Methods of treating cancer using enhanced antigen presenting cells

Abstract

The present application provides enhanced APCs for use in the treatment of HPV-associated cancers. The enhanced APC is capable of activating T cells in an HLA-agnostic manner and is derived from an input APC in which at least one nucleic acid encoding an HPV antigen has been delivered within the cell. In some aspects, the enhanced APC is administered in combination with an immune checkpoint inhibitor, such as an antagonist of PD-1 / PD-L1 and / or an antagonist of CTLA-4.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 369,752, filed on July 28, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the sequence listing are submitted electronically (name: 4821_088PC01_SequenceListing_ST26.XML; size: 35,417 bytes; and creation date: July 28, 2023) and are submitted with the application incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to methods of treating HPV-associated cancers in a subject using modified antigen presenting cells ("enhanced APCs") that are capable of activating T cells in an HLA-agnostic manner, and dosages and regimens thereof. Background Art

[0006] Human papillomavirus (HPV) is a virus that infects many people. In fact, more than 75% of women and men will be infected at some point in their lives. While most HPV infections are subclinical and do not cause any physical symptoms, in some people, the infection may lead to growths called papillomata, and even to cervical, vulvar, vaginal, penile, oropharyngeal, and anal cancers. Specifically, HPV16 and HPV18 are known to cause approximately 70% of cervical cancer cases. There is currently no treatment that targets HPV itself. Current treatment options are generally designed to treat the various ailments that may be associated with HPV infection. While vaccines (e.g., ) has been approved, but is essentially only preventive and is not without side effects. Therefore, new alternative treatment options for HPV infection are needed.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Patent publications WO 2013 / 059343, WO 2015 / 023982, WO 2016 / 070136, WO2017041050, WO2017008063, WO2017 / 192785, WO 2017 / 192786, WO 2019 / 178005, WO 2019 / 178006, WO 2020 / 072833, WO2020 / 154696 and WO 2020 / 176789, US 20180142198 and US 20180201889 are hereby expressly incorporated by reference in their entirety. Summary of the invention

[0008] Provided herein is a method for treating a human papillomavirus (HPV)-related cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner. In some aspects, the method further comprises administering to the subject an immune checkpoint inhibitor. Also provided herein is a method for treating a human papillomavirus (HPV)-related cancer in a subject in need thereof, the method comprising administering to the subject (i) a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner, and (ii) an immune checkpoint inhibitor.

[0009] In some aspects, the enhanced APC includes T cells, B cells, NK cells, monocytes or a combination thereof. In some aspects, the HPV antigen includes an HPV-16 antigen or an HPV-18 antigen. In some aspects, the HPV antigen includes a peptide derived from HPV E6 and / or HPVE7. In some aspects, the HPV antigen includes a peptide derived from HPV E6 and a peptide derived from HPV E7. In some aspects, the peptide derived from HPV E6 and / or HPV E7 is full-length E6 and / or E7. In some aspects, the HPV antigen comprises an amino acid sequence shown in any one of SEQ ID NOs: 14-17. In some aspects, the HPV antigen comprises a first antigen containing an amino acid sequence shown in SEQ ID NO: 14 and a second antigen containing an amino acid sequence shown in SEQ ID NO: 15. In some aspects, the HPV antigen comprises a first antigen containing an amino acid sequence shown in SEQ ID NO: 16 and a second antigen containing an amino acid sequence shown in SEQ ID NO: 17.

[0010] In some aspects, the enhanced APCs exhibit increased expression of co-stimulatory molecules compared to corresponding unmodified APCs ("reference APCs"). In some aspects, the co-stimulatory molecules include CD86. In some aspects, the enhanced APCs exhibit increased expression of cytokines compared to corresponding unmodified APCs ("reference APCs"). In some aspects, the cytokines include membrane-bound cytokines. In some aspects, the cytokines include IL-2, IL-12, or both.

[0011] In some aspects, enhanced APCs are prepared by passing a cell suspension comprising an input APC through a cell deformation contraction, thereby causing a disturbance in the APC, so that nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines enter the APC by disturbance when in contact with the APC, thereby producing enhanced APCs. In some aspects, a cell suspension comprising an input APC has been cultured in combination with nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines, so that nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines contact the input APC. In some aspects, nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines are mRNA. In some aspects, the diameter of the cell deformation contraction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

[0012] In any method provided herein, in some aspects, the enhanced APC has been regulated in a culture medium comprising an adjuvant. In some aspects, the enhanced APC has been regulated in a culture medium comprising an adjuvant for about 2 hours to about 10 hours. In some aspects, the enhanced APC has been regulated in a culture medium comprising an adjuvant for about 3 hours to about 6 hours. In some aspects, the enhanced APC has been regulated in a culture medium comprising an adjuvant for about 4 hours. In some aspects, the enhanced APC has been regulated in a culture medium comprising an adjuvant at about 37°C. In some aspects, the adjuvant includes CpG oligodeoxynucleotides (ODN), LPS, IFN-α, STING agonists, RIG-I agonists, poly I: C, R837, R848, TLR3 agonists, TLR4 agonists or TLR 9 agonists. In some aspects, the adjuvant is ODN.

[0013] For any method provided herein including administering an immune checkpoint inhibitor, in some aspects, the immune checkpoint inhibitor includes an antagonist of CTLA-4 and / or an antagonist of PD-1 / PD-L1. In some aspects, the immune checkpoint inhibitor is an antagonist of PD-1 / PD-L1. In some aspects, the antagonist of PD-1 / PD-L1 is an antibody that binds to PD-1 or an antibody that binds to PD-L1. In some aspects, the antagonist of CTLA-4 is an antibody that binds to CTLA-4. In some aspects, the antibody that binds to PD-1 is pembrolizumab. In some aspects, the antibody that binds to PD-1 is nivolumab. In some aspects, the antibody that binds to PD-L1 is atezolizumab. In some aspects, the antibody that binds to CTLA-4 is ipilimumab.

[0014] Provided herein is a method for treating HPV in a subject in need thereof. + A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering to a subject a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner.

[0015] For this method, in some aspects, the enhanced APC includes T cells, B cells, NK cells, monocytes or a combination thereof. In some aspects, the HPV antigen includes an HPV-16 antigen or an HPV-18 antigen. In some aspects, the HPV antigen includes a peptide derived from HPV E6 and / or HPV E7. In some aspects, the HPV antigen includes a peptide derived from HPV E6 and a peptide derived from HPV E7. In some aspects, the peptide derived from HPV E6 and / or HPV E7 is full-length E6 and / or E7. In some aspects, the HPV antigen comprises an amino acid sequence shown in any one of SEQID NO: 14-17. In some aspects, the HPV antigen comprises a first antigen containing an amino acid sequence shown in SEQ ID NO: 14 and a second antigen containing an amino acid sequence shown in SEQ ID NO: 15. In some aspects, the HPV antigen comprises a first antigen containing an amino acid sequence shown in SEQ ID NO: 16 and a second antigen containing an amino acid sequence shown in SEQ ID NO: 17.

[0016] In some aspects, the enhanced APCs exhibit increased expression of co-stimulatory molecules compared to corresponding unmodified APCs ("reference APCs"). In some aspects, the co-stimulatory molecules include CD86. In some aspects, the enhanced APCs exhibit increased expression of cytokines compared to corresponding unmodified APCs ("reference APCs"). In some aspects, the cytokines include membrane-bound cytokines. In some aspects, the cytokines include IL-2, IL-12, or both.

[0017] In some aspects, the enhanced APCs have been prepared by passing a cell suspension comprising an input APC through a cell deformation constriction, thereby causing a disturbance in the APC, such that nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and / or nucleic acids encoding cytokines enter the APCs through the disturbance when in contact with the APCs, thereby producing enhanced APCs. In some aspects, a cell suspension comprising an input APC has been cultured in combination with nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and / or nucleic acids encoding cytokines, such that nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and / or nucleic acids encoding cytokines contact the input APCs.

[0018] In some aspects, the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule, and / or the nucleic acid encoding the cytokine is mRNA.In some aspects, the diameter of the cell deformation constriction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

[0019] In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 2 hours to about 10 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 3 hours to about 6 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 4 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant at about 37°C. In some aspects, the adjuvant includes CpG oligodeoxynucleotides (ODN), LPS, IFN-α, STING agonists, RIG-I agonists, poly I:C, R837, R848, TLR3 agonists, TLR4 agonists, or TLR 9 agonists. In some aspects, the adjuvant is ODN.

[0020] For any of the above methods, in some aspects, a composition comprising an enhanced APC is administered in combination with an immune checkpoint inhibitor. In some aspects, one or more immune checkpoint inhibitors include antagonists of CTLA-4 and / or antagonists of PD-1 / PD-L1. In some aspects, the immune checkpoint inhibitor is an antagonist of PD-1 / PD-L1. In some aspects, the antagonist of PD-1 / PD-L1 is an antibody that binds to PD-1 or an antibody that binds to PD-L1. In some aspects, the antagonist of CTLA-4 is an antibody that binds to CTLA-4. In some aspects, the antibody that binds to PD-1 is pembrolizumab. In some aspects, the antibody that binds to PD-1 is nivolumab. In some aspects, the antibody that binds to PD-L1 is atezolizumab. In some aspects, the antibody that binds to CTLA-4 is ipilimumab.

[0021] In some aspects, the subject that can be treated using the methods provided herein is a human. In some aspects, the subject is resistant to human papillomavirus type 16 (HPV16 + ) is positive. In some aspects, HPV-related cancer and / or HPV + Cancers include cancer of the cervix, anus, vulva, vagina, penis, or oropharynx.

[0022] For any of the treatment methods provided herein, in some aspects, the composition comprising the enhanced APCs is present at about 0.25×10 6 cells / kg to about 7.50×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 0.5×10 6 cells / kg to about 5.0×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 0.5×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 2.5×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 5.0×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 0.25×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 1.25×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 3.25×10 6 In some aspects, the composition comprising the enhanced APC is administered in an amount of about 7.5×10 6 cells / kg.

[0023] For any treatment methods provided herein, in some aspects, the composition comprising enhanced APC is administered intravenously. When the method includes administering an immune checkpoint inhibitor, in some aspects, the immune checkpoint inhibitor is administered intravenously, orally or subcutaneously. In some aspects, the immune checkpoint inhibitor is administered intravenously.

[0024] In some aspects, pembrolizumab is administered in an amount of about 10 mg to about 400 mg. In some aspects, pembrolizumab is administered in an amount of about 200 mg to about 400 mg. In some aspects, pembrolizumab is administered in an amount of about 200 mg.

[0025] In some aspects, nivolumab is administered in an amount of about 15 mg to about 500 mg. In some aspects, nivolumab is administered in an amount of about 240 mg to about 480 mg. In some aspects, nivolumab is administered in an amount of about 360 mg.

[0026] In some aspects, atezolizumab is administered in an amount of about 75 mg to about 1700 mg. In some aspects, atezolizumab is administered in an amount of about 840 mg to about 1680 mg. In some aspects, atezolizumab is administered in an amount of about 1200 mg.

[0027] In some aspects, ipilimumab is administered in an amount of about 1 mg / kg to about 10 mg / kg. In some aspects, ipilimumab is administered in an amount of about 1 mg / kg to about 3 mg / kg.

[0028] For any of the treatment methods provided herein, in some aspects, the composition comprising the enhanced APCs is administered on day 1 of a three-week cycle. In some aspects, the composition comprising the enhanced APCs is further administered on day 2 of the first three-week cycle. In some aspects, about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg or about 7.50×10 6 In some aspects, about 0.25×10 cells / kg are administered on day 2 of the first three-week cycle. 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg or about 7.50×10 6 cells / kg.

[0029] In some aspects, antibodies that bind to PD-1, antibodies that bind to PD-L1, and / or antibodies that bind to CTLA-4 are administered once every three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 1st day of a three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 8th day of the first three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 8th day of the first three-week cycle and on the 1st day of each subsequent three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 1st day of the third three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 1st day of the third three-week cycle and on the 1st day of each subsequent three-week cycle. In some aspects, antibodies that bind to PD-1 are administered on the 1st day of the third three-week cycle and on the 1st day of each subsequent three-week cycle. In some aspects, antibodies that bind to PD-1 are pembrolizumab, wherein pembrolizumab is administered in an amount of about 200 mg. In some aspects, antibodies that bind to PD-1 are nivolumab, wherein nivolumab is administered in an amount of about 360 mg. In some aspects, antibodies that bind to CTLA-4 are administered on the 1st day of each three-week cycle. In some aspects, the antibody that binds to CTLA-4 is administered once every two three-week cycles. In some aspects, the antibody that binds to CTLA-4 is ipilimumab, wherein ipilimumab is administered at a dose of about 3 mg / kg. In some aspects, the antibody that binds to PD-L1 is administered on day 8 of the first three-week cycle and on day 1 of each subsequent cycle. In some aspects, the antibody that binds to PD-L1 is atezolizumab, wherein atezolizumab is administered at a dose of about 1200 mg.

[0030] For any of the treatment methods provided herein, in some aspects, the composition comprising enhanced APCs is administered to the subject for at least about 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months.

[0031] In some aspects, the composition comprising enhanced APCs comprises: (a) about 5×10 6 Enhanced APC to about 1x10 9 enhanced APCs, (b) a cryopreservation medium at a concentration of about 40% to about 95% (weight / weight), (c) a cryopreservation medium at a concentration of about 25% to about 35% (weight / weight), and (d) a human serum albumin solution at a concentration of 15% to about 25% (weight / weight), wherein the pH of the composition is about pH 6.0 to about pH 8.5. In some aspects, the composition comprising enhanced APCs comprises: (a) about 8.1×10 7enhanced APCs, (b) a cryopreservation medium at a concentration of about 50% (weight / weight), (c) a cryopreservation medium at a concentration of about 30% (weight / weight), and (d) a human serum albumin solution at a concentration of 20% (weight / weight), wherein the pH of the composition is about pH 7.0 to about pH 7.9. In some aspects, the composition comprising enhanced APCs comprises: (a) about 1.05×10 8 The invention relates to a method of treating a humanized APC comprising: (a) a cryopreservation medium having a concentration of about 50% (weight / weight) and (b) a cryopreservation medium having a concentration of about 30% (weight / weight), and (d) a human serum albumin solution having a concentration of 20% (weight / weight), wherein the pH of the composition is about pH 7.0 to about pH 7.9.

[0032] For any of the methods provided herein, in some aspects, the composition comprising the enhanced APC comprises about 1×10 6 Enhanced APC / mL to about 1x10 8 Enhanced APCs / mL. In some aspects, the composition comprising enhanced APCs comprises about 8.5×10 6 Enhanced APC / mL. In some aspects, the composition comprising enhanced APC comprises about 1.1×10 7 Enhanced APC / mL.

[0033] In some aspects, the cryopreservation medium is CS10. In some aspects, the cryopreservation medium is FRS.

[0034] Provided herein is a method for treating human papillomavirus (HPV)-related cancer in a subject in need thereof, the method comprising: administering to the subject: (i) a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner, and (ii) an antagonist of PD-1. Also provided herein is a method for treating HPV in a subject in need thereof. + A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), and wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner.

[0035] In some aspects, the composition comprising the enhanced APC is administered in combination with an antagonist of PD-1. In some aspects, the antagonist of PD-1 is an antibody that binds to PD-1. In some aspects, the antibody that binds to PD-1 is pembrolizumab.

