TW202317179A - Methods of treating hpv16-positive or hpv16-associated cancer in a subject - Google Patents

Abstract

Provided herein are methods of treating an HPV16-positive or HPV16-associated cancer in a subject that include adminsitering a dose of a pharmaceutical composition including human enucleated erythroid cells.

Description

Method of treating HPV16-positive or HPV16-associated cancer in an individual

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application Serial No. 63/196,615, filed June 3, 2021, the entire contents of which are incorporated herein by reference.

sequence listing

This application contains a Sequence Listing filed electronically in an ASCII text file named Sequence_Listing.txt. The size of the ASCII text file created on May 25, 2022 is 82.6 KB. The material in the ASCII text file is incorporated herein by reference in its entirety.

The present invention generally relates to a method of treating an HPV16-positive or HPV16-associated cancer in an individual comprising administering to the individual human enucleated erythroid cells.

Engineered enucleated erythroid cells, which carry or present exogenous proteins to patients in need, are currently being developed as therapeutic agents.

Provided herein are methods of treating HPV16-positive or HPV16-associated cancers in a human individual in need thereof. In some embodiments, the methods described herein comprise intravenously administering to a human subject a dose of a pharmaceutical composition comprising about 0.5 x 10 ⁹ to about 5 x 10 ¹⁰ human enucleated erythroid cells, human denucleated The surface of the nuclear erythrocytes comprises: an exogenous antigen-presenting polypeptide linked to an exogenous antigen polypeptide, wherein the exogenous antigen-presenting polypeptide is an MHC class 1 HLA-A2 single-chain fusion protein comprising a α chain, a β2-microglobulin (β2m) chain, and a membrane anchor region, and wherein the exogenous antigenic polypeptide comprises a human papillomavirus (HPV) E7 antigen; a stimulating polypeptide; and an exogenous cytokine polypeptide comprising IL-12; the frequency of administration is from about 2 weeks to about 4 weeks (eg, about 3 weeks).

In some embodiments, the HPV16-positive or HPV16-related cancer is selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, cervical cancer, vaginal cancer, penile cancer, oropharyngeal cancer, oral cavity cancer and vaginal cancer. In some embodiments, the HPV16-positive or HPV16-related cancer is selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, and cervical cancer. In some embodiments, the HPV16-positive or HPV16-related cancer is an unresectable solid tumor.

In some embodiments, the HPV16-positive or HPV16-related cancer is relapsed or refractory to standard therapy for the HPV16-positive or HPV16-related cancer. In some embodiments, the human subject has previously received chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof. In some embodiments, the human subject has previously received external beam radiation therapy, brachytherapy, or a combination thereof. In some embodiments, the human subject has previously received standard platinum or mitomycin C-based chemotherapy. In some embodiments, the human subject has previously received immunotherapy comprising a therapeutic vaccine, targeted antibody, or immune checkpoint therapy. In some embodiments, the human subject has previously received PD-1/PD-L1 therapy.

In some embodiments, the frequency is about every 14 days to about every 28 days. In some embodiments, the frequency is about once every three weeks.

In some embodiments, the dose of the pharmaceutical composition comprises about 0.5 x 10 ⁹ to about 5 x 10 ⁹ human enucleated erythroid cells. In some embodiments, the dose of the pharmaceutical composition comprises about 1 x 10 ⁹ human enucleated erythroid cells.

In some embodiments, the dose of the pharmaceutical composition comprises about 1 x 10 ⁹ to about 1 x 10 ¹⁰ human enucleated erythroid cells. In some embodiments, the dose of the pharmaceutical composition comprises about 5 x ¹⁰⁹ human enucleated erythroid cells.

In some embodiments, the dose of the pharmaceutical composition comprises about 5 x 10 ⁹ to about 5 x 10 ¹⁰ human enucleated erythroid cells. In some embodiments, the dose of the pharmaceutical composition comprises about 1 x ¹⁰¹⁰ human enucleated erythroid cells.

In some embodiments, the human enucleated erythroid cells comprise at least 1,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide. In some embodiments, the human enucleated erythroid cells comprise at least 5,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide.

In some embodiments, human enucleated erythroid cells comprise at least 1,000 copies of an exogenous costimulatory polypeptide. In some embodiments, human enucleated erythroid cells comprise at least 5,000 copies of an exogenous costimulatory polypeptide.

In some embodiments, human enucleated erythroid cells comprise at least 1,000 copies of an exogenous cytokine polypeptide. In some embodiments, the human enucleated erythroid cells comprise at least 5,000 copies of the exogenous cytokine polypeptide.

In some embodiments, the HPV E7 antigen comprises SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. In some embodiments, the HPV E7 antigen comprises SEQ ID NO:9.

In some embodiments, the β2m chain comprises SEQ ID NO: 1.

In some embodiments, the exogenous antigenic polypeptide is linked to the β2m chain via a linker. In some embodiments, the linker comprises SEQ ID NO: 33.

In some embodiments, the exogenous antigen-presenting polypeptide comprises an HLA-A2*02:01 allele polypeptide. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises a Y84A or Y84C substitution. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises SEQ ID NO: 3, 5 or 7. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises SEQ ID NO:3.

In some embodiments, the α-chain is linked to the β2m chain via a linker. In some embodiments, the linker comprises SEQ ID NO: 38.

In some embodiments, the α-chain is linked to the membrane anchor region via a linker. In some embodiments, the linker comprises SEQ ID NO: 39. In some embodiments, the membrane anchor region comprises glycophorin A (GPA) or its transmembrane domain, or a small integral membrane protein 1 (SMIM1) transmembrane domain. In some embodiments, the membrane anchor region comprises GPA. In some embodiments, the membrane anchor region comprises SEQ ID NO: 30.

In some embodiments, the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 15. In some embodiments, the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 15. In some embodiments, the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises SEQ ID NO:15.

In some embodiments, 4-1BBL comprises a sequence at least 90% identical to SEQ ID NO: 19. In some embodiments, 4-1BBL comprises SEQ ID NO: 19.

In some embodiments, the exogenous costimulatory polypeptide is fused to a membrane anchor region. In some embodiments, the exogenous co-stimulatory polypeptide is fused to the membrane anchor region via a linker. In some embodiments, the membrane anchor region comprises GPA or its transmembrane domain, or a transmembrane domain of SMIM1. In some embodiments, the GPA comprises SEQ ID NO: 30. In some embodiments, the linker comprises SEQ ID NO: 36.

In some embodiments, the exogenous costimulatory polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the exogenous costimulatory polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the exogenous co-stimulatory polypeptide comprises SEQ ID NO: 21.

In some embodiments, IL-12 comprises IL-12A protein and IL-12B protein. In some embodiments, the IL-12A protein comprises a sequence at least 90% identical to SEQ ID NO: 25, and the IL-12B protein comprises a sequence at least 90% identical to SEQ ID NO: 27. In some embodiments, the IL-12A protein comprises a sequence at least 95% identical to SEQ ID NO: 25, and the IL-12B protein comprises a sequence at least 95% identical to SEQ ID NO: 27. In some embodiments, the IL-12A protein comprises SEQ ID NO: 25, and the IL-12B protein comprises SEQ ID NO: 27.

In some embodiments, the exogenous cytokine polypeptide further comprises a linker between the IL-12A protein and the IL-12B protein. In some embodiments, the linker comprises SEQ ID NO: 33.

In some embodiments, the exogenous cytokine polypeptide is fused to a membrane anchor region. In some embodiments, the exogenous cytokine polypeptide is fused to the membrane anchor region via a linker. In some embodiments, the membrane anchor region comprises GPA or its transmembrane domain, or a transmembrane domain of SMIM1. In some embodiments, the membrane anchor region comprises SEQ ID NO: 31. In some embodiments, the linker comprises SEQ ID NO: 33.

In some embodiments, the exogenous cytokine polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 48. In some embodiments, the exogenous cytokine polypeptide comprises a sequence at least 95% identical to SEQ ID NO:48. In some embodiments, the exogenous cytokine polypeptide comprises SEQ ID NO:48.

In some embodiments, human enucleated erythroid blood cells are prepared by a process comprising: linking an exogenous antigen-presenting polypeptide encoding an exogenous antigenic polypeptide, an exogenous co-stimulatory polypeptide, and an exogenous A nucleic acid of a cytokine polypeptide, introduced into a nucleated erythroid precursor cell; and in a manner sufficient to express an exogenous antigen-presenting polypeptide linked to an exogenous antigen polypeptide, an exogenous costimulatory polypeptide, and an exogenous cytokine polypeptide Under the condition, the nucleated erythroid precursor cells are cultured, and the nucleated erythroid precursor cells are enucleated.

As used herein, an "HPV16-associated cancer" is a cancer that is HPV16-positive with a high probability (eg, at least 75%, at least 80%, or at least 85% probability). In some embodiments, HPV16-related cancers can be HPV16-positive.

The term "human engineered enucleated erythroid cells" refers to cells comprising one or more (e.g., two, three, four, five, or six) exogenous polypeptides (e.g., exemplified herein or known in the art). Any combination of exogenous polypeptides) human engineered enucleated erythroid cells. For example, a human engineered enucleated erythroid cell can have one or more exogenous polypeptides present in its cytoplasm. In some examples, human engineered enucleated erythroid cells can have one or more exogenous polypeptides present on their extracellular surface. In some examples, human engineered enucleated erythroid cells can have (i) one or more exogenous polypeptides present in their cytoplasm, and (ii) one or more exogenous polypeptides present on their extracellular surface. Non-limiting examples of human engineered enucleated erythroid cells include conjugated human enucleated erythroid cells, hypotonically loaded human enucleated erythroid cells, and human enucleated erythroid cells that have been modified by physical manipulation, such as described herein or known in the art Any of the exemplary types of manipulation) loaded human enucleated erythroid cells. Additional non-limiting aspects of human engineered enucleated erythroid cells are described herein.

The term "conjugated human enucleated erythroid cells" refers to human engineered enucleated erythroid cells having at least one exogenous polypeptide conjugated to another polypeptide (e.g., one of the endogenous human enucleated erythroid cells). Polypeptides or different exogenous polypeptides), which are present on the cell membrane of the human modified enucleated erythrocytes through the catalytic activity of enzymes and/or peptide sequences, and/or chemical reactions.

The term "hypotonically-loaded human enucleated erythroid cells" refers to a process obtained at least in part by exposing human enucleated erythroid cells or human erythroid precursor cells to a hypotonic buffer (e.g., the exemplary hypotonic erythrocytes described herein). Any of the strength buffers) containing one or more exogenous peptides to generate human engineered enucleated erythroid cells. Non-limiting examples of methods that can be used to generate hypotonic-loaded human enucleated erythroid cells are described herein. Additional methods for producing hypotonic-loaded human enucleated erythroid cells are known in the art.

The term "human enucleated erythroid cells loaded by physical manipulation" refers to human erythroid precursor cells that have been at least partially physically manipulated such that they will encode one or more exogenous polypeptides (such as any described herein or known in the art). Exemplary exogenous polypeptides), and/or one or more exogenous polypeptides (such as any exogenous polypeptides described herein) are introduced into the human erythroid precursor cells to produce human enucleated erythroid cells. Non-limiting examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous polypeptides into human erythroid precursor cells include electroporation and particle-mediated transfection. Additional examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous polypeptides into human erythroid precursor cells are known in the art.

The term "exogenous polypeptide" refers to a polypeptide that is introduced into or on a cell, or is expressed by a cell by introducing an exogenous nucleic acid encoding the polypeptide into the cell or a precursor of the cell. In some embodiments, an exogenous polypeptide is a polypeptide encoded by an exogenous nucleic acid introduced into a cell or a precursor of the cell, optionally not retained by the cell. In some embodiments, an exogenous polypeptide is one that is chemically or enzymatically attached to the extracellular surface of a cell. Examples of exogenous polypeptides that may be present in engineered human enucleated erythroid cells are described herein.

The phrase "on the extracellular surface" when used in the context of an exogenous polypeptide refers to (1) an exogenous polypeptide that is physically attached to or at least partially embedded in the cell membrane of a human enucleated erythrocyte (e.g. , a transmembrane polypeptide, a peripheral membrane polypeptide, a lipid-anchor polypeptide (e.g., a GPI-anchor, an N-myristoylated polypeptide, or an S-palmitoylated polypeptide) or (2) stably bound to its cognate receptor Exogenous polypeptide, wherein the cognate receptor is physically attached to the cell membrane of human enucleated erythrocytes (for example, a ligand that binds to its cognate receptor, wherein the cognate receptor is physically attached to The human enucleated erythroid cell membrane). Non-limiting methods for determining the presence of exogenous polypeptides on the extracellular surface of human enucleated erythroid cells include fluorescence-activated cell sorting (FACS), immunotissue Chemistry, Cell-sorting Assays and Western Blotting.

The term "human erythroid precursor cells" refers to human cells that can eventually differentiate/develop into human enucleated erythroid cells. In some embodiments, the human erythroid precursor cells are cord blood stem cells, CD34 ⁺ cells, hematopoietic stem cells/precursor cells (HSC, HSPC), spleen colony forming (CFU-S) cells, common bone marrow precursor cells (CMP), embryonic Colony-forming cells, burst-forming unit-erythroid/erythroid (BFU-E), megakaryocyte-erythroid precursor (MEP) cells, colony-forming unit erythroid, or colony-forming unit erythroid (CFU-E) , induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), or a combination thereof.

The term "adult human subject" means an individual who is 18 years of age or older (e.g., 20 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older , aged 45 or over, aged 50 or over, aged 55 or over, aged 60 or over, aged 65 or over, aged 70 or over, aged 75 or over, aged 80 or over, aged 85 or over, aged 90 or over, aged 95 or over, or aged 100 or over).

As used herein, "treating" refers to the number, severity, frequency, and/or Duration reduced.

As used herein, the term "biological sample" refers to any type of material of biological origin isolated from an individual, including, for example, DNA, RNA, lipids, carbohydrates, and proteins. The term "biological sample" includes tissues, cells and biological fluids isolated from an individual. Biological samples include, for example but not limited to, whole blood, plasma, serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, cerebrospinal fluid, bone marrow, bile, hair, muscle slices, organ tissue, or Other materials of biological origin known to those of knowledge. Biological samples can be obtained from individuals for diagnosis or research, or from healthy individuals as controls or for basic research.

Unless defined otherwise, 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; alternatively, other suitable methods and materials known in the art can be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All literature, patent applications, patents, sequences, database entries and other references mentioned in this document are hereby incorporated by reference in their entirety.

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

Provided herein is a method of treating an HPV16-positive or HPV16-associated cancer in a human subject in need thereof, comprising intravenously administering to the human subject a drug comprising about 0.5 x ¹⁰⁹ to about 5 x ¹⁰¹⁰ human enucleated erythroid cells A dose of pharmaceutical composition comprising, on the surface of human enucleated erythroid cells: an exogenous antigen-presenting polypeptide linked to an exogenous antigen polypeptide; an exogenous costimulatory polypeptide comprising 4-BBL; and an exogenous costimulatory polypeptide comprising An exogenous cytokine polypeptide of IL-12; the frequency of administration is from about 2 weeks to about 4 weeks. In some embodiments, the exogenous antigen-presenting polypeptide is an MHC class 1 HLA-A2 single-chain fusion protein comprising an α chain, a β2-microglobulin (β2m) chain, and a membrane anchoring region, And wherein the exogenous antigenic polypeptide comprises a human papillomavirus (HPV) E7 antigen. exogenous antigen - presenting polypeptide

In some embodiments, the invention features human enucleated erythroid blood cells comprising an exogenous antigen-presenting polypeptide linked to an exogenous antigen comprising a human papillomavirus (HPV) E7 antigen peptide. In some embodiments, the exogenous antigen-presenting polypeptide is an MHC class 1 HLA-A2 single chain fusion protein comprising a β2-microglobulin (β2m) chain, an α chain, and a membrane anchor region.

MHC class I molecules are heterodimers consisting of two polypeptide chains, an alpha chain and a beta2-microglobulin (beta2m) chain. Class I alpha chains consist of a single polypeptide consisting of three extracellular domains (termed α1, α2, and α3), a transmembrane region anchoring it in the cell membrane, and a short intracytoplasmic tail. β2m consists of a single molecule non-covalently bound to an α-chain. Only the α chain is polymorphic and is encoded by the HLA gene, whereas the β2m subunit is not polymorphic and is encoded by the β-2 microglobulin gene. Class I MHC molecules have a β2 subunit and are therefore only recognized by the CD8 co-receptor.

In some embodiments, the β2m strand can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1 ), or a functional fragment thereof. In some embodiments, the β2m chain comprises the sequence of SEQ ID NO: 1.

In some embodiments, the β2m strand consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) nucleic acid sequence code: ATACAAAAGAACACCAAAAATCCAGGTTTATTCAAGACACCCAGCTGAAAACGGAAAGAGCAACTTTTTGAATTGTTATGTGAGCGGTTTCCATCCTTCTGATATAGAAGTGGATCTGCTCAAAAATGGGGAAAGAATCGAAAAGGTTGAACACAGCGATCTGAGCTTTAGCAAGGATTGGTCTTTCTACCTCCTCTACTATACTGAATTTACGCCCCAGAGA AGGACGAGTATGCTTGTCGCGTGAACCACGTCACTTTGTCTCAACCGAAAATTGTGAAGTGGGATCGGGACATG (SEQ ID NO: 2).

In some embodiments, the exogenous antigen-presenting polypeptide comprises or consists of an HLA-A2*02:01 allele polypeptide. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises a Y84A or Y84C substitution.

In some embodiments, the HLA-A2*02:01 allele polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity) to a sequence Amino acid sequences that are % identical, at least 99% identical, or 100% identical): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPI (SEQ ID NO: 3), or a functional fragment thereof. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises the sequence of SEQ ID NO:3.

In some embodiments, the HLA-A2*02:01 allele polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical) Consistent, at least 99% identical, or 100% identical) nucleic acid sequence encoding: GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGAGCCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA (SEQ ID NO: 4).

In some embodiments, the HLA-A2*02:01 allele polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity) to a sequence Amino acid sequences that are % identical, at least 99% identical, or 100% identical): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPI (SEQ ID NO: 5), or a functional fragment thereof. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises the sequence of SEQ ID NO:5.

In some embodiments, the HLA-A2*02:01 allele polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical) Consistent, at least 99% identical, or 100% identical) nucleic acid sequence encoding: GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGATACTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA (SEQ ID NO: 6).

In some embodiments, the HLA-A2*02:01 allele polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity) to a sequence Amino acid sequences that are % identical, at least 99% identical, or 100% identical): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPI (SEQ ID NO: 7), or a functional fragment thereof. In some embodiments, the HLA-A2*02:01 allele polypeptide comprises the sequence of SEQ ID NO:7.

In some embodiments, the HLA-A2*02:01 allele polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical) Consistent, at least 99% identical, or 100% identical) nucleic acid sequence encoding: GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGATGCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA (SEQ ID NO: 8).

In some embodiments, the exogenous antigen-presenting polypeptide includes one or more linkers (eg, any of the exemplary linkers described herein). For example, an exogenous antigen-presenting polypeptide can include one or more linkers provided in Table 2. In some embodiments, a linker is located between the β2m chain and the α-chain in the exogenous antigen-presenting polypeptide. In some embodiments, in the exogenous antigen-presenting polypeptide, a linker is located between the β2m chain and the HLA-A2*02:01 allele polypeptide. In some embodiments, the linker comprises SEQ ID NO: 38. In some embodiments, the linker comprises SEQ ID NO: 41.

In some embodiments, the exogenous antigen-presenting polypeptide is attached to a human erythroid cell membrane (eg, a human enucleated erythroid cell membrane). In some embodiments, the membrane anchor region or transmembrane domain anchors the exogenous antigen-presenting polypeptide to a human erythroid cell membrane (eg, a human enucleated erythroid cell membrane). In some embodiments, the anchor region or transmembrane domain comprises an endogenous erythrocyte transmembrane polypeptide, or a fragment or transmembrane portion thereof. In certain embodiments, the anchor region or transmembrane domain comprises GPA or a transmembrane portion thereof. In some embodiments, the anchor region or transmembrane domain comprises small integral membrane protein 1 (SMIM1), transferrin receptor, Fas ligand (FasL), Kellor band 3, or a transmembrane portion thereof (e.g., transmembrane membrane domain). In some embodiments, the exogenous antigen-presenting polypeptide can comprise any of the anchor regions or transmembrane domains described herein. In some embodiments, the exogenous antigen-presenting polypeptide comprises an anchor region or a transmembrane domain listed in Table 1. In some embodiments, the exogenous antigen-presenting polypeptide comprises an anchor region or transmembrane domain as shown in SEQ ID NO: 30.

In some embodiments, the exogenous antigen-presenting polypeptide includes a linker between the α-chain in the exogenous antigen-presenting polypeptide and the membrane anchor region or transmembrane domain. For example, an exogenous antigen-presenting polypeptide can include one or more linkers provided in Table 2. In some embodiments, the linker comprises SEQ ID NO: 39. exogenous antigenic polypeptide

Exogenous antigenic polypeptides are polypeptides that induce an immune response. In some embodiments, the exogenous antigenic polypeptide comprising human papillomavirus (HPV) E7 antigen inhibits cancer by inducing an immune response ( e.g., reduces or alleviates the cause or symptoms of cancer), or improves parameters associated with cancer value of the peptide.

In some embodiments, the HPV E7 antigen can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity , or 100% identical) amino acid sequence: YMLDLQPET (SEQ ID NO: 9). In some embodiments, the HPV E7 antigen comprises the sequence of SEQ ID NO: 9.

In some embodiments, the HPV E7 antigen consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: TACATGCTCGACCTTCAACCCGAGACC (SEQ ID NO: 10).

In some embodiments, the HPV E7 antigen can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity , or 100% identical) amino acid sequence: YMLDLQPETT (SEQ ID NO: 11). In some embodiments, the HPV E7 antigen comprises the sequence of SEQ ID NO: 11.

In some embodiments, the HPV E7 antigen consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: TACATGCTCGACCTTCAACCCGAGACCACC (SEQ ID NO: 12).

In some embodiments, the HPV E7 antigen can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity , or 100% identical) amino acid sequence: TIHDIILECV (SEQ ID NO: 13). In some embodiments, the HPV E7 antigen comprises the sequence of SEQ ID NO: 13.

In some embodiments, the HPV E7 antigen consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: ACTATACATGACATTATACTGGAATGTGTT (SEQ ID NO: 14).

In some embodiments, the exogenous antigen polypeptide is linked to the exogenous antigen-presenting polypeptide via a linker. In some embodiments, the exogenous antigen polypeptide is linked to the β2m chain of the exogenous antigen-presenting polypeptide via a linker. For example, exogenous antigen polypeptides can be linked to exogenous antigen-presenting polypeptides via the linkers provided in Table 2. In some embodiments, the linker comprises SEQ ID NO: 33. In some embodiments, the linker comprises SEQ ID NO: 41.

In some embodiments, the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical) Amino acid sequence that is identical, at least 95% identical, at least 99% identical, or 100% identical): YMLDLQPETGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSG GGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEW LRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIGSGSGSGSEDGSGSGSGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEE ETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIEENPETSDQ (SEQ ID NO: 15), or a functional fragment thereof.

In some embodiments, the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical) Concordant, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: TACATGCTCGACCTTCAACCCGAGACCGGAGGCGGAGGGAGTGGCGGAGGAGGTAGTGGCGGCGGGGGCTCTATACAAAGAACACCAAAAAATCCAGGTTTATTCAAGACCCAGCTGAAAACGGAAAGAGCAACTTTTTGAATTGTTATGTGAGCGGTTTCCAT CCTTCTGATATAGAAGTGGATCTGCTCAAAAATGGGGAAAGAATCGAAAAGGTTGAACACAGCGATCTGAGCTTTAGCAAGGATTGGTCTTTCTACCTCCTCTACTATACTGAATTTACGCCCCACAGAGAAGGACGAGTATGCTTGTCGCGTGAACCACGTCACTTTGTCTCAACCGAAAATTGTGAAGTGGGATCGGGACATGGGTGGGGGAG GCAGCGGGGGTGGGGGGTCAGGTGGGGGAGGTAGCGGCGGAGGTGGCAGTGGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGT GGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTCACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGAGCCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCCAGGGGTTATCACCAGTATGCTTATGAT GGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGCGGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCAC GCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATAACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAG GTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCACTATACCGATAGGCAGCGGCTCTGGCTCCGGCTCCGAAGATGGCAGCGGCAGCGGAAGTGGTTCATTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATTCTCATCACAGACAAATGATACGCACAAACGGGACACATATG CAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCATCTGATGTAAAACCTCTCC CCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 16). exogenous co-stimulatory polypeptide

The human enucleated erythroid cells described herein, in addition to exogenous antigen-presenting polypeptides (such as any of the exogenous antigen-presenting polypeptides described herein), also include a protein comprising 4-1BBL or Its functional fragments have exogenous co-stimulatory polypeptides on its extracellular surface.

As used herein, 4-1BBL polypeptide refers to the amino acid sequence encoded by the tumor necrosis factor superfamily member 9 (TNFSF9 or CD137L) gene. 4-1BBL is the ligand for 4-1BB (also known as Tumor Necrosis Factor Receptor Superfamily, Member 9 (TNFRSF9) or CD137), a member of a family of receptors present on the extracellular surfaces of cells of the immune system . See Alderson et al., 1994, Eur. J. Immunol. 24:2219-2227.

In some embodiments, 4-1BBL is in native trimeric form.

In some embodiments, 4-1BBL can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity , or 100% identical) sequence: ACPWAVSGARASPPGSAASPRLREGELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 19), or a functional fragment thereof. In some embodiments, 4-1BBL comprises the sequence of SEQ ID NO: 19.

In some embodiments, 4-1BBL consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACA GTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGG CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCGAAATCCCAGCCGGACTCCCTTCACC GAGGTCGGAA (SEQ ID NO: 20).

In some embodiments, the exogenous co-stimulatory polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identical, or 100% identical) sequence: ACPWAVSGARASPPGSAASPRLREGELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ (SEQ ID NO: 21). In some embodiments, the exogenous co-stimulatory polypeptide polypeptide comprises the sequence of SEQ ID NO: 21.

In some embodiments, the exogenous co-stimulatory polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTG GTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATCCCAGCCGGACTCCCT TCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGA GCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCCATCTGATGTAAAACCTCTCCCCTCACCTGACAC AGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 57).

In some embodiments, the exogenous co-stimulatory polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTG GTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATCCCAGCCGGACTCCCT TCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCCTGAGCACCACCGAGGTGGCCATGCACACCAGCACCAGCAGCAGCGTGACCAGAGCTACATCAGCAGCCAGACCAACGACACCCACAAGCGCGACCACCTACGCCGCCACCCCCCGCGC CCACGAGGTGAGCGAGATCAGCGTGCGCACCGTGTACCCCCCGAGGAGGAGACCGGCGAGCGCGTGCAGCTGGCCACCCACTTCAGCGAGCCCGAGATCACCCCTGATCATCTTCGGCGTGATGGCCGGCGTGATCGGCACCATCCTGCTGATCAGCTACGGCATCCGCCGCCTGATCAAGAAGAGCCCCAGCGACGTGAAGCCCCCTGCCCAGCCCCGACACCG ACGTGCCCCTGAGCAGCGTGGAGATCGAGAACCCCCGAGACCAGCGACCAG (SEQ ID NO: 22).

In some embodiments, the exogenous co-stimulatory polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identical, or 100% identical) sequence: ACPWAVSGARASPPGSAASPRLREGELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQGSGEG RGSLLTCGDVEENPG (SEQ ID NO: 17). In some embodiments, the exogenous co-stimulatory polypeptide comprises the sequence of SEQ ID NO: 17.

In some embodiments, the exogenous co-stimulatory polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTG GTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATCCCAGCCGGACTCCCT TCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCCTGAGCACCACCGAGGTGGCCATGCACACCAGCACCAGCAGCAGCGTGACCAGAGCTACATCAGCAGCCAGACCAACGACACCCACAAGCGCGACCACCTACGCCGCCACCCCCCGCGC CCACGAGGTGAGCGAGATCAGCGTGCGCACCGTGTACCCCCCGAGGAGGAGACCGGCGAGCGCGTGCAGCTGGCCACCCACTTCAGCGAGCCCGAGATCACCCCTGATCATCTTCGGCGTGATGGCCGGCGTGATCGGCACCATCCTGCTGATCAGCTACGGCATCCGCCGCCTGATCAAGAAGAGCCCCAGCGACGTGAAGCCCCCTGCCCAGCCCCGACACCG ACGTGCCCCTGAGCAGCGTGGAGATCGAGAACCCCCGAGACCAGCGACCAGGGCAGCGGTGAAGGTCGGGGGAGCCTGCTGACTTGCGGAGATGTGGAAGAAAATCCTGGCCCA (SEQ ID NO: 18).

