells. Example 14 : Memory Phenotype of IL-12 Armored CD4+ TCR-T Cells

For CD4+ untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane tethered IL-12 (mt-IL12) or expressing human long membrane tethered IL-12 (lmt-IL12) L202 TCR-T PBMCs were stained for CD45RO and CCR7. As shown in Figures 14A-14D, the results demonstrate that the expression of surface-bound IL-12 biases CD4+ PBMCs towards the effector memory phenotype. Example 15 : Memory Phenotype of IL-12 Armored CD8+ TCR-T Cells

For CD8+ untransduced (UT), L202 TCR-T (L202), L202 TCR-T expressing human membrane tethered IL-12 (mt-IL12) or expressing human long membrane tethered IL-12 (lmt-IL12) L202 TCR-T PBMCs were stained for CD45RO and CCR7. As shown in Figures 15A-15D, the results demonstrate that the expression of surface-bound IL-12 biases CD8+ PBMCs towards an effector memory phenotype. Example 16 : IL-12 is not released from cells expressing membrane-tethered IL-12

Jurkat cells were either untransduced (UT), or transduced to express IL-12, NHS76-IL-12 or membrane-tethered IL-12. On day 5 post-transduction, ¹ x 106 cells were plated in 1 ml of cell culture medium in 12-well plates. After 24 hours, the cell culture was centrifuged and the supernatant was collected. The concentration of IL-12 in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). As shown in Figure 16, Jurkat cells expressing membrane-tethered IL-12 had the lowest levels of IL-12 in the cell culture medium. In contrast, Jurkat cells expressing IL-12 or NHS76-IL-12 had high levels of extracellular IL-12 in culture medium (eg, 3000 pg/mL-5000 pg/mL). The results show that membrane-tethered IL-12 with CH2 and CH3 domains and CD4 transmembrane region can be safely tethered to the cell membrane and is not released into the cell culture medium. Example 17. Construct Design

The mouse and human IL-12 expression vectors were further modified (Figures 17A-17B). Sequences encoding mouse Flt3L (mFlt3L), mouse CXCL10 (mCXCL10) or mouse XCL1 (mXCL1) were added to the construct (Figure 17A). mFlt3L, mCXCL10 or mXCL1 are separated from membrane-tethered IL-12 by a 2A self-cleaving peptide.

Similarly, sequences encoding human Flt3L (hFlt3L), human CXCL10 (mCXCL10) or human XCL1 (hXCL1) were added to the construct (Figure 17B). hFlt3L, hCXCL10 or hXCL1 are separated from membrane-tethered IL-12 by a 2A self-cleaving peptide.

The sequence encoding the TCR or CAR can be added before the sequence encoding Flt3L, CXCL10 or XCL1 or after the sequence encoding membrane-tethered CD4. These sequences can be separated by the 2A self-cleaving peptide.

In this example, sequences encoding anti-EGFRvIII CARs were added to these constructs. Anti-EGFRvIII CARs are known in the art and are described, for example, in US10570214B2, which is incorporated herein by reference in its entirety. Primary mouse lymphocytes were isolated from lymph nodes and transduced with these constructs. These cells were first labeled with the His-tagged EGFRvIII peptide (SEQ ID NO: 45) and then with a labeled secondary antibody for His-tagging. As shown in Figure 18, the transduction efficiencies of these constructs ranged from 26%-53%. Efficiency was then used to determine the number of CAR+ cells so that an equal amount of CAR+ cells was used in the experiments for comparison purposes. Example 18. EGFRvIII CAR with a single armor efficiently kills target cells