[0036] In some aspects, the enhanced APCs have been prepared by passing a cell suspension comprising an input APC through a cell deformation constriction, thereby causing a disturbance in the APC, such that nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and nucleic acids encoding cytokines enter the APCs through the disturbance when in contact with the APCs, thereby producing enhanced APCs. In some aspects, a cell suspension comprising an input APC has been cultured in combination with nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and nucleic acids encoding cytokines, such that nucleic acids encoding antigens, nucleic acids encoding co-stimulatory molecules, and nucleic acids encoding cytokines contact the input APCs.

[0037] In some aspects, the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule, and the nucleic acid encoding the cytokine are mRNA.In some aspects, the diameter of the cell deformation constriction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

[0038] In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 2 hours to about 10 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 3 hours to about 6 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant for about 4 hours. In some aspects, the enhanced APC has been conditioned in a culture medium comprising an adjuvant at about 37°C. In some aspects, the adjuvant includes CpG oligodeoxynucleotides (ODN), LPS, IFN-α, STING agonists, RIG-I agonists, poly I:C, R837, R848, TLR3 agonists, TLR4 agonists, or TLR 9 agonists. In some aspects, the adjuvant is ODN. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Modified APCs with intracellular HPV-16E6 mRNA, HPV-16E7 mRNA, CD86 mRNA, mbIL-2 mRNA, and mbIL-12 mRNA are shown. The modified APCs present various types of HLA molecules, HPV-16E6 and HPV-16E7 epitopes, CD86, mbIL-2, and mbIL-12 on their cell surfaces.

[0040] Figure 2A schematic diagram showing an exemplary mechanism of action of the enhanced APCs described herein, which are generated from peripheral blood mononuclear cells (PBMCs) treated with mRNA extrusion encoding one or more HPV antigens, co-stimulatory molecules and / or membrane-bound cytokines. The enhanced APCs migrate to lymphoid organs, where presentation of the E6 and E7 epitopes stimulates E6-specific and E7-specific T cells that are capable of generating antibodies against HPV16. + specific anti-tumor response of cells while minimizing the risk of accidentally attacking healthy tissue.

[0041] Figure 3 Anti-tumor immune response after enhanced APC treatment is shown. The top graph (i.e., bar graph) shows the percentile increase or decrease in CD8, HLA-1, E6, and PD-L1 expression in tumor cells 28 days after screening date and enhanced APC treatment. The bottom graph shows the corresponding immunohistochemical staining of CD8, HLA-1, E6, and PD-L1 in tumor biopsies 28 days after screening date and enhanced APC treatment.

[0042] Figure 4 A schematic diagram of the clinical study design is shown, wherein during the monotherapy phase, monotherapy of enhanced APC is administered at the indicated dose every three weeks (q3w), followed by a combination phase consisting of administration of enhanced APC in combination with 200 mg of pembrolizumab every three weeks (q3w); and a lead-in phase consisting of administration of enhanced APC as monotherapy for 6 weeks, followed by combination therapy of enhanced APC with 200 mg of pembrolizumab every three weeks (q3w).

[0043] Figure 5 A schematic diagram of monotherapy dosing with enhanced APCs administered on day 1 of each cycle is shown.

[0044] Figure 6 Shown is the staggered enrollment of the first two patients in each cohort in a clinical study design involving the administration of enhanced APCs.

[0045] Figure 7 A safety schematic of the clinical study design involving the administration of enhanced APC in combination therapy with pembrolizumab is shown.

[0046] Figure 8 A schematic diagram of the clinical study design involving the administration of enhanced APCs in combination therapy with pembrolizumab is shown.

[0047] Fig. 9 Shown is a schematic diagram of dosage administration modifications made in response to adverse events associated with infusion of enhanced APCs as part of the safety assessment of a clinical study of therapy involving administration of enhanced APCs. DETAILED DESCRIPTION

[0048] The present application generally relates to the use of enhanced APCs to treat human papillomavirus (HPV)-related cancers in subjects in need thereof. The enhanced APCs have been modified so that the APCs exhibit one or more enhanced properties. Non-limiting examples of such enhanced properties are provided throughout the present disclosure. Unless otherwise indicated, the terms "enhanced APCs" (or derivatives thereof) and "modified APCs" (or derivatives thereof) are used interchangeably to describe such APCs. As further described herein, the difference between enhanced APCs and other APCs is that the cells are able to activate T cells in an HLA-agnostic manner. Therefore, the methods provided herein can be used in various clinical settings and allow treatment of different subjects regardless of HLA haplotype. Other aspects of the present disclosure are further provided below.

[0049] General Technology

[0050] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely employed using conventional methods, such as, for example, the widely used methods described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, J.Wiley and Sons, 1993-8); Handbook of ExperimentalImmunology (DMWeir and CC Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011).

[0051] definition

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions) shall prevail. Unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. All publications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

[0053] Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are exemplary only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the specific embodiments and claims.

[0054] To further define the present disclosure, the following terms and definitions are provided.

[0055] Unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include plural referents. The terms "a / an" and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the terms "a / an" mean "single". In other aspects, the terms "a / an" include "two or more" or "plurality".

[0056] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents are intended to be equivalent in meaning and to be open ended, in that the items following any one of these words are not meant to be an exhaustive list of the items, nor are they meant to be limited to the listed items. For example, an article "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and its grammatical equivalents include disclosures in terms of "consisting essentially of" or "consisting of."

[0057] As used herein, the term "about" refers to the usual error range of the corresponding value that is easily known to those skilled in the art, and is used herein to indicate approximately, roughly, near or in the region. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical values ​​shown. Generally speaking, the term "about" is used herein to modify the numerical value to be higher and lower than the stated value by changing 10% upward or downward (increasing or decreasing). In addition, "about" cited herein as a value or parameter includes and describes aspects for the value or parameter itself. For example, a description referring to "about X" includes the "X" described.

[0058] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range (to the tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other intervening values ​​stated or in the stated range are encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the stated limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0059] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount or quantity of a drug or pharmaceutically active substance sufficient to elicit the desired or expected therapeutic response, or in other words, an amount sufficient to elicit a significant biological response when administered to a patient.

[0060] As used herein, the term "HLA-agnostic manner" means being independent of human leukocyte antigen (HLA) haplotypes. In general, T cell activation requires recognition of antigens (as peptide fragments) presented on "human leukocyte antigens" or "HLAs" expressed on certain cells. Due to the variability of HLA molecules, certain peptides are restricted or only bind to certain HLA molecules. Therefore, whether a specific antigenic peptide fragment induces an immune response in a subject depends largely on the specific HLA molecules present in the subject. It is apparent from the present disclosure that since the enhanced APCs described herein are able to activate T cells in an HLA-agnostic manner, they can produce therapeutic effects in larger populations.

[0061] When the term "heterologous" relates to nucleic acid sequences (such as coding sequences and control sequences), it means sequences that are not usually connected together and / or are not usually associated with a specific cell. Therefore, the "heterologous" region of a nucleic acid construct or a vector is a nucleic acid fragment that is located within or attached to another nucleic acid molecule, and the nucleic acid fragment is not associated with the other molecule in nature. For example, the heterologous region of a nucleic acid construct may include a coding sequence, the flank of which is a sequence that is not found to be associated with the coding sequence in nature. Another example of a heterologous coding sequence is a construct that the coding sequence itself is not found in nature (for example, a synthetic sequence with codons different from natural genes). Similarly, for the purposes of this disclosure, cells transformed with constructs that are not usually present in cells will be considered heterologous. Allelic variation or naturally occurring mutation events will not produce heterologous DNA as used herein.

[0062] The term "heterologous" refers to amino acid sequences (such as peptide sequences and polypeptide sequences) when it means sequences that are not usually connected together and / or are not usually associated with a specific cell. Therefore, the "heterologous" region of a peptide sequence is an amino acid fragment located within or attached to another amino acid molecule, and the amino acid fragment is not associated with the other molecule in nature. For example, the heterologous region of a peptide construct may include an amino acid sequence of a peptide, and the flanks of the amino acid sequence are sequences that are not found in nature to be associated with the amino acid sequence of the peptide. Another example of a heterologous peptide sequence is a construct that the peptide sequence itself is not found in nature (e.g., a synthetic sequence with different amino acids encoded by a natural gene). Similarly, for the purposes of this disclosure, cells transformed with vectors expressing amino acid constructs that are not usually present in cells will be considered heterologous. Allelic variation or naturally occurring mutation events will not produce heterologous peptides as used herein.

[0063] When the term "exogenous" is used to refer to an agent associated with a cell (such as an antigen or adjuvant), it refers to an agent outside the cell or an agent delivered to the cell from outside the cell. The cell may or may not have the agent already present, and may or may not produce the agent after the exogenous agent is delivered.

[0064] As used herein, the term "homologous" refers to molecules from the same organism. In some aspects, the term refers to nucleic acids or proteins that are normally found or expressed in a given organism.

[0065] As used herein, the term "autologous" refers to biological material (eg, cells or tissue) obtained from the same subject. In some aspects, a subject's "autologous" cells or tissue are placed back into the same subject.

[0066] As used herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result (including a clinical result). For the purposes of this disclosure, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving or improving the quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses reducing the pathological consequences of cancer (e.g., tumor volume). The methods of this disclosure contemplate any one or more of these therapeutic aspects. In the context of cancer, "treatment" includes killing cancer cells, inhibiting cancer cell growth, inhibiting cancer cell replication, reducing overall tumor burden, and improving any or all of one or more symptoms associated with the disease.

[0067] As used herein, the term "prophylactic treatment" refers to treatment performed when it is known or suspected that the individual suffers from a condition or is at risk for a condition but does not yet show symptoms of the condition or has minimal symptoms. An individual receiving prophylactic treatment may receive treatment before symptoms develop. In some aspects, an individual may be treated if the individual suffers from a precancerous lesion, particularly a precancerous lesion associated with HPV infection.

[0068] As used herein, the term "combination therapy" means that the first agent can be administered in combination with another agent. "Combination" refers to administering a treatment modality in addition to another treatment modality, such as administering a PMBC composition as described herein in addition to administering an immunoconjugate as described herein to the same individual. Therefore, "combination" refers to administering a treatment modality before, during, or after administering another treatment modality to an individual.

[0069] As used herein, the term "simultaneous administration" means that the first therapy and the second therapy in the combination therapy are administered at a time interval of no more than about 15 minutes, such as no more than about any of 10, 5 or 1 minute. When the first therapy and the second therapy are administered simultaneously, the first therapy and the second therapy may be included in the same composition (e.g., a composition comprising the first therapy and the second therapy) or in separate compositions (e.g., the first therapy is included in one composition and the second therapy is included in another composition).

[0070] As used herein, the term "sequential administration" means that the first therapy and the second therapy in the combination therapy are administered at a time interval greater than about 15 minutes, such as greater than about any of 20, 30, 40, 50, 60 or more minutes. The first therapy may be administered first, or the second therapy may be administered first. The first therapy and the second therapy are contained in separate compositions, which may be contained in the same or different packages or kits.

[0071] As used herein, the term "simultaneous administration" means that the administration of a first therapy and the administration of a second therapy in the combination therapy overlap with each other.

[0072] As used herein, the term "regulate" may refer to an action that changes, alters, changes, or otherwise modifies the presence or activity of a particular target. For example, regulating an immune response may refer to any action that causes a change, alteration, change, or otherwise modifies an immune response. In some aspects, "regulate" refers to enhancing the presence or activity of a particular target. In some aspects, "regulate" refers to inhibiting the presence or activity of a particular target. In other aspects, regulating the expression of a nucleic acid may include, but is not limited to, changes in nucleic acid transcription, changes in mRNA abundance (e.g., increasing mRNA transcription), corresponding changes in mRNA degradation, changes in mRNA translation, and the like.

[0073] As used herein, the term "inhibition" refers to the act of blocking, reducing, eliminating or otherwise antagonizing the presence or activity of a specific target. Inhibition may refer to partial inhibition or complete inhibition. In some aspects, inhibiting an immune response may refer to any behavior that results in blocking, reducing, eliminating or any other antagonistic immune response. In other aspects, inhibiting the expression of nucleic acids may include, but is not limited to, reducing the transcription of nucleic acids, reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, gene editing, and the like. In other aspects, inhibiting the expression of proteins may include, but is not limited to, reducing the transcription of nucleic acids encoding proteins, reducing the stability of mRNA encoding proteins, inhibiting the translation of proteins, reducing the stability of proteins, and the like. In another aspect, inhibition may refer to the act of slowing or stopping growth; for example, delaying or preventing the growth of tumor cells.

[0074] As used herein, the term "inhibit" may refer to the behavior of reducing, lowering, prohibiting, limiting, alleviating or otherwise weakening the presence or activity of a specific target. Inhibition may refer to partial inhibition or complete inhibition. For example, inhibiting an immune response may refer to any behavior that causes reduction, reduction, prohibition, limitation, alleviating or otherwise weakening an immune response. In some aspects, inhibiting the expression of nucleic acids may include but is not limited to reducing the transcription of nucleic acids, reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, and the like. In other aspects, inhibiting the expression of proteins may include but is not limited to reducing the transcription of nucleic acids encoding proteins, reducing the stability of mRNA encoding proteins, inhibiting the translation of proteins, reducing the stability of proteins, and the like.

[0075] As used herein, the term "enhancement" may refer to improving, promoting, strengthening or otherwise increasing the presence or activity of a specific target. For example, enhancing an immune response may refer to any behavior that causes improvement, promotion, strengthening or otherwise increasing an immune response. In one aspect, enhancing an immune response may refer to using antigens and / or adjuvants to improve, promote, strengthen or otherwise increase an immune response. In some aspects, enhancing the expression of nucleic acids may include but is not limited to increasing the transcription of nucleic acids, increasing mRNA abundance (e.g., increasing mRNA transcription), reducing mRNA degradation, increasing mRNA translation, etc. In other aspects, enhancing the expression of proteins may include but is not limited to increasing the transcription of nucleic acids encoding proteins, increasing the stability of mRNA encoding proteins, increasing the translation of proteins, increasing the stability of proteins, etc.

[0076] As used herein, the term "induce" may refer to an action that initiates, prompts, stimulates, establishes, or otherwise produces a result. For example, inducing an immune response may refer to any action that causes initiation, prompts, stimulates, establishes, or otherwise produces a desired immune response. In some aspects, inducing the expression of nucleic acids may include, but is not limited to, initiating transcription of nucleic acids, initiating mRNA translation, and the like. In other aspects, inducing the expression of proteins may include, but is not limited to, increasing transcription of nucleic acids encoding proteins, increasing the stability of mRNA encoding proteins, increasing the translation of proteins, increasing the stability of proteins, and the like.

[0077] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Therefore, this term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine bases and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may include sugar and phosphate groups (usually found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide may include repeating units, such as N-(2-aminoethyl)-glycine, connected by peptide bonds (i.e., peptide nucleic acids). Alternatively, the backbone of a polynucleotide may include a polymer of synthetic subunits (such as phosphoamide and thiophosphate), and therefore may be oligodeoxynucleoside phosphoamide (P-NH2) or mixed thiophosphate-phosphodiester oligomers or mixed phosphoamide-phosphodiester oligomers. Furthermore, a double-stranded polynucleotide can be obtained from a chemically synthesized single-stranded polynucleotide product by synthesizing complementary chains and annealing the chains under appropriate conditions, or by de novo synthesis of complementary chains using a DNA polymerase and appropriate primers.

[0078] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to proteins including modifications to the native sequence, such as deletions, additions and substitutions (usually of a conservative nature), as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or errors due to PCR amplification.