In some embodiments, the exogenous co-stimulatory polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identical, or 100% identical) sequence: MYGKIIFVLLLSEIVSISAACPWAVSGARASPPGSASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAF GFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPD TDVPLSSVEIENPETSDQ (SEQ ID NO: 23). In some embodiments, the exogenous co-stimulatory polypeptide comprises the sequence of SEQ ID NO: 23.

In some embodiments, the exogenous co-stimulatory polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) encoded by the nucleic acid sequence: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTT GCGCAGCTGGTGGCCCAAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTTACTATGTCTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCG CCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAAACCTCTTCTTCAGTCACAAAGTTACATC TCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTC GCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 24).

In some embodiments, the exogenous co-stimulatory polypeptide includes a leader (signal) sequence. In some embodiments, 4-1BBL or a functional fragment thereof is fused to a leader (signal) sequence. Non-limiting examples of leader (signal) sequences include the amino acid sequences provided in Table 3. In some embodiments, the leader (signal) sequence includes a GPA signal peptide. In some embodiments, the guide (signal) sequence includes the amino acid sequence of SEQ ID NO:45. In some embodiments, the exogenous co-stimulatory polypeptide comprises 4-1BBL or a functional fragment thereof, and a guide (signal) sequence such as the amino acid sequence of SEQ ID NO: 45. In some embodiments, the exogenous co-stimulatory polypeptide comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 45, and 4-1BBL, which has the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the exogenous costimulatory polypeptide is attached to a human erythroid cell membrane (eg, a human enucleated erythroid cell membrane). In some embodiments, the exogenous costimulatory polypeptide further comprises an anchor region or transmembrane domain that anchors the exogenous costimulatory polypeptide to a human erythroid cell membrane (e.g., a human enucleated erythroid cell membrane) superior. In some embodiments, the anchor region or transmembrane domain is heterologous to the exogenous costimulatory polypeptide (eg, 4-1BBL). In some embodiments, the anchor region or transmembrane domain comprises an endogenous erythrocyte transmembrane polypeptide, or a fragment or transmembrane portion thereof. In certain embodiments, the anchor region or transmembrane domain comprises GPA or a transmembrane portion thereof. In some embodiments, the anchor region or transmembrane domain comprises small integral membrane protein 1 (SMIM1), transferrin receptor, Fas ligand (FasL), Kellor band 3, or a transmembrane portion thereof (e.g., transmembrane membrane domain). In some embodiments, the exogenous co-stimulatory polypeptide can comprise any of the anchor regions or transmembrane domains described herein. In some embodiments, the exogenous co-stimulatory polypeptide comprises an anchor region or a transmembrane domain listed in Table 1. In some embodiments, the exogenous co-stimulatory polypeptide comprises an anchor region or a transmembrane domain as shown in SEQ ID NO: 30.

In some embodiments, the exogenous co-stimulatory polypeptide includes one or more linkers (eg, any of the exemplary linkers described herein). For example, an exogenous co-stimulatory polypeptide can include one or more linkers provided in Table 2. In some embodiments, the linker is located between 4-1BBL or a functional fragment thereof and the anchoring region or transmembrane domain of the exogenous co-stimulatory polypeptide. In some embodiments, the linker comprises SEQ ID NO: 36.

In some embodiments, the exogenous co-stimulatory polypeptide can further include a signal peptide (eg, any of the exemplary signal peptides described herein). For example, exogenous co-stimulatory polypeptides can include signal peptides provided in Table 3.

In some embodiments, the exogenous co-stimulatory polypeptide includes a signal peptide, 4-1BBL or a functional fragment thereof, and an anchor region. In some embodiments, the exogenous co-stimulatory polypeptide includes a signal peptide, 4-1BBL or a functional fragment thereof, a linker, and an anchor region. In some embodiments, the exogenous co-stimulatory polypeptide comprises (for example, from the N-terminus to the C-terminus) a signal peptide comprising the amino acid sequence of SEQ ID NO: 45, a signal peptide comprising the amino acid sequence of SEQ ID NO: 19 4-1BBL or a functional fragment thereof, a linker comprising the amino acid sequence of SEQ ID NO: 36 (for example, between 4-1BBL or a functional fragment thereof and the anchor region), and comprising SEQ ID NO: Anchor region of 30 amino acid sequence. In some embodiments, the exogenous co-stimulatory polypeptide comprises 4-1BBL or a functional fragment thereof, a linker, and an anchor region. In some embodiments, the exogenous co-stimulatory polypeptide comprises (eg, from N-terminus to C-terminus) 4-1BBL comprising the amino acid sequence of SEQ ID NO: 19 or a functional fragment thereof, comprising SEQ ID NO: A linker with an amino acid sequence of 36 (for example, located between 4-1BBL or its functional fragment and the anchor region), and an anchor region comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the exogenous co-stimulatory polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 21 (which is encoded by the nucleic acid sequence of SEQ ID NO: 22).

In some embodiments, the exogenous co-stimulatory polypeptide may have post-translational modification characteristics of eukaryotic cells (eg, mammalian cells, such as human cells). In some embodiments, the exogenous costimulatory polypeptide is glycosylated, phosphorylated, or both. In vitro detection of glycoproteins is routinely done on SDS-PAGE gels and Western blotting using a modified version of the Periodic Acid-Schiff (PAS) method. Cellular localization of glycoproteins can be achieved using lectin fluorescent conjugates known in the art. Phosphorylation can be assessed by Western blotting using phospho-specific antibodies.

Post-translational modifications also include attachment to hydrophobic groups (e.g., myristylation, palmitoylation, isopolyprenylation, polyprenylation, or glycosylation), attachment to cofactors (e.g., lipid Acylation, flavin moiety (e.g., FMN or FAD), heme C attachment, phosphopanthiolation, or retinylidene Schiff base formation), benzamide formation, ethanolamine phosphoglycerol attachment , putrescine lysine (hypusine) formation, acylation (eg O-acylation, N-acylation, or S-acylation), formylation, acetylation, alkylation (eg, methylation or ethylation), amidation, butyrylation, γ-carboxylation, malonylation, hydroxylation, iodination, nucleotide additions such as ADP-ribosylation, oxidation, phosphate (O-linkage) or Phosphoramidate (N-linked) formation (eg, phosphorylation or adenylation), propylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation, Sulfation, ISGylation, SUMOylation, ubiquitination, neddylation, or chemical modification of amino acids (such as citrullination, deamidation, deimidation, or carbamylation), formation of disulfide bonds, racemization (eg racemization of proline, serine, alanine or methionine). In embodiments, glycosylation comprises adding a sugar group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan , resulting in glycoproteins. In embodiments, glycosylation comprises, for example, O-linked glycosylation or N-linked glycosylation. exogenous cytokine peptide

The human enucleated erythroid cells described herein, in addition to having an exogenous antigen-presenting polypeptide (such as any of the exogenous antigen-presenting polypeptides described herein) and an exogenous costimulatory polypeptide on their extracellular surface, also It includes an exogenous cytokine polypeptide comprising IL-12 or its functional fragments on its extracellular surface.

As used herein, IL-12 refers to a protein having amino acids encoded by the interleukin 12A (IL-12A or IL-12 subunit p35) gene and the interleukin 12B (IL-12B or IL-12 subunit p40) gene sequence heterodimers. IL-12 is a ligand for IL12Rβ1 and IL12Rβ2 (also known as interleukin 12 receptor β1 subunit and interleukin 12 receptor β2 subunit, respectively), members of a family of receptors present on the extracellular surface of immune system cells one. See Vignali and Kuchroo, 2012, Nat Immunol. 13(8): 722–728.

In some embodiments, the IL-12A protein may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) sequence: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 25), or a functional fragment thereof. In some embodiments, the IL-12A protein comprises the sequence of SEQ ID NO: 25.

In some embodiments, the IL-12A protein consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical , or 100% identical) nucleic acid sequence encoded by: CGGAATCTGCCAGTGGCAACCCCAGACCCCGGAATGTTCCCATGCCTGCACCACTCTCAGAACCTGCTGAGGGCCGTGAGCAATATGCTGCAGAAGGCCCGCCAGACACTGGAGTTTTACCCTTGTACCAGCGAGGAGATCGACCACGAGGACATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGCCTGCCACTGGAGCTGACCAAGAACGAGAGCTGT CTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCAGCTGCCTGGCCTCCAGAAAGACATCTTTTATGATGGCCCTGTGCCTGTCTAGCATCTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGCTGCTGATGGACCCAAGAGGCAGATTTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCC CTGAACTTTAATTCCGAGACAGTGCCTCAGAAGTCCTCTCTGGAGGAGCCAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAATGCCTCC (SEQ ID NO: 26).

In some embodiments, the IL-12B protein may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) sequence: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNK EYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 27), or a functional fragment thereof. In some embodiments, the IL-12B protein comprises the sequence of SEQ ID NO: 27.

In some embodiments, the IL-12B protein consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical , or 100% identical) nucleic acid sequence encoded by: ATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGCGACACACCTGAGGAGGATGGCATCACCTGGACACTGGATCAGAGCAGCGAGGTGCTGGGCTCCGGCAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCCGGCCAGTACACATGTCA CAAGGGAGGAGAGGTGCTGAGCCACTCCCTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGTCTACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAACAAGACCTTCCTGCGGTGCGAGGCCAAGAATTATAGCGGCCGGTTCACCTGTTGGTGGCTGACCACAATCTCTACCGACCTGACCTTCAGCGTGAAGTCTAGCCGGGGCTCCTCT GACCCTCAGGGAGTGACATGCGGAGCAGCCACCCTGTCCGCCGAGCGGGTGAGAGGCGATAACAAGGAGTACGAGTATAGCGTGGAGTGCCAGGAGGACTCCGCCTGTCCAGCAGCAGAGGAGAGCCTGCCAATCGAAGTGATGGTGGATGCCGTGCACAAGCTGAAGTACGAGAATTATACAAGCTCCTTTCTTATCAGGGACATCATCAAGCCGATC CCCCTAAGAACCTGCAGCTGAAGCCCCTGAAGAACAGCCGGCAGGTGGAGGTGTCTTGGGAGTACCCCGACACCTGGAGCACACCTCACTCCTATTTCTCTCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAAGGACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGTCGGAAGAACGCCTCTATCAGCGTGCGGGCCC AGGATAGATACTATTCTAGCTCCTGGAGCGAGTGGGCCTCCGTGCCATGTTCT (SEQ ID NO: 28).

In some embodiments, the exogenous cytokine polypeptide can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identical, or 100% identical) sequence: MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCKGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVK EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 48). In some embodiments, the exogenous cytokine polypeptide comprises the sequence of SEQ ID NO: 48.

In some embodiments, the exogenous cytokine polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) nucleic acid sequence encoded by: ATGCAACCGCAAGAGAGTCACGTACATTTATTCAAGATGGGAAGATGGAAGTCGGGACGGTGTGTCTCTCGGCGCTGTTAGTTCAACGGAGGAAGCGTCTCGCTGTCGCCGGATAAGTCAACGCCTTTGTACGGGAAAACTGGGTATAGCTATGAAGGTCCTCGGCGGGGTGGCGTTGTTTTGGATTATCTTTATACTTGGGTATCT GACCGGTTACTATGTTCACAAGTGTAAAGGAGGTGGAGGATCAGGTGGAGGTGGTTCAGGTGGAGGAGGTAGCATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGCGACACACCTGAGGAGGATGGCATCACCTGGACACTGGATCAGAGCAGCGAG GTGCTGGGCTCCGGCAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCCGGCCAGTACACATGTCACAAGGGAGGAGAGGTGCTGAGCCACTCCCTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGTCTACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAACAAGACCTTCCTGCGGTGCGAGGCCAAGAATTATAGCGGCCGGT TCACCTGTTGGTGGCTGACCACAATCTCTACCGACCTGACCTTCAGCGTGAAGTCTAGCCGGGGCTCCTCTGACCTCTAGGGAGTGACATGCGGAGCAGCACCCTGTCCGCCGAGCGGGTGAGAGGCGATAACAAGGAGTACGAGTATAGCGTGGAGTGCCAGGAGGACTCCGCCTGTCCAGCAGCAGAGGAGAGCCTGCCAATCGAAGTGATGGTG GATGCCGTGCACAAGCTGAAGTACGAGAATTATACAAGCTCCTTCTTTATCAGGGACATCATCAAGCCCGATCCCCCTAAGAACCTGCAGCTGAAGCCCCTGAAGAACAGCCGGCAGGTGGAGGTGTCTTGGGAGTACCCCGACACCTGGAGCACACCTCACTCCTTATTTCTCTCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAAG GACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGTCGGAAGAACGCCTCTATCAGCGTGCGGGCCCAGGATAGATACTATTCTAGCTCCTGGAGCGAGTGGGCCTCCGTGCCATGTTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCAGCCGGAATCTGCCAGTGGCAACCCCAGACCCCGGAATGTTCCCATGC CTGCACCACTCTCAGAACCTGCTGAGGGCCGTGAGCAATATGCTGCAGAAGGCCCGCCAGACACTGGAGTTTTACCCTTGTACCAGCGAGGAGATCGACCACGAGGACATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGCCTGCCACTGGAGCTGACCAAGAACGAGAGCTGTCTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCAGCTGCCTGG CCTCCAGAAAGACATCTTTTATGATGGCCCTGTGCCTGTCTAGCATCTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCCAAGAGGCAGATTTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTTAATTCCGAGACAGTGCCTCAGAAGTCCTCTCTGGAGGAGC CAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAATGCCTCC (SEQ ID NO: 49).

In some embodiments, the exogenous cytokine polypeptide includes one or more linkers (eg, any of the exemplary linkers described herein). For example, an exogenous cytokine polypeptide can include one or more of the linkers provided in Table 2. In some embodiments, the linker is located between the IL-12A protein and the IL-12B protein in the exogenous cytokine polypeptide. In some embodiments, the linker comprises SEQ ID NO: 33.

In some embodiments, the exogenous cytokine polypeptide is attached to a human erythroid cell membrane (eg, a human enucleated erythroid cell membrane). In some embodiments, the exogenous cytokine polypeptide further comprises an anchor region or transmembrane domain that anchors the exogenous cytokine polypeptide to a human erythroid cell membrane (eg, a human enucleated erythroid cell membrane). In some embodiments, the anchor region or transmembrane domain is heterologous to an exogenous cytokine polypeptide (eg, IL-12). In some embodiments, the anchor region or transmembrane domain comprises an endogenous erythrocyte transmembrane polypeptide, or a fragment or transmembrane portion thereof. In certain embodiments, the anchor region or transmembrane domain comprises GPA or a transmembrane portion thereof. In some embodiments, the anchor region or transmembrane domain comprises small integral membrane protein 1 (SMIM1), transferrin receptor, Fas ligand (FasL), Kellor band 3, or a transmembrane portion thereof (e.g., transmembrane membrane domain). In some embodiments, the exogenous cytokine polypeptide can comprise any of the anchor regions or transmembrane domains described herein. In some embodiments, the exogenous cytokine polypeptide comprises an anchor region or a transmembrane domain listed in Table 1. In some embodiments, the exogenous cytokine polypeptide comprises an anchor region or a transmembrane domain as shown in SEQ ID NO: 31.

In some embodiments, the exogenous cytokine polypeptide includes a linker between the IL-12 and the anchor region or transmembrane domain of the exogenous cytokine polypeptide. In some embodiments, the linker comprises SEQ ID NO: 33.

In some embodiments, the exogenous cytokine polypeptide may have post-translational modification characteristics of eukaryotic cells (eg, mammalian cells, such as human cells). In some embodiments, the exogenous cytokine polypeptide is glycosylated, phosphorylated, or both. In vitro detection of glycoproteins is routinely done on SDS-PAGE gels and Western blotting using a modified version of the Periodic Acid-Schiff (PAS) method. Cellular localization of glycoproteins can be achieved using lectin fluorescent conjugates known in the art. Phosphorylation can be assessed by Western blotting using phospho-specific antibodies.

Post-translational modifications also include attachment to hydrophobic groups (e.g., myristylation, palmitoylation, isopolyprenylation, polyprenylation, or glycosylation), attachment to cofactors (e.g., lipid Acylation, flavin moiety (e.g., FMN or FAD), heme C attachment, phosphopantyl thioethylation, or retinylidene Schiff base formation), dibenzamide formation, ethanolamine phosphoglycerol Attachment, putrescine hypusine formation, acylation (e.g., O-acylation, N-acylation, or S-acylation), formylation, acetylation, alkylation (e.g., methyl or ethylation), amidation, butylation, γ-carboxylation, malonylation, hydroxylation, iodination, nucleotide additions such as ADP-ribosylation, oxidation, phosphate (O-linked ) or phosphoramidate (N-linked) formation (e.g., phosphorylation or adenylation), propylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation , Sulfation, ISGylation, SUMOylation, Ubiquitination, Neddylation, or chemical modification of amino acids (such as citrullination, deamidation, deimidation, or carboxylation) , formation of disulfide bonds, racemization (eg racemization of proline, serine, alanine or methionine). In embodiments, glycosylation comprises adding a sugar group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan, Thus a glycoprotein is obtained. In an embodiment, glycosylation comprises, for example, O-linked glycosylation or N-linked glycosylation. anchor / transmembrane domain

In some embodiments, the transmembrane domain comprises or consists of the transmembrane domain of a Type 1 membrane polypeptide. In some embodiments, the Type 1 membrane polypeptide is selected from the group consisting of: Glycophorin A (GPA), Glycophorin B (GPB), Basigin (also known as CD147); CD44, CD58 (also known as LFA3), intercellular adhesion molecule 4 (ICAM4), basal cell adhesion molecule (BCAM), CR1, CD99, erythroblastoma membrane-associated protein (ERMAP), junctional adhesion molecule A (JAM-A), neuroamyloid protein (NPTN) , AMIGO2, and DS-like cell adhesion molecule 1 (DSCAML1). In some embodiments, the transmembrane domain comprises or consists of the transmembrane domain of a Type 2 membrane polypeptide. In some embodiments, the type 2 membrane polypeptide is selected from the group consisting of: small integral membrane protein 1 (SMIM1), transferrin receptor (CD71); Fas ligand (FasL) transmembrane, and Kell . In some embodiments, the polypeptide sequence that anchors the exogenous polypeptide to the human enucleated erythroid cell membrane comprises, consists of, or is derived from a GPI-linked membrane polypeptide ( For example, its fragment). In some embodiments, the GPI-linked membrane polypeptide is selected from the group consisting of CD59, CD55, and Semaphorin 7A (SEMA7A).

In particular embodiments, the transmembrane domain comprises glycoprotein A (GPA) or a transmembrane portion thereof. Without being bound by theory, in certain embodiments, GPA is preferred because it has a cytoplasmic domain that interacts with the reticulocyte cytoskeleton, which functions to retain GPA during cell differentiation and maturation. In some embodiments, the transmembrane domain comprises small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchor region is selected from the amino acid sequences listed in Table 1. Table 1. Anchor sequences SEQ ID NO: sequence name sequence description amino acid sequence 29 GPA full-length GPA MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ 30 GPA GPA fragment containing a transmembrane domain LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDTDVPLSSVEIEENPETSDQ 31 SMIM1 SMIM1 MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK Linker _

In some embodiments, one or more exogenous antigen-presenting polypeptides are linked to the exogenous antigen polypeptide; an exogenous costimulatory polypeptide comprising 4-BBL; and an exogenous cytokine comprising IL-12 A polypeptide (eg, any of the exemplary polypeptides described herein) can include one or more linkers. For example, a linker can be between a cytokine polypeptide sequence (such as IL-12 or a functional fragment thereof) and a transmembrane domain sequence, or between an exogenous antigen-presenting polypeptide and an exogenous antigen polypeptide .

In some embodiments, the linker comprises or consists of a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 or more amino acids. In some embodiments, the linker comprises or consists of between about 5 and about 25 amino acids in length, between about 5 and about 20 amino acids in length, between about 10 and about 25 amino acids in length between amino acids, or between about 10 and about 20 amino acids in length. In some embodiments, linkers useful in the invention comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a preferred embodiment, the linker is non-immunogenic.

In some embodiments, the linker is selected from the amino acid sequences listed in Table 2. Table 2. Linker sequences SEQ ID NO. sequence description amino acid sequence 32 G4S linker GGGGS 33 (G4S) ₃ linker GGGGSGGGGSGGGGS 34 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS 35 Linker GGSGGSGGGGGSGGGSGGGSGGGS 36 Linker GGSGGSGGGPEDEPGSGSGGGSGGGS 37 Linker GSGSGSGSGSEDEDEDEDEDGSGSGSGSGS 38 Linker GGGGSGGGGSGGGGSGGGGS 39 Linker GSGSGSGSEDGSGSGSGS 40 Linker GSGSGSGSGSGSGSGSGSGS 41 Linker GCGGSGGGGSGGGGS 42 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA 43 Linker GGGGSGGGGSGGGGSGGGGSGGGG 44 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA

In some embodiments, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 50), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker consists of (GGGGS) _n linker (SEQ ID NO: 50), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 33). In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 35. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 39.

It can be used to link exogenous antigen-presenting polypeptides to exogenous antigen polypeptides; exogenous costimulatory polypeptides comprising 4-BBL; or exogenous cytokine polypeptides comprising IL-12 (e.g., the exemplified examples described herein). Other suitable linkers for any of the sex polypeptides are known in the art. boot ( signal ) sequence

In some embodiments, one or more exogenous antigen-presenting polypeptides are linked to an exogenous antigen polypeptide; an exogenous costimulatory polypeptide comprising 4-BBL; and an exogenous cytokine polypeptide comprising IL-12 (eg, any of the exemplary polypeptides described herein) comprise a leader (signal) sequence. In some embodiments, the leader sequence is selected from the sequences listed in Table 3. Table 3. Boot ( Signal ) Sequence SEQ ID NO. sequence description amino acid sequence 45 GPA signal peptide MYGKIIFVLLLSEIVSISA 46 Ig heavy chain V region 3 signal sequence MGWSCIILFLVATATGVHS 47 light chain leader sequence MRVPAQLLGLLLLWLPGARC Exemplary Human Enucleated Erythroid Cells

In some embodiments, human enucleated erythroid cells comprise a combination of: an exogenous antigen-presenting polypeptide (e.g., a polypeptide comprising an MHC class 1 HLA-A2 single-chain fusion protein comprising an alpha chain, a beta2 chain, and a membrane Anchoring region), which is connected to an exogenous antigenic polypeptide comprising HPV E7 antigen on its extracellular surface; an exogenous co-stimulatory polypeptide comprising 4-1BBL or a fragment thereof, which is linked to an exogenous costimulatory polypeptide on its extracellular surface Transmembrane polypeptide (such as GPA or its transmembrane fragment); And exogenous cytokine polypeptide, it comprises IL-12 or its fragment, it is linked to a transmembrane polypeptide (such as GPA or its transmembrane fragment); for example, as described in International Application Publication No. WO 2019/126818, incorporated herein by reference.

In some embodiments, human enucleated erythroid cells are not hypotonic loaded cells. In some embodiments, the human enucleated erythroid cells do not comprise a sortase-transfer marker.

In some embodiments, the human enucleated erythroid cells described herein are negative for (i.e., do not include) one or more minor blood group antigens, such as Le(a ⁻ b ⁻ ) (Lewis antigen system), Fy(a ^- b ^- ) (Duffy system), Jk(a ^- b ^- ) (Kidd system), M ^- N ^- (MNS system), K ^- k ^- (Kel Kell system), Lu(a ^- b ^- ) (Lutheran system), and H-antigen negative (Bombay phenotype), or any combination thereof. In some embodiments, human enucleated erythroid cells are also O and/or Rh ^- type. Minor blood groups are described eg in Agarwal et al., "Blood group phenotype frequencies in blood donors from a tertiary care hospital in north India," Blood Res. 48(1):51–54, 2013, and Mitra et al., "Blood groups systems,” Indian J. Anaesth. 58(5):524–528, 2014, the description of which is incorporated herein by reference.

In some embodiments, the human enucleated erythroid cells described herein exhibit an isolated, uncultured erythrocyte that does not contain an exogenous polypeptide (e.g., any exogenous polypeptide described herein or known in the art). Human enucleated erythroid cells have substantially the same permeable membrane fragility. In some embodiments, the human enucleated erythroid population has an osmotic fragility of less than 50% cell lysis at 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% NaCl. In some embodiments, osmotic fragility is determined using the method described in Example 59 of WO 2015/073587, the description of which is incorporated herein by reference.

In some embodiments, human enucleated erythroid cells have approximately the same diameter or volume as wild-type, unprocessed enucleated erythroid cells. In some embodiments, the population of human enucleated erythroid cells has a mean diameter of about 4, 5, 6, 7, 8, 9, 10, 11, or 12 microns, or about 4.0 to about 12.0 microns, about 4.0 to about 11.5 microns , about 4.0 to about 11.0 microns, about 4.0 to about 10.5 microns, about 4.0 to about 10 microns, about 4.0 to about 9.5 microns, about 4.0 to about 9.0 microns, about 4.0 to about 8.5 microns, about 4.0 to about 8.0 microns, About 4.0 to about 7.5 microns, about 4.0 to about 7.0 microns, about 4.0 to about 6.5 microns, about 4.0 to about 6.0 microns, about 4.0 to about 5.5 microns, about 4.0 to about 5.0 microns, about 4.0 to about 4.5 microns, about 4.5 to about 12.0 microns, about 4.5 to about 11.5 microns, about 4.5 to about 11.0 microns, about 4.5 to about 10.5 microns, about 4.5 to about 10.0 microns, about 4.5 to about 9.5 microns, about 4.5 to about 9.0 microns, about 4.5 to about 8.5 microns, about 4.5 to about 8.0 microns, about 4.5 to about 7.5 microns, about 4.5 to about 7.0 microns, about 4.5 to about 6.5 microns, about 4.5 to about 6.0 microns, about 4.5 to about 5.5 microns, about 4.5 to about 5.0 microns, about 5.0 to about 12.0 microns, about 5.0 to about 11.5 microns, about 5.0 to about 11.0 microns, about 5.0 to about 10.5 microns, about 5.0 to about 10.0 microns, about 5.0 to about 9.5 microns, about 5.0 to about 9.0 microns, about 5.0 to about 8.5 microns, about 5.0 to about 8.0 microns, about 5.0 to about 7.5 microns, about 5.0 to about 7.0 microns, about 5.0 to about 6.5 microns, about 5.0 to about 6.0 microns, about 5.0 to about 5.5 microns, about 5.5 to about 12.0 microns, about 5.5 to about 11.5 microns, about 5.5 to about 11.0 microns, about 5.5 to about 10.5 microns, about 5.5 to about 10.0 microns, about 5.5 to about 9.5 microns, about 5.5 to about 9.0 microns , about 5.5 to about 8.5 microns, about 5.5 to about 8.0 microns, about 5.5 to about 7.5 microns, about 5.5 to about 7.0 microns, about 5.5 to about 6.5 microns, about 5.5 to about 6.0 microns, about 6.0 to about 12.0 microns, About 6.0 to about 11.5 microns, about 6.0 to about 11.0 microns, about 6.0 to about 10.5 microns, about 6.0 to about 10.0 microns, about 6.0 to about 9.5 microns, about 6.0 to about 9.0 microns, about 6.0 to about 8.5 microns, about 6.0 to about 8.0 microns, about 6.0 to about 7.5 microns, about 6.0 to about 7.0 microns, about 6.0 to about 6.5 microns, about 6.5 to about 12.0 microns, about 6.5 to about 11.5 microns, about 6.5 to about 11.0 microns, about 6.5 to about 10.5 microns, about 6.5 to about 10.0 microns, about 6.5 to about 9.5 microns, about 6.5 to about 9.0 microns, about 6.5 to about 8.5 microns, about 6.5 to about 8.0 microns, about 6.5 to about 7.5 microns, about 6.5 to About 7.0 microns, about 7.0 to about 12.0 microns, about 7.0 to about 11.5 microns, about 7.0 to about 11.0 microns, about 7.0 to about 10.5 microns, about 7.0 to about 10.0 microns, about 7.0 to about 9.5 microns, about 7.0 to about 9.0 microns, about 7.0 to about 8.5 microns, about 7.0 to about 8.0 microns, about 7.0 to about 7.5 microns, about 7.5 to about 12.0 microns, about 7.5 to about 11.5 microns, about 7.5 to about 11.0 microns, about 7.5 to about 10.5 microns, about 7.5 to about 10.0 microns, about 7.5 to about 9.5 microns, about 7.5 to about 9.0 microns, about 7.5 to about 8.5 microns, about 7.5 to about 8.0 microns, about 8.0 to about 12.0 microns, about 8.0 to about 11.5 microns , about 8.0 to about 11.0 microns, about 8.0 to about 10.5 microns, about 8.0 to about 10.0 microns, about 8.0 to about 9.5 microns, about 8.0 to about 9.0 microns, about 8.0 to about 8.5 microns, about 8.5 to about 12.0 microns, About 8.5 to about 11.5 microns, about 8.5 to about 11.0 microns, about 8.5 to about 10.5 microns, about 8.5 to about 10.0 microns, about 8.5 to about 9.5 microns, about 8.5 to about 9.0 microns, about 9.0 to about 12.0 microns, about 9.0 to about 11.5 microns, about 9.0 to about 11.0 microns, about 9.0 to about 10.5 microns, about 9.0 to about 10.0 microns, about 9.0 to about 9.5 microns, about 9.5 to about 12.0 microns, about 9.5 to about 11.5 microns, about 9.5 to about 11.0 microns, about 9.5 to about 10.5 microns, about 9.5 to about 10.0 microns, about 10.0 to about 12.0 microns, about 10.0 to about 11.5 microns, about 10.0 to about 11.0 microns, about 10.0 to about 10.5 microns, about 10.5 to about 12.0 microns, about 10.5 to about 11.5 microns, about 10.5 to about 11.0 microns, about 11.0 to about 12.0 microns, about 11.0 to about 11.5 microns, or about 11.5 to about 12.0 microns, and optionally the standard deviation of the population is less than 1, 2, or 3 microns. Human enucleated erythroid diameter can be measured using, for example, an Advia 120 hematology system or a Moxi Z cytometer (Orflo).