Mouse lymphocytes were encoded with (1) anti-EGFRvIII CAR ("EGFRvIII"), (2) IL12 and anti-EGFRvIII CAR ("EGFRvIII-IL12"), (3) CXCL10 and anti-EGFRvIII CAR ("EGFRvIII-CxCL10"), (4) Flt3L and anti-EGFRvIII CAR ("EGFRvIII-FLt3L"), (5) CXCL10, IL12 and anti-EGFRvIII CAR ("EGFRvIII-CxCL10-IL12") or (5) Flt3L, IL12 and anti-EGFRvIII CAR ("EGFRvIII- Flt3L-IL12") construct for transduction. Transduced lymphocytes were co-cultured with KLUC and KLuc-EGFRvIII target cells at different effector to target cell ratios for 24 hours. As shown in Figure 19, all transduced lymphocytes efficiently killed target cells. Example 19. Cytokinin secretion in co-culture

Transduced mouse lymphocytes (CAR+) were cultured with equal numbers of Kluc or Kluc-EGFRvIII ("Kluc vIII") tumor cells. Interferon secretion was measured. IFNg secretion was higher when CAR+ cells were co-cultured with KLUC vIII target cells (Figure 20A). Similarly, IL12 secretion was higher when CAR+ cells were co-cultured with KLUC vIII target cells (FIG. 20B). CXCL10 secretion was also measured (Figure 20C). Example 20. In vivo efficacy studies of EGFRvIII armored CARs

On day -39, C57BL/6 mice were inoculated with ⁶ x 106 KLUC cells and KLUC vIII cells. On day -4, mice were then injected intraperitoneally with 150 mg/kg cyclophosphamide in a dosing volume of 10 mL/kg. On day 0, cells transduced with various constructs were transferred into mice (Figure 21). Before mice were treated with these CAR-T cells, the average tumor size was about 106 mm ³ .

22A to 22F show the results of the experiments. As shown in Figures 22A-22F, T cells expressing anti-EGFRvIII CAR, CXCL10 and IL12 (Figure 22B), T cells expressing anti-EGFRvIII CAR, Flt3 and IL12 (Figure 22C) and T cells expressing anti-EGFRvIII CAR and IL12 (FIG. 22F) Effectively kills tumor cells. In contrast, T cells that did not express IL12 were not very effective. Figure 23 shows survival curves of different groups of mice. In Figure 23, the EGFRvIII+CxCl10+IL12 group and the EGFRvIII+flt3+IL12 group have the same curve.

Of those mice shown in Figures 22B, 22C and 22F, most of them became tumor free. On day 94 (ie, 94 days after T cell transfer), all tumor-free mice were rechallenged with 2.5 x 106 ^KLUC cells on the right and 2.5 x 106 ^KLUCvIII cells on the left. These mice were tumor free for approximately 66 days prior to challenge. Table 1 Group size tumor cell type number of tumor cells not processed 4 KLUC/KLUCvIII 5x10 ⁶ EGFRvIII Car+CxCl10+IL12 6 KLUC/KLUCvIII 5x10 ⁶ EGFRvIII Car+flt3+IL12 6 KLUC/KLUCvIII 5x10 ⁶ EGFRvIII Car+IL12 5 KLUC/KLUCvIII 5x10 ⁶

Figure 24A shows the percentage of antigen-negative (EGFRvIII negative) tumor-free animals at day 52 after tumor cell implantation in mice. In these mice, group C001 was the control group. The mice had not been previously treated with cell therapy. Mice in the C001-IL12 group were previously treated with cells expressing IL12 and an anti-EGFRvIII CAR ("C001-IL12"). Mice in the C001-CXCL10-IL12 group were previously treated with cells expressing CXCL10, IL12 and anti-EGFRvIII CAR. Mice in the C001-Flt3L-IL12 group were previously treated with cells expressing Flt3L, IL12 and anti-EGFRvIII CAR. The results showed that CXCL10 and Flt3L could further improve the immune response. Without wishing to be bound by theory, it is hypothesized that CXCL10 and Flt3L may increase the activity of antigen presenting cells. Antigen-presenting cells can present some other tumor antigens to host immune cells, such that the host immune cells will recognize these tumor antigens and kill these tumor cells, even if these tumor cells do not express the antigen recognized by CAR or TCR. Figure 24B shows survival curves for a rechallenge study after mice were engrafted with tumor cells. The results again showed that CXCL10 and Flt3L further improved the immune response. Other implementations

It should be understood that while the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

without

Exemplary embodiments are shown in the accompanying drawings. The embodiments and figures disclosed herein are intended to be regarded in an illustrative rather than a restrictive sense.