[0079] As used herein, the term "adjuvant" refers to a substance that modulates and / or causes an immune response. Generally, administration of an adjuvant in conjunction with an antigen enhances the immune response to the antigen compared to administration of the antigen alone. Various adjuvants are described herein.

[0080] The terms "CpG oligodeoxynucleotide", "CpG ODN" and "ODN" herein refer to DNA molecules of 10 to 30 nucleotides in length, which contain cytosine and guanine dinucleotides separated by phosphate (also referred to herein as "CpG" dinucleotides or "CpG"). The CpG ODN of the present disclosure contains at least one unmethylated CpG dinucleotide. That is, the cytosine in the CpG dinucleotide is not methylated (i.e., not 5-methylcytosine). The CpG ODN may have a partial or complete phosphorothioate (PS) backbone.

[0081] As used herein, the term "pharmaceutically acceptable" or "pharmacologically compatible" means that a substance is non-biological or undesirable, for example, the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicology and manufacturing testing and / or are included in the inactive ingredient guidelines established by the U.S. Food and Drug Administration.

[0082] As used herein, the term "peripheral blood mononuclear cell" or "PBMC" refers to a heterogeneous blood cell population with a round nucleus. Examples of cells that can be found in a PBMC population include lymphocytes, such as T cells, B cells, NK cells (including natural killer T cells (NKT cells) and cytokine-induced killer cells (CIK cells)) and monocytes, such as macrophages and dendritic cells.

[0083] The term "enhanced antigen presenting cell" or "eAPC" refers to an immune cell that has been modified to exhibit enhanced function. For example, as described herein, the eAPC described herein is derived from PBMCs that have been modified using extrusion treatment to contain one or more nucleic acid constructs (e.g., mRNA encoding HPV antigens, mRNA encoding co-stimulatory molecules, and / or mRNA encoding cytokines). The intracellular delivery of these constructs changes one or more properties of PBMCs (e.g., expressing encoded antigens on their surfaces so that they can activate antigen-specific T cells and show increased expression of co-stimulatory molecules and / or cytokines), so that after delivery, the modified cells are different in structure and / or function from PBMCs, so that they can enhance T cell responses to current antigens.

[0084] As used herein, "microfluidic system" refers to a system for processing low volume (e.g., mL, nL, pL, fL) fluids to achieve discrete processing of small volumes of liquids. Certain implementations described herein include multiplexing, automation, and high-throughput screening. Fluids (e.g., buffers, solutions, solutions containing payloads, or cell suspensions) can be moved, mixed, separated, or otherwise processed. In some aspects, microfluidic systems are used to apply mechanical constrictions to cells suspended in buffers, thereby causing disturbances (e.g., holes) in cells so that payloads or compounds can enter the cytosol of cells.

[0085] As used herein, a "constriction" may refer to a portion of a microfluidic channel defined by an inlet portion, a center point, and an outlet portion, wherein the center point is defined by width, length, and depth. In other examples, a constriction may refer to a hole, or may be a portion of a hole. The hole may be contained on a surface (e.g., a filter and / or a membrane).

[0086] For any of the structural and functional features described herein, methods of determining such features are known in the art.

[0087] Treatment

[0088] In some aspects, a method of treating an HPV-related disease in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising an enhanced APC, wherein the enhanced APC comprises an HPV antigen and is capable of activating T cells in an HLA-agnostic manner. In some aspects, the method further comprises administering to the subject an immune checkpoint inhibitor.

[0089] In some aspects, provided are methods of treating an HPV-related cancer in a subject in need thereof, the methods comprising administering to the subject (i) a composition comprising an enhanced APC, wherein the enhanced APC comprises an HPV antigen and is capable of activating T cells in an HLA-agnostic manner, and (ii) an immune checkpoint inhibitor.

[0090] In some aspects, a method of treating an HPV-related disease in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising an enhanced APC, wherein the enhanced APC is administered at a concentration of 0.25×10 6 cells / kg to about 7.50×10 6 The method further comprises administering to the subject an amount of 10 cells / kg, and wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner. In some aspects, the method further comprises administering to the subject an immune checkpoint inhibitor.

[0091] In some aspects, a method for treating HPV in a subject in need thereof is provided. +A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering to a subject a composition comprising an enhanced APC, wherein the enhanced APC comprises an HPV antigen and is capable of activating T cells in an HLA-agnostic manner. In some aspects, the composition comprising the enhanced APC is administered in combination with an immune checkpoint inhibitor.

[0092] In some aspects, HPV-related cancers include cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, or oropharyngeal cancer. + Cancers include cancer of the cervix, anus, vulva, vagina, penis, or oropharynx.

[0093] In some aspects, the subject is a human. In some aspects, the subject is an adult. In other aspects, the subject is an adolescent. In other aspects, the subject is a child. In some aspects, the subject is resistant to human papillomavirus type 16 (HPV16 + ) was positive.

[0094] In some aspects, the enhanced APCs include T cells, B cells, NK cells, monocytes, or a combination thereof.

[0095] In some aspects, the enhanced APC comprises an HPV antigen, and the HPV antigen comprises an HPV-16 antigen. In some aspects, the enhanced APC comprises an HPV antigen, and the HPV antigen comprises an HPV-18 antigen. In some aspects, the HPV antigen comprises a peptide derived from HPVE6 (e.g., an E6 protein of HPV-16 or HPV-18, each of which has a length of 158 amino acids). In some aspects, the HPV antigen comprises a peptide derived from HPV E7 (e.g., an E7 protein of HPV-16, having a length of 98 amino acids, or an E7 protein of HPV-18, having a length of 105 amino acids). In other aspects, the HPV antigen comprises a peptide derived from HPV E6 and a peptide derived from HPV E7. In some aspects, the peptide derived from HPV E6 is a full-length E6 (e.g., an E6 protein of HPV-16 or HPV-18, each of which has a length of 158 amino acids). In some aspects, the peptide derived from HPV E7 is full-length E7 (e.g., the E7 protein of HPV-16, which is 98 amino acids in length, or the E7 protein of HPV-18, which is 105 amino acids in length). In other aspects, the peptide derived from HPV E6 is full-length E6, and the peptide derived from HPV E7 is full-length HPV E7.

[0096] The amino acid sequence of the full-length E6 protein of HPV-16 is shown in SEQ ID NO: 14 (see Table A below). The amino acid sequence of the full-length E7 protein of HPV-16 is shown in SEQ ID NO: 15 (see Table A below). The amino acid sequence of the full-length E6 protein of HPV-18 is shown in SEQ ID NO: 16 (see Table A below). The amino acid sequence of the full-length E7 protein of HPV-18 is shown in SEQ ID NO: 17 (see Table A below).

[0097] Table A. Exemplary HPV E6 and E7 protein sequences

[0098]

[0099]

[0100] Thus, in some aspects, the methods provided herein include administering a composition comprising an enhanced APC, the enhanced APC comprising an HPV antigen, wherein the HPV antigen comprises the amino acid sequence shown in SEQ ID NO: 14, and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner. In some aspects, the methods provided herein include administering a composition comprising an enhanced APC, the enhanced APC comprising an HPV antigen, wherein the HPV antigen comprises the amino acid sequence shown in SEQ ID NO: 15, and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner. In some aspects, the methods provided herein include administering a composition comprising an enhanced APC, the enhanced APC comprising an HPV antigen, wherein the HPV antigen comprises the amino acid sequence shown in SEQ ID NO: 16, and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner. In some aspects, the methods provided herein include administering a composition comprising an enhanced APC, the enhanced APC comprising an HPV antigen, wherein the HPV antigen comprises the amino acid sequence shown in SEQ ID NO: 17, and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner.

[0101] In some aspects, the HPV antigens include variants of the full-length HPV E6 protein ("HPV E6 variant"). In some aspects, the HPV E6 variant is a fragment of the full-length HPV E6 protein. For example, in some aspects, the HPV E6 variant comprises about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or about 155 amino acid fragments of SEQ ID NO: 14 or SEQ ID NO: 16. In some aspects, the HPV E7 variant comprises about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 95 amino acid fragments of SEQ ID NO: 15. In some aspects, the HPV E7 variant comprises a fragment of about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids of SEQ ID NO:17.

[0102] In some aspects, the HPV E6 variant comprises one or more amino acid substitutions compared to the corresponding wild-type HPV E6 protein. Thus, in some aspects, the HPV E6 variant comprises one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 14. In some aspects, the HPV E6 variant comprises one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 16. In some aspects, the HPV E6 variant comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, or about 99% sequence identity with the sequence shown in SEQ ID NO: 14. In some aspects, the HPV E6 variant comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, or about 99% sequence identity with the sequence shown in SEQ ID NO: 16. Similarly, in some aspects, the HPV E7 variant comprises one or more amino acid substitutions compared to the corresponding wild-type HPV E7 protein. In some aspects, the HPV E7 variant comprises one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 15. In some aspects, the HPV E7 variant comprises one or more amino acid substitutions compared to the sequence set forth in SEQ ID NO: 17. In some aspects, the HPV E7 variant comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, or about 99% sequence identity to the sequence set forth in SEQ ID NO: 15. In some aspects, the HPV E7 variant comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, or about 99% sequence identity to the sequence set forth in SEQ ID NO: 17.

[0103] In some aspects, the enhanced APCs of the methods provided herein comprise a variety of antigens. In some aspects, the enhanced APCs comprise HPV antigens and non-HPV antigens. For example, in some aspects, the enhanced APCs comprise a variety of HPV antigens. In some aspects, the enhanced APCs comprise at least two, at least three, at least four, or at least five HPV antigens. In some aspects, the enhanced APCs comprise two HPV antigens, wherein the first HPV antigen is an HPV E6 protein (e.g., a full-length E6 protein of HPV-16) and the second HPV antigen is an HPV E7 protein (e.g., a full-length E7 protein of HPV-16).

[0104] In some aspects, any antigen described herein can be introduced into an input APC using any suitable method known in the art to produce an enhanced APC. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to cells include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof. In some aspects, antigens (e.g., HPV antigens) are introduced into input APCs using the constriction-mediated delivery described herein. As further described elsewhere in this disclosure, when cells pass through a constriction, they are temporarily deformed, causing the cell membrane of the cell to be disturbed. Disturbances within the cell membrane can allow various payloads (e.g., nucleic acids encoding HPV antigens, co-stimulatory molecules, and / or cytokines) to enter the cell through disturbance (e.g., by diffusion). The specific process in which cells pass through a constriction and are temporarily deformed is referred to herein as "extrusion processing." 、 "Extrusion delivery" or "extrusion".

[0105] Thus, in some aspects, the enhanced cells described herein (e.g., comprising HPV antigens) have been passed through a constriction under a set of parameters, thereby causing a perturbation within the APC such that the antigen can enter the APC through the perturbation when in contact with the APC. More specifically, in some aspects, the enhanced APCs of the methods described herein have been passed through a constriction under a set of parameters, thereby causing a perturbation within the APC such that a nucleic acid encoding an HPV E6 protein (e.g., a full-length E6 protein of HPV-16) enters the APC when in contact with the APC. In some aspects, the enhanced APCs of the methods described herein have been passed through a constriction under a set of parameters, thereby causing a perturbation within the APC such that a nucleic acid encoding an HPV E7 protein (e.g., a full-length E7 protein of HPV-16) enters the APC when in contact with the APC. In some aspects, the enhanced APC of the methods described herein has been passed through a constriction under a set of parameters, thereby causing a disturbance within the APC, so that a nucleic acid encoding an HPV E6 protein (e.g., a full-length E6 protein of HPV-16) and a nucleic acid encoding an HPV E7 protein (e.g., a full-length E7 protein of HPV-16) enter the APC when in contact with the APC. In some aspects, the nucleic acid encoding the HPV E6 protein comprises mRNA. In some aspects, the nucleic acid encoding the HPV E7 protein comprises mRNA. In other aspects, the nucleic acid encoding the HPV E6 protein and the nucleic acid encoding the HPV E6 protein each comprise mRNA.

[0106] In some aspects, the enhanced APCs useful in the present disclosure also exhibit increased expression of co-stimulatory molecules compared to a corresponding APC that has not been modified as described herein ("reference APC"). In some aspects, the enhanced APCs described herein also exhibit increased expression of cytokines compared to the reference APC. In some aspects, the enhanced APCs described herein also exhibit increased expression of co-stimulatory molecules and cytokines compared to the reference APC.

[0107] As is generally understood in the art, multiple signals are required for optimal activation of T cells: (1) "Signal 1": an antigen-specific signal provided by binding of the antigenic peptide complexed with the TCR to the MHC; (2) "Signal 2": mediated by binding of co-stimulatory molecules (such as CD80 and CD86) on antigen presenting cells (APCs); and (3) "Signal 3": mediated by cytokines (e.g., IL-2 and / or IL-12). Thus, methods comprising administering a composition comprising the enhanced APCs described herein can induce a greatly enhanced immune response compared to methods comprising administering a composition comprising a reference APC that has not been modified as described herein (e.g., does not exhibit increased expression of co-stimulatory molecules and / or cytokines). In some aspects, the enhanced immune response comprises: (i) an increase in the magnitude of the induced immune response compared to an immune response induced by a method comprising administering a composition comprising a reference APC, (ii) an increase in the breadth of the induced immune response compared to an immune response induced by a method comprising administering a composition comprising a reference APC, (iii) an increase in the duration of the induced immune response compared to an immune response induced by a method comprising administering a composition comprising a reference APC, or (iv) any combination of (i) to (iii).

[0108] In some aspects, the cytokine includes a type I cytokine. In some aspects, the cytokine includes IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-1α, IL-1β, IL-1ra, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36ra, IL-37, IL-38, IL-3, IL-5, IL-6, IL-11, IL-13, IL-23, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), stem cell factor (SCFA), leukemia inhibitory factor (LIF ... Cytokine (SCF), thrombopoietin (TPO), macrophage-colony stimulating factor (M-CSF), erythropoietin (EPO), Flt-3, IFN-α, IFN-β, IFN-γ, IL-19, IL-20, IL-22, IL-24, TNF-α, TNF-β, BAFF, APRIL, lymphotoxin β (TNF-γ), IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-25, TSLP, IL-35, IL-27, TGF-β or a combination thereof. In some aspects, cytokines include IL-12. In some aspects, cytokines include IL-2. In some aspects, cytokines include IL-12 and IL-2. In some aspects, cytokines include membrane-bound forms of cytokines ("membrane-bound cytokines"). The amino acid sequences of exemplary membrane-bound cytokines are shown in SEQ ID NO:7-10 and 13.

[0109] In some aspects, the costimulatory molecules include OX40, OX40L, CD27, CD70, CD40, CD40L, 4-1BB, 4-1BBL, CD28, CD80, CD86, ICOS, ICOSL or its related molecules or any combination thereof. In some aspects, the costimulatory molecules include CD80. In some aspects, the costimulatory molecules include CD86.

[0110] Thus, at least as apparent from the above disclosure, a method is provided herein, comprising administering a composition comprising an enhanced APC comprising an HPV antigen, wherein the enhanced APC exhibits increased expression of co-stimulatory molecules and / or cytokines compared to a corresponding APC that is not modified (e.g., as described herein), and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner. More specifically, in some aspects, the method as described herein comprises administering a composition comprising an enhanced APC comprising an HPV E6 protein or a variant thereof (e.g., a full-length HPV-16 E6 protein) and / or an HPV E7 protein or a variant thereof (e.g., a full-length HPV-16 E7 protein), wherein the enhanced APC exhibits increased expression of co-stimulatory molecules (e.g., CD86) and cytokines (e.g., membrane-bound IL-2 and / or membrane-bound IL-12) compared to a corresponding reference APC that is not modified (e.g., as described herein), and wherein the enhanced APC is capable of activating T cells in an HLA-agnostic manner.