In some embodiments, the human enucleated erythroid cells have a mean particle volume of about 10 fL to about 175 fL, about 10 fL to about 160 fL, about 10 fL to about 140 fL, about 10 fL to about 120 fL, about 10 fL to about 100 fL, about 10 fL to about 95 fL, about 10 fL to about 90 fL, about 10 fL to about 85 fL, about 10 fL to about 80 fL, about 10 fL to about 75 fL, about 10 fL to about About 70 fL, about 10 fL to about 65 fL, about 10 fL to about 60 fL, about 10 fL to about 55 fL, about 10 fL to about 50 fL, about 10 fL to about 45 fL, about 10 fL to about 40 fL, about 10 fL to about 35 fL, about 10 fL to about 30 fL, about 10 fL to about 25 fL, about 10 fL to about 20 fL, about 10 fL to about 15 fL, about 15 fL to about 175 fL, About 15 fL to about 160 fL, about 15 fL to about 140 fL, about 15 fL to about 120 fL, about 15 fL to about 100 fL, about 15 fL to about 95 fL, about 15 fL to about 90 fL, about 15 fL to about 85 fL, about 15 fL to about 80 fL, about 15 fL to about 75 fL, about 15 fL to about 70 fL, about 15 fL to about 65 fL, about 15 fL to about 60 fL, about 15 fL to about About 55 fL, about 15 fL to about 50 fL, about 15 fL to about 45 fL, about 15 fL to about 40 fL, about 15 fL to about 35 fL, about 15 fL to about 30 fL, about 15 fL to about 25 fL fL, about 15 fL to about 20 fL, about 20 fL to about 175 fL, about 20 fL to about 160 fL, about 20 fL to about 140 fL, about 20 fL to about 120 fL, about 20 fL to about 100 fL, About 20 fL to about 95 fL, about 20 fL to about 90 fL, about 20 fL to about 85 fL, about 20 fL to about 80 fL, about 20 fL to about 75 fL, about 20 fL to about 70 fL, about 20 fL to about 65 fL, about 20 fL to about 60 fL, about 20 fL to about 55 fL, about 20 fL to about 50 fL, about 20 fL to about 45 fL, about 20 fL to about 40 fL, about 20 fL to about About 35 fL, about 20 fL to about 30 fL, about 20 fL to about 25 fL, about 25 fL to about 175 fL, about 25 fL to about 160 fL, about 25 fL to about 140 fL, about 25 fL to about 120 fL, about 25 fL to about 100 fL, about 25 fL to about 95 fL, about 25 fL to about 90 fL, about 25 fL to about 85 fL, about 25 fL to about 80 fL, about 25 fL to about 75 fL, About 25 fL to about 70 fL, about 25 fL to about 65 fL, about 25 fL to about 60 fL, about 25 fL to about 55 fL, about 25 fL to about 50 fL, about 25 fL to about 45 fL, about 25 fL to about 40 fL, about 25 fL to about 35 fL, about 25 fL to about 30 fL, about 30 fL to about 175 fL, about 30 fL to about 160 fL, about 30 fL to about 140 fL, about 30 fL to about About 120 fL, about 30 fL to about 100 fL, about 30 fL to about 95 fL, about 30 fL to about 90 fL, about 30 fL to about 85 fL, about 30 fL to about 80 fL, about 30 fL to about 75 fL, about 30 fL to about 70 fL, about 30 fL to about 65 fL, about 30 fL to about 60 fL, about 30 fL to about 55 fL, about 30 fL to about 50 fL, about 30 fL to about 45 fL, About 30 fL to about 40 fL, about 30 fL to about 35 fL, about 35 fL to about 175 fL, about 35 fL to about 160 fL, about 35 fL to about 140 fL, about 35 fL to about 120 fL, about 35 fL to about 100 fL, about 35 fL to about 95 fL, about 35 fL to about 90 fL, about 35 fL to about 85 fL, about 35 fL to about 80 fL, about 35 fL to about 75 fL, about 35 fL to about About 70 fL, about 35 fL to about 65 fL, about 35 fL to about 60 fL, about 35 fL to about 55 fL, about 35 fL to about 50 fL, about 35 fL to about 45 fL, about 35 fL to about 40 fL, about 40 fL to about 175 fL, about 40 fL to about 160 fL, about 40 fL to about 140 fL, about 40 fL to about 120 fL, about 40 fL to about 100 fL, about 40 fL to about 95 fL, About 40 fL to about 90 fL, about 40 fL to about 85 fL, about 40 fL to about 80 fL, about 40 fL to about 75 fL, about 40 fL to about 70 fL, about 40 fL to about 65 fL, about 40 fL to about 60 fL, about 40 fL to about 55 fL, about 40 fL to about 50 fL, about 40 fL to about 45 fL, about 45 fL to about 175 fL, about 45 fL to about 160 fL, about 45 fL to About 140 fL, about 45 fL to about 120 fL, about 45 fL to about 100 fL, about 45 fL to about 95 fL, about 45 fL to about 90 fL, about 45 fL to about 85 fL, about 45 fL to about 80 fL, about 45 fL to about 75 fL, about 45 fL to about 70 fL, about 45 fL to about 65 fL, about 45 fL to about 60 fL, about 45 fL to about 55 fL, about 45 fL to about 50 fL, About 50 fL to about 175 fL, about 50 fL to about 160 fL, about 50 fL to about 140 fL, about 50 fL to about 120 fL, about 50 fL to about 100 fL, about 50 fL to about 95 fL, about 50 fL to about 90 fL, about 50 fL to about 85 fL, about 50 fL to about 80 fL, about 50 fL to about 75 fL, about 50 fL to about 70 fL, about 50 fL to about 65 fL, about 50 fL to about About 60 fL, about 50 fL to about 55 fL, about 60 fL to about 175 fL, about 60 fL to about 160 fL, about 60 fL to about 140 fL, about 60 fL to about 120 fL, about 60 fL to about 100 fL fL, about 60 fL to about 95 fL, about 60 fL to about 90 fL, about 60 fL to about 85 fL, about 60 fL to about 80 fL, about 60 fL to about 75 fL, about 60 fL to about 70 fL, About 60 fL to about 65 fL, about 70 fL to about 175 fL, about 70 fL to about 160 fL, about 70 fL to about 140 fL, about 70 fL to about 120 fL, about 70 fL to about 100 fL, about 70 fL to about 95 fL, about 70 fL to about 90 fL, about 70 fL to about 85 fL, about 70 fL to about 80 fL, about 70 fL to about 75 fL, about 80 fL to about 175 fL, about 80 fL to about About 160 fL, about 80 fL to about 140 fL, about 80 fL to about 120 fL, about 80 fL to about 100 fL, about 80 fL to about 95 fL, about 80 fL to about 90 fL, about 80 fL to about 85 fL, about 100 fL to about 175 fL, about 100 fL to about 160 fL, about 100 fL to about 140 fL, about 100 fL to about 120 fL, about 120 fL to about 175 fL, about 120 fL to about 160 fL, From about 120 fL to about 140 fL, from about 140 fL to about 175 fL, from about 140 fL to about 160 fL, or from about 160 fL to about 175 fL, and where the standard deviation of the population is less than 50, 40, 30, 20, 10, 5, or 2 fL. The mean particle volume can be measured using, for example, a hematology analysis instrument such as a Coulter counter, Moxi Z cell counter (Orflo) or a Sysmex hematology analyzer.

In some embodiments, the human enucleated erythroid cells described herein have one or more (e.g., 2, 3, 4, or more) of the physical properties described herein, such as osmotic fragility, cell size, heme concentration, or phospholipids Amylserine content. While not wishing to be bound by theory, in some embodiments, human enucleated erythroid cells expressing an exogenous polypeptide have physical characteristics similar to wild-type, unprocessed human enucleated erythroid cells. In contrast, hypotonically loaded human enucleated erythroid cells sometimes display abnormal physical features such as increased osmotic fragility, altered cell size, reduced hemoglobin concentration, or phosphatidylserine levels on the outer leaflet of the cell membrane Increase.

In some embodiments, the human enucleated erythroid cells comprise an exogenous polypeptide encoded by an exogenous nucleic acid that is not retained by the cell, has not been purified, or is not present entirely outside the human enucleated erythrocytes. In some embodiments, human enucleated erythroid cells are in a composition lacking a stabilizer.

In some embodiments, the human enucleated erythroid cells have a heme content similar to wild-type, unprocessed human enucleated erythroid cells. In some embodiments, the human enucleated erythroid cells comprise greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or greater than 10% fetal heme. In some embodiments, human enucleated erythroid blood cells comprise at least about 20, 22, 24, 26, 28, or 30 pg, and optionally up to about 30 pg, of total hemoglobin. In some embodiments, the hemoglobin level is determined using the Drabkin reagent method of Example 33 of WO2015/073587.

In some embodiments, the human enucleated erythroid cells have about the same phosphatidylserine content on the outer leaflet of their cell membranes as wild-type, untreated human enucleated erythroid cells. Phosphatidylserine is mainly present in the inner lobe of the cell membrane of wild-type, untreated human enucleated erythrocytes, and hypotonic loading can cause phosphatidylserine to partition to the outer lobe, thereby triggering an immune response. In some embodiments, the population of human enucleated erythroid cells comprises less than about 30, 25, 20, 15, 10, 9, 8, 6, 5, 4, 3, 2, or 1% of cells that respond to annexin V Stain was positive. In some embodiments, phosphatidylserine exposure is assessed by staining for Annexin-V-FITC preferentially bound to PS and measuring FITC fluorescence by flow cytometry, e.g. using Example 54 of WO2015/073587 method.

In some embodiments, the population of human enucleated erythroid cells comprises at least 50%, 60%, 70%, 80%, 90%, or 95% (and optionally up to 90 or 100%) of the cells that are positive for GPA. In some embodiments, the presence of GPA is detected using FACS.

In some embodiments, the human enucleated erythroid cells have a half-life in a human subject of at least 30, 45, or 90 days.

In some embodiments, the cell population comprising human enucleated erythroid cells comprises less than about 10, 5, 4, 3, 2, or 1% echinocytes.

In some embodiments, a pharmaceutical composition comprising a plurality of human enucleated erythroid cells can be administered to a human individual (eg, any of the individuals described herein). In such embodiments, greater than 50%, 60%, 70%, 80%, or 90% of the human cells in the pharmaceutical composition may be enucleated. In some embodiments, cells, such as human enucleated erythroid cells, contain non-functional (eg, inactivated) nuclei.

In some embodiments of any of the compositions described herein, the human enucleated erythroid cells are engineered human enucleated erythroid cells. In some examples, the human engineered enucleated erythroid cells comprise a single exogenous polypeptide. In other examples, human engineered enucleated erythroid cells comprise two or more exogenous polypeptides (eg, any of the exemplary exogenous polypeptides described herein).

In some embodiments, the exogenous polypeptide present on the membrane of a human engineered enucleated erythrocyte can be the product of a click chemistry reaction (e.g., the exogenous polypeptide can be joined to the membrane present on the cell membrane using any of the methods described herein). (e.g., additional exogenous polypeptides or endogenous polypeptides). In some embodiments, exogenous polypeptides present on human engineered enucleated erythroid cell membranes may be conjugation reactions using sortase Products (e.g., exogenous polypeptides can be ligated to polypeptides present on human cell membranes (e.g., additional exogenous polypeptides or endogenous polypeptides), using any of the methods described herein). Non-limiting examples can be found in U.S. Patent No. 10,260,038 and U.S. Patent Publication No. 2016/0082046 A1. In some embodiments, the exogenous polypeptide present on the engineered human enucleated red blood cell membrane can be a lipid-anchored polypeptide , such as a GPI-anchor region, an N-myristylated polypeptide, or an S-palmitoylated polypeptide. In some embodiments, the exogenous polypeptide present on the membrane of human engineered enucleated erythrocytes may be a transmembrane polypeptide ( For example, a single- or multi-spanning transmembrane polypeptide) or a peripheral membrane polypeptide. In some embodiments, the exogenous polypeptide present on the membrane of a human engineered enucleated erythroid cell may be a fusion polypeptide comprising a transmembrane domain (e.g., a fusion polypeptide comprising the transmembrane domain of small integral membrane protein 1 (SMIM1) or glycophorin A (GPA).) In some embodiments, the exogenous The exogenous polypeptide does not have any amino acids that protrude into the cytoplasm of the engineered enucleated erythrocyte. In some embodiments, the exogenous polypeptide present on the cell membrane of the engineered human enucleated erythrocyte has a protruding into the extracellular space and amino acids protruding to the cytoplasm of the engineered human enucleated erythroid cells. Exemplary methods for producing human enucleated erythroid cells using sorting are described in WO2014/183071 or WO2014/183066, wherein each One is incorporated herein by reference.

Human enucleated erythroid cells can be engineered by introducing one or more nucleic acids (e.g., DNA) encoding one or more exogenous polypeptides (e.g., any of the exogenous polypeptides described herein or known in the art). expression vector or mRNA), introduced into human erythroid precursor cells (eg, any of the human erythroid precursor cells described herein or known in the art). Exemplary methods of introducing DNA expression vectors into human erythroid precursor cells include, but are not limited to, liposome-mediated transfer, transformation, biolistics, transfection, and transduction, such as virus-mediated gene transfer (e.g. , using viral vectors including adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, herpesvirus vectors, and retrovirus-based vectors). Additional exemplary methods for introducing DNA expression vectors into human erythroid precursor cells include using, for example, naked DNA, _CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, and microinjection of cells.

Human erythroid precursor cells can optionally be cultured, eg, before and/or after introduction of one or more nucleic acids encoding one or more exogenous polypeptides, under appropriate conditions that allow differentiation into human engineered enucleated erythroid cells. In some embodiments, the resulting engineered human enucleated erythroid cells comprise polypeptides associated with mature human erythrocytes, such as heme (e.g., adult heme and/or fetal heme), glycophorin A, and exogenous polypeptides , which can be verified and quantified by standard methods (eg, Western blot or FACS analysis).

In some embodiments, two or more exogenous polypeptides are encoded in a single nucleic acid, eg, in a single vector. In embodiments, a single vector has separate promoters for each gene, with two polypeptides initially transcribed into a single polypeptide with a protease cleavage site in the middle, such that subsequent proteolytic processing yields two exogenous polypeptide, or any other suitable configuration. In some embodiments, two or more polypeptides are encoded in two or more nucleic acids, eg, each vector encodes one of the exogenous polypeptides.

A nucleic acid can be, for example, DNA or RNA. A variety of viruses are available as gene transfer vectors, including retroviruses, Moloney murine leukemia virus (MMLV), adenoviruses, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency Virus 1 (HIV 1), and spumaviruses such as spumaviruses.

In some embodiments, the human enucleated erythroid lineage is expanded at least 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000-fold (and optionally up to 100,000, 200,000, or 500,000-fold). In some embodiments, cell number is measured using an automated cell counter.

In some examples, human enucleated erythroid cells or human erythroid precursor cells can be transfected with mRNA encoding an exogenous polypeptide to produce engineered human enucleated erythroid cells. Messenger RNA can be derived from in vitro translation of a cDNA plastid construct containing a sequence encoding an exogenous polypeptide. For example, a cDNA sequence encoding an exogenous polypeptide can be inserted into a cloning vector containing a promoter sequence compatible with a particular RNA polymerase. For example, the cloning vector ZAP Express® pBK-CMV (Stratagene, La Jolla, Calif., USA) contains T3 and T7 promoter sequences compatible with T3 and T7 RNA polymerases, respectively. For in vitro translation of the primary strand mRNA, the plastids are linearized at a restriction site downstream of the stop codon corresponding to the terminus of the sequence encoding the exogenous polypeptide. mRNA was transcribed from this linear DNA template using the commercially available kit RNAMaxx® High Yield Transcription Kit (obtained from Stratagene, La Jolla, Calif., USA). In certain instances, it may be desirable to produce 5'-m7GpppG-capped mRNA. Thus, transcription of linearized cDNA templates can be performed using, for example, the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit from Ambion (Austin, Tex., USA). Transcription can be performed at 37 °C for 30 minutes to 4 hours in reaction volumes ranging from 20 to 100 μL. Transcribed mRNA was purified from the reaction mixture by brief treatment with DNase I to eliminate the linearized DNA template, followed by precipitation in 70% ethanol in the presence of lithium chloride, sodium acetate, or ammonium acetate. The integrity of transcribed mRNA can be assessed using electrophoresis with agar-formaldehyde gels or commercially available Novex preformed TBE gels (Novex, Invitrogen, Carlsbad, Calif., USA).

A messenger RNA encoding an exogenous polypeptide can be introduced into human enucleated erythroid cells or human erythroid precursor cells using a variety of methods including, for example, lipofection and electroporation (van Tandeloo et al., Blood 98:49-56, 2001 ). For liposomes, for example, 5 μg of in vitro transcribed mRNA in Opti-MEM (Invitrogen, Carlsbad, Calif., USA) and cationic lipid DMRIE-C (Invitrogen) in a 1:4 ratio for 5 to 15 minutes .

Alternatively, there are a variety of other cationic lipids or cationic polymers that can be used to transfect human erythroid precursor cells with mRNA, including, for example, DOTAP, various forms of polyethyleneimine, and poly-L-lysine (Sigma-Aldrich, Saint Louis, Mo., USA) and Superfect (Qiagen Corporation, Valencia, Calif., USA; see eg Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001). The resulting mRNA/lipid complexes were incubated with cells (1 to 2 x ¹⁰⁶ cells/mL) for 2 hours at 37°C, washed and returned to culture. For electroporation, for example, about 5 to 20 x ¹⁰ cells in 500 μL of Opti-MEM (Invitrogen, Carlsbad, Calif., USA), are mixed with about 20 μg of in vitro transcribed mRNA, and treated using, for example, The Easyject Plus device (EquiBio, Kent, UK) was used for electroporation in 0.4-cm colorimetric tubes. In some instances, it may be necessary to test various voltages, capacitances, and electroporation volumes to determine suitable conditions for transfection of a particular mRNA into human erythroid precursor cells. In general, the electroporation parameters required to efficiently transfect cells with mRNA appear to be less harmful to cells than those required for DNA electroporation (van Tandeloo et al., Blood 98:49-56, 2001) .

Alternatively, peptide-mediated RNA delivery strategies can be used to transfect mRNA into human enucleated erythroid cells or human erythroid precursor cells (see, e.g., Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001) . For example, the cationic lipid polyethyleneimine 2 kDA (Sigma-Aldrich, Saint Louis, Mo., USA) can be combined with melittin (Alta Biosciences, Birmingham, UK) to increase the efficiency of mRNA transfection , especially in primary cells after mitosis. Conjugation Melittin can be conjugated to PEI using a disulfide crosslinker such as, for example, the hetero-bifunctional crosslinker succinimidyl 3-(2-pyridyldithio)propionate. In vitro transcribed mRNA was pre-incubated with melittin-PEI for 5 to 15 minutes to form RNA/peptide/lipid complexes. The complex is then added to cells in serum-free medium for 2 to 4 hours at 37°C in a humidified environment of 5% CO ₂ and then removed, and the transfected cells are further cultured.

In some embodiments, the human enucleated erythroid cell line is engineered by encoding one or more exogenous polypeptides (eg, any exogenous polypeptide or any combination of exogenous polypeptides described herein) Nucleic acid, produced by introduction into human erythroid precursor cells. In some embodiments, the exogenous polypeptide is encoded by a DNA that is introduced into human erythroid precursor cells. In some embodiments, the exogenous polypeptide is encoded by an RNA that is introduced into human erythroid precursor cells.

Nucleic acids encoding one or more exogenous polypeptides can be introduced into human erythroid precursor cells using a variety of DNA techniques including, for example, transient or stable transfection and gene therapy, prior to final differentiation into human enucleated erythroid cells.

Viral gene transfer can be used to transfect cells with nucleic acids encoding one or more exogenous polypeptides. A variety of viruses are available as gene transfer vectors, including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency virus 1 (HIV1) , and foamy viruses such as foamy viruses (see eg Osten et al., HEP 178:177-202, 2007). For example, retroviruses efficiently transduce mammalian cells, including human cells, and integrate into chromosomes, conferring stable gene transfer.

Nucleic acids encoding one or more exogenous polypeptides can be transfected into human erythroid precursor cells. A suitable vector is a Moloney murine leukemia virus (MMLV) vector (Malik et al., Blood 91:2664-2671, 1998). Vectors based on MMLV, an oncogenic retrovirus, are currently used in gene therapy clinical trials (Hassle et al., News Physiol. Sci. 17:87-92, 2002). For example, a DNA construct containing a cDNA encoding an exogenous polypeptide can be generated in a MMLV vector backbone using standard molecular biology techniques. The constructs are transfected into packaging cell lines, such as, for example, PA317 cells, and viral supernatants are used to transfect producer cells, such as, for example, PG13 cells. PG13 viral supernatants were incubated with human erythroid precursor cells. If the exogenous polypeptide is present on the engineered human enucleated erythroid cell membrane, its expression can be monitored using FACS analysis (fluorescence-activated cell sorting), eg, with fluorescently labeled antibodies directed against the exogenous polypeptide. A similar approach can be used to render exogenous polypeptides present in the cytoplasm of engineered human enucleated erythroid cells.

Optionally, nucleic acids encoding fluorescent tracking molecules such as, for example, green fluorescent protein (GFP) can be transfected into human erythroid precursor cells using virus-based methods (Tao et al., Stem Cells 25:670-678, 2007 ). Cells containing DNA encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (e.g. DsRed-Express) are encapsulated using encapsulating cells such as, for example, the Phoenix-Eco cell line (from Orbigen, San Diego, Calif.). Ectopic retroviral vector. Encapsulating cell line stably expressing viral polypeptides required for proper viral packaging, including e.g. gag, pol and env. Supernatant from Phoenix-Eco cells into which virions have been shed for transfection Induction of human erythroid precursor cells. In some instances, transduction can be performed on specially coated surfaces such as, for example, recombinant fibronectin fragments to enhance retrovirus-mediated gene transfer (e.g. RetroNectin, Takara Bio USA, Madison , Wis.). Cells were cultured in RetroNectin-coated dishes with retroviral Phoenix-Eco supernatant and appropriate cofactors. Transduction could be repeated the next day. In this example, expression of EGFP or DsRed -Express the percentage of human erythroid precursor cells can be assessed by FACS. Other reporter genes that can be used to assess transduction efficiency include, for example, β-galactosidase, chloramphenicol acetyltransferase and luciferase, and low Affinity nerve growth factor receptor (LNGFR), and human cell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613, 1999).

Non-viral vectors can be used to introduce nucleic acids encoding one or more exogenous polypeptides into human erythroid precursor cells to produce engineered human enucleated erythroid cells. Non-viral vectors can be introduced into human erythroid precursor cells using a variety of delivery methods, including chemical and physical methods.

Non-viral vectors encoding exogenous polypeptides can be introduced into human erythroid precursor cells using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). Cationic liposomes form complexes with DNA via, for example, charge interactions. The positively charged DNA/lipid complex binds to the negatively charged cell surface and is taken up by the cell by endocytosis. This method can be used, for example, to transfect hematopoietic cells (see Keller et al., Gene Therapy 6:931-938, 1999). For human erythroid precursor cells, plastid DNA (in serum-free medium such as OptiMEM (Invitrogen, Carlsbad, Calif.)) is combined with cationic liposomes (in serum-free medium) such as the commercially available transfection reagent Lipofectamine™ (Invitrogen, Carlsbad, Calif.) and let stand for at least 20 minutes to form the complex. The DNA/liposome complexes are added to human erythroid precursor cells and cultured for 5 to 24 hours, after which the transgene expression of the exogenous polypeptide can be measured. Alternatively, other commercially available lipofectamines (eg, In vivo GeneSHUTTLE™, Qbiogene, Carlsbad, Calif.) can be used.

Optionally, cationic polymers, such as, for example, polyethyleneimine (PEI), can be used to efficiently transfect human erythroid precursor cells, such as hematopoietic and cord blood-derived CD34 ⁺ cells (see e.g., Shin et al., Biochim. Biophys. Acta 1725:377-384, 2005). Human CD34 ⁺ cells were isolated from human cord blood and cultured in Iscove's modified Dulbecco's medium supplemented with 200 ng/ml stem cell factor and 20% heat-inactivated serum. Plastid DNA encoding exogenous polypeptides is grown with branched or linear PEI ranging in size from 0.8 K to 750 K (Sigma Aldrich, Saint Louis, Mo., USA; Fermetas, Hanover, Md., USA). A 4.2 mg/mL stock solution of PEI was prepared in distilled water and slightly acidified to pH 5.0 with HCl. DNA can be bound to PEI at various nitrogen/phosphate ratios based on 1 μg DNA containing 3 nmol phosphate and 1 μL PEI stock solution containing 10 nmol amine nitrogen for 30 minutes at room temperature. Isolated CD34 ⁺ cells were inoculated with DNA/cation complexes, centrifuged at 280 xg for 5 minutes, and incubated in culture medium for 4 hours or more until expression of exogenous polypeptide was assessed.

Plastid vectors can be introduced into suitable human erythrocytes using physical methods such as particle-mediated transfection, "gene guns", biolistic or particle bombardment techniques (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). In precursor cells,. In this example, DNA encoding an exogenous polypeptide was absorbed onto gold particles and delivered to cells by a particle gun. This method can be used, for example, to transfect human erythroid precursor cells, such as hematopoietic stem cells derived from umbilical cord blood (see, eg, Verma et al., Gene Therapy 5:692-699, 1998). Therefore, cord blood lines were isolated and diluted three-fold in phosphate-buffered saline. CD34 ⁺ cells were purified using an anti-CD34 monoclonal antibody and magnetic beads coated with secondary antibodies and a magnetic separation system (eg, Miltenyi MiniMac System, Auburn, Calif., USA). CD34 ⁺ enriched cells can be cultured as described herein. For transfection, plastid DNA encoding the exogenous polypeptide is precipitated onto particles such as gold microbeads by treatment with calcium chloride and spermtriamine. After washing the DNA-coated beads with ethanol, the beads can be delivered into cultured cells using, for example, the Biolistic PDS-1000/He system (Bio-Rad, Hercules, Calif., USA). Reporter genes such as β-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein can be used to assess transfection efficiency.

Optionally, electroporation methods can be used to introduce plastid vectors into human erythroid precursor cells. Electroporation creates transient pores in the cell membrane that allow the introduction of various molecules into the cell, including, for example, DNA and RNA. Therefore, CD34 ⁺ cell lines were isolated and cultured as described herein. Immediately prior to electroporation, cells are detached by centrifugation at 250 x g for 10 minutes at room temperature and resuspended in electroporation buffer at 0.2 to 10 x ¹⁰ viable cells/ml, e.g. supplemented with 1.0% human serum X-VIVO 10 with albumin (HSA). Add plastid DNA (1 to 50 µg) with 500 µL of the cell suspension to an appropriate electroporation cuvette.

Electroporation can be performed using, for example, an ECM 600 electroporator (Genetronics, San Diego, Calif., USA) with a voltage range of 200 V to 280 V and a pulse length of 25 to 70 milliseconds. Several other electroporation instruments are commercially available and can be used for this purpose (eg, Gene Pulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo, Thermo Science, Milford, Mass.). Alternatively, efficient electroporation of isolated CD34 ⁺ cells was performed using the following parameters: 4 mm cuvette, 1600 μE, 550 V/cm, and 10 μg of DNA per 500 μL cells at 1× ¹⁰ cells/mL (Oldak et al., Acta Biochim. Polonica 49:625-632, 2002).

Nucleofection (a form of electroporation) can also be used to transfect human erythroid precursor cells. In this example, transfection is performed using electrical parameters in cell-type-specific solutions that allow DNA (or other reagents) to be delivered directly into the nucleus, thus reducing the risk of its possible degradation in the cytoplasm. For example, the Human CD34 Cell Nucleofector™ Kit (from Amaxa Inc.) can be used to transfect human erythroid precursor cells. In this example, 1 to 5 x ¹⁰⁶ cell lines in Human CD34 Cell Nucleofector™ solution were mixed with 1 to 5 μg DNA and transfected in the Nucleofector™ instrument using preprogrammed settings determined by the manufacturer.