Figure 1A is a schematic diagram showing a mouse IL-12 expression vector. P2A encodes 2A self-cleaving peptide; mSP encodes mouse message peptide; mP40 encodes mouse IL-12B (beta chain); linker encodes peptide linker; mP35 encodes mouse IL-12A (alpha chain); hinge encodes immunoglobulin hinge region ; mCH2 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH2; mCH3 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH3; mCD4 TM encodes mouse CD4 transmembrane region; NHS-76 vH encodes target tumor necrosis The heavy chain variable region (VH) of human IgG1 (NHS-76); and NHS-76 vL encodes the light chain variable region (VL) of NHS-76.

Figure IB is a schematic diagram showing a human IL-12 expression vector. P2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 (human mutated CH2) encodes mutated Human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 (human CH3) encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the VH of NHS-76; And NHS-76 vL encodes the VL of NHS-76.

Figure 1C is a schematic diagram showing a vector expressing human IL-12 in combination with TCR. TCR encodes a T cell receptor (eg, L202); P2A encodes a 2A self-cleaving peptide; hSP encodes a human message peptide; hP40 encodes human IL-12B; linker encodes a peptide linker; hP35 encodes human IL-12A; hinge encodes an immunoglobulin hinge Region; hmCH2 encodes the mutated human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 encodes the human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the NHS-76 VH; and NHS-76 vL encodes the VL of NHS-76.

Figure ID is a schematic diagram showing a vector expressing human IL-12 in combination with a CAR. CAR encodes chimeric antigen receptor; P2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 encodes Mutated human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region; NHS-76 vH encodes the VH of NHS-76; and NHS -76 vL encodes the VL of NHS-76.

Figure 2A is a bar graph showing mouse IL-12 concentration in culture medium as determined by ELISA (enzyme-linked immunosorbent assay). Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus mouse membrane-tethered IL-12 (sample name: mt12) , or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells), or alone (control).

Figure 2B is a bar graph showing mouse IFNy concentrations in culture medium as determined by ELISA. Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus mouse membrane-tethered IL-12 (sample name: mt12) , or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells), or alone (control).

Figure 3 is a bar graph showing mouse IFNy concentrations in culture medium as determined by ELISA. Purified mouse lymphocytes were not transduced (sample name: UT), or transduced to express L202 TCR alone (sample name: L202), L202 plus membrane-tethered IL-12 (sample name: mt12), or L202 plus NHS76-IL-12 (sample name: NHS76). Lymphocytes were co-cultured with Jurkat cells (target cells + mouse lymphocytes), or alone (control).

Figure 4A is a graph showing cell surface IL-12 expression in untransduced primary mouse lymphocytes (sample name: untransduced).

Figure 4B is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202) transduced to express L202 TCR.

Figure 4C is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202+mtIL12) transduced to express L202 TCR and human membrane-tethered IL-12.

Figure 4D is a graph showing cell surface IL-12 expression in primary mouse lymphocytes (sample name: L202+NHS-IL12) transduced to express L202 TCR and NHS76-IL12.

Figure 5A is a graph showing primary mouse lymphocytes either untransduced (sample name: NHS76-12 UT) or transduced to express NHS76-IL-12 (sample name: NHS76-12 ) is a graph of human Fab (hFab) performance. NHS76-IL12 was detected by staining for human Fab.

Figure 5B is a graph showing primary mouse lymphocytes either untransduced (sample name: NHS76-12 UT) or transduced to express NHS76-IL-12 (sample name: NHS76-12 ) in the graph of IL-12 expression. NHS76-IL12 exhibited a peak shift when stained for IL-12.

Figure 6A is a graph showing TCRb surface staining of untransduced human PBMC (sample name: untransduced).