[0111] Not bound by any theory, in some aspects, nucleic acids encoding costimulatory molecules and / or nucleic acids encoding cytokines can be introduced into APC to modify them, so as to show that the expression of costimulatory molecules and / or cytokines increases. In some aspects, such nucleic acids can be introduced into APC using the delivery mediated by the contraction as described herein. In some aspects, the enhanced cells described herein (for example, comprising HPV antigens) have passed through the contraction under a set of parameters, thereby causing disturbances in APC, so that any of the following nucleic acids enter APC by disturbances when contacting with APC: (i) nucleic acids encoding HPV E7 proteins (for example, the full-length E7 proteins of HPV-16), (ii) nucleic acids encoding HPV E6 proteins (for example, the full-length E6 proteins of HPV-16), (iii) nucleic acids encoding costimulatory molecules (for example, CD86), (iv) nucleic acids encoding cytokines (for example, membrane-bound IL-2 and / or membrane-bound IL-12), or (v) (i) to (iv) any combination. In some aspects, nucleic acids include mRNA.

[0112] In some aspects, any of the enhanced APCs provided herein (e.g., comprising HPV antigens and / or exhibiting increased expression of co-stimulatory molecules and / or cytokines) can be modulated such that the APC exhibits improved properties compared to an unmodulated corresponding APC. It will be apparent from this disclosure that in some aspects, the enhanced APCs described herein have been incubated in the presence of an adjuvant such that the APCs are modulated. In some aspects, the enhanced APCs described herein have been incubated with an adjuvant for about 2 hours to about 10 hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours to about 4 hours, about 3 hours to about 10 hours, about 3 hours to about 9 hours, about 3 hours to about 8 hours, about 3 hours to about 7 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, about 3 hours to about 4 hours, about 4 hours to about 10 hours, about 4 hours to about 9 hours, about 4 hours to about 8 hours, about 4 hours to about 7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 3 hours to about 4 hours, about 4 hours to about 10 hours, about 4 hours to about 9 hours, about 4 hours to about 8 hours, about 4 hours to about 7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours. In some aspects, the enhanced APCs described herein have been incubated with the adjuvant for about 3 hours to about 6 hours. In some aspects, the enhanced APCs described herein have been incubated with the adjuvant for about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. In some aspects, the enhanced APCs described herein have been incubated with the adjuvant for about 4 hours. In some aspects, the incubation is performed at about 37°C.

[0113] Non-limiting examples of adjuvants that can be used in the present disclosure include: interferon gene stimulator (STING) agonists, retinoic acid-induced gene I (RIG-I) agonists, and agonists of TLR3, TLR4, TLR7, TLR8, TLR9, CpG ODN, interferon-α (IFN-α), IFN-β, IFN-γ, α-galactosylceramide, polyinosinic acid: polycytidylic acid (polyI: C), imiquimod (R837), resiquimod (R848), cyclic dinucleotide (CDN) or lipopolysaccharide (LPS). In some aspects, CpG ODN includes A-class CpG ODN, B-class CpG ODN or C-class CpG ODN. In some aspects, CpG ODN includes CpG ODN 1018, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CPG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362, CpG ODN D-SL03.

[0114] In some aspects, the enhanced APC of the methods described herein is prepared by passing a cell suspension containing an input APC through a cell deformation contraction, thereby causing a disturbance in the APC, so that a nucleic acid encoding an HPV E6 protein (e.g., a full-length E6 protein of HPV-16) enters the APC when in contact with the APC. In some aspects, the enhanced APC of the methods described herein is prepared by passing a cell suspension containing an input APC through a cell deformation contraction, thereby causing a disturbance in the APC, so that a nucleic acid encoding an HPV E7 protein (e.g., a full-length E7 protein of HPV-16) enters the APC when in contact with the APC. In some aspects, the enhanced APC of the methods described herein is prepared by passing a cell suspension containing an input APC through a cell deformation contraction, thereby causing a disturbance in the APC, so that a nucleic acid encoding an HPV E6 protein (e.g., a full-length E6 protein of HPV-16) and a nucleic acid encoding an HPV E7 protein (e.g., a full-length E7 protein of HPV-16) enters the APC when in contact with the APC. In some aspects, a cell suspension comprising an input APC has been cultured in combination with nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines, such that nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines are contacted with the input APCs, thereby generating enhanced APCs capable of inducing an enhanced immune response. In some aspects, nucleic acids encoding antigens, nucleic acids encoding costimulatory molecules, and / or nucleic acids encoding cytokines are mRNAs.

[0115] In some aspects, a cell suspension comprising an input APC is passed through a plurality of cell deformation constrictions, wherein the plurality of cell deformation constrictions are arranged in series and / or in parallel. In some aspects, the cell deformation constriction comprises a diameter of about 4.2 μm to about 6 μm, about 4.2 μm to about 5.8 μm, about 4.2 μm to about 5.6 μm, about 4.2 μm to about 5.4 μm, about 4.2 μm to about 5.2 μm, about 4.2 μm to about 5.0 μm, or about 4.2 μm to about 4.8 μm. In some aspects, the cell deformation constriction comprises a diameter of about or less than any of: 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm, 9.5μm, 10μm, 10.5μm, 11μm, 11.5μm, 12μm, 12.5μm, 13μm, 13.5μm, 14μm, 14.5μm, or 15μm. In some aspects, the cell deformation constriction comprises a diameter of about or less than any of 3.0 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4.0 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 4.9 μm, or 5.0 μm. In some aspects, the cell deformation constriction comprises a diameter of about 4.5 μm.

[0116] Dosage and regimen

[0117] In some aspects, a method for treating an HPV-related cancer in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising an enhanced APC, wherein the enhanced APC is administered at a concentration of 0.25×10 6 cells / kg to about 7.50×10 6 The method further comprises administering an immune checkpoint inhibitor.

[0118] In some aspects, the enhanced APCs are administered in an amount of any of the following: about 0.25×10 6 cells / kg to about 7.50×10 6 cells / kg, about 0.25×10 6 cells / kg to about 5.0×10 6 cells / kg, about 0.25×10 6 cells / kg to about 3.25×10 6 cells / kg, about 0.25×106 cells / kg to about 2.5×10 6 cells / kg, about 0.25×10 6 cells / kg to about 1.25×10 6 cells / kg, about 0.25×10 6 cells / kg to about 0.5×10 6 cells / kg, about 0.5×10 6 cells / kg to about 7.50×10 6 cells / kg, about 0.5×10 6 cells / kg to about 5.0×10 6 cells / kg, about 0.5×10 6 cells / kg to about 3.25×10 6 cells / kg, about 0.5×10 6 cells / kg to about 2.5×10 6 cells / kg, about 0.5×10 6 cells / kg to about 1.25×10 6 cells / kg, about 1.25×10 6 cells / kg to about 7.50×10 6 cells / kg, about 1.25×10 6 cells / kg to about 5.0×10 6 cells / kg, about 1.25×10 6 cells / kg to about 3.25×10 6 cells / kg, about 1.25×10 6 cells / kg to about 2.5×10 6 cells / kg, about 2.5×10 6 cells / kg to about 7.50×10 6 cells / kg, about 2.5×10 6 cells / kg to about 5.0×10 6 cells / kg, about 2.5×10 6 cells / kg to about 3.25×10 6 cells / kg, about 3.25×10 6 cells / kg to about 7.50×10 6 cells / kg, about 3.25×10 6 cells / kg to about 5.0×10 6 cells / kg and about 5.0×10 6 cells / kg to about 7.50×10 6In some aspects, the enhanced APCs are present at about 0.25×10 6 cells / kg to about 7.50×10 6 In other aspects, the enhanced APCs are administered in an amount of about 0.5×10 6 cells / kg to about 5.0×10 6 In some aspects, the enhanced APCs are administered in an amount of any of the following: about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg and about 7.50×10 6 cells / kg.

[0119] In some aspects, when an immune checkpoint inhibitor is administered to a subject, the immune checkpoint inhibitor includes an antagonist of PD-1 (such as, but not limited to, pembrolizumab), an antagonist of PD-L1 (such as, but not limited to, atezolizumab), and / or an antagonist of CTLA-4 (such as, but not limited to, ipilimumab). Therefore, in some aspects, disclosed herein is a method for treating human papillomavirus (HPV)-related cancers in a subject in need, comprising administering to the subject a composition comprising an enhanced APC as described herein and an antagonist of PD-1. In some aspects, disclosed herein is a method for treating human papillomavirus (HPV)-related cancers in a subject in need, comprising administering to the subject a composition comprising an enhanced APC as described herein and an antagonist of PD-L1. In some aspects, disclosed herein is a method for treating human papillomavirus (HPV)-related cancers in a subject in need, comprising administering to the subject a composition comprising an enhanced APC as described herein and an antagonist of CTLA-4. In some aspects, the antagonist of PD-1 is an antibody that binds to PD-1. In some aspects, the antagonist of PD-L1 is an antibody that binds PD-L1. In some aspects, the antagonist of CTLA-4 is an antibody that binds CTLA-4.

[0120] In some aspects, the antibody that binds to PD-1 is pembrolizumab. In some aspects, pembrolizumab is administered in an amount of any one of the following: about 10 mg to about 400 mg, about 10 mg to about 390 mg, about 10 mg to about 380 mg, about 10 mg to about 370 mg, about 10 mg to about 360 mg, about 10 mg to about 350 mg, about 10 mg to about 340 mg, about 10 mg to about 330 mg, about 10 mg to about 320 mg, about 10 mg to about 310 mg, about 10 mg to about 300 mg, about 10 mg to about 290 mg, about 10 mg to about 280 mg, about 10 mg to about 270 mg, about 10 mg to about 260 mg, about 10 mg to about 250 mg, about 10 mg to about 240 mg. 0 mg, about 10 mg to about 230 mg, about 10 mg to about 220 mg, about 10 mg to about 210 mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 10 mg to about 50 mg, about 50 mg to about 400 mg, about 50 mg to about 390 mg, about 50 mg to about 380 mg, about 50 mg to about 370 mg, about 50 mg to about 360 mg, about 50 mg to about 350 mg, about 50 mg to about 340 mg, about 50 mg to about 330 mg, about 50 mg to about 320 mg, about 50 mg to about 310 mg, about 50 mg to about 300 mg, about 50 mg to about about 290 mg, about 50 mg to about 280 mg, about 50 mg to about 270 mg, about 50 mg to about 260 mg, about 50 mg to about 250 mg, about 50 mg to about 240 mg, about 50 mg to about 230 mg, about 50 mg to about 220 mg, about 50 mg to about 210 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 400 mg, about 100 mg to about 390 mg, about 100 mg to about 380 mg, about 100 mg to about 370 mg, about 100 mg to about 360 mg, about 100 mg to about 350 mg, about 100 mg to about 3 40mg, about 100mg to about 330mg, about 100mg to about 320mg, about 100mg to about 310mg, about 100mg to about 300mg, about 100mg to about 290mg, about 100mg to about 280mg, about 100mg to about 270mg, about 100mg to about 260mg, about 100mg to about 250mg, about 100mg to about 240mg, about 100mg to about 230mg, about 100mg to about 220mg, about 100mg to about 210mg, about 100mg to about 200mg, about 100mg to about 150mg, about 150mg to about 400mg, about 150mg to about 390mg,about 150 mg to about 380 mg, about 150 mg to about 370 mg, about 150 mg to about 360 mg, about 150 mg to about 350 mg, about 150 mg to about 340 mg, about 150 mg to about 330 mg, about 150 mg to about 320 mg, about 150 mg to about 310 mg, about 150 mg to about 300 mg, about 150 mg to about 290 mg, about 150 mg to about 280 mg, about 150 mg to about 270 mg, about 150 mg to about 260 mg, about 150 mg to about 250 mg, about 150 mg to about 240 mg, about 150 mg to about 230 mg, about 150 mg to about 220 mg, about 150 mg to about 21 0mg, about 150mg to about 200mg, about 200mg to about 400mg, about 200mg to about 390mg, about 200mg to about 380mg, about 200mg to about 370mg, about 200mg to about 360mg, about 200mg to about 350mg, about 200mg to about 340mg, about 200mg to about 330mg, about 200mg to about 320mg, about 200mg to about 310mg, about 200mg to about 300mg, about 200mg to about 290mg, about 200mg to about 280mg, about 200mg to about 270mg, about 200mg to about 260mg, about 200mg to about 250mg, about 200mg to about about 240 mg, about 200 mg to about 230 mg, about 200 mg to about 220 mg, about 200 mg to about 210 mg, about 225 mg to about 400 mg, about 225 mg to about 390 mg, about 225 mg to about 380 mg, about 225 mg to about 370 mg, about 225 mg to about 360 mg, about 225 mg to about 350 mg, about 225 mg to about 340 mg, about 225 mg to about 330 mg, about 225 mg to about 320 mg, about 225 mg to about 310 mg, about 225 mg to about 300 mg, about 225 mg to about 290 mg, about 225 mg to about 280 mg, about 225 mg to about 270 mg, about 225 mg to about 360 mg From about 250 mg to about 300 mg, from about 250 mg to about 275 mg, from about 275 mg to about 400 mg, from about 250 mg to about 390 mg, from about 250 mg to about 380 mg, from about 250 mg to about 370 mg, from about 250 mg to about 360 mg, from about 250 mg to about 350 mg, from about 250 mg to about 340 mg, from about 250 mg to about 330 mg, from about 250 mg to about 320 mg, from about 250 mg to about 310 mg, from about 250 mg to about 300 mg, from about 250 mg to about 275 mg, from about 275 mg to about 400 mg, from about 275 mg to about 390 mg, from about 275 mg to about 380 mg, from about 275 mg to about 370 mg,about 275 mg to about 360 mg, about 275 mg to about 350 mg, about 275 mg to about 340 mg, about 275 mg to about 330 mg, about 275 mg to about 320 mg, about 275 mg to about 310 mg, about 275 mg to about 300 mg, about 300 mg to about 400 mg, about 300 mg to about 390 mg, about 300 mg to about 380 mg, about 300 mg to about 370 mg, about 300 mg to about 360 mg, about 300 mg to about 360 mg, about 300 mg to about about 350 mg, about 300 mg to about 325 mg, about 325 mg to about 400 mg, about 325 mg to about 390 mg, about 325 mg to about 380 mg, about 325 mg to about 370 mg, about 325 mg to about 360 mg, about 325 mg to about 350 mg, about 350 mg to about 400 mg, about 350 mg to about 390 mg, about 350 mg to about 380 mg, about 350 mg to about 375 mg and about 375 mg to about 400 mg. In some aspects, pembrolizumab is administered in an amount of about 10 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 275 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 325 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 375 mg, about 380 mg, about 390 mg, and about 400 mg. In some aspects, pembrolizumab is administered in an amount of about 200 mg.