Human erythroid precursor cells can be non-virally transfected with traditional expression vectors that cannot replicate themselves in mammalian cells unless integrated into the gene body. Alternatively, human erythroid precursor cells can be transfected with an episomal vector, which can be retained in the host nucleus as an autonomously replicating genetic unit that does not integrate into the chromosome (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). These vectors utilize genetic elements derived from viruses that normally replicate extrachromosomally in cells following latent infection, such as, for example, EBV, human polyomavirus BK, bovine papillomavirus-1 (BPV-1), simplex Herpesvirus-1 (HSV), and Simian virus 40 (SV40). Mammalian artificial chromosomes can also be used for non-viral gene transfer (Vanderbyl et al., Exp. Hematol. 33:1470-1476, 2005).

Exogenous nucleic acids encoding one or more exogenous polypeptides can be assembled into expression vectors by standard molecular biology methods known in the art (e.g., restriction cleavage, overlap-extension PCR, and Gibson assembly). .

The exogenous nucleic acid may comprise a gene encoding an exogenous polypeptide not normally present on the extracellular surface of, for example, human enucleated erythroid cells, fused to a gene encoding an endogenous or native membrane polypeptide such that The exogenous polypeptide is expressed on the extracellular surface. For example, an exogenous gene encoding an exogenous polypeptide can be colonized at the N-terminus of a type 1 membrane polypeptide following the leader sequence, at the C-terminus of a type 2 membrane polypeptide, or at the GPI attachment site of a GPI-linked membrane polypeptide point upstream.

Standard breeding methods can be used to introduce elastic amino acid linkers between the two fusion genes. For example, the elastic linker is a polyglycine poly-serine linker, such as [Gly ₄ Ser] ₃ (SEQ ID NO: 33), which is commonly used to generate single-chain antibody fragments from full-length antibodies (Antibody Engineering : Methods & Protocols, B. Lo ed., Humana Press, 2004, p. 576), or Ala-Gly-Ser-Thr polypeptides, such as those used to generate single-chain Arc suppressors (Robinson & Sauer, Proc. Nat'l. Acad. Sci. USA 95: 5929-34, 1998). In some embodiments, a flexible linker provides a more flexible and sterically free exogenous polypeptide than an equivalent construct without a flexible linker. This added flexibility is useful in applications requiring binding to a target, such as an antibody or polypeptide, or in an enzymatic reaction of a polypeptide whose active site must accommodate a substrate (eg, a target).

In some embodiments, methods are provided comprising delivering a large nucleic acid, particularly RNA, such as mRNA, into a human erythroid precursor cell by contacting the human erythroid precursor cell with the nucleic acid in an effective manner to deliver the nucleic acid to Nucleic acid is introduced by electroporation under cellular conditions, such as those described herein. Suitable electroporators include, but are not limited to, the Bio-Rad GENE PULSER and GENE PULSER II; the Life Technologies NEON; the BTX GEMINI system; and the MAXCYTE electroporator. These methods do not require viral delivery or the use of viral vectors. Suitable nucleic acids include RNA, such as mRNA. Suitable nucleic acids also include DNA, including transposable elements, stable episomes, plastid DNA or linear DNA.

Conditions for electroporation of cell lines have been described in the literature, eg Van Tendeloo et al., Blood 98(1):49-56, 2001. Suitable electroporation conditions for the methods described herein include, for the Life Technologies Neon Transfection System: pulse voltages in the range of about 500 to about 2000 V, about 800 to about 1800 V, or about 850 to about 1700 V; pulse widths in the range From about 5 to about 50 msec, or from about 10 to about 40 msec; the number of pulses ranges from 1 to 2 pulses, 1 to 3 pulses, 1 to 4 pulses, or 1 to 5 pulses.

Electroporation conditions particularly suitable for human erythroid precursor cells include, for example, for 4 days: a) pulse voltage 1300 to 1400, pulse width: 10 to 20 msec, pulse number: 1 to 3; b) pulse voltage 1400, pulse width : 10 msec, pulse number: 3; c) pulse voltage 1400, pulse width: 20 msec, pulse number: 1; and d) pulse voltage 1300, pulse width: 10 msec, pulse number: 3.

Electroporation conditions particularly suitable for human erythroid precursor cells include, for example, for 8 to 9 days: a) pulse voltage: 1400 to 1600, pulse width: 20, pulse number: 1; b) pulse voltage: 1100 to 1300, pulse Width: 30, pulse number: 1; c) pulse voltage: 1000 to 1200, pulse width: 40, pulse number: 1; d) pulse voltage: 1100 to 1400, pulse width: 20, pulse number: 2; e) pulse Voltage: 950 to 1150, pulse width: 30, pulse number: 2; f) pulse voltage: 1300 to 1600, pulse width: 10, pulse number: 3. These conditions typically result in a transfection efficiency of at least about 60% or greater (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least about 97% or greater), and A cell viability of at least about 70% or greater (eg, at least about 75%, 80%, 85%, 90%, 95%, or at least about 97% or greater).

Particularly suitable for electroporation conditions of cultured human erythroid precursor cells under differentiation conditions, including, for example, for 12 to 13 days: a) pulse voltage: 1500 to 1700, pulse width: 20, pulse number: 1; b) Pulse voltage: 1500 to 1600, pulse width: 10, pulse number: 3. These conditions typically result in at least about 50% or greater (e.g., at least about 55%, about 60%, about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least about 97% or more High) transfection efficiency, and at least about 70% or higher (eg, at least about 75%, 80%, 85%, 90%, 95%, or at least about 97% or more) cell viability.

The conditions disclosed herein with respect to the Life Technologies Neon system can be readily adjusted by one of ordinary skill in the art only by routine experimentation to suit different electroporators and/or different electroporation settings, and the specific electroporation described herein There is no limitation with respect to the disclosed methods.

In some embodiments, cultured human erythroid precursor cells are subjected to a first electroporation using the electroporation conditions described herein, followed by incubation for a desired period of time (optionally under differentiation conditions), followed by a second electroporation. Secondary electroporation. In some embodiments, cultured human erythroid precursor cells are subjected to a first electroporation using the electroporation conditions described herein, followed by incubation for a desired period of time (optionally under differentiation conditions), followed by a second electroporation. Second, third, fourth, fifth, or sixth electroporation. The period of incubation between the first and second, second and third, etc. electroporations may vary, as appropriate. For example, the time period between each electroporation can be adjusted as needed, for example, the time period can be 30 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 21 days. For example, human erythroid precursor cells can be expressed at day 1 and day 2, day 1 and day 3, day 1 and day 4, day 1 and day 5, day 1 and day 6, day 1 Day 1 and Day 7, Day 1 and Day 8, Day 1 and Day 9, Day 1 and Day 10, Day 1 and Day 11, Day 1 and Day 12, Day 1 And electroporation was performed on day 13, day 1 and day 14, day 1 and day 15, or day 1 and day 16. In another example, the cells can be treated at day 2 and day 3, day 2 and day 4, day 2 and day 5, day 2 and day 6, day 2 and day 7, day 2 2 days and 8 days, 2 days and 9 days, 2 days and 10 days, 2 days and 11 days, 2 days and 12 days, 2 days and 13 days, 2 days And electroporation was performed on day 14, day 2 and day 15, or day 2 and day 16. In yet another example, the human erythroid precursor cells can be expressed at day 3 and day 4, day 3 and day 5, day 3 and day 6, day 3 and day 7, day 3 and day 8 days, 3 days and 9 days, 3 days and 10 days, 3 days and 11 days, 3 days and 12 days, 3 days and 13 days, 3 days and 14 days , day 3 and day 15, or day 3 and day 16 for electroporation. In yet another example, the cells can be treated at day 4 and day 5, day 4 and day 6, day 4 and day 7, day 4 and day 8, day 4 and day 9, day Day 4 and Day 10, Day 4 and Day 11, Day 4 and Day 12, Day 4 and Day 13, Day 4 and Day 14, Day 4 and Day 15, or Day 4 Electroporation was performed on day 1 and day 16. In yet another example, the cells can be treated at day 5 and day 6, day 5 and day 7, day 5 and day 8, day 5 and day 9, day 5 and day 10, day 5 Electroporation on days 5 and 11, days 5 and 12, days 5 and 13, days 5 and 14, days 5 and 15, or days 5 and 16 . In yet another example, human erythroid precursor cells can be expressed at days 6 and 7, days 6 and 8, days 6 and 9, days 6 and 10, days 6 and 11 Electroporation was performed on day 1, day 6 and day 12, day 6 and day 13, day 6 and day 14, day 6 and day 15, or day 6 and day 16. In yet another example, the human erythroid precursor cells can be expressed at day 7 and day 8, day 7 and day 9, day 7 and day 10, day 7 and day 11, day 7 and day Electroporation was performed on day 12, day 7 and day 13, day 7 and day 14, day 7 and day 15, or day 7 and day 16. In yet another example, the human erythroid precursor cells can be expressed at day 8 and day 9, day 8 and day 10, day 8 and day 11, day 8 and day 12, day 8 and day Electroporation was performed on day 13, day 8 and day 14, day 8 and day 15, or day 8 and day 16. In yet another example, the human erythroid precursor cells can be expressed at day 9 and day 10, day 9 and day 11, day 9 and day 12, day 9 and day 13, day 9 and day Electroporation was performed on day 14, day 9 and day 15, or day 9 and day 16. In yet another example, the human erythroid precursor cells can be expressed at day 10 and day 11, day 10 and day 12, day 10 and day 13, day 10 and day 14, day 10 and day Electroporation was performed on day 15, or on day 10 and day 16. In yet another example, the human erythroid precursor cells can be detected at day 11 and day 12, day 11 and day 13, day 11 and day 14, day 11 and day 15, or day 11 and day 15. Electroporation was performed on day 16. In yet another example, the human erythroid precursor cells can be electroporated on days 12 and 13, days 12 and 14, days 12 and 15, or days 12 and 16. In yet another example, the human erythroid precursor cells can be electroporated on days 13 and 14, days 13 and 15, or days 13 and 16. In yet another example, the human erythroid precursor cells can be electroporated on day 14 and day 15, or day 14 and day 16. Optionally, human erythroid precursor cells may be electroporated more than two times, eg three, four, five or six times, and the intervals may be chosen as desired at any point in the differentiation process of the cells.

In some embodiments, using the electroporation conditions described herein, cultured human erythroid precursor cells are differentiated at stages 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15 or 16 days for electroporation.

In some embodiments, the engineered human enucleated erythroid cells can be click-engaged engineered human enucleated erythroid cells. The catalytic bond-forming polypeptide domain can be expressed on or within, for example, a human erythroid precursor cell, be present in its cytoplasm or be present on the cell membrane. A number of catalytic bond-forming polypeptides exist, including transpeptidases, sortases, and isopeptidases, including those derived from Spy0128, a polypeptide isolated from Streptococcus pyogenes . It was demonstrated that the autocatalytic isopeptide bond-forming subunit (CnaB2 domain) of Spy0128 splits into two distinct polypeptides that retain catalytic activity and are specific for each other. The polypeptides in this system are referred to as SpyTag and SpyCatcher. Upon mixing, SpyTag and SpyCatcher undergo an isopeptide bond formation process between Aspl 17 on the SpyTag and Lys31 on the SpyCatcher (Zakeri and Howarth, JACS 132:4526, 2010). This reaction is compatible with the cellular environment and is highly specific for protein/peptide conjugation (Zakeri et al., Proc. Natl. Acad. Sci. USA 109:E690-E697, 2012). SpyTag and SpyCatcher are known to direct post-translational topological modifications of elastin. For example, placement of a SpyTag at the N-terminus and a SpyCatcher at the C-terminus induces the formation of a circular elastin-like protein (Zhang et al., J. Am. Chem. Soc. 2013).

The components SpyTag and SpyCatcher are interchangeable such that a system in which molecule A is fused to SpyTag and molecule B is fused to SpyCatcher is functionally equivalent to a system in which molecule A is fused to SpyCatcher and molecule B is fused to SpyTag. For purposes of the present invention, when using SpyTag and SpyCatcher it is understood that complementary molecules may take their place.

Catalytic bond-forming polypeptides such as the SpyTag/SpyCatcher system can be used to attach exogenous polypeptides to, for example, the extracellular surface of human erythroid precursor cells or human enucleated erythroid cells. The SpyTag polypeptide sequence can be expressed on the extracellular surface of erythroid precursor cells or human enucleated erythroid cells. The SpyTag polypeptide can be, for example, fused to the N-terminus of a type 1 or type 3 transmembrane polypeptide (e.g., glycoprotein A), fused to the C-terminus of a type 2 transmembrane polypeptide (e.g., Kell), inserted in frame into a multiple membrane-penetrating polypeptide (eg band 3), fused to a GPI-receptor polypeptide (such as CD55 or CD59), fused to a lipid-chain-anchor polypeptide, or fused to a peripheral membrane polypeptide. Exogenous polypeptides can be fused to SpyCatcher. Nucleic acids encoding SpyCatcher fusions can be expressed and secreted by the same human erythroid precursor cells or human enucleated erythroid cells expressing the SpyTag fusion. Alternatively, nucleic acid sequences encoding SpyCatcher fusions can be produced exogenously, for example in bacterial, fungal, insect, mammalian or cell-free production systems. When the SpyTag and SpyCatcher polypeptides react, a covalent bond will be formed linking the exogenous polypeptide to the extracellular surface of human erythroid precursor cells or human enucleated erythroid cells.

In one embodiment, the SpyTag polypeptide can be expressed as a fusion with the N-terminus of glycophorin A in human erythroid cells under the control of the Gatal promoter. The exogenous polypeptide fused to the SpyCatcher polypeptide sequence can be expressed in the same class of erythrocytes under the control of the Gatal promoter. Upon expression of the two fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, and a covalent bond will be formed between the human erythroid cell surface (extracellular surface of human erythrocytes) and the exogenous polypeptide.

In another embodiment, the SpyTag polypeptide can be expressed as a fusion with the N-terminus of glycophorin A in human erythroid precursor cells or human enucleated erythroid cells under the control of the Gatal promoter. The exogenous polypeptide fused to the SpyCatcher polypeptide sequence can be expressed in a suitable mammalian cell expression system, such as HEK293 cells. When the SpyTag fusion polypeptide is expressed on human erythroid precursor cells or human enucleated erythroid cells, the SpyCatcher fusion polypeptide engages the cell. Under suitable reaction conditions, an isopeptide bond will be formed between SpyTag and SpyCatcher polypeptide, and a covalent bond will be formed between the surface of human erythroid precursor cells or the surface of human enucleated erythroid cells and exogenous polypeptide.

Catalytic bond-forming polypeptides such as the SpyTag/SpyCatcher system can be used to anchor exogenous polypeptides to the intracellular space of human erythroid precursor cells or human enucleated erythroid cells. SpyTag polypeptide sequences can be expressed in the intracellular space of human erythroid precursor cells or human enucleated erythroid cells by a variety of methods, including direct expression of transgenes, fusion to endogenous intracellular polypeptides (such as heme), fusion To the intracellular domain of endogenous cell surface polypeptides (eg band 3, glycophorin A, Kell), or fused to structural components of the cytoskeleton. The SpyTag sequence is not limited to the end of the polypeptide, and can be integrated into the internal sequence of the endogenous polypeptide, so that the translation and positioning of the polypeptide are not disturbed. Exogenous polypeptides can be fused to SpyCatcher. The nucleic acid sequence encoding the SpyCatcher fusion can be expressed in the same human erythroid precursor cells or human enucleated erythroid cells that express the SpyTag fusion. When the SpyTag and SpyCatcher polypeptides react, a covalent bond is formed which acts to anchor the exogenous polypeptide into the intracellular space of the human erythroid precursor or human enucleated erythroid cells.

In one embodiment, human erythroid precursor cells or human enucleated erythroid cells can express the fusion of SpyTag and heme β in the cells. Human erythroid precursor cells or human enucleated erythroid cells can be genetically modified with a gene sequence that includes a heme promoter, a β-globin gene, and a SpyTag sequence that fuses the C-terminus of the intracellular β-globin during translation to SpyTag. In addition, human erythroid precursor cells or human enucleated erythroid cells express a Gatal promoter-directed gene encoding SpyCatcher-driven polypeptide expression (e.g., phenylalanine hydroxylase (PAH) expression), such that after translation, The N-terminus of an intracellular polypeptide (eg PAH) is fused to SpyCatcher. After expression of the two fusion polypeptides, the SpyTag-bound β-globin is linked via an isopeptide bond to a SpyCatcher-binding polypeptide (e.g., PAH) in the intracellular space, allowing the polypeptide (e.g., PAH) to be anchored to the β-globin and retained during maturation .

In another embodiment, the SpyTag polypeptide can be expressed as a fusion with an exogenous polypeptide fused to human erythroid precursor cells or human enucleated erythroid cells. The SpyCatcher polypeptide can be expressed as a fusion to the C-terminus (intracellular) of glycophorin A in the same human erythroid precursor cell or human enucleated erythroid cells. Upon expression of the two fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, and a covalent bond will be formed between the membrane-anchored endogenous erythroid polypeptide and the exogenous polypeptide.

Other molecular fusions can be formed between polypeptides and include direct or indirect linkages. Polypeptides can be joined to each other directly or indirectly via a linker. Linkers can be peptides, polymers, aptamers or nucleic acids. Polymers can be, for example, natural, synthetic, linear or branched. The exogenous polypeptide may comprise a heterologous fusion polypeptide comprising a first polypeptide and a second polypeptide, wherein the fusion polypeptide comprises polypeptides directly linked to each other or having intervening linker sequences and/or other sequences at one or both ends. Attachment to the linker can be by covalent or ionic bonding.

In some embodiments, the engineered human enucleated erythroid cells are human enucleated erythroid cells that have been hypotonically loaded. For hypotonic loading/lysis, human erythroid precursor cells or human enucleated erythroid lineages are exposed to a low ionic strength buffer, causing their rupture. Exogenous polypeptides are distributed in cells. Human enucleated erythroid cells or human erythroid precursors can be lysed hypotonically by adding a 30 to 50-fold excess volume of 5 mM phosphate buffer (pH 8) to the isolated enucleated erythroid pellet. The resulting lysed cell membranes were separated by centrifugation. The lysed cell membrane pellet is resuspended and incubated, eg, for 30 minutes, in a low ionic strength buffer in the presence of the exogenous polypeptide. Alternatively, lysed cell membranes can be incubated with exogenous polypeptides for as little as a minute or as long as several days, depending on optimal conditions determined for efficient loading of human enucleated erythroid cells or human erythroid precursor cells. For hypotonic loading of nucleic acids encoding one or more exogenous polypeptides (e.g., any of the exemplary exogenous polypeptides described herein or known in the art), the nucleic acids can be suspended in a hypotonic Tris-HCl solution ( pH 7.0) and injected into human erythroid precursor cells. The concentration of Tris-HCl may be from about 20 mmol/l to about 150 mmol/l, depending on optimal conditions determined for efficient loading of human enucleated erythroid cells.

Alternatively, human erythroid precursor cells or human enucleated erythroid cells can be loaded using controlled dialysis against a hypotonic solution to expand the cells and create pores in the cell membrane (see, e.g., U.S. Pat. Nos. 4,327,710; 5,753,221; 6,495,351 and 10,046,009). For example, resuspend the cell pellet in 10 mM HEPES, 140 mM NaCl, 5 mM glucose, pH 7.4, in a solution containing 10 mM NaH ₂ P0 ₄ , 10 mM NaHCO ₃ , 20 mM glucose, and 4 mM MgCl ₂ , Dialysis against a low ionic strength buffer, pH 7.4. After 30 to 60 minutes, the cells were further dialyzed against 16 mM NaH ₂ P0 ₄ , pH 7.4 solution (which contained exogenous polypeptide) for 30 to 60 minutes. All these procedures can preferably be performed at 4°C. In some instances, loading into large numbers of human erythroid precursor cells or human enucleated erythroid cells can be advantageous by means of dialysis, and specific equipment designed for this purpose can be used (see, e.g., U.S. Pat. Nos. 4,327,710, 6,139,836 and 6,495,351).

Exemplary methods of making human enucleated erythroid cells (e.g., reticulocytes or erythrocytes) comprising exogenous polypeptides are described, for example, in WO2015/073587, WO2015/153102, WO2020/243006, and WO2020/219909, each of which Incorporated herein by reference in its entirety.

In some embodiments, the population of human enucleated erythrocytes contains less than 1% live human enucleated cells, eg, no detectable live human enucleated cells. In some embodiments, enucleation is measured by FACS using nuclear staining. In some embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionally up to about 70, 80, 90, or 100%) comprise one or more (eg 2, 3, 4 or more) exogenous polypeptides. In some embodiments, expression of an exogenous polypeptide can be measured by FACS using a labeled antibody raised against the polypeptide. In some embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionally up to about 70, 80, 90, or 100%) of a population of cells go to nucleated and comprise one or more (eg, 2, 3, 4 or more) exogenous polypeptides. Method of treating HPV16- positive or HPV16- associated cancer in a human subject

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from about 0.5 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 0.5 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 0.5 x 10 ⁹ to about 9 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 8 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 7 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 6 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 5 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 4 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 3 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 2 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 1 x 10 ⁹ , from about 1 x 10 ⁹ to about 5 x 10 ¹⁰ , from about 1 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 1 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 1 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 1 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 1 x 10 ⁹ to about 9 x 10 ⁹ , from about 1 x 10 ⁹ to about 8 x 10 ⁹ , from about 1 x 10 ⁹ to about ⁷ x 10 ⁹ , from about 1 x 10 ⁹ to about 6 x 10 9 , from about 1 x 10 ⁹ to about 5 x 10 ⁹ , from about 1 x 10 ⁹ to about 4 x 10 ⁹ , from about 1 x 10 ⁹ to about 3 x 10 ⁹ , from about 1 x 10 ⁹ to about 2 x 10 ^{9 , from about 2 x 10 9 to} about 5 x 10 ¹⁰ , From about 2 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 2 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 2 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 2 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 2 x 10 ⁹ to about 9 x 10 ⁹ , from about 2 x 10 ⁹ to about 8 x 10 ⁹ , from about 2 x 10 ⁹ to about 7 x 10 ⁹ , from about 2 x 10 ⁹ to about 6 x 10 ⁹ , from about 2 x 10 ⁹ to about 5 x 10 ⁹ , from about 2 x 10 ⁹ to about 4 x 10 ⁹ , from about 2 x 10 ⁹ to about 3 x 10 ⁹ , from about 3 x 10 ⁹ to About 5 x 10 ¹⁰ , from about 3 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 3 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 3 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 3 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 3 x 10 ⁹ to about 9 x 10 ⁹ , from about 3 x 10 ⁹ to about 8 x 10 ⁹ , from about 3 x 10 ⁹ to about 7 x 10 ⁹ , from about 3 x 10 ⁹ to about 6 x 10 ⁹ , from about 3 x 10 ⁹ to about 5 x 10 ⁹ , from about 3 x 10 ⁹ to about 4 x 10 ⁹ , from about 4 x 10 ⁹ to about 5 x 10 ¹⁰ , from From about 4 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 4 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 4 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 4 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 4 x 10 ⁹ to about 9 x 10 ⁹ , from about 4 x 10 ⁹ to about 8 x 10 ⁹ , from about 4 x 10 ⁹ to about 7 x 10 ⁹ , from about 4 x 10 ⁹ to about 6 x 10 ⁹ , from about 4 x 10 ⁹ to about 5 x 10 ⁹ , from about 5 x 10 ⁹ to about 5 x 10 ¹⁰ , from about 5 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 5 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 5 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 5 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 5 x 10 ⁹ to about 9 x 10 ⁹ , from about 5 x 10 ⁹ to about 8 x 10 ⁹ , from about 5 x 10 ⁹ to about 7 x 10 ⁹ , from about 5 x 10 ⁹ to about 6 x ¹⁰ 9 , from about 6 x 10 ⁹ to about 5 x 10 ¹⁰ , from about 6 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 6 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 6 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 6 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 6 x 10 ⁹ to about 9 x 10 ⁹ , from about 6 x 10 ⁹ to about 8 x 10 ⁹ , from about 6 x 10 ⁹ to about 7 x 10 ^{9 , from about 7 x 10 9 to} about 5 x 10 ¹⁰ , From about 7 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 7 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 7 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 7 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 7 x 10 ⁹ to about 9 x 10 ⁹ , from about 7 x 10 ⁹ to about 8 x 10 ⁹ , from about 8 x 10 ⁹ to about 5 x 10 ¹⁰ , from about 8 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 8 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 8 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 8 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 8 x 10 ⁹ to About 9 x 10 ⁹ , from about 9 x 10 ⁹ to about 5 x 10 ¹⁰ , from about 9 x 10 ⁹ to about 4 x 10 ¹⁰ , from about 9 x 10 ⁹ to about 3 x 10 ¹⁰ , from about 9 x 10 ⁹ to about 2 x 10 ¹⁰ , from about 9 x 10 ⁹ to about 1 x 10 ¹⁰ , from about 1 x 10 ¹⁰ to about 5 x 10 ¹⁰ , from about 1 x 10 ¹⁰ to about 4 x 10 ¹⁰ , from about 1 x 10 ¹⁰ to about 3 x 10 ¹⁰ , from about 1 x 10 ¹⁰ to about 2 x 10 ¹⁰ , from about 2 x 10 ¹⁰ to about 5 x 10 ¹⁰ , from about 2 x 10 ¹⁰ to about 4 x 10 ¹⁰ , from From about 2 x 10 ¹⁰ to about 3 x 10 ¹⁰ , from about 3 x 10 ¹⁰ to about 5 x 10 ¹⁰ , from about 3 x 10 ¹⁰ to about 4 x 10 ¹⁰ , or from about 4 x 10 ¹⁰ to about 5 x 10 ¹⁰ human enucleated erythroid cells comprising on their surface: (i) an exogenous antigen-presenting polypeptide linked to an exogenous antigen polypeptide; (ii) an exogenous costimulatory polypeptide comprising 4-BBL; (iii) ) an exogenous cytokine polypeptide comprising IL-12, administered at a frequency of about 2 weeks to about 4 weeks. In some embodiments, the exogenous antigen-presenting polypeptide is an MHC class 1 HLA-A2 single-chain fusion protein comprising an α chain, a β2-microglobulin (β2m) chain, and a membrane anchoring region, And wherein the exogenous antigenic polypeptide comprises a human papillomavirus (HPV) E7 antigen.

In some embodiments of any of these methods, the human subject has a cancer selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, cervical cancer, vaginal cancer, penile cancer, oral Pharyngeal cancer, tongue cancer, tonsil cancer, oral cancer, vaginal cancer or their metastases. In some embodiments of any of these methods, the human subject has a cancer selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, and cervical cancer. In some embodiments, the oropharyngeal cancer is tongue cancer or tonsil cancer. In some embodiments of any of the methods described herein, the solid tumor is a locally advanced solid tumor. In some embodiments of any of the methods described herein, the solid tumor is recurrent or refractory to standard therapy for solid tumors. In some embodiments, the human subject has cervical cancer, squamous cell carcinoma of the head and neck, or squamous cell carcinoma of the anal canal that is not amenable to curative treatment.

In some embodiments of any of these methods, the human subject is positive for HLA-A*02:01. In some embodiments of any of these methods, the human subject has clinical history or documentation indicating that the subject has an HPV16-positive or HPV16-associated tumor. In some embodiments, the human subject has a clinical history or documentation indicating that the subject has an HPV16-positive or HPV16-associated tumor (e.g., head and neck squamous cell carcinoma or cervical cancer HPV16-positive or HPV16-associated tumor ).