Figure 6B is a graph showing TCRb surface staining of human PBMC (sample name: L202+long mtIL12) transduced to express the combination of L202 TCR and human long-membrane-tethered IL-12.

Figure 6C is a graph showing TCRb surface staining of human PBMCs (sample name: L202+mtIL12) transduced to express L202 TCR in combination with human membrane-tethered IL-12.

Figure 6D is a graph showing TCRb surface staining of human PBMC (sample name: L202) transduced to express the L202 TCR alone.

Figure 6E is a graph showing human IL-12 surface staining of untransduced human PBMC (sample name: untransduced).

Figure 6F is a graph showing human IL-12 surface staining of human PBMC (sample name: L202+long mtIL12) transduced to express the combination of L202 TCR and long mt-IL-12.

Figure 6G is a graph showing human IL-12 surface staining of human PBMC (sample name: L202+mtIL12) transduced to express the combination of L202 TCR and mt-IL-12.

Figure 6H is a graph showing human IL-12 surface staining of human PBMC (sample name: L202) transduced to express the L202 TCR alone.

Figure 7A is a graph showing CD69 expression in human PBMC co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells. Samples included untransduced human PBMC (sample name: UT A375LLW); human PBMC transduced to express L202 TCR (sample name: L202 A375LLW); human PBMC transduced to express L202 plus human membrane-tethered IL-12 ( Sample name: smt A375LLW); and human PBMC (sample name: lmt A375LLW) transduced to express L202 plus human long membrane tethered IL-12 (lmt).

Figure 7B is a graph showing CD69 expression in human PBMC without co-culture. Samples included untransduced human PBMC (sample name: UT); human PBMC transduced to express L202 TCR (sample name: L202); human PBMC transduced to express L202 plus human membrane-tethered IL-12 (sample name) : smt); and human PBMCs (sample name: lmt) transduced to express L202 plus human long-membrane-tethered IL-12.

Figure 8A shows flow cytometry results showing human IFNy (hlFNy) expression in untransduced human PBMCs.

Figure 8B shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR.

Figure 8C shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR plus membrane-tethered IL-12.

Figure 8D shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR lengthening membrane tethered IL-12.

Figure 8E shows flow cytometry results showing human IFNy (hlFNy) expression in untransduced human PBMCs. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

Figure 8F shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

Figure 8G shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR plus membrane-tethered IL-12. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

Figure 8H shows flow cytometry results showing human IFNy (hlFNy) expression in human PBMCs transduced to express L202 TCR lengthening membrane-tethered IL-12. PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.

Figure 9A is a graph showing the expression of CD69 in the mTCRb+ population after generating a mixed population of 50% L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as controls) and 50% untransduced human PBMCs (mTCRb-) picture. Samples included untransduced PBMCs (sample name: UT+A375); PBMCs transduced to express L202 TCR (sample name: L202+A375); transduced to express L202 plus short membrane tethered IL-12 (smt) PBMCs (sample name: smt+A375); and PBMCs transduced to express L202 lengthening membrane-tethered IL-12 (sample name: lmt+A375).

Figure 9B is a graph showing CD69 expression in the mTCRb population after generating a mixed population of 50% L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as controls) and 50% untransduced PBMCs (mTCRb-) . Samples included untransduced PBMCs (sample name: UT+A375); PBMCs transduced to express L202 TCR (sample name: L202+A375); transduced to express L202 plus short membrane tethered IL-12 (smt) PBMCs (sample name: smt+A375); and PBMCs transduced to express L202 lengthening membrane-tethered IL-12 (sample name: lmt+A375).

Figure 10A shows flow cytometry results showing human IFNγ (hIFNγ) expression in mTCRb+ cells from untransduced PBMCs

Figure 10B shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR.

Figure 1OC shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12.

Figure 10D shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12.

Figure 10E shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from untransduced PBMCs. PBMCs were co-cultured with A375-LLW target cells.

Figure 10F shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-LLW target cells.