[0121] In some aspects, the antibody that binds to PD-1 is nivolumab. In some aspects, nivolumab is administered in an amount of any of the following: about 15 mg to about 500 mg, about 15 mg to about 480 mg, about 15 mg to about 440 mg, about 15 mg to about 400 mg, about 15 mg to about 360 mg, about 15 mg to about 320 mg, about 15 mg to about 300 mg, about 15 mg to about 280 mg, about 15 mg to about 240 mg, about 15 mg to about 200 mg, about 15 mg to about 160 mg, about 15 mg to about 120 mg, about 15 mg to about 100 mg, about 15 mg to about 40 mg, about 40 mg to about 500 mg, about 40 mg to about 480 mg, about 40 mg to about 440 mg , about 40 mg to about 400 mg, about 40 mg to about 360 mg, about 40 mg to about 320 mg, about 40 mg to about 300 mg, about 40 mg to about 280 mg, about 40 mg to about 240 mg, about 40 mg to about 200 mg, about 40 mg to about 160 mg, about 40 mg to about 120 mg, about 40 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 480 mg, about 100 mg to about 440 mg, about 100 mg to about 400 mg, about 100 mg to about 360 mg, about 100 mg to about 320 mg, about 100 mg to about 300 mg, about 100 mg to about 280 mg, about 100 mg to about 240 mg, about 100 mg to about 200 mg, about 100 mg to about 160 mg, about 100 mg to about 120 mg, about 120 mg to about 500 mg, about 120 mg to about 480 mg, about 120 mg to about 440 mg, about 120 mg to about 400 mg, about 120 mg to about 360 mg, about 120 mg to about 320 mg, about 120 mg to about 300 mg, about 120 mg to about 280 mg, about 120 mg to about 240 mg, about 120 mg to about 200 mg, about 120 mg to about 160 mg, about 160 mg to about 500 mg, about 160 mg to about 480 mg, about 160 mg to about about 440 mg, about 160 mg to about 400 mg, about 160 mg to about 360 mg, about 160 mg to about 320 mg, about 160 mg to about 300 mg, about 160 mg to about 280 mg, about 160 mg to about 240 mg, about 160 mg to about 200 mg, about 200 mg to about 500 mg, about 200 mg to about 480 mg, about 200 mg to about 440 mg, about 200 mg to about 400 mg, about 200 mg to about 360 mg, about 200 mg to about 320 mg, about 200 mg to about 300 mg, about 200 mg to about 280 mg, about 200 mg to about 240 mg, about 240 mg to about 500 mg,From about 240 mg to about 480 mg, from about 240 mg to about 440 mg, from about 240 mg to about 400 mg, from about 240 mg to about 360 mg, from about 240 mg to about 320 mg, from about 240 mg to about 300 mg, from about 240 mg to about 280 mg, from about 280 mg to about 500 mg, from about 280 mg to about 480 mg, from about 280 mg to about 440 mg, from about 280 mg to about 400 mg, from about 280 mg to about 360 mg, from about 280 mg to about 320 mg, from about 280 mg to about 300 mg, from about 300 mg to about 500 mg, from about 300 mg to about 480 mg, from about 300 mg to about 440 mg, from about 300 mg to about 400 mg, from about 300 mg to about 360 mg, from about 300 mg to about about 320 mg, about 320 mg to about 500 mg, about 320 mg to about 480 mg, about 320 mg to about 440 mg, about 320 mg to about 400 mg, about 320 mg to about 360 mg, about 360 mg to about 500 mg, about 360 mg to about 480 mg, about 360 mg to about 440 mg, about 360 mg to about 400 mg, about 360 mg to about 500 mg, about 360 mg to about 480 mg, about 360 mg to about 440 mg, about 360 mg to about 400 mg, about 400 mg to about 500 mg, about 400 mg to about 480 mg, about 400 mg to about 440 mg, about 440 mg to about 500 mg, about 440 mg to about 480 mg, or about 480 mg to about 500 mg. In some aspects, nivolumab is administered in an amount of any of about 15 mg, about 40 mg, about 100 mg, about 120 mg, about 160 mg, about 200 mg, about 240 mg, about 280 mg, about 300 mg, about 320 mg, about 360 mg, about 400 mg, about 440 mg, about 480 mg, and about 500 mg. In some aspects, nivolumab is administered in an amount of about 360 mg.

[0122] In some aspects, the antibody that binds to PD-L1 is atezolizumab. In some aspects, atezolizumab is administered in an amount of any of about 75 mg to about 1700 mg, about 75 mg to about 1680 mg, about 75 mg to about 1600 mg, about 75 mg to about 1500 mg, about 75 mg to about 1400 mg, about 75 mg to about 1300 mg, about 75 mg to about 1200 mg, about 75 mg to about 1080 mg, about 75 mg to about 900 mg, about 75 mg to about 840 mg, about 75 mg to about 720 mg, about 75 mg to about 600 mg, about 75 mg to about 480 mg, about 75 mg to about 360 mg, about 75 mg to about 300 mg, about 75 mg to about 240 mg, about 75 mg to about 120 mg, about 120 mg to about 1700 mg, about 120 mg to about 1680 mg, about 120 mg to about 1600 mg, about 120 mg to about 1500 mg, about 120 mg to about 1400 mg, about 120 mg to about 1300 mg, about 120 mg to about 1200 mg, about 120 mg to about 1080 mg, about 120 mg to about 900 mg, about 120 mg to about 840 mg, about 120 mg to about 720 mg, about 120 mg to about 600 mg, about 120 mg to about 480 mg, about 120 mg to about 360 mg, about 120 mg to about 300 mg, about 120 mg to about 240 mg g, about 240 mg to about 1700 mg, about 240 mg to about 1680 mg, about 240 mg to about 1600 mg, about 240 mg to about 1500 mg, about 240 mg to about 1400 mg, about 240 mg to about 1300 mg, about 240 mg to about 1200 mg, about 240 mg to about 1080 mg, about 240 mg to about 900 mg, about 240 mg to about 840 mg, about 240 mg to about 720 mg, about 240 mg to about 600 mg, about 240 mg to about 480 mg, about 240 mg to about 360 mg, about 240 mg to about 300 mg, about 300 mg to about 1700 mg, about 300 mg to about about 1680 mg, about 300 mg to about 1600 mg, about 300 mg to about 1500 mg, about 300 mg to about 1400 mg, about 300 mg to about 1300 mg, about 300 mg to about 1200 mg, about 300 mg to about 1080 mg, about 300 mg to about 900 mg, about 300 mg to about 840 mg, about 300 mg to about 720 mg, about 300 mg to about 600 mg, about 300 mg to about 480 mg, about 300 mg to about 360 mg, about 360 mg to about 1700 mg, about 360 mg to about 1680 mg, about 360 mg to about 1600 mg, about 360 mg to about 1500 mg,about 360 mg to about 1400 mg, about 360 mg to about 1300 mg, about 360 mg to about 1200 mg, about 360 mg to about 1080 mg, about 360 mg to about 900 mg, about 360 mg to about 840 mg, about 360 mg to about 720 mg, about 360 mg to about 600 mg, about 360 mg to about 480 mg, about 480 mg to about 1700 mg, about 480 mg to about 1680 mg, about 480 mg to about 1600 mg, about 480 mg to about 1500 mg, about 480 mg to about 1400 mg, about 480 mg to about 1300 mg, about 480 mg to about 1200 mg, about 480 mg to about 1080 mg , about 480 mg to about 900 mg, about 480 mg to about 840 mg, about 480 mg to about 720 mg, about 480 mg to about 600 mg, about 600 mg to about 1700 mg, about 600 mg to about 1680 mg, about 600 mg to about 1600 mg, about 600 mg to about 1500 mg, about 600 mg to about 1400 mg, about 600 mg to about 1300 mg, about 600 mg to about 1200 mg, about 600 mg to about 1080 mg, about 600 mg to about 900 mg, about 600 mg to about 840 mg, about 600 mg to about 720 mg, about 720 mg to about 1700 mg, about 720 mg to about 1680 mg, about 720 mg to about 1600 mg, about 720 mg to about 1500 mg, about 720 mg to about 1400 mg, about 720 mg to about 1300 mg, about 720 mg to about 1200 mg, about 720 mg to about 1080 mg, about 720 mg to about 900 mg, about 720 mg to about 840 mg, about 840 mg to about 1700 mg, about 840 mg to about 1680 mg, about 840 mg to about 1600 mg, about 840 mg to about 1500 mg, about 840 mg to about 1400 mg, about 840 mg to about 1300 mg, about 840 mg to about 1200 mg, about 840 mg to about 1080 mg, about 840 mg to about 900 mg. g, about 900 mg to about 1700 mg, about 900 mg to about 1680 mg, about 900 mg to about 1600 mg, about 900 mg to about 1500 mg, about 900 mg to about 1400 mg, about 900 mg to about 1300 mg, about 900 mg to about 1200 mg, about 900 mg to about 1080 mg, about 1080 mg to about 1700 mg, about 1080 mg to about 1680 mg, about 1080 mg to about 1600 mg, about 1080 mg to about 1500 mg, about 1080 mg to about 1400 mg, about 1080 mg to about 1300 mg, about 1080 mg to about 1200 mg, about 1200 mg to about 1700 mg,about 1200 mg to about 1680 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1700 mg, about 1300 mg to about 1680 mg, about 1300 mg to about 1600 mg, about 1300 mg to about 1500 mg, about 1300 mg to about 1400 mg, From about 1400 mg to about 1700 mg, from about 1400 mg to about 1680 mg, from about 1400 mg to about 1600 mg, from about 1400 mg to about 1500 mg, from about 1500 mg to about 1700 mg, from about 1500 mg to about 1680 mg, from about 1500 mg to about 1600 mg, from about 1600 mg to about 1700 mg, from about 1600 mg to about 1680 mg, or from about 1680 mg to about 1700 mg. In some aspects, atezolizumab is administered in an amount of any of about 75 mg, about 120 mg, about 240 mg, about 300 mg, about 360 mg, about 480 mg, about 600 mg, about 720 mg, about 840 mg, about 900 mg, about 1080 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1680 mg, and about 1700 mg. In some aspects, atezolizumab is administered in an amount of about 1200 mg.

[0123] In some aspects, the antibody that binds to CTLA-4 is ipilimumab. In some aspects, ipilimumab is administered in an amount of any of about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 9 mg / kg, about 1 mg / kg to about 8 mg / kg, about 1 mg / kg to about 7 mg / kg, about 1 mg / kg to about 6 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 4 mg / kg, about 1 mg / kg to about 3 mg / kg, about 1 mg / kg to about 2 mg / kg, about 2 mg / kg to about 10 mg / kg g, about 2 mg / kg to about 9 mg / kg, about 2 mg / kg to about 8 mg / kg, about 2 mg / kg to about 7 mg / kg, about 2 mg / kg to about 6 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 4 mg / kg, about 2 mg / kg to about 3 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3 mg / kg to about 9 mg / kg, about 3 mg / kg to about 8 mg / kg, about 3 mg / kg to about 7 mg / kg, about 3 ... kg to about 6 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 4 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4 mg / kg to about 9 mg / kg, about 4 mg / kg to about 8 mg / kg, about 4 mg / kg to about 7 mg / kg, about 4 mg / kg to about 6 mg / kg, about 4 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 9 mg / kg, about 5 mg / kg to about 8 mg / kg. In some aspects, ipilimumab is used in an amount of about 1 mg / kg to about 3 mg / kg. In some aspects, ipilimumab is used in an amount of any one of the following: about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 ... In some aspects, ipilimumab is administered in an amount of about 3 mg / kg.

[0124] In some aspects, according to any of the methods described herein, the composition comprising the enhanced APC is administered intravenously. In some aspects, the immune checkpoint inhibitor is administered intravenously, orally, or subcutaneously.

[0125] In some aspects, the methods described herein include administering to a subject in need a plurality of (e.g., any one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) cycles of a composition comprising an enhanced APC as described herein. In some aspects, an enhanced APC is administered to a subject in need 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In some aspects, the duration between any two consecutive administrations of a composition comprising an enhanced APC is at least about 1 day (e.g., at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more, including any ranges between these values).

[0126] In some aspects, the composition comprising the enhanced APC is administered in any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 week cycles. In some aspects, the composition comprising the enhanced APC is administered on the first day of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 week cycles. In some aspects, the composition comprising the enhanced APC is administered in a 3-week cycle. In some aspects, the composition comprising the enhanced APC is administered in a 6-week cycle. In some aspects, the composition comprising the enhanced APC is administered on one or more days of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days of the treatment cycle. In some aspects, the composition comprising the enhanced APC is administered on the first day of the treatment cycle. In other aspects, the composition comprising the enhanced APC is administered on the second day of the treatment cycle. In some aspects, the composition comprising enhanced APCs is administered on day 1 and day 2 of the treatment cycle. In some aspects, the composition comprising enhanced APCs is administered on day 1 and day 3 of the treatment cycle. In some aspects, the composition comprising enhanced APCs is administered on day 8 of the treatment cycle. In some aspects, the composition comprising enhanced APCs is administered on day 1 of a three-week cycle. In some aspects, the composition comprising enhanced APCs is further administered on day 2 of a three-week cycle. In some aspects, the composition comprising enhanced APCs is administered in a three-week cycle until the supply of the composition of enhanced APCs is exhausted, or for one year. In some aspects, the composition comprising enhanced APCs is administered to the subject for at least about 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months.

[0127] In some aspects, enhanced APCs are administered on day 1 of each three-week cycle at any of: about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg and about 7.50×10 6 In some aspects, about 0.25×10 cells / kg are administered on day 1 of each three-week cycle. 6 cells / kg to about 7.5×10 6 cells / kg enhanced APC. In some aspects, about 0.5×10 cells / kg enhanced APC are administered on day 1 of each three-week cycle. 6 cells / kg to about 5.0×10 6 cells / kg enhanced APC. In some aspects, about 0.5×10 cells / kg enhanced APC are administered on day 1 of each three-week cycle. 6 cells / kg, about 2.5×10 6 cells / kg or about 5.0×10 6 cells / kg enhanced APC. In some aspects, enhanced APC is administered on day 2 of each three-week cycle at any of the following: about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg and about 7.50×10 6 In some aspects, about 0.25×10 cells / kg are administered on day 2 of each three-week cycle. 6 cells / kg to about 7.5×10 6 cells / kg enhanced APC. In some aspects, about 0.5×10 cells / kg enhanced APC are administered on day 2 of each three-week cycle. 6 cells / kg to about 5.0×10 6 cells / kg enhanced APC. In some aspects, about 0.5×10 cells / kg enhanced APC are administered on day 2 of each three-week cycle. 6 cells / kg, about 2.5×10 6 cells / kg or about 5.0×10 6 cells / kg enhanced APC.

[0128] In some aspects, 0.5×10 6 cells / kg enhanced APC, and 0.5×10 6 cells / kg enhanced APC. In some aspects, 0.5×10 6 cells / kg enhanced APC, and 0.5×10 6 cells / kg enhanced APC. In some aspects, 2.5×10 6 cells / kg enhanced APC, and 2.5×10 6 cells / kg enhanced APC. In some aspects, 2.5×10 6 cells / kg enhanced APC, and 2.5×10 6 cells / kg enhanced APC. In some aspects, 5.0×10 6 cells / kg enhanced APC, and 5.0×10 6 cells / kg enhanced APC. In some aspects, 5.0×10 6 cells / kg enhanced APC, and 5.0×10 6 cells / kg enhanced APC. In some aspects, 0.25×10 6 cells / kg enhanced APC, and 0.25×10 6 cells / kg enhanced APC. In some aspects, 0.25×10 6 cells / kg enhanced APC, and 0.25×10 6 cells / kg enhanced APC. In some aspects, 1.25×10 6 cells / kg enhanced APC, and 1.25×10 6 cells / kg enhanced APC. In some aspects, 1.25×10 6 cells / kg enhanced APC, and 1.25×10 6cells / kg enhanced APC. In some aspects, 3.25×10 6 cells / kg enhanced APC, and 3.25×10 6 cells / kg enhanced APC. In some aspects, 3.25×10 6 cells / kg enhanced APC, and 3.25×10 6 cells / kg enhanced APC. In some aspects, 7.50×10 6 cells / kg enhanced APC, and 7.50×10 6 cells / kg enhanced APC. In some aspects, 7.50×10 6 cells / kg enhanced APC, and 7.50×10 6 cells / kg enhanced APC.