In some embodiments of any of the methods described herein, the frequency of administration is about every 14 days to about every 28 days (such as about every 14 days to about every 27 days, about every 14 days to about every 26 days, about every 14 days every 14 days to about every 25 days, about every 14 days to about every 24 days, about every 14 days to about every 23 days, about every 14 days to about every 22 days, about every 14 days to about every 21 days, about every 14 days day to about every 20 days, about every 14 days to about every 19 days, about every 14 days to about every 18 days, about every 14 days to about every 17 days, about every 14 days to about every 16 days, about every 14 days every day to about every 15 days, about every 15 days to about every 28 days, about every 15 days to about every 27 days, about every 15 days to about every 26 days, about every 15 days to about every 25 days, about every 15 days every day to about every 24 days, about every 15 days to about every 23 days, about every 15 days to about every 22 days, about every 15 days to about every 21 days, about every 15 days to about every 20 days, about every 15 days every day to about every 19 days, about every 15 days to about every 18 days, about every 15 days to about every 17 days, about every 15 days to about every 16 days, about every 16 days to about every 28 days, about every 16 days every day to about every 27 days, about every 16 days to about every 26 days, about every 16 days to about every 25 days, about every 16 days to about every 24 days, about every 16 days to about every 23 days, about every 16 days day to about every 22 days, about every 16 days to about every 21 days, about every 16 days to about every 20 days, about every 16 days to about every 19 days, about every 16 days to about every 18 days, about every 16 days every 17 days to about every 17 days, about every 17 days to about every 28 days, about every 17 days to about every 27 days, about every 17 days to about every 26 days, about every 17 days to about every 25 days, about every 17 days day to about every 24 days, about every 17 days to about every 23 days, about every 17 days to about every 22 days, about every 17 days to about every 21 days, about every 17 days to about every 20 days, about every 17 days day to about every 19 days, about every 17 days to about every 18 days, about every 18 days to about every 28 days, about every 18 days to about every 27 days, about every 18 days to about every 26 days, about every 18 days every 18 days to about every 25 days, about every 18 days to about every 24 days, about every 18 days to about every 23 days, about every 18 days to about every 22 days, about every 18 days to about every 21 days, about every 18 days day to about every 20 days, about every 18 days to about every 19 days, about every 18 days to about every 18 days, about every 18 days to about every 17 days, about every 18 days to about every 16 days, about every 18 days day to about every 15 days, about every 19 days to about every 28 days, about every 19 days to about every 27 days, about every 19 days to about every 26 days, about every 19 days to about every 25 days, about every 19 days day to about every 24 days, about every 19 days to about every 23 days, about every 19 days to about every 22 days, about every 19 days to about every 21 days, about every 19 days to about every 20 days, about every 20 days every 20 days to about every 28 days, about every 20 days to about every 27 days, about every 20 days to about every 26 days, about every 20 days to about every 25 days, about every 20 days to about every 24 days, about every 20 days day to about every 23 days, about every 20 days to about every 22 days, about every 20 days to about every 21 days, about every 21 days to about every 28 days, about every 21 days to about every 27 days, about every 21 days every 21 days to about every 26 days, about every 21 days to about every 25 days, about every 21 days to about every 24 days, about every 21 days to about every 23 days, about every 21 days to about every 22 days, about every 22 days every 22 days to about every 28 days, about every 22 days to about every 27 days, about every 22 days to about every 26 days, about every 22 days to about every 25 days, about every 22 days to about every 24 days, about every 22 days every 23 days to about every 23 days, about every 23 days to about every 28 days, about every 23 days to about every 27 days, about every 23 days to about every 26 days, about every 23 days to about every 25 days, about every 23 days day to about every 24 days, about every 24 days to about every 28 days, about every 24 days to about every 27 days, about every 24 days to about every 26 days, about every 24 days to about every 25 days, about every 25 days day to about every 28 days, about every 25 days to about every 27 days, about every 25 days to about every 26 days, about every 26 days to about every 28 days, about every 26 days to about every 27 days, or about every 27 days to approximately every 28 days).

In some embodiments of these methods, the dosage of the pharmaceutical composition comprises about 0.5 x 10 ⁹ to about 5 x 10 9 , from about 0.5 x ^{10 9 to} about 4 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 3 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 2 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 1 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 0.9 x 10 ⁹ , from about 0.5 x 10 ⁹ to about 0.7 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 5 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 4 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 3 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 2 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 1 x 10 ⁹ , from about 0.7 x 10 ⁹ to about 0.9 x 10 ⁹ , from about 0.9 x 10 ⁹ to about 5 x 10 ⁹ , from about 0.9 x 10 ⁹ to about 4 x 10 ⁹ , from about 0.9 x 10 ⁹ to about 3 x 10 ⁹ , from about 0.9 x 10 ⁹ to about 2 x 10 ⁹ , from about 0.9 x 10 ⁹ to about 1 x 10 ⁹ , from about 1 x 10 ⁹ to about 5 x 10 ⁹ , from about 1 x 10 ⁹ to about 4 x 10 ⁹ , from about 1 x 10 ⁹ to about 3 x 10 ⁹ , from about 1 x 10 ⁹ to about 2 x 10 ⁹ , From about 2 x 10 ⁹ to about 5 x 10 ⁹ , from about 2 x 10 ⁹ to about 4 x 10 ⁹ , from about 2 x 10 ⁹ to about 3 x 10 ⁹ , from about 3 x 10 ⁹ to about 5 x 10 ^9. From about 3 x ¹⁰⁹ to about 4 x ¹⁰⁹ , from about 4 x ¹⁰⁹ to about 5 x ¹⁰⁹ human enucleated erythroid cells (eg, any of the exemplary human enucleated erythroid cells described herein). In some embodiments, the dosage of the pharmaceutical composition is administered at a frequency of about once every three weeks.

In some embodiments of these methods, the dosage of the pharmaceutical composition comprises about 1 x 10 ⁹ to about 1 x 10 ¹⁰ (eg, about 1.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 2 x 10 ⁹ to about 1 x 10 ¹⁰ , about 2.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 3 x 10 ⁹ to about 1 x 10 ¹⁰ , about 3.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 4 x 10 ⁹ to about 1 x 10 ¹⁰ , About 4.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 5.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 6 x 10 ⁹ to about 1 x 10 ¹⁰ , about 6.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 7 x 10 ⁹ to about 1 x 10 ¹⁰ , about 7.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 8 x 10 ⁹ to about 1 x 10 ¹⁰ , about 8.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 9 x 10 ⁹ to about 1 x 10 ¹⁰ , about 9.5 x 10 ⁹ to about 1 x 10 ¹⁰ , about 1.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 2 x 10 ⁹ to About 9.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 3 x 10 ⁹ to about 9.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 4 x 10 ⁹ to about 9.5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 5.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 6 x 10 ⁹ to about 9.5 x 10 ⁹ , about 6.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 7 x 10 ⁹ to about 9.5 x 10 ⁹ , about 7.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 8 x 10 ⁹ to about 9.5 x 10 ⁹ , About 8.5 x 10 ⁹ to about 9.5 x 10 ⁹ , about 9 x 10 ⁹ to about 9.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 9 x 10 ⁹ , about 2 x 10 ⁹ to about 9 x 10 ⁹ , about 2.5 x 10 ⁹ to about 9 x 10 ⁹ , about 3 x 10 ⁹ to about 9 x 10 ⁹ , about 3.5 x 10 ⁹ to about 9 x 10 ⁹ , about 4 x 10 ⁹ to about 9 x 10 ⁹ , about 4.5 x 10 ⁹ to about 9 x 10 ⁹ , about 5 x 10 ⁹ to about 9 x 10 ⁹ , about 5.5 x 10 ⁹ to about 9 x 10 ⁹ , about 6 x 10 ⁹ to about 9 x 10 ⁹ , about 6.5 x 10 ⁹ to About 9 x 10 ⁹ , about 7 x 10 ⁹ to about 9 x 10 ⁹ , about 7.5 x 10 ⁹ to about 9 x 10 ⁹ , about 8 x 10 ⁹ to about 9 x 10 ⁹ , about 8.5 x 10 ⁹ to about 9 x 10 ⁹ , about 9 x 10 ⁹ to about 9 x 10 ⁹ , about 1.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 2 x 10 ⁹ to about 8.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 3 x 10 ⁹ to about 8.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 4 x 10 ⁹ to about 8.5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 8.5 x 10 ⁹ , About 5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 5.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 6 x 10 ⁹ to about 8.5 x 10 ⁹ , about 6.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 7 x 10 ⁹ to about 8.5 x 10 ⁹ , about 7.5 x 10 ⁹ to about 8.5 x 10 ⁹ , about 8 x 10 ⁹ to about 8.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 8 x 10 ⁹ , about 2 x 10 ⁹ to about 8 x 10 ⁹ , about 2.5 x 10 ⁹ to about 8 x 10 ⁹ , about 3 x 10 ⁹ to about 8 x 10 ⁹ , about 3.5 x 10 ⁹ to about 8 x 10 ⁹ , about 4 x 10 ⁹ to About 8 x 10 ⁹ , about 4.5 x 10 ⁹ to about 8 x 10 ⁹ , about 5 x 10 ⁹ to about 8 x 10 ⁹ , about 5.5 x 10 ⁹ to about 8 x 10 ⁹ , about 6 x 10 ⁹ to about 8 x 10 ⁹ , about 6.5 x 10 ⁹ to about 8 x 10 ⁹ , about 7 x 10 ⁹ to about 8 x 10 ⁹ , about 7.5 x 10 ⁹ to about 8 x 10 ⁹ , about 1.5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 2 x 10 ⁹ to about 7.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 3 x 10 ⁹ to about 7.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 7.5 x 10 ⁹ , About 4 x 10 ⁹ to about 7.5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 5.5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 6 x 10 ⁹ to about 7.5 x 10 ⁹ , about 6.5 x 10 ⁹ to about 7.5 x 10 ⁹ , about 7 x 10 ⁹ to about 7.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 7 x 10 ⁹ , about 2 x 10 ⁹ to about 7 x 10 ⁹ , about 2.5 x 10 ⁹ to about 7 x 10 ⁹ , about 3 x 10 ⁹ to about 7 x 10 ⁹ , about 3.5 x 10 ⁹ to about 7 x 10 ⁹ , about 4 x 10 ⁹ to About 7 x 10 ⁹ , about 4.5 x 10 ⁹ to about 7 x 10 ⁹ , about 5 x 10 ⁹ to about 7 x 10 ⁹ , about 5.5 x 10 ⁹ to about 7 x 10 ⁹ , about 6 x 10 ⁹ to about 7 x 10 ⁹ , about 6.5 x 10 ⁹ to about 7 x 10 ⁹ , about 1.5 x 10 ⁹ to about 6.5 x 10 ⁹ , about 2 x 10 ⁹ to about 6.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 6.5 x 10 ⁹ , about 3 x 10 ⁹ to about 6.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 6.5 x 10 ⁹ , about 4 x 10 ⁹ to about 6.5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 6.5 x 10 ⁹ , About 5 x 10 ⁹ to about 6.5 x 10 ⁹ , about 5.5 x 10 ⁹ to about 6.5 x 10 ⁹ , about 6 x 10 ⁹ to about 6.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 6 x 10 ⁹ , about 2 x 10 ⁹ to about 6 x 10 ⁹ , about 2.5 x 10 ⁹ to about 6 x 10 ⁹ , about 3 x 10 ⁹ to about 6 x 10 ⁹ , about 3.5 x 10 ⁹ to about 6 x 10 ⁹ , about 4 x 10 ⁹ to about 6 x 10 ⁹ , about 4.5 x 10 ⁹ to about 6 x 10 ⁹ , about 5 x 10 ⁹ to about 6 x 10 ⁹ , about 5.5 x 10 ⁹ to about 6 x 10 ⁹ , about 1.5 x 10 ⁹ to About 5.5 x 10 ⁹ , about 2 x 10 ⁹ to about 5.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 5.5 x 10 ⁹ , about 3 x 10 ⁹ to about 5.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 5.5 x 10 ⁹ , about 4 x 10 ⁹ to about 5.5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 5.5 x 10 ⁹ , about 5 x 10 ⁹ to about 5.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 5 x 10 ⁹ , about 2 x 10 ⁹ to about 5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 5 x 10 ⁹ , about 3 x 10 ⁹ to about 5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 5 x 10 ⁹ , About 4 x 10 ⁹ to about 5 x 10 ⁹ , about 4.5 x 10 ⁹ to about 5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 4.5 x 10 ⁹ , about 2 x 10 ⁹ to about 4.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 4.5 x 10 ⁹ , about 3 x 10 ⁹ to about 4.5 x 10 ⁹ , about 3.5 x 10 ⁹ to about 4.5 x 10 ⁹ , about 4 x 10 ⁹ to about 4.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 4 x 10 ⁹ , about 2 x 10 ⁹ to about 4 x 10 ⁹ , about 2.5 x 10 ⁹ to about 4 x 10 ⁹ , about 3 x 10 ⁹ to about 4 x 10 ⁹ , about 3.5 x 10 ⁹ to About 4 x 10 ⁹ , about 1.5 x 10 ⁹ to about 3.5 x 10 ⁹ , about 2 x 10 ⁹ to about 3.5 x 10 ⁹ , about 2.5 x 10 ⁹ to about 3.5 x 10 ⁹ , about 3 x 10 ⁹ to about 3.5 x 10 ⁹ , about 1.5 x 10 ⁹ to about 3 x 10 ⁹ , about 2 x 10 ⁹ to about 3 x 10 ⁹ , about 2.5 x 10 ⁹ to about 3 x 10 ⁹ , about 1.5 x 10 ⁹ to about 2.5 x 10 ⁹ , about 2 x 10 ⁹ to about 2.5 x 10 ⁹ , or about 1.5 x 10 ⁹ to about 2 x 10 ⁹ ) human enucleated erythroid cells (eg, any of the exemplary human enucleated erythroid cells described herein). In some embodiments, the dosage of the pharmaceutical composition is administered at a frequency of about once every three weeks.

In some embodiments of these methods, the dosage of the pharmaceutical composition comprises about 5 x 10 ⁹ to about 3 x 10 ¹⁰ (eg, about 6 x 10 ⁹ to about 3 x 10 ¹⁰ , about 7 x 10 ⁹ to about 3 x 10 ¹⁰ , about 8 x 10 ⁹ to about 3 x 10 ¹⁰ , about 9 x 10 ⁹ to about 3 x 10 ¹⁰ , about 1 x 10 ¹⁰ to about 3 x 10 ¹⁰ , about 2 x 10 ¹⁰ to about 3 x 10 ¹⁰ , About 6 x 10 ⁹ to about 2 x 10 ¹⁰ , about 7 x 10 ⁹ to about 2 x 10 ¹⁰ , about 8 x 10 ⁹ to about 2 x 10 ¹⁰ , about 9 x 10 ⁹ to about 2 x 10 ¹⁰ , about 1 x 10 ¹⁰ to about 2 x 10 ¹⁰ , about 6 x 10 ⁹ to about 1 x 10 ¹⁰ , about 7 x 10 ⁹ to about 1 x 10 ¹⁰ , about 8 x 10 ⁹ to about 1 x 10 ¹⁰ , about 9 x 10 ⁹ to about 1 x 10 ¹⁰ , about 6 x 10 ⁹ to about 9 x 10 ⁹ , about 7 x 10 ⁹ to about 9 x 10 ⁹ , about 8 x 10 ⁹ to about 9 x 10 ⁹ , about 6 x 10 ⁹ to About 8 x 10 ⁹ , about 7 x 10 ⁹ to about 8 x 10 ⁹ , or about 6 x 10 ⁹ to about 7 x 10 ⁹ ) human enucleated erythroid cells (e.g., any of the exemplary human enucleated human cells described herein red blood cells). In some embodiments, the dosage of the pharmaceutical composition is administered at a frequency of about once every three weeks.

Some embodiments of any of the methods described herein further comprise administering to the human individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. Some embodiments of any of the methods described herein may further comprise combining different treatment modalities to treat each individual suffering from cancer. Such combination treatments or therapies can include, for example, surgery and radiation therapy, in addition to the compositions provided herein. In some embodiments, radiation therapy includes external beam radiation therapy or brachytherapy. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutics, Therapeutic antibodies, therapeutic vaccines, chimeric antigen receptor-T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a human subject substantially simultaneously with any of the compositions provided herein. In some embodiments, one or more additional therapeutic agents can be administered to the human subject before or after administration of any of the compositions described herein.

For example, in some embodiments, the one or more additional therapeutic agents include agents that block, reduce and/or inhibit EGFR and/or VEGFR. Non-limiting examples include bevacizumab, nilotinib, dasatinib, erlotinib, and gefitinib.

In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting either PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD -1 Agents that bind to PD-L2 (as non-limiting examples, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA , MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing, and/or inhibiting the activity of CTLA-4, and/or CTLA-4 and its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A) A reagent for combining one or more of them. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or trememe Tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55) , CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2, and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7). In some embodiments, the one or more additional therapeutic agents are selected from pembrolizumab and nivolumab.

In some embodiments, the human subject has previously received chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof (e.g., has previously received chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof prior to administration of pharmaceutical composition). For example, in some embodiments, the human subject has previously received external beam radiation therapy and/or brachytherapy. In some embodiments, the human subject has previously received a chemotherapeutic agent, such as standard platinum-based chemotherapy or mitomycin C-based chemotherapy. In some embodiments, the immunotherapy is selected from a targeted antibody, a therapeutic vaccine, or an immune checkpoint therapy (eg, any of the immune checkpoint therapies described herein).

Any of the pharmaceutical compositions described herein can be formulated as described in WO 2020/219909 (incorporated by reference herein).

Methods of administering human enucleated erythroid cells (e.g., reticulocytes) comprising (e.g., expressing) an exogenous agent (e.g., a polypeptide) are described, e.g., in WO2015/073587, WO2015/153102, and WO 2019/173798, Each of which is incorporated by reference in its entirety.

Some embodiments of any of the methods described herein further comprise assessing an immune response in the individual. Methods that can be used to assess an individual's immune response include, for example, ELISPOT assays (cellular immune responses), ICS (intracellular cytokine staining assays), and major histocompatibility complex (MHC) tetramer assays to detect Detection and quantification of antigen-specific T cells, antigen-specific CD4+ T cells or E7-antigen-specific CD8+ T cells. Some embodiments of any of the methods described herein may further comprise assessing an individual's innate immune response (e.g., NK cells or monocytes) and/or adaptive responses (e.g., CD4 ⁺ Th1 cells and E7-antigen-specific CD8 ⁺ T cells).

Some embodiments of the methods described herein further comprise assessing a population of immune cells in a biological sample obtained from the individual. In some embodiments, E7-specific CD8+ T cells are assessed in a biological sample from an individual. In some embodiments, E7-specific CD8 ⁺ T cell lines are assessed by a tetramer assay using peripheral whole blood obtained from an individual. Tetramer assays can include, for example, transgenic T cells for the E7 T cell receptor (TCR-T) incorporated into PBMC preparations and stained for tetramers and assessed by flow cytometry.

In some embodiments, E7-specific CD8 ⁺ T cell lines are assessed by ELISpot analysis of isolated live peripheral blood mononuclear cells (PBMCs) obtained from the individual.

In some embodiments, a biological sample from an individual is immunophenotyped. In some embodiments, immunophenotyping is performed using immunofluorescence from the tumor microenvironment in a biological sample from an individual. In some embodiments, the biological sample is a tumor slide sample. In some embodiments, immunophenotyping is performed using immunofluorescence analysis of the tumor microenvironment in paired tumor section samples from an individual.

In some embodiments, the tumor microenvironment in a biological sample from an individual is identified using Nanostring 10 plate analysis. In some embodiments, the biological sample is a tumor slide sample. In some embodiments, the tumor microenvironment in paired tumor sections from an individual is identified using Nanostring 10 plate analysis. set

Also provided herein are kits comprising any dosage of the pharmaceutical compositions provided herein, and instructions for performing any of the methods described herein. In some embodiments, the human enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide. In some embodiments, the human enucleated erythroid cells comprise from about 1,000 replicas to about 100,000 replicas, from about 1,000 replicas to about 80,000 replicas, from about 1,000 replicas to about 60,000 replicas, from about 1,000 replicas to about 50,000 replicas 1,000 to 40,000 copies, 1,000 to 30,000 copies, 1,000 to 20,000 copies, 1,000 to 10,000 copies, 1,000 to 5,000 copies 5,000 to 100,000 copies, 5,000 to 80,000 copies, 5,000 to 60,000 copies, 5,000 to 50,000 copies, 5,000 to 40,000 copies 5,000 to 30,000 copies, 5,000 to 20,000 copies, 5,000 to 10,000 copies, 10,000 to 100,000 copies, 10,000 to 80,000 copies 10,000 to 60,000 copies, 10,000 to 50,000 copies, 10,000 to 40,000 copies, 10,000 to 30,000 copies, 10,000 to 20,000 copies 20,000 to 100,000 copies, 20,000 to 80,000 copies, 20,000 to 60,000 copies, 20,000 to 50,000 copies, 20,000 to 40,000 copies 20,000 to 30,000 copies, 30,000 to 100,000 copies, 30,000 to 80,000 copies, 30,000 to 60,000 copies, 30,000 to 50,000 copies 30,000 to 40,000 copies, 40,000 to 100,000 copies, 40,000 to 80,000 copies, 40,000 to 60,000 copies, 40,000 to 50,000 copies 50,000 to 100,000 copies, 50,000 to 80,000 copies, 50,000 to 60,000 copies, 60,000 to 100,000 copies, 60,000 to 80,000 copies copies, or about 80,000 copies to about 100,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide.

In some embodiments, the human enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of an exogenous costimulatory polypeptide. In some embodiments, the human enucleated erythroid cells comprise from about 1,000 replicas to about 100,000 replicas, from about 1,000 replicas to about 80,000 replicas, from about 1,000 replicas to about 60,000 replicas, from about 1,000 replicas to about 50,000 replicas 1,000 to 40,000 copies, 1,000 to 30,000 copies, 1,000 to 20,000 copies, 1,000 to 10,000 copies, 1,000 to 5,000 copies 5,000 to 100,000 copies, 5,000 to 80,000 copies, 5,000 to 60,000 copies, 5,000 to 50,000 copies, 5,000 to 40,000 copies 5,000 to 30,000 copies, 5,000 to 20,000 copies, 5,000 to 10,000 copies, 10,000 to 100,000 copies, 10,000 to 80,000 copies 10,000 to 60,000 copies, 10,000 to 50,000 copies, 10,000 to 40,000 copies, 10,000 to 30,000 copies, 10,000 to 20,000 copies 20,000 to 100,000 copies, 20,000 to 80,000 copies, 20,000 to 60,000 copies, 20,000 to 50,000 copies, 20,000 to 40,000 copies 20,000 to 30,000 copies, 30,000 to 100,000 copies, 30,000 to 80,000 copies, 30,000 to 60,000 copies, 30,000 to 50,000 copies 30,000 to 40,000 copies, 40,000 to 100,000 copies, 40,000 to 80,000 copies, 40,000 to 60,000 copies, 40,000 to 50,000 copies 50,000 to 100,000 copies, 50,000 to 80,000 copies, 50,000 to 60,000 copies, 60,000 to 100,000 copies, 60,000 to 80,000 copies copies, or about 80,000 copies to about 100,000 copies of an exogenous costimulatory polypeptide.

In some embodiments, the human enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of an exogenous cytokine polypeptide. In some embodiments, the human enucleated erythroid cells comprise from about 1,000 replicas to about 100,000 replicas, from about 1,000 replicas to about 80,000 replicas, from about 1,000 replicas to about 60,000 replicas, from about 1,000 replicas to about 50,000 replicas 1,000 to 40,000 copies, 1,000 to 30,000 copies, 1,000 to 20,000 copies, 1,000 to 10,000 copies, 1,000 to 5,000 copies 5,000 to 100,000 copies, 5,000 to 80,000 copies, 5,000 to 60,000 copies, 5,000 to 50,000 copies, 5,000 to 40,000 copies 5,000 to 30,000 copies, 5,000 to 20,000 copies, 5,000 to 10,000 copies, 10,000 to 100,000 copies, 10,000 to 80,000 copies 10,000 to 60,000 copies, 10,000 to 50,000 copies, 10,000 to 40,000 copies, 10,000 to 30,000 copies, 10,000 to 20,000 copies 20,000 to 100,000 copies, 20,000 to 80,000 copies, 20,000 to 60,000 copies, 20,000 to 50,000 copies, 20,000 to 40,000 copies 20,000 to 30,000 copies, 30,000 to 100,000 copies, 30,000 to 80,000 copies, 30,000 to 60,000 copies, 30,000 to 50,000 copies 30,000 to 40,000 copies, 40,000 to 100,000 copies, 40,000 to 80,000 copies, 40,000 to 60,000 copies, 40,000 to 50,000 copies 50,000 to 100,000 copies, 50,000 to 80,000 copies, 50,000 to 60,000 copies, 60,000 to 100,000 copies, 60,000 to 80,000 copies copies, or about 80,000 copies to about 100,000 copies of the exogenous cytokine polypeptide.

Any dose of the pharmaceutical composition described herein may be formulated for administration to a human subject as described in WO 2020/219909 (incorporated by reference herein).

Also provided herein are kits comprising one or more sterile containers containing any of the compositions described herein (e.g., sterile conical tubes, sterile Petri dishes, sterile vials (e.g., borosilicate glass vials) and sterile plastic bags (di-2-ethylhexyl phthalate (DEHP)-plasticized polyvinyl chloride (PVC) bags, or n-butyryl-tris(n-hexyl)-citric acid (BTHC)-plasticized PVC bag). In some embodiments, any of the kits provided herein can further include instructions for intravenously administering any of the pharmaceutical compositions to a human subject in need thereof.