Figure 10G shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

Figure 10H shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb+ cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

Figure 11A shows flow cytometry results showing human IFNγ (hIFNγ) expression in mTCRb-cells from untransduced PBMCs

Figure 11B shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR.

Figure 11C shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12.

Figure 1 ID shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12.

Figure 11E shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from untransduced PBMCs. PBMCs were co-cultured with A375-LLW target cells.

Figure 11F shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR. PBMCs were co-cultured with A375-LLW target cells.

Figure 11G shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human membrane-tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

Figure 11H shows flow cytometry results showing human IFNy (hlFNy) expression in mTCRb-cells from PBMCs transduced to express L202 TCR plus human long membrane tethered IL-12. PBMCs were co-cultured with A375-LLW target cells.

Figure 12A shows flow cytometry results showing CD4 and CD8 expression in untransduced PBMC (UT).

Figure 12B shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMCs (L202).

Figure 12C shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

Figure 12D shows flow cytometry results showing CD4 and CD8 expression in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

Figure 13 shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in untransduced PBMCs.

Figure 14A shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in untransduced PBMC (UT).

Figure 14B shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (L202).

Figure 14C shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

Figure 14D shows flow cytometry results showing the percentage of effector memory CD4+ cells and central memory CD4+ cells in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

Figure 15A is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in untransduced PBMC (UT).

Figure 15B is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (L202).

Figure 15C is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (mt-IL12) expressing human membrane-tethered IL-12.

Figure 15D is an image of flow cytometry results showing the percentage of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMC (lmt-IL12) expressing human long membrane tethered IL-12.

Figure 16 shows the expression of human PBMC (sample name: UT), transduced to express human IL-12 (sample name: IL-12), transduced to express human NHS76-IL-12 IL-12 concentration in cell culture medium of human PBMC (sample name: NHS76-IL-12) or human PBMC (sample name: Mt-IL-12) transduced to express human membrane-tethered IL-12.

Figure 17A is a schematic diagram showing a mouse IL-12 expression vector with mouse Flt3L (mFlt3L), mouse CXCL10 (mCXCL10) or mouse XCL1 (mXCL1). 2A encodes 2A self-cleaving peptide; mSP encodes mouse message peptide; mP40 encodes mouse IL-12B (beta chain); linker encodes peptide linker; mP35 encodes mouse IL-12A (alpha chain); hinge encodes immunoglobulin hinge region mCH2 encodes the mouse immunoglobulin IgG4 heavy chain constant domain CH2; mCH3 encodes the mouse immunoglobulin IgG4 heavy chain constant domain CH3; and mCD4 TM encodes the mouse CD4 transmembrane region.

Figure 17B is a schematic diagram showing human IL-12 expression vectors with human Flt3L (hFlt3L), human CXCL10 (mCXCL10) or human XCL1 (hXCL1). 2A encodes 2A self-cleaving peptide; hSP encodes human message peptide; hP40 encodes human IL-12B; linker encodes peptide linker; hP35 encodes human IL-12A; hinge encodes immunoglobulin hinge region; hmCH2 (human mutated CH2) encodes mutated Human immunoglobulin IgG4 heavy chain constant domain CH2; hCH3 (human CH3) encodes human immunoglobulin IgG4 heavy chain constant domain CH3; hCD4 TM encodes the human CD4 transmembrane region.

Figure 18 is a graph showing CAR performance in untransduced primary mouse lymphocytes and primary mouse lymphocytes transduced with various constructs. Among them, Cxcl10-IL12 refers to the construct encoding CXCL10, IL12 and anti-EGFRvIII CAR; Flt3L-IL12 refers to the construct encoding Flt3L, IL12 and anti-EGFRvIII CAR; IL12 refers to the construct encoding IL12 and anti-EGFRvIII CAR EGFRvIII refers to the construct encoding anti-EGFRvIII CAR; CxCl10 refers to the construct encoding CXCL10 and anti-EGFRvIII CAR; Flt3L refers to the construct encoding Flt3L and anti-EGFRvIII CAR.