[0129] In some aspects, the method further comprises administering an immune checkpoint inhibitor directed against PD-1, PD-L1, and / or CTLA-4. In some aspects, antibodies binding to PD-1, antibodies binding to PD-L1, and / or antibodies binding to CTLA-4 are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per cycle. In some aspects, antibodies binding to PD-1 are administered once every three-week cycle. In some aspects, antibodies binding to PD-L1 are administered once every three-week cycle. In some aspects, antibodies binding to CTLA-4 are administered once every three-week cycle. In some aspects, antibodies binding to PD-1 are administered once every two three-week cycles. In some aspects, antibodies binding to PD-L1 are administered once every two three-week cycles. In some aspects, antibodies binding to CTLA-4 are administered once every two three-week cycles.

[0130] In some aspects, according to any of the methods described herein, the immune checkpoint inhibitor is administered in any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 week cycles. In some aspects, the immune checkpoint inhibitor is administered on day 1 of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 week cycles. In some aspects, the immune checkpoint inhibitor is administered once or more on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the treatment cycle.

[0131] In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 8 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 8 of the first three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 8 of the first three-week cycle and on day 1 of each subsequent three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is administered on day 1 of the third three-week cycle and on day 1 of each subsequent three-week cycle.

[0132] In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 8 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 8 of the first three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 8 of the first three-week cycle and on day 1 of each subsequent three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is pembrolizumab, wherein pembrolizumab is administered at a dose of about 200 mg on day 1 of the third three-week cycle and on day 1 of each subsequent three-week cycle.

[0133] In other aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on the first day of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on the first day of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on the 8th day of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on the 8th day of the first three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on day 8 of the first three-week cycle and on day 1 of each subsequent three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-1, wherein the antibody that binds to PD-1 is nivolumab, wherein nivolumab is administered at a dose of about 360 mg on day 1 of the third three-week cycle and on day 1 of each subsequent three-week cycle.

[0134] In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-L1, wherein the antibody that binds to PD-L1 is administered on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-L1, wherein the antibody that binds to PD-L1 is administered on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-L1, wherein the antibody that binds to PD-L1 is administered on day 8 of the first three-week cycle and on day 1 of each subsequent cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-L1, wherein the antibody that binds to PD-L1 is atezolizumab, wherein atezolizumab is administered at a dose of about 1200 mg on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to PD-L1, wherein the antibody that binds to PD-L1 is atezolizumab, wherein atezolizumab is administered at a dose of about 1200 mg on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds PD-L1, wherein the antibody that binds PD-L1 is atezolizumab, wherein atezolizumab is administered at a dose of about 1200 mg on day 8 of the first three-week cycle and day 1 of each subsequent cycle.

[0135] In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is administered on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is administered on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is administered up to four doses. In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is administered once every two three-week cycles. In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is ipilimumab, wherein ipilimumab is administered at a dose of about 3 mg / kg on day 1 of a three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds to CTLA-4, wherein the antibody that binds to CTLA-4 is ipilimumab, wherein ipilimumab is administered at a dose of about 3 mg / kg on day 1 of each three-week cycle. In some aspects, the immune checkpoint inhibitor is an antibody that binds CTLA-4, wherein the antibody that binds CTLA-4 is ipilimumab, wherein ipilimumab is administered at a dose of about 3 mg / kg for up to four doses. In some aspects, the immune checkpoint inhibitor is an antibody that binds CTLA-4, wherein the antibody that binds CTLA-4 is ipilimumab, wherein ipilimumab is administered once every two three-week cycles at a dose of about 3 mg / kg.

[0136] Composition of enhanced APC

[0137] In some aspects, according to the methods described herein, the composition comprising enhanced APCs comprises about 1.0×10 6 to about 1.0x10 8 Enhanced APC / mL. In some aspects, the composition comprising enhanced APC comprises about 1.0×10 6 About 0.5×10 7 , about 0.5×10 7 About 1.0×10 7 , about 1.0×10 7 About 0.5×10 8 and about 0.5×10 8 About 1.0×10 8 In some aspects, the composition comprising enhanced APCs comprises about 1.0×10 6 , about 0.2×10 7 , about 0.3×10 7 , about 0.4×10 7 , about 0.5×10 7 , about 0.6×107 , about 0.7×10 7 , about 0.8×10 7 , about 0.9×10 7 , about 1.0×10 7 , about 0.2×10 8 , about 0.3×10 8 , about 0.4×10 8 , about 0.5×10 8 , about 0.6×10 8 , about 0.7×10 8 , about 0.8×10 8 , about 0.9×10 8 and about 1.0×10 8 In some aspects, the composition comprising the enhanced APCs comprises about 8.5×10 6 In other aspects, the composition comprising enhanced APCs comprises about 1.1×10 7 Enhanced APC / mL.

[0138] In some aspects, the composition comprises about 5.0×10 6 to about 1.0x10 9 In some aspects, the composition comprises about 5.0×10 6 About 1.0×10 7 , about 1.0×10 7 About 0.5×10 8 , about 0.5×10 8 About 1.0×10 8 , about 1.0×10 8 About 0.5×10 9 and about 0.5×10 9 About 1.0×10 9 In some aspects, the composition comprises about 5.0×10 6 , about 6.0×10 6 , about 7.0×10 6 , about 8.0×10 6 , about 9.0×10 6 , about 1.0×10 7 , about 2.0×10 7 , about 3.0×10 7 , about 4.0×10 7 , about 5.0×10 7 , about 6.0×10 7 , about 7.0×10 7 , about 8.0×107 , about 9.0×10 7 , about 1.0×10 8 , about 2.0×10 8 , about 3.0×10 8 , about 4.0×10 8 , about 5.0×10 8 , about 6.0×10 8 , about 7.0×10 8 , about 8.0×10 8 , about 9.0×10 8 and about 1.0×10 9 In some aspects, the composition comprises about 8.1×10 7 In other aspects, the composition comprises about 1.05×10 8 An enhanced APC.

[0139] In some aspects, according to the methods described herein, the composition comprising enhanced APC comprises a cryopreservation medium. In some aspects, the composition comprising enhanced APC comprises a cryopreservation medium at a concentration of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% (weight / weight). In some aspects, the composition comprising enhanced APC comprises a cryopreservation medium at a concentration of about 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80% or 80% to 85% (weight / weight).

[0140] In some aspects, according to the methods described herein, the composition comprising enhanced APCs comprises cryopreservation medium. In some aspects, the composition comprising enhanced APCs comprises a cryopreservation medium at a concentration of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% (weight / weight). In some aspects, the composition comprising enhanced APCs comprises a cryopreservation medium at a concentration of about 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65% or 65% to 70% (weight / weight).

[0141] In some aspects, according to the methods described herein, the composition comprising the enhanced APC comprises human serum albumin at a concentration of about 2%, 3%, 4%, 5%, 8% or 10% (weight / weight). In some aspects, the composition comprising the enhanced APC comprises human serum albumin at a concentration of about 2% to 3%, 3% to 5%, 5% to 8% or 8% to 10% (weight / weight). In some aspects, human serum albumin is added to the composition as a human serum albumin solution. In some aspects, the concentration of the human serum albumin solution in the composition is about 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% (weight / weight). In some aspects, the concentration of the human serum albumin solution in the composition is about any of 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, or 45% to 50% (weight / weight).

[0142] In some aspects, according to the methods described herein, the pH of the composition is from about 5.0 to about 9.5. In some aspects, the pH of the composition is from about 6.0 to about 8.5. In some aspects, the pH of the composition is from about 7.0 to about pH 7.9. In some aspects, the pH of the composition is about 7.4. In some aspects, the pH of the composition is about any one of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. In other aspects, the pH of the composition is about any one of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9. In some aspects, the pH of the composition is about any one of 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10. In some aspects, the pH of the composition is any of about 7 to about 7.1, about 7.1 to about 7.2, about 7.2 to about 7.3, about 7.3 to about 7.4, about 7.4 to about 7.5, about 7.5 to about 7.6, about 7.6 to about 7.7, about 7.7 to about 7.8, or about 7.8 to about 7.9.

[0143] In some aspects, the cryopreservation medium comprises CS10. In some aspects, the composition comprising the enhanced APC comprises About 5.0×10 in CS10 6 About 1.0×10 9 In some aspects, the composition comprising the enhanced APC comprises About 5×10 in CS10 6 About 5×10 7 An enhanced APC.

[0144] In some aspects, the composition comprising enhanced APCs comprises: a) about 5×10 6 Enhanced APC to about 1x10 9 enhanced APCs; b) a cryopreservation medium at a concentration of about 40% to about 95% (weight / weight); c) a cryopreservation medium at a concentration of about 25% to about 35% (weight / weight); and d) a human serum albumin solution at a concentration of 15% to about 25% (weight / weight), wherein the pH of the composition is about pH 6.0 to about pH 8.5.

[0145] In some aspects, the composition comprising enhanced APCs comprises: a) about 8.1×10 7 enhanced APCs; b) a cryopreservation medium at a concentration of about 50% (weight / weight); c) a cryopreservation medium at a concentration of about 30% (weight / weight); and d) a human serum albumin solution at a concentration of 20% (weight / weight), wherein the pH of the composition is about pH 7.0 to about pH 7.9.

[0146] In other aspects, the composition comprising the enhanced APC comprises: a) about 1.05×10 8 enhanced APCs; b) a cryopreservation medium at a concentration of about 50% (weight / weight); c) a cryopreservation medium at a concentration of about 30% (weight / weight); and d) a human serum albumin solution at a concentration of 20% (weight / weight), wherein the pH of the composition is about pH 7.0 to about pH 7.9.

[0147] In some aspects, provided herein is a method for treating a human papillomavirus (HPV)-related cancer in a subject in need thereof, the method comprising: administering to the subject: (i) a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner, and (ii) an antagonist of PD-1.

[0148] In some aspects, provided herein is a method for treating HPV in a subject in need thereof. +A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), and wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner.

[0149] Example

[0150] Those skilled in the art will recognize that within the scope and spirit of the present disclosure, several aspects are possible. The present disclosure will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present disclosure, but of course should not be interpreted as limiting its scope in any way.

[0151] Example 1. Clinical Protocol

[0152] A Phase 1 / 2, first-in-human, multicenter, open-label study was conducted in patients with HPV16+ recurrent, locally advanced or metastatic solid tumors using the enhanced APC described herein as monotherapy and in combination with immune checkpoint inhibitors.

[0153] As described herein, the enhanced APCs used in the above experiments have been generated by extrusion delivery of the following nucleic acid constructs into PBMCs: (1) mRNA encoding full-length HPV-16 E6 protein (SEQ ID NO: 14), (2) mRNA encoding full-length HPV-16 E7 protein (SEQ ID NO: 17), (3) mRNA encoding CD86, (4) mRNA encoding membrane-bound IL-2 and mRNA encoding membrane-bound IL-12. (See Figure 1 Before use, the enhanced APC was already contained in 50% (wt / wt) CS10, 30% (w / w) FRS and 20% (w / w) 25% human serum albumin (HSA) were prepared to a target concentration of 11x10 6 The preparation is prepared by freezing the vials at a temperature of ≤-170°C, and the temperature of the vials is maintained at ≤-140°C during different stages of the development process (e.g., production and storage). Unless otherwise specified, the term "drug substance" as used herein refers to the enhanced APC of the present disclosure. Unless otherwise specified, the term "drug product" refers to a preparation containing a drug substance that is ultimately administered to a subject to be treated.

[0154] Overview

[0155] The study population consists of adult patients with HPV16+ recurrent, locally advanced, or metastatic solid tumors (head and neck, cervix, anus, vulva, penis) who meet key eligibility criteria and have been diagnosed with HPV16+ tumor status by local standard of care (SOC) methods of oncology. HPV16+ status can be confirmed by local reports reviewed by the sponsor. If the sponsor believes that the local documentation to establish HPV16+ tumor status is insufficient, circulating HPV DNA analysis should be performed using whole blood, or central confirmation should be performed from archival tumor tissue or fresh tumor biopsies collected at screening.

[0156] Alternatively, whole blood supply for tumor tissue-modified virus (TTMV) HPV16 (cfDNA) or tumor tissue (archival or fresh) can be provided for central laboratory analysis. Screening should be completed 7-14 days before leukapheresis, i.e. within 28 days.

[0157] Eligible patients must undergo leukapheresis. The leukapheresis product is sent to the manufacturer to create each patient's personalized autologous cell therapy. Frozen vials of drug product are then sent to the study site for administration.

[0158] This study is conducted in 2 parts. The initial part, Part 1 (1A and 1B), will determine the recommended Phase 2 dose (RP2D) of the drug product (as a monotherapy) and evaluate the safety and preliminary efficacy of patients with HPV+ solid tumors who have progressed from existing therapies (including ICIs) when combined with pembrolizumab. In Part 1A, monotherapy is established through a dose escalation cohort in a BOIN design. In Part 1B, the safety and preliminary efficacy of the drug product in combination with pembrolizumab at the RP2D or a dose level calibrated based on overall safety data will be further evaluated. Patients receive a dose level of the drug product at the RP2D or lower and a standard dose (200 mg) of pembrolizumab.

[0159] Part 2 starts ICI therapy for HPV16+ recurrent, locally advanced or metastatic tumors in patients who have progressed after standard of care treatment. When a patient is diagnosed with an HPV16+ solid tumor and has progressed after standard of care (SOC), but has not yet received PD-1, PD-L1 or CTLA-4 inhibitors or any other agent targeting T cell co-stimulation or checkpoint pathways, he or she is an ICI therapy-naive patient. The goal of combining the drug product with pembrolizumab as an introduction strategy is to change the tumor microenvironment by increasing the amount of tumor-infiltrating lymphocytes, which are associated with improved clinical responses to checkpoint inhibitor immunotherapy. Patients received the drug product at RP2D (from Part 1) and were administered a standard dose (200 mg) of pembrolizumab 6 weeks after the introduction of the drug product monotherapy.

[0160] Each study uses a Bayesian optimal interval (BOIN) design to find the maximum tolerated dose (MTD) of the drug product with a target DLT rate of 30%. According to the BOIN design, each monotherapy cohort will enroll a minimum of 3 (unless the first 2 patients both have DLT) and a maximum of 12 patients, and a dose level can be declared safe when ≤30% of patients experience DLT. If the toxicity level is higher than 30% DLT, the dose can be downgraded. If there is a lack of immunogenic effects and no safety signals, the cohort can be declared safe. There is an option to expand the cohort to more than 6 patients to further study the safety and tolerability, immunogenic effects, and anti-tumor activity at dose levels that may be RP2D. The final sample size depends on the dosing regimen, the number of DLTs, and the total number of treatment cohorts to determine the RP2D. A maximum of approximately 60 patients who can be evaluated for safety can be enrolled.

[0161] Prior to starting study treatment, eligible patients undergo leukapheresis at the study site. Leukopak is sent to the manufacturer to produce personalized autologous cell therapy for each patient. Frozen vials of drug product are then sent to the study site for administration. The study protocol is as follows: Figure 4 shown.

[0162] Part 1A: Dose escalation (monotherapy)

[0163] To determine the safety, preliminary efficacy and RP2D of the drug product as a monotherapy, three dose levels of the drug product need to be evaluated according to the following cohorts shown in Table 1.