Some embodiments of the kits described herein include a suitable single dosage form of any of the pharmaceutical compositions described herein. For example, a single dosage form of any of the pharmaceutical compositions described herein can have a volume of, for example, about 0.5 mL to about 2 L, about 0.5 mL to about 1800 mL, about 0.5 mL to about 1500 mL, about 0.5 mL to about 1200 mL, about 0.5 mL to about 1000 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about 600 mL, about 0.5 mL to about 500 mL, about 0.5 mL to about 450 mL, about 0.5 mL to about 400 mL, about 0.5 mL to about 350 mL, about 0.5 mL to about 300 mL, about 0.5 mL to about 250 mL, about 0.5 mL to about 200 mL, about 0.5 mL to about 180 mL, about 0.5 mL to about 160 mL, about 0.5 mL to about 140 mL, about 0.5 mL to about 120 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 60 mL, about 0.5 mL to about 40 mL , about 0.5 mL to about 20 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 1.0 mL, about 1.0 mL to about 2 L, about 1.0 mL to about 1800 mL, about 1.0 mL to about 1500 mL, about 1.0 mL to about 1200 mL, about 1.0 mL to about 1000 mL, about 1.0 mL to about 800 mL, about 1.0 mL to about 600 mL, about 1.0 mL to about 500 mL, about 1.0 mL to about 450 mL, about 1.0 mL to about 400 mL, about 1.0 mL to about 350 mL, about 1.0 mL to about 300 mL, about 1.0 mL to about 250 mL, about 1.0 mL to about 200 mL, about 1.0 mL to about 180 mL, about 1.0 mL to about 160 mL, about 1.0 mL to about 140 mL, about 1.0 mL to about 120 mL, about 1.0 mL to about 100 mL, about 1.0 mL to about 80 mL, about 1.0 mL to about 60 mL , about 1.0 mL to about 40 mL, about 1.0 mL to about 20 mL, about 1.0 mL to about 10 mL, about 1.0 mL to about 5 mL, about 5 mL to about 2 L, about 5 mL to about 1800 mL, about 5 mL to about 1500 mL, about 5 mL to about 1200 mL, about 5 mL to about 1000 mL, about 5 mL to about 800 mL, about 5 mL to about 600 mL, about 5 mL to about 1800 mL, about 5 mL to about 500 mL, about 5 mL to about 450 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL to about 180 mL, about 5 mL to about 160 mL, about 5 mL to about 140 mL, about 5 mL to about 120 mL, about 5 mL to about 100 mL, about 5 mL to about 80 mL , about 5 mL to about 60 mL, about 5 mL to about 40 mL, about 5 mL to about 20 mL, about 5 mL to about 10 mL, about 10 mL to about 2 L, about 10 mL to about 1800 mL, about 10 mL to about 1500 mL, about 10 mL to about 1200 mL, about 10 mL to about 1000 mL, about 10 mL to about 800 mL, about 10 mL to about 600 mL, about 10 mL to about 500 mL, about 10 mL to about 450 mL, about 10 mL to about 400 mL, about 10 mL to about 350 mL, about 10 mL to about 300 mL, about 10 mL to about 250 mL, about 10 mL to about 200 mL, about 10 mL to about 180 mL, about 10 mL to about 160 mL, about 10 mL to about 140 mL, about 10 mL to about 120 mL, about 10 mL to about 100 mL, about 10 mL to about 80 mL, about 10 mL to about 60 mL , about 10 mL to about 40 mL, about 10 mL to about 20 mL, about 20 mL to about 2 L, about 20 mL to about 1800 mL, about 20 mL to about 1500 mL, about 20 mL to about 1200 mL, about 20 mL to about 1000 mL, about 20 mL to about 800 mL, about 20 mL to about 600 mL, about 20 mL to about 500 mL, about 20 mL to about 450 mL, about 20 mL to about 400 mL, about 20 mL to about 350 mL, about 20 mL to about 300 mL, about 20 mL to about 250 mL, about 20 mL to about 200 mL, about 20 mL to about 180 mL, about 20 mL to about 160 mL, about 20 mL to about 140 mL, about 20 mL to about 120 mL, about 20 mL to about 100 mL, about 20 mL to about 80 mL, about 20 mL to about 60 mL, about 20 mL to about 40 mL, about 40 mL to about 2 L , about 40 mL to about 1800 mL, about 40 mL to about 1500 mL, about 40 mL to about 1200 mL, about 40 mL to about 1000 mL, about 40 mL to about 800 mL, about 40 mL to about 600 mL, about 40 mL to about 500 mL, about 40 mL to about 450 mL, about 40 mL to about 400 mL, about 40 mL to about 350 mL, about 40 mL to about 300 mL, about 40 mL to about 250 mL, about 40 mL to about 200 mL, about 40 mL to about 180 mL, about 40 mL to about 160 mL, about 40 mL to about 140 mL, about 40 mL to about 120 mL, about 40 mL to about 100 mL, about 40 mL to about 80 mL, about 40 mL to about 60 mL, about 60 mL to about 2 L, about 60 mL to about 1800 mL, about 60 mL to about 1500 mL, about 60 mL to about 1200 mL, about 60 mL to about 1000 mL , about 60 mL to about 800 mL, about 60 mL to about 600 mL, about 60 mL to about 500 mL, about 60 mL to about 450 mL, about 60 mL to about 400 mL, about 60 mL to about 350 mL, about 60 mL to about 300 mL, about 60 mL to about 250 mL, about 60 mL to about 200 mL, about 60 mL to about 180 mL, about 60 mL to about 160 mL, about 60 mL to about 140 mL, about 60 mL to about 120 mL, about 60 mL to about 100 mL, about 60 mL to about 80 mL, about 80 mL to about 2 L, about 80 mL to about 1800 mL, about 80 mL to about 1500 mL, about 80 mL to about 1200 mL, about 80 mL to about 1000 mL, about 80 mL to about 800 mL, about 80 mL to about 600 mL, about 80 mL to about 500 mL, about 80 mL to about 450 mL, about 80 mL to about 400 mL , about 80 mL to about 350 mL, about 80 mL to about 300 mL, about 80 mL to about 250 mL, about 80 mL to about 200 mL, about 80 mL to about 180 mL, about 80 mL to about 160 mL, about 80 mL to about 140 mL, about 80 mL to about 120 mL, about 80 mL to about 100 mL, about 100 mL to about 2 L, about 100 mL to about 1800 mL, about 100 mL to about 1500 mL, about 100 mL to about 1200 mL, about 100 mL to about 1000 mL, about 100 mL to about 800 mL, about 100 mL to about 600 mL, about 100 mL to about 500 mL, about 100 mL to about 450 mL, about 100 mL to about 400 mL, about 100 mL to about 350 mL, about 100 mL to about 300 mL, about 100 mL to about 250 mL, about 100 mL to about 200 mL, about 100 mL to about 180 mL, about 100 mL to about 160 mL , about 100 mL to about 140 mL, about 100 mL to about 120 mL, about 120 mL to about 2 L, about 120 mL to about 1800 mL, about 120 mL to about 1500 mL, about 120 mL to about 1200 mL, about 120 mL to about 1000 mL, about 120 mL to about 800 mL, about 120 mL to about 600 mL, about 120 mL to about 500 mL, about 120 mL to about 450 mL, about 120 mL to about 400 mL, about 120 mL to about 350 mL, about 120 mL to about 300 mL, about 120 mL to about 250 mL, about 120 mL to about 200 mL, about 120 mL to about 180 mL, about 120 mL to about 160 mL, about 120 mL to about 140 mL, about 140 mL to about 2 L, about 140 mL to about 1800 mL, about 140 mL to about 1500 mL, about 140 mL to about 1200 mL, about 140 mL to about 1000 mL, about 140 mL to about 800 mL , about 140 mL to about 600 mL, about 140 mL to about 500 mL, about 140 mL to about 450 mL, about 140 mL to about 400 mL, about 140 mL to about 350 mL, about 140 mL to about 300 mL, about 140 mL to about 250 mL, about 140 mL to about 200 mL, about 140 mL to about 180 mL, about 140 mL to about 160 mL, about 160 mL to about 500 mL, about 160 mL to about 450 mL, about 160 mL to about 400 mL, about 160 mL to about 350 mL, about 160 mL to about 300 mL, about 160 mL to about 250 mL, about 160 mL to about 200 mL, about 160 mL to about 180 mL, about 180 mL to about 2 L, about 180 mL to about 1800 mL, about 180 mL to about 1500 mL, about 180 mL to about 1200 mL, about 180 mL to about 1000 mL, about 180 mL to about 800 mL, about 180 mL to about 600 mL , about 180 mL to about 500 mL, about 180 mL to about 450 mL, about 180 mL to about 400 mL, about 180 mL to about 350 mL, about 180 mL to about 300 mL, about 180 mL to about 250 mL, about 180 mL to about 200 mL, about 200 mL to about 2 L, about 200 mL to about 1800 mL, about 200 mL to about 1500 mL, about 200 mL to about 1200 mL, about 200 mL to about 1000 mL, about 200 mL to about 800 mL, about 200 mL to about 600 mL, about 200 mL to about 500 mL, about 200 mL to about 450 mL, about 200 mL to about 400 mL, about 200 mL to about 350 mL, about 200 mL to about 300 mL, about 200 mL to about 250 mL, about 250 mL to about 2 L, about 250 mL to about 1800 mL, about 250 mL to about 1500 mL, about 250 mL to about 1200 mL, about 250 mL to about 1000 mL , about 250 mL to about 800 mL, about 250 mL to about 600 mL, about 250 mL to about 500 mL, about 250 mL to about 450 mL, about 250 mL to about 400 mL, about 250 mL to about 350 mL, about 250 mL to about 300 mL, about 300 mL to about 2 L, about 300 mL to about 1800 mL, about 300 mL to about 1500 mL, about 300 mL to about 1200 mL, about 300 mL to about 1000 mL, about 300 mL to about 800 mL, about 300 mL to about 600 mL, about 300 mL to about 500 mL, about 300 mL to about 450 mL, about 300 mL to about 400 mL, about 300 mL to about 350 mL, about 350 mL to about 2 L, about 350 mL to about 1800 mL, about 350 mL to about 1500 mL, about 350 mL to about 1200 mL, about 350 mL to about 1000 mL, about 350 mL to about 800 mL, about 350 mL to about 600 mL , about 350 mL to about 500 mL, about 350 mL to about 450 mL, about 350 mL to about 400 mL, about 400 mL to about 2 L, about 400 mL to about 1800 mL, about 400 mL to about 1500 mL, about 400 mL to about 1200 mL, about 400 mL to about 1000 mL, about 400 mL to about 800 mL, about 400 mL to about 600 mL, about 400 mL to about 500 mL, about 400 mL to about 450 mL, about 450 mL to about 2 L, about 450 mL to about 1800 mL, about 450 mL to about 1500 mL, about 450 mL to about 1200 mL, about 450 mL to about 1000 mL, about 450 mL to about 800 mL, about 450 mL to about 600 mL, about 450 mL to about 500 mL, about 500 mL to about 2 L, about 500 mL to about 1800 mL, about 500 mL to about 1500 mL, about 500 mL to about 1200 mL, about 500 mL to about 1000 mL , about 500 mL to about 800 mL, about 500 mL to about 600 mL, about 600 mL to about 2 L, about 600 mL to about 1800 mL, about 600 mL to about 1500 mL, about 600 mL to about 1200 mL, about 600 mL to about 1000 mL, about 600 mL to about 800 mL, about 800 mL to about 2 L, about 800 mL to about 1800 mL, about 800 mL to about 1500 mL, about 800 mL to about 1200 mL, about 800 mL to about 1000 mL, about 1000 mL to about 2 L, about 1000 mL to about 1800 mL, about 1000 mL to about 1500 mL, about 1000 mL to about 1200 mL, about 1200 mL to about 2 L, about 1200 mL to about 1800 mL, about 1200 mL to about 1500 mL, about 1500 mL to about 2 L, about 1500 mL to about 1800 mL, or about 1800 mL to about 2 L. EXAMPLES Example 1. Generation of different versions of HLA-A2 (HPV E7 ) expressed on RCT .

DNA constructs (HLA-A2 wild type, HLA-A2 Y84A, and HLA-A2 Y84C+L2C) using three different versions of HLA-A2 were expressed as described in Figure 1A and HPV P1-2 (HPV16 EE7 11-19). HPV P1 (HPV16 EE7 11-20). The sequences of these constructs are described below. See, eg, Hansen et al., 2010, Trends Immunol. 31(10): 363–369. HLA-A2v1 (wild type): * (SEQ ID NO: 51) HLA-A2v2 (Y84A): * (SEQ ID NO: 52) HLA-A2v3 (Y84C and L2C): * (SEQ ID NO: 53) These 3個版本的HLA-A2用於表現單鏈變體HPV P1-2-HLA-A2-GPA： MSRSVALAVLALLSLSGLEAYMLDLQPETGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIGSGSGSGSEDGSGSGSGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 54)製造慢病毒載體

Construct the gene of HPV P1-HLA-A2-GPA fusion protein and 4-1BBL. The genes encoding these proteins were cloned into the multiple colonization site of the lentiviral vector pCDH, which has the MSCV promoter sequence (obtained from System Biosciences), allowing genes for both exogenous proteins to be included in one vector. Lentiviruses were produced in 293T cells by transfecting them with pPACKH1 (System Biosciences) and pCDH lentiviral vectors containing HPV P1-HLA-A2-GPA and 4-1BBL genes. Alternatively, separate vectors containing the genes for HPV P1-HLA-A2-GPA and 4-1BBL, respectively, can be used. Place cells in fresh medium. Forty-eight hours after the exchange, the virus supernatant was collected by centrifugation at 1,500 rpm for 5 minutes. Collect the supernatant and freeze in aliquots at -80 °C. transduced erythroid precursor cells

Expansion and differentiation of erythroid cells were performed according to Example 5 using the lentiviral vectors described above. Erythroid precursor cells were transduced during step 1 of the culture procedure. Erythrocytes were combined with lentiviral supernatant and polybrene in culture medium. Infection was achieved by spin-inoculation, spinning the plates at 2000 rpm for 90 minutes at room temperature. After spin-seeding, cells were incubated overnight at 37°C. antibody binding

The expression of HPV P1-HLA-A2-GPA fusions in engineered erythroid cells was verified using binding to APC-labeled or PE-labeled anti-β2 microglobulin antibodies as described in Example 5. As also described in Example 5, the binding of APC-labeled or PE-labeled anti-MHCI antibodies was used to verify the expression of MHCI antigen-presenting polypeptides in engineered erythroid cells. Binding of APC-labeled or PE-labeled anti-4-1BBL antibody (R&D Systems) was used to verify the expression of 4-1BBL in engineered erythroid cells. Binding of either antibody was measured by flow cytometry for APC fluorescence or PE fluorescence. Circles were set based on stained non-transduced cells. Example 2. In Vitro Activity of HLA-A2 (HPV E7) Stimulated HPV16- Specific T Cells Expressed on RCT

Human RCTs expressing 4-1BBL, HLA-A2 HPV PI, or both 4-1BBL and HLA-A2 HPV PI were prepared essentially as described in Example 18. The ability of these RCTs co-expressed with HPV P1-HLA-A2-GPA fusion protein and 4-1BBL co-stimulatory polypeptide to stimulate initial activation and proliferation of primary CD8+ T cells was tested essentially as described in Example 6. T2 cells pulsed with CMV or HPV peptides served as positive controls.

Primary human CD8+ cells were purchased from Astarte Biologics. Wells containing 4E4 or 2E4 CD8+ T cells were seeded with varying amounts of human RCT in cRPMI medium at RCT:T cell ratios of 10:1, 2:1, and 0.4:1 (from left to right, for each 1 RCT, shown in Figures 1B and 1C ), and cultured at 37°C for two days.

Cellular levels of Nur77 and IFN-γ were measured using intracellular staining. Briefly, cells were fixed with 200 µL of IC fixation buffer and incubated for 20 to 60 minutes at room temperature in the dark. Samples were centrifuged at 400 to 600 x g for 5 min at room temperature. Discard the supernatant and add 200 µL of permeabilization buffer. Samples were centrifuged at 400 to 600 x g for 5 min at room temperature. Discard the supernatant and resuspend the pellet in approximately 100 µL of 1X Permeabilization Buffer. Without washing, directly conjugated antibodies (anti-Nur77 PE and anti-IFN-γ APC) were added, followed by incubation at room temperature for at least 30 minutes in the dark. Nur77 and IFN-γ were analyzed 2, 6 and 24 hours after RCT cultured with primary CD8+ T cells.

Flow cytometry analysis showed that all tested versions of HLA-A2 (HPV E7) were well represented on the surface of human RCT ( data not shown ). Furthermore, all three versions resulted in the activation of HPV E7-specific T cells as determined by Nur77 expression ( Fig. IB ) and IFNγ secretion ( Fig. 1C ). These results demonstrate the ability to efficiently and selectively activate antigen-specific T cells, such as HPV-specific T cells, using engineered enucleated cells as described herein. Example 3. Generation of Erythroid Cells Presenting MHCI ( Ovalbumin ) , 4-1BBL and IL-12 (OVA-H2Kb+4-1BBL+IL12)

Modifications using click methodology (click chemistry for functionalizing erythroid cells is described in International Application No. PCT/US2018/000042, which asserts U.S. Provisional Application No. 62/460589 filed February 17, 2017 and July 8, 2017 No. 62/542142, the priority of U.S. Provisional Application No. 62/542142, the entire content of which is incorporated herein by reference), combines murine erythrocytes with MHCI presenting ovalbumin peptide (OVA-H2Kb), 4-1BBL, and IL-12. Each joins. Triple click cells were made with the following reagents: Fc-mIL12Fc (labeled with TCO-PEG4-NHS ester); m4-1BBL (DBCO sulfur linker) and OVA-H2Kb (DBCO sulfur linker). Briefly, the reaction was performed as follows: OVA-4-1BBL-IL12 (MTZ-TCO)

Mouse peripheral blood was filtered through a PAL de-leukocyte filter, 1 x 10 ⁸ mRCT with 0.04 mM of 6' azido-NHS-ester and 0.02 mM of methyltetrazine-PEG-NHS-ester (MTZ), in The labeling reaction was carried out in PBS pH 9 for 30 minutes at room temperature. 5 ul of 20 μM Fc-mIL12Fc-TCO-PEG4-NHS ester and 5 ul of 50 μM OVA-H2Kb-DBCO-sulfur linker were added and incubated for 1 hour at room temperature. 5 ul of 50 μM m4-1BBL-DBCO-sulfur linker was added to the cells, and the mixture was incubated overnight at 4°C. OVA-4-1BBL

Mouse peripheral blood was filtered through a PAL de-leukocyte filter, 1 x 10 ⁸ mRCT with 0.04 mM of 6' azido-NHS-ester and 0.02 mM of methyltetrazine-PEG-NHS-ester (MTZ), Labeling reactions were carried out in PBS pH 9 for 30 minutes at room temperature. Azido-labeled mRCT was incubated with 5 ul of 50 μM m4-1BBL-DBCO-sulfur linker and 5 ul of 50 μM OVA-H2Kb-DBCO-sulfur linker, and the mixture was incubated overnight at 4°C.

The copy numbers of IL12, OVA, and 4-1BBL were determined in OVA-4-1BBL-IL12 triple-clicked mRCTs and OVA-4-1BBL double-clicked mRCTs. (See Table 4 below). Table 4. Number of replicas group Number of IL12 Replicas# Number of OVA Copies# 4-1BBL Replica Number# OVA-4-1BBL-IL12 90,277 12,451 72,579 OVA-4-1BBL -- 26,397 105,807 Example 4. Generation and testing of RTX-321

RTX-321 was developed for the treatment of patients with HPV16 ⁺ cancer. RTX-321 comprises allogeneic, cultured human enucleated erythrocytes engineered to express a single chain comprising a fusion of HLA-A*02:01 and β2M with the HPV16 E7 _11–19 peptide (HLA-A2-HPV) Proteins, proteins comprising 4-1BBL, and proteins comprising IL-12 are on the cell surface.

The sequence of the mature single-chain protein comprising HLA-A*02:01, β2M, and HPV16 E7 _11–19 peptide (HLA-A2-HPV) is shown below (with individual domains indicated): HPVE7(11-19)/B2M/ HLA-A2 YMLDLQPETGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPI連接子GSGSGSGSEDGSGSGSGS人類血型糖蛋白 A LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 15)

The nucleic acid sequence encoding the mature single-chain protein comprising HLA-A*02:01, β2M, and HPV16 E7 _11-19 peptide is shown below (each encoded domain is shown): HPVE7(11-19)/B2M/HLA- A2 Linker GGCAGCGGCTCTGGCTCCGGCTCCGAAGATGGCAGCGGCAGCGGAAGTGGTTCA Human Glycophorin A TTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGT TAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGA AATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 16)

The protein sequence comprising 4-1BBL is shown below (domains indicated): 4-1BBL ACPWAVSGARASPPGSAASPRLREGPELSPDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQG RLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Linker V17 GGSGGSGGGPEDEPGSGSGGGSGGGS Human Glycophorin A LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRR LIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 21)

The nucleic acid sequence encoding the protein comprising 4-1BBL is shown below (each encoded domain is indicated): 4-1BBLGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGT CCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCC GCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA Linker V17GGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCC human blood glycoproteinTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGGTTACATTCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCAT TATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 57)

The sequence of the protein comprising IL-12 is shown below (each domain is indicated): Humanity SMIM1MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK LinkerGGGGSGGGGSGGGGS IL-12(p40)IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESL PIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS LinkerGGGGSGGGGSGGGGS IL-12(p35)RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPFYKTKIKLCILLHAFRIRAVTIDRVMSYL NAS (SEQ ID NO: 48)

編碼包含IL-12的蛋白質的核酸序列如下所示(標明各經編碼的結構域)：人類 SMIM1 ATGCAACCGCAAGAGAGTCACGTACATTATTCAAGATGGGAAGATGGAAGTCGGGACGGTGTGTCTCTCGGCGCTGTTAGTTCAACGGAGGAAGCGTCTCGCTGTCGCCGGATAAGTCAACGCCTTTGTACGGGAAAACTGGGTATAGCTATGAAGGTCCTCGGCGGGGTGGCGTTGTTTTGGATTATCTTTATACTTGGGTATCTGACCGGTTACTATGTTCACAAGTGTAAA連接子GGAGGTGGAGGATCAGGTGGAGGTGGTTCAGGTGGAGGAGGTAGC IL-12(p40) ATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGCGACACACCTGAGGAGGATGGCATCACCTGGACACTGGATCAGAGCAGCGAGGTGCTGGGCTCCGGCAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCCGGCCAGTACACATGTCACAAGGGAGGAGAGGTGCTGAGCCACTCCCTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGTCTACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAACAAGACCTTCCTGCGGTGCGAGGCCAAGAATTATAGCGGCCGGTTCACCTGTTGGTGGCTGACCACAATCTCTACCGACCTGACCTTCAGCGTGAAGTCTAGCCGGGGCTCCTCTGACCCTCAGGGAGTGACATGCGGAGCAGCCACCCTGTCCGCCGAGCGGGTGAGAGGCGATAACAAGGAGTACGAGTATAGCGTGGAGTGCCAGGAGGACTCCGCCTGTCCAGCAGCAGAGGAGAGCCTGCCAATCGAAGTGATGGTGGATGCCGTGCACAAGCTGAAGTACGAGAATTATACAAGCTCCTTCTTTATCAGGGACATCATCAAGCCCGATCCCCCTAAGAACCTGCAGCTGAAGCCCCTGAAGAACAGCCGGCAGGTGGAGGTGTCTTGGGAGTACCCCGACACCTGGAGCACACCTCACTCCTATTTCTCTCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAAGGACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGTCGGAAGAACGCCTCTATCAGCGTGCGGGCCCAGGATAGATACTATTCTAGCTCCTGGAGCGAGTGGGCCTCCGTGCCATGTTCT連接子GGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCAGC IL-12(p35) CGGAATCTGCCAGTGGCAACCCCAGACCCCGGAATGTTCCCATGCCTGCACCACTCTCAGAACCTGCTGAGGGCCGTGAGCAATATGCTGCAGAAGGCCCGCCAGACACTGGAGTTTTACCCTTGTACCAGCGAGGAGATCGACCACGAGGACATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGCCTGCCACTGGAGCTGACCAAGAACGAGAGCTGTCTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCAGCTGCCTGGCCTCCAGAAAGACATCTTTTATGATGGCCCTGTGCCTGTCTAGCATCTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCAAGAGGCAGATTTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTTAATTCCGAGACAGTGCCTCAGAAGTCCTCTCTGGAGGAGCCAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAATGCCTCC (SEQ ID NO: 49)

Each gene encoding a protein was cloned into multiple selection sites of the lentiviral vector under the control of the murine stem cell virus promoter sequence. Lentivirus was produced in Lenti-X 293T cells, and the titer was determined in K562 cells. Human CD34+ hematopoietic precursor cells were derived from normal human donors. Human CD34+ hematopoietic precursor cells were initially cultured in Stem Cell Growth Medium (CellGenix® Inc., NH, USA) supplemented with StemSpan™ CC100 (STEMCELL Technologies, Inc., BC, Canada). Subsequently, the cells were cultured in Iscove's modified Dulbecco medium (IMDM, Thermo Fisher Scientific), which included 10 µg/mL recombinant human insulin (Sigma-Aldrich®, Inc., MO, USA), 200 µg/mL saturated human Transferrin (BBI Solutions, ME, USA), 1 ng/mL recombinant human IL-3 (PeproTech, Inc., NJ, USA), 100 ng/mL stem cell factor (PeproTech, Inc., NJ, USA), and 50 ng/mL erythropoietin (PeproTech, Inc.). Cells were then cultured in IMDM containing 10 µg/mL recombinant human insulin, 200 µg/mL saturated human transferrin, 10 ng/mL stem cell factor, and 16.7 ng/mL erythropoietin. Finally, cells were cultured in IMDM containing 10 µg/mL recombinant human insulin, 1 mg/mL saturated human transferrin, and 50 ng/mL erythropoietin. _Incubate the cells at 37 °C, 5% CO. Erythroid precursor cells are transduced with lentiviral vectors to express the gene of interest.

The following antibodies were used to measure expression on the surface of engineered RBCs: anti-human β2M (1:50, 2M2, BioLegend), anti-human 4-1BBL (1:100, 5F4, BioLegend), and anti-human IL- 12 p40/p70 (1:50, C11.5, BD Biosciences, CA, USA). Cells were washed and analyzed on a NovoCyte® 3000 flow cytometer (Acea Biosciences™, Inc., CA, USA). Flow cytometry data were analyzed using FlowJo ^™ software (BD Biosciences). Flow cytometry data confirmed a single-chain protein comprising a fusion of HLA-A*02:01 and β2M with HPV16 E7 _11–19 peptide (HLA-A2-HPV), a protein comprising 4-1BBL, and a protein comprising IL- Expression of 12 proteins on the surface of RTX-321 (FIG. 2A).

CD8 ⁺ T cells (Cellero™, MA, USA) from a human HLA-A*02:01 positive donor, collected with Advarra IRB approval and informed consent, were lined with Dynabeads human T-activator CD3/CD28 beads ( Themo Fisher Scientific) were stimulated at a ratio of 1:1 in X-VIVO™15 medium (PeproTech, NJ, USA) containing 100 IU/mL human IL-2. The next day, the beads were removed and CD8 ⁺ T cells transduced with lentivirus encoding the HPV E711-19-specific TCR were activated in X-VIVO ¹⁵ medium containing 100 IU/mL human IL-2. Medium was changed on days 1, 4 and 8 after transduction. On day 10, 8×10 ⁴ non-transduced T cells or TCR-modified primary CD8 ⁺ T cells with about 20% TCR expression were mixed with 3.2×10 ⁵ RTX-321 or control RBC in 200 μL Cultured together in X-VIVO 15 medium (without human IL-2). Cells were harvested at 2 hours, surface stained, fixed, and permeabilized using the Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific), and stained for Nur77 expression. On day 1, cells were treated with brefeldin A (Bio-Legend) for 4 hours before harvesting and stained for CD69 and granzyme B expression using a similar protocol. On day 5, cells were collected for flow cytometric analysis of HPV E7 _11-19 tetramer ⁺ (1:10, MBL International Corporation) CD8 ⁺ T cell numbers and live CD8 ⁺ T cell phenotype. The following antibodies were used after exclusion of dead cells via live/dead fixed aqueous dead cell staining: anti-CD8 (1:200, RPA-T8, Bio-Legend), anti-mouse TCRβ (1:100, H57-597, Bio-Legend ), anti-Nur77 (1:100, 12.14, Thermo Fisher Scientific), anti-CD69 (1:200, FN50, BioLegend), anti-4-1BB (1:200, 4B4-1, BioLegend), anti-CCR7 (1:200, G043H7, BioLegend), anti-CD45RO (1;200, UCHL1, BioLegend), and anti-Granzyme B (1:200, QA16A02, BioLegend). Flow cytometry data were collected using BD FACSDiva v8.0.2 on a BD LSRFortessa flow cytometer and analyzed using FlowJo 10.5.3 software. After 5 days, cell culture supernatants were collected and secreted IFNγ was measured using the LEGENDplex Human CD8/NK Assay Panel (13-plex) multiplex assay (BioLegend). The assay was performed according to the manufacturer's instructions.

To confirm that RTX-321 can engage and activate HPV16 E7 _11–19 -specific T cells, CD8 ⁺ T cells from HLA-A*02:01 donors were lentivirally transduced to express HPV E7-specific TCR29 (E7-TCR cells) (FIG. 2B) and co-cultured with RTX-321 or engineered RBCs expressing components of RTX-321. Furthermore, we assessed antigen specificity by testing RCT-aAPCs presenting the cytomegalovirus (CMV) peptide but not the HPV 16 E7 _11-19 peptide (RCT-CMV-4-1BBL-IL-12).

Co-incubation with RTX-321 for 2 or 24 hours significantly increased the expression of Nur77 and CD69 on E7-TCR cells, respectively, indicating efficient TCR engagement (Fig. 2C and 2D). TCR signaling was independent of expression of 4-1BBL and IL-12, as only engineered RBCs expressing HLA-A2-HPV were able to induce these activation markers (Figures 2C and 2D), as well as 4-1BB (Figure 2E). Although HLA-A2-HPV alone is sufficient for T cell activation, HLA-A2-HPV, 4-1BBL-containing proteins, and IL-12-containing proteins are still required for T cell expansion (Fig. 2F), which is accompanied by Induction of granzyme B and IFNγ secretion (Fig. 2G). Co-culture with RTX-321, but not RCT-CMV-4-1BBL-IL-12, resulted in expansion of E7-TCR cells, demonstrating that expansion is antigen-specifically driven. CD8 ⁺ T cells expanded in response to RTX-321 consisted of T _CM and _TEM cells (Fig. 2H). The increase of T _CM cells is mainly dependent on HLA-A2-HPV and proteins comprising 4-1BBL, while the increase of _TEM cells by each of HLA-A2-HPV, proteins comprising 4-1BBL and proteins comprising IL-12 All contribute. These data demonstrate that RTX-321 promotes the expansion of primary CD8 ⁺ antigen-specific T cells, induces production of effector molecules, and increases T cell activation and generation of _TEM and _TCM phenotypes in an antigen-specific manner. Example 5. Evaluation of the safety and tolerability of RTX-321

Engineered to express HPV 16 oncoprotein E7 peptide 11-19 and β2 microglobulin (β2M; HLA-A2-HPV), 4-1BB ligand (4-1BBL; tumor necrosis factor superfamily member 9), and a fusion protein of interleukin 12 (IL-12) p40 and p35 subunits on the cell surface of human Enucleated erythroid cells were produced as described in Example 4 (RTX-321).

The safety and tolerability of RTX-321 in patients with HLA-A*02:01-positive solid tumors associated with the HPV 16 virus is currently being evaluated in an open-label, multicenter, multiple ascending-dose, first-in-class trial of RTX-321 In humans, Phase 1 trial in patients with persistent, recurrent or metastatic, unresectable HPV 16+ cancer. In addition, optimal doses and dosing regimens for RTX-321 monotherapy were also determined. The trial will enroll approximately 63 patients.

This test consists of 2 parts: Approximately 18 patients will be enrolled in Part 1 of the trial. Part 1 will determine the Phase 2 recommended dose (RP2D) and characterize the preliminary safety, tolerability, PK, PD and antitumor activity of RTX-321 monotherapy. Part 2 will target cervical cancer (expansion cohort 1), HNSCC (expansion cohort 2), and anal cancer (expansion cohort 3), enrolling approximately 15 patients in each expansion cohort. Part 2 will further characterize the safety, tolerability, PK, PD and preliminary antitumor activity of RTX-321 monotherapy under RP2D for these indications.

Both parts of the trial will include pre-screening, screening, treatment and long-term follow-up periods. The experimental design is depicted in Figure 3. pre-screening

All patients must be confirmed to be HLA-A*02:01 positive before entering the screening period. For patients with cervical cancer or HNSCC, confirmation of HPV 16+ tumors is required in addition to positive HLA-A*02:01 results. The pre-screening period begins with the signing of an individual informed consent form (ICF) specific to pre-screening and ends upon completion of the assessments required for pre-screening. A flowchart of the pre-screening visit is provided in Figure 3.