Figure 19 shows the percentage of competitive killing when untransduced lymphocytes ("UT") or transduced lymphocytes were co-cultured with target tumor cells at different effector to target cell ratios.

Figure 20A shows mIFNg secretion when untransduced lymphocytes or transduced lymphocytes are co-cultured with Kluc cells or Kluc-EGFRvIII ("Kluc vIII") cells. Lymphocytes were untransduced ("UT") or treated with encoding (1) anti-EGFRvIII CAR ("EGFRvIII CAR"), (2) IL12 and anti-EGFRvIII CAR ("EGFRvIII-IL12"), (3) CXCL10 and Anti-EGFRvIII CAR ("EGFRvIII-CxCL10"), (4) Flt3L and anti-EGFRvIII CAR ("EGFRvIII-FLt3L"), (5) CXCL10, IL12 and anti-EGFRvIII CAR ("EGFRvIII-Cx12") or (5) Flt3L, Constructs transduction of IL12 and anti-EGFRvIII CAR ("EGFRvIII-Ft12").

Figure 20B shows mIL12 secretion when untransduced lymphocytes or transduced lymphocytes are co-cultured with Kluc cells or Kluc-EGFRvIII cells.

Figure 20C shows CXCL10 secretion when untransduced lymphocytes or transduced lymphocytes were co-cultured with Kluc cells or Kluc-EGFRvIII cells.

Figure 21 is a diagram illustrating the study design of the in vivo testing model.

Figures 22A-22F show tumor volumes in different mice following treatment with different lymphocytes transduced with different constructs.

Figure 23 shows the survival curves of different mice after treatment with different transduced lymphocytes.

Figure 24A shows the percentage of tumor-free animals in the rechallenge study.

Figure 24B shows survival curves for different mice in the rechallenge study.

Figure 25 lists the sequences described in this disclosure.

Claims (81)