[0164] While the traditional 3+3 design is designed to assess safety and tolerability, it is prudent to treat up to 6 additional patients in the same cohort to further investigate safety and tolerability, immunogenic effects, and antitumor activity. In this modified 3+3 design, each cohort will have a maximum of 12 patients. A minimum of 6 patients is required.

[0165] Table 1. Summary of planned monotherapy cohorts during the escalation phase

[0166] queue Dose of drug product (cells / kg) <![CDATA[Total dose a > 1 <![CDATA[0.5×10 6 ]]> ≥3 2 <![CDATA[2.5×10 6 ]]> ≥3 3 <![CDATA[5.0×10 6 ]]> ≥3

[0167] DLT = dose-limiting toxicity; iRECIST = modified RECIST criteria for inclusion in solid tumor studies of immunotherapy; RECIST = Response Evaluation Criteria in Solid Tumors, version 1.1

[0168] a. Continue to administer the drug product every 3 weeks until treatment discontinuation criteria are met, or until the drug product supply is exhausted, or for up to 1 year (whichever comes first). Patients who experience disease progression according to RECIST 1.1 may continue to receive dosing if the treating investigator determines that this is in the best interest of the patient to confirm disease progression; i.e., iCPD according to iRECIST (Seymour et al., 2017).

[0169] b. In Cycle 1, patients received drug product on Day 1.

[0170] After review of the safety, efficacy, and pharmacodynamics data from the existing cohort of patients by the Safety Studies Committee (SSC), the SSC may determine that an evaluation of a dose above 5.0x10 6 mbIL-2 (CD45+ cells / kg body weight) or less than 0.5x10 6 Dose levels of mbIL-2 (CD45+ cells / kg body weight). If deemed necessary by the SSC based on review of existing safety data, the following interim cohorts may be opened prior to opening Part 1B. See Table 2.

[0171] Table 2. Summary of planned expansions of the monotherapy cohort during the escalation phase

[0172]

[0173] The SSC may decide to open Part 1B without opening any of the interim cohorts listed above. The dose cohort selected by the SSC for the RP2D must have at least 6 patients treated with the drug product as monotherapy.

[0174] Patients must have enough autologous drug product to achieve at least 3 full doses of the drug product to be administered in the currently enrolled cohort; otherwise, they will be assigned to a lower dose cohort. The dose may be downgraded if the toxicity level is above 30% DLT. Patients are evaluated according to the BOIN design and will participate in the study in cohorts of at least 3 patients per dose level and up to 12 patients. Additional patients (up to 12 total) may be treated in optional exploratory cohorts to further study the immunogenic effects and anti-tumor activity of the drug product as a monotherapy.

[0175] like Figure 5 As indicated, the drug product is administered to patients as monotherapy on day 1 of each 3-week treatment cycle (i.e., C1D1, C2D1, C3D1, etc.) for up to 1 year, or until the drug product supply is exhausted or the treatment discontinuation criteria are met (whichever occurs first). All adverse events of special interest (AESI) should be reduced to ≤ Grade 1 before starting a new treatment cycle. Other study drug-related adverse events (AEs) should be reduced to ≤ Grade 2 before starting a new treatment cycle. Patients are monitored for the occurrence of DLTs within 28 days after the first dose of the drug product. In order to be evaluable for DLT assessment, patients must complete a 28-day safety observation period (unless a DLT occurs during the observation period). Patients who are considered unevaluable for DLT assessment will be replaced. Patients who experience disease progression according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1) may continue dosing to confirm disease progression if the treating investigator deems this to be in the best interest of the patient. The first 2 patients in each cohort are observed for at least 23 hours after the first administration of the drug product.

[0176] like Figure 6 As shown, enrollment of the first 2 patients was staggered with 21 days between each patient. As an additional safety measure, all patients in the study were observed for at least 4 hours after each administration of the drug product.

[0177] Part 1: Escalation phase (combination of drug product with immune checkpoint inhibitors)

[0178] To evaluate the safety and preliminary efficacy of the drug product in combination with pembrolizumab, at least 3 and up to 12 patients will be enrolled and evaluated according to the BOIN design. Patients will receive the drug product at the RP2D or lower dose level defined by the SSC and the sponsor on Day 1 of Cycle 1 (C1D1) and the standard dose (200 mg) of pembrolizumab on Day 8 of Cycle 1 (C1D8). In subsequent cycles, patients will first receive the drug product and then the standard dose of pembrolizumab on Day 1 of each cycle (C2D1, C3D1, etc.). Patients receive 21-day treatment cycles for up to 1 year on the drug product or until the autologous drug product is exhausted and up to 2 years on pembrolizumab or until the treatment discontinuation criteria are met (whichever comes first), such as Figure 7 The first 2 patients in each cohort will be observed for at least 23 hours after the first administration of drug product. Enrollment of the first 2 patients is staggered, with 21 days between each patient. As an additional safety measure, all patients in the study will be observed for at least 4 hours after each administration of drug product.

[0179] If a lower starting dose of drug product is chosen, 3 patients are initially enrolled and treated with the drug product and pembrolizumab. These first 3 patients are evaluated for safety for 42 days before additional patients are enrolled. If none of the first 3 patients experience a DLT during the 42-day safety evaluation period (0 / 3), the drug product dose may be escalated to the RP2D defined in Part 1A. If 1 DLT is observed in the first 3 patients (1 / 3), additional patients (up to a total of 12) are accrued using a BOIN design with a 42-day safety evaluation period.

[0180] A minimum of 6 patients will be required to determine the final RP2D for use in Part 1B and will be based on a review of available safety, tolerability, immunogenicity and other pharmacodynamic and anti-tumor data.

[0181] Patients who experience disease progression per Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1) may continue dosing for confirmed disease progression if the treating investigator determines that this is in the best interest of the patient. Patients in Part 1A and Part 1B were enrolled in a staggered manner across all cohorts (i.e., no less than 21 days between enrollment of the first 2 patients in each cohort). All AESI should recover to ≤ Grade 1 before starting a new treatment cycle. Other study drug-related AEs should resolve to ≤ Grade 2 before starting a new treatment cycle.

[0182] Part 2: Introducing drug products in combination with immune checkpoint inhibitors

[0183] To further evaluate the safety and preliminary efficacy of the drug product in combination with pembrolizumab, ICI-naive HPV16+ solid tumor patients will receive the drug product as a monotherapy at the RP2D (from Part 1B) on Day 1 of each treatment cycle. Pembrolizumab treatment will begin in Cycle 3. Starting in Cycle 3 and in all subsequent cycles, a standard dose of pembrolizumab will be administered on Day 1 after drug product administration, such as Figure 8 As shown. Patients were treated in 21-day cycles, with drug products for up to 1 year or until autologous drug products were exhausted, and pembrolizumab for up to 2 years or until treatment discontinuation criteria were met (whichever occurred first). A BOIN design was used to accumulate and evaluate patients. All AESI should recover to ≤ Grade 1 before starting a new treatment cycle. Other study drug-related AEs should be reduced to ≤ Grade 2 before starting a new treatment cycle.

[0184] If 1 DLT is observed in the first 3 patients (1 / 3), additional (up to 12 total) patients will be accrued using a BOIN design with a safety evaluation period of 42 days. If a DLT rate ≥ 30% is found at any time, all available overall safety, pharmacodynamic, and efficacy data will be reviewed. Based on the data review, it may be determined that a lower dose level of the drug product should be explored (dose de-escalation) or a decision may be made to stop enrollment altogether.

[0185] Patients who experience disease progression according to RECIST 1.1 may continue dosing if the treating investigator deems it to be in the best interest of the patient to allow for confirmation of disease progression.

[0186] Dose-limiting toxicity

[0187] Patients were considered evaluable for DLT evaluation if they: a) experienced a DLT during the DLT evaluation period (28 days for Part 1A and 42 days for Parts 1B and 2), regardless of the dose administered; or b) did not experience a DLT during the DLT evaluation period after receiving 2 doses of the drug product (Part 1A) and completed a 42-day drug product monotherapy lead-in period and at least 1 dose of pembrolizumab (Parts 1B and 2).

[0188] DLT requires a minimum number of mbIL-2 in CD45+ cells of 70% of the target dose at each dose to be considered evaluable. Patients who manufacture cell doses less than 70% of the target dose will be excluded from DLT evaluation. However, these patients will be included in safety, dose feasibility, and exploratory studies.

[0189] Patients who experience a DLT other than an IRR will be discontinued from the study. If continued treatment with the investigational product is in the best interest of the patient, subsequent treatment will be determined by the investigator in consultation with the sponsor. For IRRs, premedication or administration rates will be adjusted to enable the patient to remain on study. Patients who experience a Grade 3 IRR may continue to receive study treatment if the event resolves to ≤ Grade 2 within 72 hours. Premedication or administration rates may be adjusted to enable the patient to remain on study.

[0190] DLTs are defined as AEs or abnormal laboratory values ​​assessed by the principal investigator and confirmed by the SSC to be unrelated to disease, disease progression, intercurrent illness, concomitant medications / procedures, or environmental factors, but related to the drug product (alone or in combination), occurring within the first 28 days of monotherapy or within the first 42 days of combination therapy, and meeting any of the predefined criteria listed using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. CRS and neurotoxicity will be graded using the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading.

[0191] Non-hematologic toxicity

[0192] Level 4 or 5

[0193] Grade 3 toxicity that does not resolve to < Grade 2 or baseline within 7 days despite best supportive care, except for grade 3 CRS or neurologic toxicity that does not resolve to ≤ Grade 2 within 24 hours

[0194] Grade 3 autoimmune or hypersensitivity reaction

[0195] Grade 3 infusion-related reaction that does not resolve to ≤ Grade 2 within 72 hours

[0196] Grade ≥3 hepatotoxicity lasting for >48 hours, with the following exceptions: For patients with Grade 2 abnormalities in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and / or alkaline phosphatase at baseline, only increases to >8×ULN lasting for >48 hours were considered DLTs.

[0197] Grade 3 laboratory values ​​persisting for >7 days and requiring medical intervention

[0198] Abnormal liver function tests meeting Hy's law criteria

[0199] Hematological toxicity

[0200] Any level 5 toxicity

[0201] Any grade 4 anemia

[0202] Any grade ≥ 3 febrile neutropenia

[0203] ≥Grade 4 neutropenia (absolute neutrophil count <500 / microL) lasting >7 days

[0204] ≥Grade 4 thrombocytopenia (<25,000 / μL)

[0205] Grade ≥3 thrombocytopenia (<50,000 / microL) persisting for >7 days with clinically significant bleeding

[0206] Adverse events at least possibly related to the drug product (alone or in combination) leading to permanent discontinuation or delay for >14 days of scheduled administration of the drug product on Cycle 2 Day 1

[0207] Any other event judged by the investigator and the sponsor was considered a DLT.

[0208] The following events are not considered DLT:

[0209] Grade 3 lipase value alone, not associated with a grade ≥ 3 amylase value or clinical symptoms or radiographic evidence of pancreatitis

[0210] Grade 3 CRS, improved to ≤ Grade 2 within 24 hours, regardless of symptomatic treatment

[0211] Grade 3 rash that resolves to ≤ Grade 2 within 7 days regardless of appropriate supportive care

[0212] Immediate hypersensitivity reactions that occur within 2 hours of cell product administration and are reversible to < Grade 2 within 24 hours

[0213] Grade 1 or 2 electrolyte abnormalities corrected within 72 hours without clinical sequelae

[0214] Hair loss

[0215] Grade 3 IRRs that can be adequately managed by adding premedication or adjusting the rate of administration will not be considered DLTs unless, based on the SSC's recommendation, these changes are considered applicable to all subsequent patients enrolled in the study, e.g. Fig. 9 If the modification applies to all subsequent patients, the cohort will restart for DLT evaluation. Patients who experience a Grade 3 IRR may continue in the study with modification of premedication or administration rate only if the Grade 3 IRR recovers to ≤ Grade 2 within 72 hours.

[0216] The incidence of late immune-mediated toxicities will be considered when determining the RP2D for the drug product, either as monotherapy or in combination with an ICI. If the MTD is not reached in any cohort, cohort 3P may be tested. If an AE occurs that meets the definition of a DLT but is not related to the drug product, the SSC will discuss the findings.

[0217] Cohort stopping criteria, stop of dose escalation or cohort progression, and study termination

[0218] The BOIN design will drive the decision to declare a cohort safe (Section 3.1.1). The minimum number of patients required to confirm a cohort is 3 with 0 DLTs. A cohort is considered safe if a maximum of 12 patients are enrolled and DLTs are ≤ 30% of patients (e.g., DLTs < 2 in 6 patients, DLTs < 3 in 9 patients, or DLTs < 4 in 12 patients).

[0219] AEs that meet the definition of a DLT and occur outside the DLT window will not be considered as DLTs but will be considered in the overall safety assessment and selection of the RP2D regimen for a given cohort.

[0220] Cohort stopping rules are based on DLT thresholds above a given sample size, as shown below. If a stopping rule is triggered, the SSC may declare the previously tolerated dose level to be the MTD; recommend testing an intermediate dose level; stop enrollment and / or the study; and / or recommend a protocol modification. After review by the SSC, patients may be discontinued based on the following general safety-related criteria:

[0221] Any SAE considered potentially life-threatening and assessed by the Medical Monitor to be related to the investigational product

[0222] Any other clinically significant changes that indicate a significant safety risk to the investigator or sponsor

[0223] Dosing regimen and study duration

[0224] All patients received a single leukapheresis prior to initiating treatment. Patients underwent leukapheresis at the study site, typically 8 to 14 days prior to the first administration of drug product. The first administration of drug product was scheduled based on site location and transportation logistics.

[0225] One cycle was defined as a 21-day treatment period.

[0226] The duration of treatment for monotherapy dose escalation (Part 1A) depends on the RP2D regimen selected. For some patients in Part 2, tumor growth kinetics may initially exceed the rate of antitumor immune activity. Over time, antitumor activity will dominate and become clinically apparent. Therefore, it is important to assess RECISTv.1.1 and iRECIST42 in parallel at each time point. Therefore, patients can continue study treatment after initial RECIST v.1.1-defined progression if the following criteria are met, allowing for confirmation of disease progression according to iRECIST:

[0227] Investigator-assessed clinical benefit without rapid disease progression

[0228] Study drug tolerance as defined by the investigator

[0229] Stable behavioral state

[0230] Treatment after disease progression does not interfere with preventing serious consequences of rapid disease progression Lack of complications of disease progression (eg, central nervous system [CNS] metastases)

[0231] The assessment of clinical benefit should consider whether the subject's clinical condition has worsened and is unlikely to derive further benefit from continued treatment.

[0232] The safety observation period corresponds to the DLT (28 days for drug product monotherapy and 42 days for combination).

[0233] After completion of safety follow-up (EODW6), patients will enter the long-term follow-up (LTFU) and overall survival (OS) periods. Patients who experience persistent AEs related to study treatment should continue to be followed as clinically indicated until the AE resolves to Grade 1 or baseline or is deemed irreversible. Patients who complete treatment or discontinue study treatment for reasons other than disease progression will continue to receive tumor assessments according to the protocol-specified schedule until documented disease progression or subsequent treatment is started. For OS assessments, patients will be contacted (by telephone) every 3 months for up to 2 years, or until death, withdrawal of consent, loss to follow-up, initiation of additional anticancer therapy, or end of the study (whichever occurs first).