Both HLA and HPV 16+ tests will be performed at the central laboratory. For the HPV 16 assay, archival formalin-fixed, paraffin-embedded (FFPE) slides must be submitted from all cervical cancer patients who have not documented HPV 16+ tumors or HNSCC using an FDA-approved test. A "Research Use Only" assay at the central laboratory will be used to determine HPV 16 status in patient samples and to determine eligibility. Use of the HPV 16 test to determine the use of RTX-321 therapy is also under investigation and is therefore part of the investigational program. Complete instructions for collection, processing, handling and shipping of all samples can be found in the testing laboratory manual.

Patients confirmed to be HLA-A*02:01 positive at the time of pre-screening and have a previous history of HPV 16+ tumors using an FDA-approved test can enter the screening period without additional HPV 16 testing. HPV 16 testing allows retrospective testing of these patients.

Since anal cancer is presumed to be HPV 16 positive (>80% of patients are), prescreening testing is not required during prescreening for this indication. Anal cancer patients confirmed to be HLA-A*02:01 positive during pre-screening can enter the screening phase without additional HPV 16 testing. HPV 16 testing will be done retrospectively in patients with anal cancer. screening period

After confirming HLA-A*02:01 positive status and providing HPV 16+ tumor records (cervical cancer and HNSCC only), patients will sign the main trial ICF to enter the screening period. The screening period begins with the first screening assessment and lasts up to 28 days. Screening assessments are provided in the assessment schedule (Figures 4A-4B). Patients who completed the screening assessment and met the patient inclusion criteria (described below) were considered eligible for inclusion.

Screening failure occurs when a patient does not meet the eligibility requirements for the specified time frame of the Screening Period. Patients can be re-screened if the reason for the screening failure has been resolved and an appropriate inclusion interval can be provided. The screening interval may be extended by up to 7 days at the discretion of the trial sponsor's medical monitor before any re-screening assessment is required, except for any assessment that resulted in a screening failure. treatment period

The treatment period begins with the first dose of RTX-321 and ends at the end-of-treatment (EOT) visit. The first day of the treatment period was designated as Day 1 of Cycle 1 (C1D1). The treatment regimens are detailed below (see Dosage Types section titles). Assessments performed during the treatment period are provided in the assessment schedule (Figures 4A-4B) and are described further below. long-term follow-up

The long-term follow-up (LTFU) period began immediately after the EOT visit and ended at the end-of-study (EOS). LTFU includes liaising with patients to assess the incidence of potential long-term effects associated with RTX-321 treatment. Patients will be followed for a maximum of 15 years, or until death or withdrawal of consent to participate in the study. Patients who had entered the LTFU period were allowed to receive subsequent medical care and/or investigational treatment without any concomitant treatment ban applicable to the treatment period.

LTFU assessments are listed in the assessment timeline (Figures 4A to 4B). Patient Inclusion Criteria

Patients must meet the following criteria to be included in the trial. 1. Provide written informed consent. 2. Male or female aged ≥18 years. 3. The physical status of East Coast Cancer Clinical Research Collaborative Organization is 0 or 1. 4. Persistent, recurrent or metastatic, unresectable cervical cancer (squamous, glandular or glandular squamous histology), HNSCC or anal canal squamous cell carcinoma confirmed by local laboratory . 5. All patients must experience disease progression after receiving platinum-based chemotherapy or mitomycin C-based chemotherapy in persistent, recurrent or metastatic conditions. 6. All PD-L1+ cervical cancer patients and HNSCC patients must have received or been judged to be ineligible for immunotherapy with PD-1 or PD-L1 inhibitors. 7. All patients with cervical cancer will have received or been judged to be ineligible for bevacizumab treatment. 8. Confirm HLA-A*02:01 positive status through center test. 9. In patients with cervical cancer or HNSCC, confirm intratumoral HPV 16 from historical pathology results (using FDA-approved HPV assays, cervical cancer patients only) or based on central laboratory analysis of tumor samples. Patients with anal cancer did not need to be pre-tested for HPV 16+ before inclusion. 10. Measurable disease according to RECIST version 1.1. Investigators should contact the sponsor medical monitor to discuss patients with unmeasurable, evaluable disease in Part 1. 11. According to the National Cancer Institute Common Adverse Event Evaluation Criteria Version 5.0 (NCI CTCAE v5.0), the acute adverse reaction from the previous therapy has recovered to ≤ Grade 1 or the baseline period. Well-controlled endocrine abnormalities (eg, hypothyroidism) or leukoplakia on prior checkpoint inhibitor therapy are acceptable after discussion with the sponsor's medical supervisor. 12. Completion of previous anticancer therapy ≥ 28 days, or 5 half-lives of previous treatment before trial treatment (whichever is shorter). Radiation therapy must be completed ≥28 days prior to trial treatment. 13. With adequate organ function, defined as follows: a. AST and ALT ≤3 × upper limit of normal (ULN) b. Total bilirubin ≤1.5 × except in confirmed cases of Gilbert syndrome ULN c. Serum albumin ≥ 2.5 g/dL d. Serum or plasma creatinine calculated by Cockcroft-Gault formula ≤ 1.5 × ULN, and/or glomerular filtration rate ≥ 50 mL/min/1.73 14. Appropriate blood count , defined as: a. Absolute neutrophil count ≥1 × 103/μL without bone marrow growth factor support for ≥1 week b. Platelet count ≥100 × 103/μL without platelet transfusion for ≥1 week c. Hemoglobin ≥ 9 g/dL, no red blood cell transfusion ≥ 2 weeks 15. Agree to use highly effective (female) or acceptable (male) contraceptive methods during the trial and 6 months after the last dose of trial treatment (if fertile). Patient Exclusions

Patients who meet any of the following conditions will be excluded from participation in this trial. 1. The patient involves central nervous system (CNS) disease. Patients were eligible for the trial after consultation with the trial sponsor's medical monitor if they met the following 3 criteria. a. Completed previous treatment for CNS metastases (radiation therapy and/or surgery) b. CNS tumors were clinically stable at the time of inclusion c. Patients did not require management of CNS metastases with corticosteroids or antiepileptic therapy 2. Known for trials Hypersensitivity to any component of treatment, including excipients 3. Clinically significant, active, uncontrolled infection, including HIV, or hepatitis B or C virus (HBV; HCV). Testing is not necessary unless clinically indicated. The following are controlled infections: a. HIV patients must receive effective antiretroviral therapy for ≥ 4 weeks before inclusion, and the HIV virus volume is <400 copies/μL, and they have not suffered from acquired immunity in the past 12 months Deficiency syndrome-defined opportunistic infection with CD4+ count ≥350 cells/μL. b. Patients with chronic HBV must be receiving antiviral therapy, and the amount of HBV virus must be below the detection limit. c. HCV patients must have completed antiviral therapy, and the HCV viral load must be below the detection limit. 4. Patients with clinically significant, uncontrolled cardiac disease, and/or recent cardiac events, including any of the following: a. Angina pectoris, coronary artery bypass grafting, symptomatic History of pericarditis or myocardial infarction b. Documented history of congestive heart failure (New York Heart Association functional class III-IV) c. Documented cardiomyopathy d. e. Any cardiac arrhythmia (eg, ventricular tachycardia), complete left bundle branch block, high-grade atrioventricular (AV) block (eg, History of bifascicular block, Mobitz type II, and third-degree AV block), supraventricular arrhythmias, nodular arrhythmias, or conduction abnormalitiesf. Uncontrolled hypertension, defined as systolic blood pressure >160 mmHg And/or diastolic blood pressure > 100 mmHg, with or without antihypertensive drugs. Initiation or adjustment of blood pressure-lowering medications prior to screening was permitted. 5. Other malignancies occurred during the 3-year trial treatment period, excluding non-melanoma skin cancer, stage 1 uterine cancer, or other malignancies with similar results. 6. Active autoimmune hemolytic anemia or other active autoimmune diseases requiring immunosuppressive therapy. 7. Concomitant medical conditions requiring active immunosuppression, including immune-related adverse events (irAEs), or a history of clinically significant grade 3 or higher irAEs from previous immunotherapy. For patients with a history of clinically significant irAEs Grade 3 or higher, investigators should consult with the sponsor's medical monitor prior to inclusion. 8. Pregnancy or breastfeeding. 9. Major surgery was performed 4 weeks before inclusion. 10. Any medical, social or other conditions that the researchers believe will hinder safety, ethics and non-compliance with the requirements of the trial. Test drug, dosage and administration method

RTX-321 is an allogeneic gene-cultured cell therapy consisting of human enucleated red blood cells expressing HLA-A2-HPV, 4-1BBL, and IL-12 on the cell surface. RTX-321 will be administered intravenously every 3 weeks. Other dosing intervals could be explored following the guidance of the Bayesian logistic regression model (BLRM) and the recommendations of the trial monitoring committee (SMC).

The dose levels planned for Part 1 are shown in Table 5. Based on emerging data, the trial monitoring committee (SMC) may assess higher, lower or intermediate dose levels as appropriate. Dose levels above the highest planned dose level can be assessed at intervals of up to half a log. Table 5: Part 1, planned dose levels for RTX-321 monotherapy dose level RTX-321 cells / dose 1 1 × 10 ⁹ 2 5 × 10 ⁹ 3 1 × 10 ¹⁰ 4 5 × 10 ¹⁰ dose escalation

Dose escalation during part 1 will employ a 2-parameter adaptive BLRM in dose-toxicity following the escalation of excess control (EWOC) principle (Babb 1998, Neuenschwander 2008), using a less informative prior to make dose recommendations And estimate the maximum tolerated dose (MTD) and/or find out the RP2D of RTX-321 monotherapy.

At least 3 patients evaluable for DLT will receive a dose level prior to dose escalation. The first patient enrolled in dose tier 1 will receive 2 doses of RTX-321, 3 weeks apart, and be observed for safety 28 days after the first dose before the second patient will be admitted. A third patient in this cohort can be enrolled 7 days after the second patient received the first dose of RTX-321. After completing the first cohort, there is no waiting period for patient enrollment.

In this trial, the DLT-evaluation period for each patient was 28 days after the first dose of RTX-321. Patients evaluable for DLT were defined as those who received at least 1 dose of RTX-321 and were monitored for 28 days after the first dose of RTX-321, or who discontinued trial treatment within 28 days due to a DLT. Patients who did not receive a second dose of RTX-321 for reasons other than safety (eg, disease progression) would be considered evaluable for DLT if 28 days had passed after the first dose of RTX-321 was administered.

After determining the DLT status of all patients at a dose level (28 days after the last patient received the first dose of RTX-321), a post hoc assignment of the probability of DLT at the different dose levels will be obtained. The results of this analysis, summarized in estimated probabilities, will be performed at each of the following intervals based on the true potential DLT probability at each dose level: • Below dosing interval: (0, 16%) • Target-Toxicity Interval: (16%, 33%) • Overdose-toxicity/overdose interval: (33%, 100%)

Dose levels for the next population group are not considered acceptable according to EWOC principles unless they have a ≤25% probability of overdose based on current data.

During the escalation period, the SMC may be determined to have achieved if the current estimated MTD based on cumulative data is within the range of dose levels investigated in the trial and there is at least a 50% probability of a DLT within the target toxicity interval of 16% to 33%. MTD. dose - limiting toxicity

Dose-limiting toxicity (DLT) was defined as any of the following non-hematologic or hematologic adverse events (AE ): Any Grade ≥3 non-hematologic toxicity, exceptions noted in Table 6. Table 6: Exceptions to Nonhematologic Dose-Limiting Toxicity Criteria toxicity Exceptions CRS If the third level of CRS lasts > 2 days, it is DLT fatigue If the third level of fatigue persists for more than 7 days, it is DLT gastrointestinal toxicity Grade 3 nausea, vomiting, or diarrhea that does not resolve within 3 days of optimal antiemetic or antidiarrheal management is a DLT Abnormal laboratory data Level 3 laboratory data abnormality is a DLT only if it is clinically significant CRS = cytokine release syndrome; DLT = dose - limiting toxicity.

Any Grade 4 hematologic toxicity lasting ≥7 days, except for exceptions noted in Table 7 or any Grade 5 hematologic toxicity. Table 7: Exceptions to Hematologic Dose-Limiting Toxicity Criteria toxicity Exceptions Neutropenia with fever Grade 4 neutropenia with fever is a DLT regardless of duration lymphopenia Grade 4 lymphopenia lasting >7 days is a DLT only if it is associated with a clinically relevant infection Thrombocytopenia Grade 4 thrombocytopenia is a DLT regardless of duration. Thrombocytopenia associated with clinically significant bleeding is a DLT, even at grade 3. DLT = dose - limiting toxicity.

In addition, any clinically significant RTX-321-related AE resulting in a delay of ≥ 2 weeks in subsequent RTX-321 doses will be considered a DLT. maximum tolerated dose

The MTD was estimated as the dose with the highest probability of being in the target toxicity interval (DLT probability between 16% and 33%) among the doses meeting the EWOC criteria (≤25% probability of overdose). Phase 2 Recommended Dosage

RP2D is the dose selected for further studies based on all available data including safety, tolerability, antitumor activity, PK/PD and manufacturing feasibility. The SMC is responsible for deciding on RP2D. RP2D must be equal to or lower than MTD. The final decision on dose selection will be made by the sponsor. Dose escalation in patients

Dose escalation in individual patients was possible after the SMC determined that higher doses were safe in Part 1 of the study. For individual patients, investigators and sponsor medical monitors should determine whether patient-specific dose escalation is appropriate after the SMC has determined the safety of higher doses. Dosing escalations should be documented in the RTX-321 Dosing eCRF. dose modification

For patients who cannot tolerate the dosing schedule specified in the trial protocol, a dose adjustment may be allowed to allow the patient to continue receiving the trial treatment. If possible, such adjustments should be discussed with the sponsor's medical monitor prior to implementation. Dose interruptions and adjustments must be documented in the dose management record eCRF. RTX-321 dose reduction

When a dose reduction of RTX-321 is indicated, the dose will be reduced to approximately 50% of the RTX-321 target cell number. Allow up to 2 dose reductions. If a patient achieves clinical benefit but requires a dose reduction, the investigator should contact the trial sponsor's medical monitor. Safety and Tolerability Assessment

During treatment, safety will be monitored at each visit by performing a physical examination, assessing vital signs, and/or assessing laboratory parameters according to the assessment schedule (Figures 4A-4B). Safety will also be followed across LTFU to assess the long-term effects of RTX-321 treatment. See the section below entitled "Adverse Events and Serious Adverse Events " for more information on the collection and reporting of AEs. Pharmacokinetic, pharmacodynamic and immunogenicity assessment

The mechanism of action of RTX-321 is to induce tumor-specific responses by selectively activating and expanding tumor-specific T cells against HPV 16 target antigens. In addition to target antigen-specific responses, nonclinical studies showed that RTX-321 was able to induce the proliferation, expansion and activation of NK cells and non-HPV 16 E7 antigen-specific CD8+ T cells (data not shown). Nonclinical studies also demonstrated the potential for memory formation and epitope diffusion when rechallenged with tumor cells with or without proteins targeted by mRBC-321 (data not shown).

In this FIH study, PD studies were designed to address aspects of RTX-321's mechanism of action, as seen in nonclinical studies: • Amplified peripheral target antigen-specific by HLA-A*02:01 HPV 16 E7 peptide 11-19 dextromorphic staining on CD8+ T cells and by exploratory interferon-gamma enzyme-linked ImmunoSpot® assay Exploratory Evidence for Sexual T Cells • Detection of expansion and/or activation of non-HPV 16 E7 antigen-specific CD8+ T cells in the periphery by flow cytometry immunophenotyping, and by exploratory multiplex immunohistochemistry (IHC) and expression analysis in selected Evidence of expansion and/or activation of CD8+ T cells in tumors • Evidence of NK cell expansion and/or activation in the periphery by flow cytometry immunophenotyping and in select tumors by exploratory multiplex IHC and expression analysis These assessments will be performed in peripheral blood and optionally paired tumor sections.

Serum, plasma and whole blood will be collected for assessment of PK, PD and immunogenicity at pre-specified time points. Table 8 summarizes the sample types collected for each assay. Where feasible, selected tumor sections will be obtained to assess PD effects in the tumor microenvironment.

Of note, pre-infusion PK and PD samples must be obtained prior to RTX-321 infusion for a new cycle. If the new cycle is not started within 3 days of the planned start date, pre-infusion PK and PD specimens scheduled for cycle day 1 should still be obtained. Table 8. Evaluate sample type analyze describe pharmacokinetics Whole blood RTX-321 triple positive cells (HLA-A2-HPV, IL-12 and 4-1BBL) The PK of RTX-321 will be assessed using an extracellular flow cytometry assay to detect triple positive expression of RTX-321 (HLA-A2-HPV, IL-12, and 4-1BBL) Pharmacodynamics Whole blood Immunophenotyping Flow cytometric analysis of various immunophenotypic subpopulations in whole blood, including but not limited to: NK cells and CD8 T cells and their proliferative activation and cytotoxicity status. Antigen-specific CD8 T cells will also be assessed serum 4-1BB Shedding - Exploratory 4-1BB circulation level MSD assessment plasma Cytokines – Exploratory MSD-based multiplex analysis to assess circulating levels of cytokines in plasma PBMC IFN-γ ELISpot – Exploratory ELISpot-based assessment to identify the presence of HPV 16 E7 peptide 11-19-specific or HPV 16-specific reactive T cells Immunogenicity serum anti-RTX-321 antibody Serum sampling will be performed to assess anti-RTX-321 antibodies exploratory whole blood for DNA Biomarkers associated with RTX-321 were identified that may indicate or predict clinical response, drug resistance, safety or pharmacodynamic activity in circulating and FFPE tumors. Whole blood can be used for TCR sequencing to understand the clonality of peripheral T cells over time. PBMCs were used retrospectively to advance the understanding of the biology of RTX-321 based on available trial data. FFPE tumor samples can be used for multiplex IHC analysis, RNA-expression analysis, and TCR sequencing to understand the microenvironment at baseline and changes over time due to treatment. PBMC FFPE tumors Efficacy evaluation

If evidence of pseudo-progression is found, the investigator will assess treatment response in parts 1 and 2 of the trial according to RECIST v.1.1 and subsequent assessments according to iRECIST (Table 9). Baseline imaging will be available within 28 days of enrollment (contact sponsor medical monitor for out-of-window imaging evaluation, sponsor medical monitor may extend screening evaluation window). Response will be assessed at 8-week intervals with the same imaging modality starting 8 weeks after the first day of trial treatment. After 12 months of trial treatment without evidence of disease progression, this interval may be extended to 12 weeks at the investigator's discretion. Table 9. Time Point Response (TPR) response criteria Immune-related complete response (iCR) ^a Same criteria as RECIST v.1.1: disappearance of target lesions and non-target lesions. Any pathological lymph nodes (target or non-target) must be reduced in size to <10 mm. Need confirmation of response after at least 4 weeks Immune-related partial response (iPR) ^a The same standard as RECIST v.1.1: Taking the sum of the baseline diameters as the reference benchmark, the sum of the measured values of the target lesions is reduced by at least 30%. Non-target lesions must be free of disease progression. Need confirmation of response after at least 4 weeks Immune-related stable disease (iSD) ^a Same criteria as RECIST v.1.1: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for disease progression, referenced to the smallest sum of diameters in the trial Immune-related unidentified progressive disease (iUPD) ^b The same standard as RECIST v.1.1: Taking the smallest sum of diameters as a reference, the sum of the diameters of the measured lesions has increased by at least 20%, and the absolute value has increased by ≥ 5 mm. The appearance of new lesions ^c will also constitute progressive disease. Immune-related confirmed progressive disease (iCPD) ^d Sustained increase in tumor burden (from iUPD) when the iUPD definition is met in target, non-target, or new lesions, based on evidence of one or more of the following, compared to the last assessment. Progression of target lesions Worsening with an absolute increase in the sum of at least 5 mm Continuing clear progression of non-target disease with increasing tumor burden Previously identified new lesions (considered a target new lesion with a sum of absolute mm) or an increase in the size of additional new lesions in previously unrecognized progressive lesion types (target or non-target or new lesions), including the appearance of additional new lesions, meeting RECIST v.1.1 (iUPD) criteria. ^a Prior to iCR, iPR, or iSD, there may be iUPD (1 or more conditions), but no iCPD ^b Multiple assignments of iUPD may be made as long as iCPD has not been confirmed at the next assessment. ^cNew lesions nTrials are classified as target or non-target lesions ^dIf progressive disease is not confirmed, an appropriate response will be assigned at that time point (if conditions are still eligible for iUPD but have not worsened, or if conditions are Comply with iSD, iPR or iCR compared to base period).

Confirmatory imaging will be performed per RECIST v.1.1 and iRECIST requirements to confirm complete response (CR), partial response (PR) or disease progression. Edited copies of baseline and all subsequent imaging may be collected by the sponsor and submitted for central review during the trial.

Disease progression during the trial was defined by RECIST v.1.1. If the investigator determines that ongoing trial treatment is not in the best interest of the patient due to evidence of clinical disease progression not consistent with RECIST v.1.1 disease progression, the reason for discontinuation of trial treatment should be recorded as "clinical disease progression." Patients with clinical disease progression may continue to collect scans, which will be collected into the imaging eCRF, until radiographic disease progression is documented.

Continue trial treatment after disease progression at the investigator's discretion, after discussion with the sponsor's medical supervisor. Adverse Events and Serious Adverse Events

An adverse event (AE) was defined as the occurrence of an undesired sign, symptom, or medical condition (or aggravation of a pre-existing condition) after obtaining individual consent from the patient. Abnormal laboratory values or test results that occur after informed consent constitute an AE if these adverse events induce clinical signs or symptoms, are considered clinically significant, and require treatment (eg, hematological abnormalities requiring blood transfusion or hematologic stem cell support ), or need to change the trial drug (including treatment interruption).

An adverse drug reaction is an unwanted or harmful reaction that occurs after administration of a drug and is suspected or known to be caused by the drug. Adverse drug reactions have traditionally been classified as pharmacological (predicted according to the pharmacology of the drug) or idiopathic (not predicted according to pharmacology).

A serious adverse event is defined as any AE with 1 of the following: 1. deadly 2. Life-threatening 3. Causes persistent or significant disability or incapacity 4. Constituting a congenital malformation/birth defect 5. Medically significant, defined as an event that endangers the patient or may require medical or surgical intervention to prevent any of the outcomes listed above 6. Hospitalization or prolonged hospitalization is required, unless the hospitalization is for: • Usual treatment or monitoring for the trial indication, independent of any exacerbation of the condition • Elective or preplanned treatment of a pre-existing condition unrelated to the indication being tested and that has not worsened since signing the individual's consent • Treatment of urgent outpatient events that do not meet any of the above SAE definitions and do not result in hospitalization • Social welfare justification and respite care when the patient's general condition is not worsening 7. Deaths due to disease progression should not be reported as SAEs if recorded using appropriate methods (eg, according to RECIST, version 1.1). Any AE that occurs due to disease progression should be reported in an appropriate manner.

The investigator will interpret the observed AE as related to the disease, trial drug, trial procedure, or other concomitant therapy or pathology. For the purpose of assessing the relationship of an AE to the study drug, the following terms are defined: • Related—a direct causal relationship between the study treatment and the AE is likely • Possibly related—a causal relationship between the study treatment and the AE is currently unproven and unlikely, but Nor is it impossible • Not relevant – there is no doubt that the AE is absolutely not related to the trial treatment All "related" and "possibly related" AEs and SAEs will be defined as being related to the trial drug. Statistical Analysis

Data will be summarized using descriptive statistics. A data list will be created to support each table and present all data. Categorical data will be summarized using frequency counts and percentages. For continuous data, the mean, standard deviation, median, minimum and maximum values are presented. Time-correspondence-event data will be summarized using the Kaplan-Meier method. Where appropriate, confidence intervals around point estimates will be presented, and estimates for the median and other quantiles will be produced, as well as for each time point (for time-to-event data).

Unless otherwise stated, the following methods will be used in this test: • Single proportion for binomial endpoints—two-sided confidence intervals will be calculated using the Clopper and Pearson method • Median time-correspondence-event—The median time-correspondence-event will be estimated using the Kaplan-Meier method; two-sided confidence intervals will be calculated using the Brookmeyer and Crowley method

The final clinical trial report will be based on all patient data until all patients complete at least 6 treatment cycles, transition to an additional regimen is completed, or the trial is discontinued.

In Part 1 of the trial, presentation will be by dose level and all data pooled for all patients in Part 1. In Part 2, the pharmacodynamic data of expanded population 1, expanded population 2, and expanded population 3 will be presented separately. Data from patients with the same tumor type in Part 1 who underwent RP2D treatment in Part 2 can be combined with the expanded patients for selected presentations. All other data will be presented separately for each expansion population and pooled together. Partial presentation will be detailed in the Statistical Analysis Plan (SAP), which will pool all patients treated with RP2D in Part 1 and Part 2.

Unless otherwise specified, results will be reported according to the following rules: • Screen failure patients are patients who signed individual consent forms but never initiated trial treatment for any reason. For these patients, collected eCRF data will not be included in the analysis but will be reported in the Clinical Study Report (CSR) as a separate tabulation • Baseline is defined as the Last assessment • Additional details about data reporting not described here will be detailed in the SAP for this trial Primary analysis

The primary endpoint was the incidence of DLT. Dose escalation during part 1 will employ a 2-parameter adaptive BLRM for dose-toxicity, following the EWOC principle (Babb 1998, Neuenschwander 2008), using a less informative prior to make dose recommendations and estimate the maximum Tolerated Dose (MTD) and/or find RP2D for RTX-321 monotherapy.

At least 3 patients evaluable for DLT will receive a dose level prior to dose escalation.

After determining the DLT status of all patients at a dose level, a post hoc assignment of DLT probabilities at different dose levels will be obtained. The results of this analysis are summarized in estimated probabilities, which will be performed at each of the following intervals based on the true potential DLT probability at each dose level: • Interval below administered dose: (0, 16%) • Target Toxicity Interval: (16%, 33%) • Overdose Toxicity/Overdose Interval: (33%, 100%)

Dose levels for the next population group are not considered acceptable according to EWOC principles unless they have a ≤25% probability of overdose based on current data.

During the escalation period, the SMC may determine that the MTD has been reached if the current estimated MTD based on cumulative data is within the range of dose levels investigated in the trial and there is at least a 50% probability of a DLT within the target toxicity interval of 16% to 33% .

The MTD was estimated as the dose with the highest probability of being in the target toxicity interval (DLT probability between 16% and 33%) among the doses meeting the EWOC criteria (≤25% probability of overdose).