a cell that expresses (a) exogenous T cell receptor (TCR) or chimeric antigen receptor (CAR); and (b) IL-12. The cell of claim 1, wherein the IL-12 is membrane-tethered IL-12. The cell of claim 2, wherein the membrane-tethered IL-12 comprises a CD4 transmembrane region. The cell of claim 3, wherein the membrane-tethered IL-12 comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain and a CD4 transmembrane region. The cell of claim 3, wherein the membrane-tethered IL-12 comprises two or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a CD4 transmembrane region. The cell of claim 4 or 5, wherein the immunoglobulin CH2 domain is a wild-type immunoglobulin constant domain. The cell of claim 4 or 5, wherein the amino acid at position 235 (EU numbering) of the immunoglobulin CH2 domain is Glu, and the immunoglobulin CH2 domain at position 297 (EU numbering) numbering) of the amino acid residue is GIn. The cell of any one of claims 2-7, wherein the membrane-tethered IL-12 further comprises an immunoglobulin hinge region. The cell of any one of claims 4-8, wherein the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain, and the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain, And the CD4 transmembrane region is a human CD4 transmembrane region. The cell of claim 1, wherein the IL-12 is soluble IL-12. The cell of any one of claims 1-10, wherein the IL-12 is linked to a tumor-targeting antibody or antigen-binding fragment thereof. The cell of claim 11, wherein the tumor-targeting antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). The cell of claim 11 or 12, wherein the tumor-targeting antibody is NHS76. The cell of any one of claims 1-13, wherein the cell further expresses CXCL10, XCL1 or Flt3L. The cell of any one of claims 1 to 14, wherein the TCR or CAR targets BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (IL-3R), CEA, IL13Ra2, ALPP, EBV associated Antigens (eg, LMP2), EGFR, EGFRvIII, GD2, GPC3, HER2, HPV-associated antigens (eg, E6, E7), MAGE (eg, MAGE-A3), mesothelin, MUC-1, NY-ESO-1 , PSCA, PSMA, ROR1, WT1 or Claudin 18.2. The cell of any one of claims 1-15, wherein the CAR comprises an extracellular domain, wherein the extracellular domain is a single-chain variable fragment (scFv), a ligand (eg, a receptor binding ligand) body) or antibody mimetics. A carrier comprising: a) a first nucleic acid sequence encoding an IL-12α subunit and an IL-12β subunit; b) a second nucleic acid sequence encoding one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence. The vector of claim 17, wherein the first linker sequence encodes an immunoglobulin hinge polypeptide sequence. The vector of claim 17 or 18, wherein the transmembrane region is a CD4 transmembrane region. The vector of claim 17 or 18, wherein the transmembrane region is an immunoglobulin transmembrane region. The vector of any one of claims 17-20, wherein the IL-12α subunit and the IL-12β subunit are linked by a linker sequence. The vector of any of claims 17-21, wherein the vector further comprises a sequence encoding a message peptide. The vector of any one of claims 17-22, wherein the IL-12α subunit is a human IL-12α subunit, the IL-12β subunit is a human IL-12β subunit, and the immunoglobulin The protein CH2 domain is a human immunoglobulin CH2 domain, and the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain. The vector of any of claims 17-23, wherein the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The vector of claim 24, wherein the third nucleic acid sequence is linked to the first nucleic acid through a second linker sequence. The vector of claim 25, wherein the second linker sequence encodes a 2A sequence (eg, a P2A sequence). The vector of any one of claims 17-26, wherein the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter); or wherein the first nucleic acid, the second nucleic acid The second nucleic acid and the third nucleic acid are under the control of regulatory elements such as promoters. The vector of any one of claims 17-27, wherein the vector further comprises a sequence encoding CXCL10, XCL1 or Flt3L. A carrier comprising: a) a first nucleic acid sequence encoding the variable heavy (VH) and variable light (VL) regions of the tumor-targeting antibody; b) a second nucleic acid sequence encoding the IL-12α subunit and the IL-12β subunit, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence. The method of claim 29, wherein the tumor-targeting antibody targets a tumor-associated antigen. The method of claim 30, wherein the tumor-targeting antibody is NHS76. The vector of any one of claims 29-31, wherein the vector further comprises a nucleic acid encoding a message peptide. The vector of claim 32, wherein the message peptide is a human message peptide, the IL-12α subunit is a human IL-12α subunit, and the IL-12β subunit is a human IL-12β subunit. The vector of any of claims 29-33, wherein the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The vector of claim 34, wherein the third nucleic acid sequence is linked to the 5' of the first nucleic acid through a second linker sequence. The vector of claim 35, wherein the second linker sequence encodes a P2A sequence. The vector of any one of claims 29-36, wherein the first nucleic acid and the second nucleic acid are under the control of a regulatory element (eg, a promoter); or wherein the first nucleic acid, the second nucleic acid The second nucleic acid and the third nucleic acid are under the control of regulatory elements such as promoters. The vector of any one of claims 29-37, wherein the vector further comprises a sequence encoding CXCL10, XCL1 or