DLT will be aggregated by dose level, and RP2D will be aggregated and combined for Part 1, Expansion Cohort 1, Expansion Cohort 2, and Expansion Cohort 3, respectively. A list will be made of participants who have experienced the DLT. This list will include the reported AE term, system organ class and/or preferred term, severity, timing, and assigned dose level. Secondary Analysis Efficacy Variables

For all efficacy endpoints, data will be presented, pooled, or analyzed for each dose-escalation treatment arm in Part 1, and data for the expansion cohort will be presented separately. Data from patients with the same tumor type in Part 1 who underwent RP2D treatment in Part 2 can be combined with the expanded patients for selected presentations. No null-hypothesized significance tests were planned for any of the secondary efficacy endpoints. objective response rate

The primary efficacy endpoint was best overall response (BOR) as assessed by investigators according to RECIST v.1.1 and iRECIST. The summary measure was ORR, which was defined as the proportion of BOR patients in CR or PR. ORR will be analyzed separately for each expanded population. Defining PR or CR as BO requires confirmation of the response. ORR will be aggregated with accompanying 95% confidence intervals. ORR will be summarized separately according to RECIST version 1.1 and iRECIST. Disease control rate, tumor size change

Disease control rates, defined as the proportion of patients achieving CR, PR, or stable disease (SD) >16 weeks, will be pooled with accompanying 95% confidence intervals. The optimal overall change in tumor size (defined as the largest percent decrease, or the smallest increase without decrease) will be presented in a waterfall plot, while the change in tumor size over time will be presented in a spider plot. response duration

Duration of response (DOR) will be defined as the time from the date of first documented response to the date of documented progression or death in the absence of disease progression. Time to initial response will be defined as the latest of the dates leading to PR or CR for the first clinic response. If the participant has not progressed after the response, their DOR will be capped at the last scan. For participants with no recorded responses, no DOR will be defined for these participants. The DOR for participants who experienced CR or PR at any time during the trial will be listed by participant. A Kaplan-Meier plot of the DOR will also be produced if a reasonable proportion of participants achieve a response, and presents the median DOR and the corresponding 95% confidence interval. Progression Free Survival (PFS)

PFS was defined as the time elapsed from the date of inclusion to the date of first documented progression or death from any cause. If the patient is event-free, PFS will be limited to the date of the last appropriate tumor assessment (RECIST v.1.1). Clinical deterioration without objective radiographic evidence was not considered documented disease progression. The primary analysis for PFS will be regional radiological assessment according to RECIST v.1.1. The median PFS time and the proportion of patients alive and progression-free at 3, 6, 9, and 12 months will be estimated. Overall survival (OS)

OS will be presented graphically using Kaplan-Meier plots including all patients treated with RP2D. Median OS times and the proportion of patients alive at 3, 6, 9, and 12 months will be estimated. initial results

Three dose cohorts (n = 9) were completed, including one patient with anal squamous cell carcinoma who had stable disease at 16 weeks, the highest dose cohort to date of 1e10 administered every three weeks. Before inclusion, patients experienced disease progression after receiving anti-PD-1 therapy. RTX-321 was generally well tolerated, with no DLTs or grade 3/4 treatment-related adverse events. Consistent with the combined mechanism of action of IL-12 and 4-1BBL, increases in activated CD4 ⁺ T cells, activated CD8 ⁺ T cells, and activated NK cells were observed at higher dose levels. other embodiments

It should be understood that while the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and modifications belong to the scope of the following claims. sequence listing SEQ ID NO: identifier sequence SEQ ID NO: 1 β2m chain IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM SEQ ID NO: 2 Nucleic acid sequence encoding β2m chain (SEQ ID NO: 1) ATACAAAAGAACACCAAAAATCCAGGTTTATTCAAGACACCCAGCTGAAAACGGAAAGAGCAACTTTTTGAATTGTTATGTGAGCGGTTTCCATCCTTCTGATATAGAAGTGGATCTGCTCAAAAATGGGGAAAGAATCGAAAAGGTTGAACACAGCGATCTGAGCTTTAGCAAGGATTGGTCTTTCTACCTCCTCTACTATACTGAATTTACGCCCCAGAGA AGGACGAGTATGCTTGTCGCGTGAACCACGTCACTTTGTCTCAACCGAAAATTGTGAAGTGGGATCGGGACATG SEQ ID NO: 3 HLA-A2*02:01 allele polypeptide GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPI SEQ ID NO: 4 Nucleic acid sequence encoding HLA-A2*02:01 allele polypeptide (SEQ ID NO: 3) GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGAGCCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA SEQ ID NO: 5 HLA-A2*02:01 allele polypeptide GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE PSSQPTIPI SEQ ID NO: 6 Nucleic acid sequence encoding HLA-A2*02:01 allele polypeptide (SEQ ID NO: 5) GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGATACTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA SEQ ID NO: 7 HLA-A2*02:01 allele polypeptide GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE PSSQPTIPI SEQ ID NO: 8 Nucleic acid sequence encoding HLA-A2*02:01 allele polypeptide (SEQ ID NO: 7) GGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAAGTTAAGGCCCATTC ACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGATGCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCTGCCGACATGGC GGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTACCCTGCGGAAATA ACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCCACTATACCGATA SEQ ID NO: 9 HPV E7 antigen YMLDLQPET SEQ ID NO: 10 Nucleic acid encoding HPV E7 antigen (SEQ ID NO: 9) TACATGCTCGACCTTCAACCCGAGACC SEQ ID NO: 11 HPV E7 antigen YMLDLQPETT SEQ ID NO: 12 Nucleic acid encoding HPV E7 antigen (SEQ ID NO: 11) TACATGCTCGACCTTCAACCCGAGACCACC SEQ ID NO: 13 HPV E7 antigen TIHDIILECV SEQ ID NO: 14 Nucleic acid encoding HPV E7 antigen (SEQ ID NO: 13) ACTATACATGACATTATACTGGAATGTGTT SEQ ID NO: 15 Exogenous antigen-presenting polypeptide linked to exogenous antigen polypeptide YMLDLQPETGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQF VRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFY PAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIGSGSGSGSEDGSGSGSGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIK KSPSDVKPLPSPDTDVPLSSVEIENPETSDQ SEQ ID NO: 16 Nucleic acid encoding an exogenous antigen-presenting polypeptide (SEQ ID NO: 15) linked to an exogenous antigen polypeptide TACATGCTCGACCTTCAACCCGAGACCGGAGGCGGAGGGAGTGGCGGAGGAGGTAGTGGCGGCGGGGGCTCTATACAAAGAACACCAAAAAATCCAGGTTTATTCAAGACACCCAGCTGAAAACGGAAAGAGCAACTTTTTGAATTGTTATGTGAGCGGTTCCATCCTTCTGATATAGAAGTGGATCTGCTCAAAAATGGGGAAAGAATCGAAA AGGTTGAACACAGCGATCTGAGCTTTAGCAAGGATTGGTCTTTCTACCTCCTCTACTATACTGAATTTACGCCCCACAGAGAAGGACGAGTATGCTTGTCGCGTGAACCACGTCACTTTGTCTCAACCGAAAATTGTGAAGTGGGATCGGGACATGGGTGGGGGAGGCAGCGGGGGTGGGGGGTCAGGTGGGGGAGGTAGCGGC GGAGGTGGCAGTGGTTCACACAGTATGCGATACTTTTTCACCTCAGTAAGTAGACCAGGCCGGGGCGAGCCGAGGTTTATCGCAGTTGGATACGTTGACGACACTCAATTCGTCCGATTCGACTCCGATGCCGCTAGCCAGCGGATGGAACCACGGGCCCCGTGGATCGAACAGGAAGGTCCAGAGTACTGGGATGGGGAAACCCGGAAA GTTAAGGCCCATTCACAGACCCACCGGGTCGATTTGGGGACGTTGCGCGGAGCCTACAACCAGAGCGAAGCGGGGAGCCACACGGTCCAAAGGATGTATGGATGCGACGTCGGGTCCGATTGGCGGTTTCTCAGGGGTTATCACCAGTATGCTTATGATGGCAAGGACTATATCGCCTTGAAAGAGGATCTTAGATCCTGGACAGCT GCCGACATGGCGGCGCAAACGACAAAGCATAAATGGGAAGCAGCACACGTTGCTGAACAACTTCGCGCCTACTTGGAAGGGACGTGTGTCGAATGGTTGAGAAGGTACTTGGAAAATGGGAAAGAGACCTTGCAGAGGACCGATGCCCCAAAAACGCATATGACACACCACGCTGTGTCCGATCACGAAGCCACTTTGCGCTGTTGGGCCCTTTCATTTTA CCCTGCGGAAATAACGCTCACTTGGCAAAGGGATGGCGAGGACCAAACGCAGGACACTGAATTGGTCGAGACGCGACCGGCAGGAGACGGTACTTTTCAGAAATGGGCAGCAGTTGTCGTACCATCAGGACAGGAACAACGGTATACCTGTCATGTTCAACACGAAGGTTTGCCAAAACCCTTGACCCTTCGATGGGAACCGTCCAGCCAGCCCACTA TACCGATAGGCAGCGGCTCTGGCTCCGGCTCCGAAGATGGCAGCGGCAGCGGAAGTGGTTCATTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCC TCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAG ACAAGTGATCAA SEQ ID NO: 17 exogenous co-stimulatory polypeptide ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQGSGEGRGSLLTCGDVEENPG SEQ ID NO: 18 Nucleic acid encoding exogenous co-stimulatory polypeptide (SEQ ID NO: 17) GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGT CCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCC GCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTG GCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCCTGAGCACCACCGAGGTGGCCATGCACACCAGCACCAGCAGCAGCGTGACCAAGAGCTACATCAGCAGCCAGACCAACGACACCCACAAGCGCGACACCTACGCCGCCACCCCCCGCGCCCACGAGGTGAGCGAGATCAGCGTGCGCA CCGTGTACCCCCCCCGAGGAGGAGACCGGCGAGCGCGTGCAGCTGGCCCACCACTTCAGCGAGCCCGAGATCACCCCTGATCATCTTCGGCGTGATGGCCGGCGTGATCGGCACCATCCTGCTGATCAGCTACGGCATCCGCCGCCTGATCAAGAAGAGCCCCAGCGACGTGAAGCCCCTGCCCAGCCCCGACACCGACGTGCCCCTGAGCAGCGTGGAGATCGAGA ACCCCGAGACCAGCGACCAGGGCAGCGGTGAAGGTCGGGGGAGCCTGCTGACTTGCGGAGATGTGGAAGAAAATCCTGGCCCA SEQ ID NO: 19 4-1BBL ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGLPSPRSE SEQ ID NO: 20 Nucleic acid encoding 4-1BBL (SEQ ID NO: 19) GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGT CCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCC GCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA SEQ ID NO: 21 exogenous co-stimulatory polypeptide ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ SEQ ID NO: 22 Nucleic acid encoding exogenous co-stimulatory polypeptide (SEQ ID NO: 21) GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGT CCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCC GCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTG GCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCCTGAGCACCACCGAGGTGGCCATGCACACCAGCACCAGCAGCAGCGTGACCAAGAGCTACATCAGCAGCCAGACCAACGACACCCACAAGCGCGACACCTACGCCGCCACCCCCCGCGCCCACGAGGTGAGCGAGATCAGCGTGCGCA CCGTGTACCCCCCCCGAGGAGGAGACCGGCGAGCGCGTGCAGCTGGCCCACCACTTCAGCGAGCCCGAGATCACCCCTGATCATCTTCGGCGTGATGGCCGGCGTGATCGGCACCATCCTGCTGATCAGCTACGGCATCCGCCGCCTGATCAAGAAGAGCCCCAGCGACGTGAAGCCCCTGCCCAGCCCCGACACCGACGTGCCCCTGAGCAGCGTGGAGATCGAGA ACCCCGAGACCAGCGACCAG SEQ ID NO: 23 exogenous co-stimulatory polypeptide MYGKIIFVLLLSEIVSISAACPWAVSGARASPPGSASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIENPETSDQ SEQ ID NO: 24 Nucleic acid encoding exogenous co-stimulatory polypeptide (SEQ ID NO: 23) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTG CTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTCTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCT CTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATCCATCACAGACAAATGATACGCACAAACG GGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCCATCTGAT GTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA SEQ ID NO: 25 IL-12A protein RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPFYKTKIKLCILLHAFRIRAVTIDRVMSYL NAS SEQ ID NO: 26 Nucleic acid encoding IL-12A protein (SEQ ID NO: 25) CGGAATCTGCCAGTGGCAACCCCAGACCCCGGAATGTTCCCATGCCTGCACCACTCTCAGAACCTGCTGAGGGCCGTGAGCAATATGCTGCAGAAGGCCCGCCAGACACTGGAGTTTTACCCTTGTACCAGCGAGGAGATCGACCACGAGGACATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGCCTGCCACTGGAGCTGACCAAGAACGAGAGCTGT CTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCAGCTGCCTGGCCTCCAGAAAGACATCTTTTATGATGGCCCTGTGCCTGTCTAGCATCTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGCTGCTGATGGACCCAAGAGGCAGATTTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCC CTGAACTTTAATTCCGAGACAGTGCCTCAGAAGTCCTCTCTGGAGGAGCCAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAATGCCTCC SEQ ID NO: 27 IL-12B protein IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESL PIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS SEQ ID NO: 28 Nucleic acid encoding IL-12B protein (SEQ ID NO: 27) ATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGCGACACACCTGAGGAGGATGGCATCACCTGGACACTGGATCAGAGCAGCGAGGTGCTGGGCTCCGGCAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCCGGCCAGTACACATGTCA CAAGGGAGGAGAGGTGCTGAGCCACTCCCTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGTCTACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAACAAGACCTTCCTGCGGTGCGAGGCCAAGAATTATAGCGGCCGGTTCACCTGTTGGTGGCTGACCACAATCTCTACCGACCTGACCTTCAGCGTGAAGTCTAGCCGGGGCTCCTCT GACCCTCAGGGAGTGACATGCGGAGCAGCCACCCTGTCCGCCGAGCGGGTGAGAGGCGATAACAAGGAGTACGAGTATAGCGTGGAGTGCCAGGAGGACTCCGCCTGTCCAGCAGCAGAGGAGAGCCTGCCAATCGAAGTGATGGTGGATGCCGTGCACAAGCTGAAGTACGAGAATTATACAAGCTCCTTTCTTATCAGGGACATCATCAAGCCGATC CCCCTAAGAACCTGCAGCTGAAGCCCCTGAAGAACAGCCGGCAGGTGGAGGTGTCTTGGGAGTACCCCGACACCTGGAGCACACCTCACTCCTATTTCTCTCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAAGGACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGTCGGAAGAACGCCTCTATCAGCGTGCGGGCCC AGGATAGATACTATTCTAGCTCCTGGAGCGAGTGGGCCTCCGTGCCATGTTCT SEQ ID NO: 29 full-length GPA MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ SEQ ID NO: 30 GPA fragment containing a transmembrane domain LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDTDVPLSSVEIEENPETSDQ SEQ ID NO: 31 SMIM1 MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK SEQ ID NO: 32 G4S linker GGGGS SEQ ID NO: 33 (G4S) ₃ linker GGGGSGGGGSGGGGS SEQ ID NO: 34 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS SEQ ID NO: 35 Linker GGSGGSGGGGGSGGGSGGGSGGGS SEQ ID NO: 36 Linker GGSGGSGGGPEDEPGSGSGGGSGGGS SEQ ID NO: 37 Linker GSGSGSGSGSSEEDEDEDEDGSGSGSGSGS SEQ ID NO: 38 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 39 Linker GSGSGSGSEDGSGSGSGS SEQ ID NO: 40 Linker GSGSGSGSGSGSGSGSGSGS SEQ ID NO: 41 Linker GCGGSGGGGSGGGGS SEQ ID NO: 42 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA SEQ ID NO: 43 Linker GGGGSGGGGSGGGGSGGGGSGGGG SEQ ID NO: 44 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA SEQ ID NO: 45 GPA signal peptide MYGKIIFVLLLSEIVSISA SEQ ID NO: 46 Ig heavy chain V region 3 signal sequence MGWSCIILFLVATATGVHS SEQ ID NO: 47 light chain leader sequence MRVPAQLLGLLLLWLPGARC SEQ ID NO: 48 exogenous cytokine peptide MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCKGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLL HKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRV FTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAV IDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS SEQ ID NO: 49 Nucleic acid encoding exogenous cytokine polypeptide (SEQ ID NO: 48) ATGCAACCGCAAGAGAGTCACGTACATTTATTCAAGATGGGAAGATGGAAGTCGGGACGGTGTGTCTCTCGGCGCTGTTAGTTCAACGGAGGAAGCGTCTCGCTGTCGCCGGATAAGTCAACGCCTTTGTACGGGAAAACTGGGTATAGCTATGAAGGTCCTCGGCGGGGTGGCGTTGTTTTGGATTATCTTTATACTTGGGTATCT GACCGGTTACTATGTTCACAAGTGTAAAGGAGGTGGAGGATCAGGTGGAGGTGGTTCAGGTGGAGGAGGTAGCATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGCGACACACCTGAGGAGGATGGCATCACCTGGACACTGGATCAGAGCAGCGAG GTGCTGGGCTCCGGCAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCCGGCCAGTACACATGTCACAAGGGAGGAGAGGTGCTGAGCCACTCCCTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGTCTACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAACAAGACCTTCCTGCGGTGCGAGGCCAAGAATTATAGCGGCCGGT TCACCTGTTGGTGGCTGACCACAATCTCTACCGACCTGACCTTCAGCGTGAAGTCTAGCCGGGGCTCCTCTGACCTCTAGGGAGTGACATGCGGAGCAGCACCCTGTCCGCCGAGCGGGTGAGAGGCGATAACAAGGAGTACGAGTATAGCGTGGAGTGCCAGGAGGACTCCGCCTGTCCAGCAGCAGAGGAGAGCCTGCCAATCGAAGTGATGGTG GATGCCGTGCACAAGCTGAAGTACGAGAATTATACAAGCTCCTTCTTTATCAGGGACATCATCAAGCCCGATCCCCCTAAGAACCTGCAGCTGAAGCCCCTGAAGAACAGCCGGCAGGTGGAGGTGTCTTGGGAGTACCCCGACACCTGGAGCACACCTCACTCCTTATTTCTCTCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAAG GACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGTCGGAAGAACGCCTCTATCAGCGTGCGGGCCCAGGATAGATACTATTCTAGCTCCTGGAGCGAGTGGGCCTCCGTGCCATGTTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCAGCCGGAATCTGCCAGTGGCAACCCCAGACCCCGGAATGTTCCCATGC CTGCACCACTCTCAGAACCTGCTGAGGGCCGTGAGCAATATGCTGCAGAAGGCCCGCCAGACACTGGAGTTTTACCCTTGTACCAGCGAGGAGATCGACCACGAGGACATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGCCTGCCACTGGAGCTGACCAAGAACGAGAGCTGTCTGAACAGCCGGGAGACCAGCTTCATCACCAACGGCAGCTGCCTGG CCTCCAGAAAGACATCTTTTATGATGGCCCTGTGCCTGTCTAGCATCTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCCAAGAGGCAGATTTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTTAATTCCGAGACAGTGCCTCAGAAGTCCTCTCTGGAGGAGC CAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACCGCGTGATGAGCTATCTGAATGCCTCC SEQ ID NO: 50 Linker (GGGGS)n SEQ ID NO: 51 HLA-A2v1 (wild type): * SEQ ID NO: 52 HLA-A2v2 (Y84A): * SEQ ID NO: 53 HLA-A2v3 (Y84C and L2C) * SEQ ID NO: 54 HPV P1-2-HLA-A2-GPA MSRSVALAVLALLSLSGLEAYMLDLQPETGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPG RGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTH MTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIGSGSGSGSEDGSGSGSGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFG VMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ SEQ ID NO: 55 Linker YMLDLQPETGGGGS(G ₄ S) ₂ SEQ ID NO: 56 Linker (G ₄ S) ₄ SEQ ID NO: 57 Nucleic acid encoding SEQ ID NO: 21 GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGT CCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCC GCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTG GCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAAC TGTTTACCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGA AAATCCAGAGACAAGTGATCAA

Figure 1A is a schematic diagram showing different versions of HLA-A2 (HPV E7) expressed on RCT. Figure 1A reveals that "YMLDLQPETGGGGS(G ₄ S) ₂ " is SEQ ID NO: 55, and "(G ₄ S) ₄ " is SEQ ID NO: 56. Figure 1B is a graph showing the activity of HLA-A2 (HPV E7) expressed on RCT to stimulate HPV16-specific T cells in vitro. Figure 1C is a graph showing the activity of HLA-A2 (HPV E7) expressed on RCT to stimulate HPV16-specific T cells in vitro. Figure 2A shows RTX-321 expression stained with anti-IL12, anti-4-1BBL and anti-β2M. Figure 2B shows a representative flowchart of TCR performance of E7-TCR cells compared to non-transduced T cells prior to co-culture. Figure 2C shows that in cells with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-HPV-4-1BBL, RCT-4-1BBL-IL-12, RTX-321, or RCT - Bar graph of the percentage of Nur77+ E7-TCR cells after CMV-4-1BBL-11-12 cultured for 2 hours. Figure 2D shows that in cells with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-HPV-4-1BBL, RCT-4-1BBL-IL-12, RTX-321, or RCT - Bar graph of the percentage of CD69+E7-TCR cells after CMV-4-1BBL-11-12 cultured for 24 hours. Fig. 2E shows that in cells with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-HPV-4-1BBL, RCT-4-1BBL-IL-12, RTX-321, or RCT - Bar graph of the percentage of 4-1BB+ E7-TCR cells after 5 days of CMV-4-1BBL-11-12 culture. Figure 2F shows a representative flowchart of E7-TCR ⁺ expression of RCT-CTRL- and RTX-321-treated E7-TCR cells at day 5, and shows the expression of E7-TCR cells and untransduced Bar graph of fold expansion of CD8 ⁺ T cells relative to media-treated controls. Figure 2G shows a histogram of granzyme B+ % of E7-TCR cells at day 1, and a histogram of IFNγ concentration in supernatants at day 5. Figure 2H shows a histogram of _TCM for E7-TCR cell numbers at day 5, and a histogram of T _EM for E7-TCR cell numbers at day 5. Figure 3 is a schematic diagram of the study design of the Phase 1 study of RTX-321 for the treatment of cancer. Part 1 includes dose escalation of RTX-321 monotherapy and Part 2 includes population expansion of RTX-321 monotherapy. Figure 4A is a timeline depicting the evaluation of a study of RTX-321 in the treatment of cancer. Figure 4B is a timeline depicting the evaluation of a study of RTX-321 in the treatment of cancer (continued).

Claims (69)

A method of treating HPV16-positive or HPV16-associated cancer in a human subject in need thereof, comprising intravenously administering to the human subject a dose of a pharmaceutical composition comprising about 0.5 x ¹⁰⁹ to about 5 x 10 ¹⁰ human enucleated erythroid cells comprising on their surface: an exogenous antigen-presenting polypeptide linked to an exogenous antigen polypeptide, wherein the exogenous antigen-presenting polypeptide is MHC class 1 HLA-A2 single-chain fusion protein comprising an α chain, a β2-microglobulin (β2m) chain, and a membrane anchor region, and wherein the exogenous antigenic polypeptide comprises a human papillomavirus (HPV) E7 antigen; an exogenous costimulatory polypeptide comprising 4-1BBL; and an exogenous cytokine polypeptide comprising IL-12; the frequency of administration is about 2 weeks to about 4 weeks. The method of claim 1, wherein the HPV16-positive or HPV16-associated cancer is selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, cervical cancer, vaginal cancer, penile cancer, oral Pharyngeal, oral and vaginal cancers. The method of claim 2, wherein the HPV16-positive or HPV16-related cancer is selected from the group consisting of head and neck squamous cell carcinoma, anal cancer, and cervical cancer. The method according to any one of claims 1 to 3, wherein the HPV16-positive or HPV16-associated cancer is an unresectable solid tumor. The method of any one of claims 1 to 4, wherein the HPV16-positive or HPV16-related cancer is relapsed or refractory to standard therapy for the HPV16-positive or HPV16-related cancer. The method of any one of claims 1 to 5, wherein the human subject has previously received chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof. The method of any one of claims 1 to 6, wherein the human subject has previously received external beam radiation therapy. The method of any one of claims 1 to 7, wherein the human subject has previously received brachytherapy. The method of any one of claims 1 to 8, wherein the human subject has previously received standard platinum or mitomycin C-based chemotherapy. The method of any one of claims 1 to 9, wherein the human subject has previously received immunotherapy comprising a therapeutic vaccine, targeted antibody, or immune checkpoint therapy. The method according to claim 10, wherein the immune checkpoint therapy is PD-1/PD-L1 therapy. The method of any one of claims 1 to 11, wherein the frequency is about every 16 days to about every 26 days. The method of claim 12, wherein the frequency is about once every three weeks. The method of any one of claims 1 to 13, wherein the dose of the pharmaceutical composition comprises about 0.5 x 10 ⁹ to about 5 x 10 ⁹ human enucleated erythroid cells. The method of claim 14, wherein the dose of the pharmaceutical composition comprises about 1 x 10 ⁹ human enucleated erythroid cells. The method of any one of claims 1 to 13, wherein the dose of the pharmaceutical composition comprises about 1 x 10 ⁹ to about 1 x 10 ¹⁰ human enucleated erythroid cells. The method of claim 16, wherein the dose of the pharmaceutical composition comprises about 5 x 10 ⁹ human enucleated erythroid cells. The method of any one of claims 1 to 13, wherein the dose of the pharmaceutical composition comprises about 5 x 10 ⁹ to about 5 x 10 ¹⁰ human enucleated erythroid cells. The method of claim 18, wherein the dose of the pharmaceutical composition comprises about 1 x 10 ¹⁰ human enucleated erythroid cells. The method of any one of claims 1 to 19, wherein the human enucleated erythroid blood cells comprise at least 1,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide. The method of claim 20, wherein the human enucleated erythroid blood cells comprise at least 5,000 copies of the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide. The method of any one of claims 1 to 21, wherein the human enucleated erythroid blood cells comprise at least 1,000 copies of the exogenous costimulatory polypeptide. The method of claim 22, wherein the human enucleated erythroid cells comprise at least 5,000 copies of the exogenous costimulatory polypeptide. The method of any one of claims 1 to 23, wherein the human enucleated erythroid blood cells comprise at least 1,000 copies of the exogenous cytokine polypeptide. The method of claim 24, wherein the human enucleated erythroid cells comprise at least 5,000 copies of the exogenous cytokine polypeptide. The method according to any one of claims 1 to 25, wherein the HPV E7 antigen comprises SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The method of claim 26, wherein the HPV E7 antigen comprises SEQ ID NO: 9. The method according to any one of claims 1 to 27, wherein the β2m chain comprises SEQ ID NO: 1. The method according to any one of claims 1 to 28, wherein the exogenous antigen polypeptide is linked to the β2m chain via a linker. The method as claimed in claim 29, wherein the linker comprises SEQ ID NO: 33. The method according to any one of claims 1 to 30, wherein the alpha chain comprises an HLA-A2*02:01 polypeptide. The method of claim 31, wherein the HLA-A2*02:01 polypeptide comprises a Y84A or Y84C substitution. The method according to claim 31 or 32, wherein the HLA-A2*02:01 allele polypeptide comprises SEQ ID NO: 3, 5 or 7. The method of claim 33, wherein the HLA-A2*02:01 polypeptide comprises SEQ ID NO: 3. The method according to any one of claims 1 to 34, wherein the α-chain is connected to the β2m chain via a linker. The method as claimed in claim 35, wherein the linker comprises SEQ ID NO: 38. The method of claim 36, wherein the α-chain is connected to the membrane anchor region via a linker. The method as claimed in claim 37, wherein the linker comprises SEQ ID NO: 39. The method according to any one of claims 1 to 38, wherein the membrane anchor region comprises glycophorin A (GPA) or its transmembrane domain, or the transmembrane structure of a small integral membrane protein 1 (SMIM1) area. The method of claim 39, wherein the membrane anchor region comprises GPA. The method of claim 40, wherein the membrane anchor region comprises SEQ ID NO: 30. The method of any one of claims 1 to 41, wherein the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 15. The method of claim 42, wherein the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 15. The method according to claim 43, wherein the exogenous antigen-presenting polypeptide linked to the exogenous antigen polypeptide comprises the sequence of SEQ ID NO: 15. The method according to any one of claims 1 to 44, wherein the 4-1BBL comprises a sequence at least 90% identical to SEQ ID NO: 19. The method of claim 45, wherein the 4-1BBL comprises SEQ ID NO: 19. The method of claim 45 or 46, wherein the exogenous co-stimulatory polypeptide is fused to a membrane anchoring region. The method according to claim 47, wherein the exogenous co-stimulatory polypeptide is linked to the membrane anchor region via a linker. The method according to claim 47 or 48, wherein the membrane anchor region comprises GPA or its transmembrane domain, or a SMIM1 transmembrane domain. The method of claim 49, wherein the GPA comprises SEQ ID NO: 30. The method according to any one of claims 48 to 50, wherein the linker comprises SEQ ID NO: 36. The method according to any one of claims 1 to 51, wherein the exogenous co-stimulatory polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 21. The method of claim 52, wherein the exogenous co-stimulatory polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 21. The method of claim 53, wherein the exogenous co-stimulatory polypeptide comprises SEQ ID NO: 21. The method according to any one of claims 1 to 54, wherein the IL-12 comprises IL-12A protein and IL-12B protein. The method of claim 55, wherein the IL-12A protein comprises a sequence at least 90% identical to SEQ ID NO: 25, and the IL-12B protein comprises a sequence at least 90% identical to SEQ ID NO: 27. The method of claim 56, wherein the IL-12A protein comprises a sequence at least 95% identical to SEQ ID NO: 25, and the IL-12B protein comprises a sequence at least 95% identical to SEQ ID NO: 27. The method of claim 57, wherein the IL-12A protein comprises SEQ ID NO: 25, and the IL-12B protein comprises SEQ ID NO: 27. The method according to any one of claims 55 to 58, wherein the exogenous cytokine polypeptide further comprises a linker between the IL-12A protein and the IL-12B protein. The method of claim 59, wherein the linker comprises SEQ ID NO: 33. The method of any one of claims 1 to 60, wherein the exogenous cytokine polypeptide is fused to a membrane anchoring region. The method of claim 61, wherein the exogenous cytokine polypeptide is linked to the membrane anchor region via a linker. The method according to claim 61 or 62, wherein the membrane anchor region comprises GPA or its transmembrane domain, or a transmembrane domain of SMIM1. The method of claim 63, wherein the membrane anchor region comprises SEQ ID NO: 31. The method according to any one of claims 62 to 64, wherein the linker comprises SEQ ID NO: 33. The method according to any one of claims 1 to 65, wherein the exogenous cytokine polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 48. The method of claim 66, wherein the exogenous cytokine polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 48. The method of claim 67, wherein the exogenous cytokine polypeptide comprises SEQ ID NO: 48. The method according to any one of claims 1 to 68, wherein the human enucleated erythroid blood cells are prepared by a process comprising: introducing into nucleated erythroid precursor cells nucleic acids encoding an exogenous antigen-presenting polypeptide linked to the exogenous antigenic polypeptide, the exogenous costimulatory polypeptide, and the exogenous cytokine polypeptide; and culturing the nucleated erythroid precursor cells under conditions sufficient to express the exogenous antigen-presenting polypeptide, the exogenous costimulatory polypeptide, and the exogenous cytokine polypeptide linked to the exogenous antigen polypeptide, and denucleate the nucleated erythroid precursor cells.

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