Flt3L. A vector comprising in the 5' to 3' direction a) a first nucleic acid sequence encoding a foreign T cell receptor (TCR) or a chimeric antigen receptor (CAR); b) a second nucleic acid sequence encoding the message peptide IL-12α subunit and IL-12β subunit; wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a linker sequence. The vector of claim 39, wherein the IL-12α subunit and the IL-12β subunit are linked by a linker sequence. The vector of claim 39 or 40, wherein the linker sequence encodes a P2A sequence. A fusion polypeptide comprising a) a first region comprising an IL-12α subunit and an IL-12β subunit, wherein the IL-12α subunit and the IL-12β subunit are connected by a linker sequence, b) A second region comprising one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains and a transmembrane region. The polypeptide of claim 42, wherein the first region and the second region are linked by an immunoglobulin hinge peptide. A fusion polypeptide comprising a) a first region comprising an IL-12α subunit and an IL-12β subunit, wherein the IL-12α subunit and the IL-12β subunit are connected by a first linker sequence, b) a second region comprising a heavy chain variable region (VH) and a light chain variable region (VL) of a tumor-targeting antibody, wherein said VH and said VL are linked by a second linker peptide sequence, wherein the first region and the second region are linked by a third linker peptide sequence. The polypeptide of claim 44, wherein the third linker polypeptide sequence has a sequence that is at least 80% identical to SEQ ID NO: 17. A nucleic acid encoding the polypeptide of any one of claims 42-45. A vector comprising the nucleic acid of claim 46. A cell expressing the fusion polypeptide of any one of claims 42-45. A cell comprising the vector of any one of claims 17-41 and 47. The cell of claim 49, wherein the cell secretes a higher level of interleukin than the same cell differing in that the cell does not contain the vector. The cell of claim 49, wherein the cell stimulates one or more cells in the vicinity of the cell to secrete interferons. The cell of claim 50 or 51, wherein the interferon is IFNγ. The cell of claim 49, wherein the cell exhibits a higher level of early TCR activation markers, and/or stimulates a One or more cells express markers of early TCR activation. The cell of claim 53, wherein the activation marker is CD69. The cell of any one of claims 1-16 and 48-54, wherein the cell is a cell line. The cell of any one of claims 1-16 and 48-54, wherein the cell is a primary cell obtained from a subject (eg, a human subject). The cell of any one of claims 1-16 and 48-54, wherein the cell is an immune cell (eg, a lymphocyte). The cell of any one of claims 1-16 and 48-54, wherein the cell is a tumor-infiltrating lymphocyte (TIL) or a NK cell (eg, a CAR-NK cell). The cell of any one of claims 1-16 and 48-54, wherein the cell is a T cell. The cell of claim 59, wherein the T cells are isolated from a human subject. The cell of claim 59 or 60, wherein the T cells are CD8+. The cell of any of claims 59-61, wherein the T cells are CD4+. The vector of any one of claims 17-41 and 47, wherein the vector is an expression vector, a viral vector, a retroviral vector or a lentiviral vector. The vector of claim 63, wherein the retroviral vector is pMP71. A method for producing a cell, the method comprising introducing the vector of any one of claims 17-41, 47, 63 and 64 into the cell in vitro or ex vivo. The method of claim 65, wherein the vector is introduced into the cell by transduction. A method of treating a subject suffering from cancer, the method comprising administering to a subject in need thereof an effective amount of a cell of any one of claims 1-16 and 48-62. The method of claim 67, wherein the cancer is a heterogeneous cancer. The method of claim 67, wherein the cancer is a homogeneous cancer. The method of any of claims 67-69, wherein the cells are isolated from peripheral blood mononuclear cells (PBMCs) of the subject. A method of treating a subject with cancer, the method comprising administered to a subject in need, a) an effective amount of cells expressing T cell receptor (TCR) or chimeric antigen receptor (CAR); and b) An effective amount of a protein comprising IL-12 and a tumor-targeting antibody or antigen-binding fragment thereof. The method of claim 71, wherein the cells are isolated from peripheral blood mononuclear cells of the subject. A method of treating a human subject suffering from cancer, the method comprising providing cells collected from the human subject or a different human subject; introducing the vector of any one of claims 17-41 and 47 into the cell; culturing and expanding the cells; and An effective amount of a composition comprising the cells is administered to the subject. The method of claim 73, wherein the cancer is a heterogeneous cancer. The method of claim 73, wherein the cancer is a homogeneous cancer. The method of any of claims 73-75, wherein the cells are peripheral blood mononuclear cells (PBMCs). The method of any of claims 73-75, wherein the cells are tumor-infiltrating lymphocytes and the vector comprises a nucleic acid encoding IL-12. The method of claim 77, wherein the IL-12 is membrane-tethered IL-12. The method of any one of claims 73-75, wherein the cell is a T cell and the vector comprises a nucleic acid encoding a TCR or CAR. The method of claim 79, wherein the vector further comprises a nucleic acid encoding IL-12. The method of claim 80, wherein the IL-12 is membrane-tethered IL-12.

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