[0234] Study Group

[0235] The study population will consist of eligible adult patients with HPV16+ solid tumors (head and neck, cervix, anus, vulva, penis) who have been diagnosed with HPV16+ tumor status by local standard of care (SOC) methods (and documentation provided to the sponsor for confirmation). If the sponsor deems that local documentation to establish HPV16+ tumor status is insufficient, central confirmation may be performed using fresh (or archived) tumor biopsy tissue or whole blood circulating cell-free (cf) DNA analysis collected at screening (liquid biopsy). Eligibility criteria for manufacturing autologous drug products must be met prior to leukapheresis.

[0236] Number of patients

[0237] The number of patients depends on safety and observed immunogenic effects. Up to 60 In the monotherapy escalation phase (Part 1A), 9 to 36 evaluable patients are expected to be enrolled. If all planned cohorts do not indicate that the MTD has been reached, additional dose levels or regimens may be tested according to the SSC. In the combined safety phase (Part 1B), a total of up to 12 evaluable patients will be enrolled, and in the introduction of drug product monotherapy into combination therapy (Part 2), a total of up to 12 patients will be enrolled.

[0238] Inclusion criteria

[0239] 1. Male or female patients aged ≥18 years

[0240] 2. Willing and able to provide a signed and dated ICF, agreeing to comply with the treatment, research procedures, and visit schedule as per the protocol.

[0241] 3. Confirmed as HPV16 by histology or HPV16 molecular analysis + , and histologically confirmed incurable or metastatic solid tumors (including but not limited to cervical and head and neck tumors).

[0242] 4. Agree to undergo leukapheresis to produce autologous blood products through venous access and be willing to insert a central line if venous access is problematic.

[0243] 5. Patients with biopsy-amenable lesions (unresectable or metastatic solid tumors) of acceptable clinical risk and consent to fresh biopsies at screening and on Day 8 (±2 days) of Cycle 2. Every effort should be made to obtain a second biopsy sample from the same lesion biopsied at screening. Lesions in areas previously treated with radiation therapy may be biopsied as long as there is objective evidence of lesion progression prior to study entry.

[0244] 6. At least 1 measurable lesion according to RECIST 1.1. Lesions biopsied for enrollment should not be followed for radiographic response.

[0245] 7. Eastern Cooperative Oncology Group (ECOG) performance status (PS) is 0 to 1.

[0246] 8. Adequate organ function and bone marrow reserve as demonstrated by the following laboratory assessments performed within 14 days prior to leukapheresis to manufacture autologous blood products:

[0247] a. Bone marrow function: absolute neutrophil count ≥1000 / μL; hemoglobin ≥9g / dL; platelet count ≥75,000 / μL. Note: Blood transfusions for autologous blood product manufacturing are not allowed within 4 weeks before blood draw; however, patients can receive blood transfusions after blood draw based on clinical indications.

[0248] b. Liver function: serum total bilirubin ≤1.5×upper limit of normal (ULN); serum aspartate aminotransferase (AST) / alanine aminotransferase (ALT) ≤2.5×ULN (≤5×ULN in case of liver metastasis), alkaline phosphatase <2.5×ULN, except for the following:

[0249] Patients with liver and bone involvement: Alkaline phosphatase ≤ 5 × ULN

[0250] Patients with inherited disorders of bilirubin metabolism (discuss with sponsor)

[0251] c. Renal function: serum creatinine ≤2.5×ULN or creatinine clearance ≥50 mL / min based on urine collection or Cockcroft-Gault estimation.

[0252] d. Coagulation characteristics: prothrombin time (PT), international normalized ratio (INR), and partial thromboplastin time (PTT) ≤ 1.5 × ULN. If the investigator considers the patient suitable for participation in the study, the PT / INR measurement of patients who have received stable maintenance anticoagulation therapy for at least 30 days before leukapheresis to manufacture autologous blood products may be > 1.5 × ULN. Adequate justification must be provided to the sponsor before enrollment.

[0253] 9. Female patients of childbearing potential must: a) have a negative serum beta-human chorionic gonadotropin (β-hCG) pregnancy test at screening; and b) agree to use highly effective contraception from the date of informed consent until at least 5 months after the last dose of pembrolizumab or medicinal product (Clinical Trials Facilitation and Coordination Group, CTFG, 2020).

[0254] 10. Male patients who have not undergone vasectomy must be willing to use condoms from the time of informed consent until at least 5 months after the last dose of the medicinal product or pembrolizumab.

[0255] Patients in Part 1A and Part 1B

[0256] 11. For cervical cancer that is not curatively treatable with surgery, radiation, and / or chemoradiotherapy, patients must have disease progression after prior therapy, including at least 1 prior systemic chemotherapy treatment with a platinum-based regimen and an ICI (received or offered) in the adjuvant or recurrent setting. Patients who relapse after definitive chemoradiotherapy or adjuvant chemoradiotherapy with a platinum-containing regimen are eligible if platinum rechallenge at relapse is not considered to be of benefit. For safety concerns regarding inability to tolerate platinum-based systemic chemotherapy for recurrent disease, the reason must be documented.

[0257] 12. For head and neck cancer that is not curatively treatable with surgery, radiation, and / or chemoradiotherapy, the cancer must have progressed after at least 1 platinum-based chemotherapy in the primary, adjuvant, or recurrent setting and have received or been offered checkpoint immunotherapy. Patients who relapse after definitive or adjuvant chemoradiotherapy with a platinum-containing regimen are eligible if platinum rechallenge at relapse is not considered to be of benefit. For safety concerns regarding inability to tolerate platinum-based systemic chemotherapy for recurrent disease, the reason must be documented.

[0258] 13. For incurable or metastatic cancers other than cervical or head and neck cancer, patients must have been offered or progressed from all available standard of care (SOC) or are intolerant to standard therapy or have a tumor for which no standard therapy exists according to the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines (www.nccn.org / guidelines / guidelines-process / development-and-update-of-guidelines).

[0259] 14. Patients with immune-mediated endocrine disease who require hormone replacement therapy after receiving immune checkpoint inhibitors are eligible. Note: Patients who require prednisone as part of hormone replacement therapy are eligible if the daily dose does not exceed 10 mg.

[0260] Patients in Part 2

[0261] 15. Patients who have not received immune checkpoint inhibitors. Patients may receive other anticancer therapies to treat the disease under study.

[0262] Exclusion criteria

[0263] 1. Received anticancer therapy, including study therapy, within 2 weeks prior to leukapheresis for the manufacture of autologous blood products. For prior therapy with a half-life of more than 3 days, the interval must be at least 28 days before the first administration of study therapy.

[0264] 2. AEs of grade >1 related to previous anticancer therapy or investigational therapy according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 (except grade 2 neuropathy, ototoxicity, mucositis, fatigue, alopecia, and endocrine disorders treated with hormone replacement therapy), and not resolved (i.e., <grade 2) within at least 2 weeks before leukapheresis for the manufacture of autologous blood products.

[0265] 3. AEs of grade ≥3 related to previous surgery, radiotherapy or disease that have not been relieved to grade ≤2, and any opportunistic or active systemic infection that has not been relieved before enrollment.

[0266] 4. Received non-corticosteroid immunosuppressive therapy within the last 6 months before leukapheresis.

[0267] 5. Patients with active, known or suspected autoimmune diseases (except autoimmune-related hypothyroidism receiving thyroid replacement therapy), controlled type 1 diabetes, and patients with eczema, psoriasis, lichen simplex or vitiligo with only skin manifestations.

[0268] 6. Patients who have previously received allogeneic bone marrow or solid organ transplantation.

[0269] 7. Administering a live virus vaccine within 4 weeks prior to undergoing leukapheresis to produce autologous blood products.

[0270] 8. Systemic treatment with corticosteroids (>10 mg prednisone or equivalent per day) or other immunosuppressive medications within 14 days prior to leukapheresis for the manufacture of autologous blood products. Inhaled, intranasal, intra-articular, and topical (including ocular) steroids are permitted. Steroid replacement is permitted in patients with adrenal insufficiency. Fludrocortisone is permitted as a substitute for mineralocorticoids in patients with adrenal insufficiency.

[0271] 9. Known active CNS metastases and / or carcinomatous meningitis. Patients with previously treated brain metastases may participate provided that their disease is stable (no signs of progression on imaging studies and any neurological symptoms have returned to baseline for at least 4 weeks prior to the first dose of the investigational product), there are no signs of new or enlarging brain metastases, and no steroid use has been performed for at least 7 days prior to leukapheresis to create autologous blood products. This exception does not include carcinomatous meningitis, which is excluded regardless of clinical status.

[0272] 10. Patients with active interstitial lung disease and any history of myocarditis.

[0273] 11. Clinically significant cardiac disease, including unstable angina, acute myocardial infarction within 6 months prior to leukapheresis for production of autologous blood products, New York Heart Association Class III or IV congestive heart failure, and arrhythmias requiring treatment.

[0274] 12. Systemic arterial thrombotic or embolic events, such as cerebrovascular accident (including ischemic attack), occurred within 1 month before leukocyte apheresis to produce autologous blood products.

[0275] 13. Systemic venous thrombotic events (e.g., deep vein thrombosis) or pulmonary artery events (e.g., pulmonary embolism) within 1 month before leukapheresis for autologous blood product production. Patients who had a venous thrombotic event before leukapheresis for autologous blood product production and were receiving stable anticoagulation therapy were eligible.

[0276] 14. History of or presence of clinically significant abnormal electrocardiogram (ECG).

[0277] 15. Left ventricular ejection fraction (LVEF) < 50%.

[0278] 16. Major surgery within 2 weeks of leukapheresis for autologous blood product production. All surgical wounds must be healed and free of infection or dehiscence after major surgery > 2 weeks before leukapheresis for autologous blood product production.

[0279] 17. Any other clinically significant comorbidity, such as active infection, known psychiatric or neurological disorder, or any other illness that, in the judgment of the investigator, may impair compliance with the protocol, interfere with the interpretation of the study results, or put the patient at risk for safety.

[0280] 18. Known human immunodeficiency virus (HIV) infection, or active hepatitis B, hepatitis C, or Mycobacterium tuberculosis infection.

[0281] 19. Female patients who are breastfeeding or have a positive serum pregnancy test at the screening visit.

[0282] 20. History of allergic or hypersensitivity reaction to any component of the drug product.

[0283] History of alcohol and / or illegal drug abuse within 12 months prior to signing the ICF.

[0284] Patients in Part 1B.

[0285] 22. History of severe allergic reaction to chimeric, human or humanized antibodies or infused proteins.

[0286] 23. Known hypersensitivity to pembrolizumab, Chinese hamster ovary cell product, or any component of pembrolizumab formulation.

[0287] 24. Any history of grade 3 immune-related AE (irAE) during previous immunotherapy (patients with endocrinopathies receiving replacement therapy or asymptomatic elevation of serum amylase or lipase are eligible), any irAE leading to permanent discontinuation of previous ICI.

[0288] Patients in Part 2

[0289] 25. Prior ICI treatment (including but not limited to anti-PD-1, anti-PD-L1, anti-CTLA4 or anti-LAG3, etc.). Other investigational immunotherapeutic agents must be discussed.

[0290] 26. History of severe allergic reaction to chimeric, human or humanized antibodies or infused proteins.

[0291] Leukapheresis

[0292] The goal of leukapheresis is to provide a PBMC yield of approximately 10 to 14 × 109 cells per patient to support a full course of therapy. If a lower yield is anticipated, efforts should be made to adjust the procedure. Depending on local procedures, a white blood cell (WBC) or complete blood cell (CBC) count should be performed during leukapheresis to increase the amount of blood processed. If a WBC or CBC count cannot be performed during leukapheresis, a sample should be collected at the end of leukapheresis to determine the WBC count in leukopak. Results should be processed as quickly as possible and provided to the sponsor in real time. Concomitant medications should be collected.

[0293] Tumor Response Assessment and Timeline

[0294] Tumor assessments will be performed by the study center at screening (baseline), every 6 weeks (±7 days) for the first 2 tumor assessments, every 8 weeks thereafter until the end of the first year, and every 12 weeks thereafter for the second year, or until disease progression confirmed by RECIST1.1 and iRECIST, initiation of subsequent treatment, unacceptable toxicity, withdrawal of consent, or death (whichever occurs first). This will be done by imaging. Patients who experience disease progression according to RECIST 1.1 may continue dosing, provided that the treating investigator deems it to be in the best interest of the patient to confirm disease progression, i.e., iCPD according to iRECIST.

[0295] Fresh tissue tumor biopsies obtained during screening should not be followed for radiologic response.

[0296] If a patient discontinues the investigational product for reasons other than disease progression, imaging of the patient should continue on the schedule above. If a patient discontinues treatment due to clinical worsening, AEs related to clinical progression should be documented on the AE page. Radiographic assessments should be obtained and documented in the eCRF according to the assessment and procedure schedule.

[0297] At screening and all subsequent time points, computed tomography (CT) of the trunk (chest, abdomen, and pelvis) and all known sites of disease are required for cervical, anal / rectal, vulvar / vaginal, and penile cancers; CT of the head, neck, chest, and other known areas of involvement is required for oropharyngeal cancer. Magnetic resonance imaging (MRI) is permitted if CT scanning is not available or if adequate tumor assessment is not possible. However, the radiographic procedures used for tumor assessment at screening should be used throughout the study. For all other advanced solid tumor types, the investigator should image all known sites of disease using the imaging modality that the investigator deems most appropriate for the tumor type in question.

[0298] All patients with a history of brain metastases will undergo a brain MRI at screening and can be repeated at subsequent time points for any patient with a history of brain metastases and / or who develops symptoms suggestive of brain metastases. CT scans can be used if patients cannot tolerate MRI or have contraindications to MRI.

[0299] The assessment should be performed by the same reviewer to ensure internal consistency at each visit. If PD is suspected, the CT scan should be repeated at any time at the discretion of the investigator. For patients who achieve a partial response (PR) or complete response (CR), tumor assessment should be repeated after 4 weeks to confirm the response.

[0300] Pharmacodynamic evaluation, including immunogenicity measurement sample collection plan

[0301] Tumor biopsy

[0302] Prior to starting treatment (Cycle 1, Day 1), patients will undergo a tumor biopsy (primary or metastatic), which may also be obtained from sites that have previously received radiation therapy and have active tumor growth. Fresh tissue tumor biopsies obtained during screening should not be followed for radiological response. All patients will undergo an additional tumor biopsy on Cycle 2 Day 8 (± 2 days) from the same primary or metastatic tumor.

[0303] If the patient progresses or discontinues treatment, an optional tumor biopsy (pre-dose) is required. If possible, another mass should be obtained from the same anatomic location if it is not possible to biopsy the same site as the screening biopsy. These unscheduled biopsies may also be obtained at other time points if the patient consents to further characterize the mechanism of response.

[0304] Tumor tissue should be of good quality based on gross and viable tumor content. If needed or applicable, archived tumor tissue can be provided for central confirmation of HPV-16 positivity.

[0305] Pharmacodynamic evaluation

[0306] Baseline and post-treatment blood and tumor samples will be collected according to the assessment and program schedule for pharmacodynamic and biomarker assessments. Tests include, but are not limited to, immunophenotyping, measurement of T cell biomarkers and cytokine production, endogenous immune responses, and HPV-16 cfDNA. Changes in circulating blood cells and cellular responses in tumor biopsies may also be evaluated. Tumor tissue will be analyzed by immunohistochemistry and / or other related techniques to assess biomarkers associated with cancer, inflammation, immune response, changes in the tumor microenvironment, and related areas of concern. Throughout the clinical study, other pharmacodynamic assessments and biomarkers discovered may be studied.

[0307] Optional pharmacogenomic analysis

[0308] Optional pharmacogenomic assessments may be performed on blood samples and / or tumor tissue samples. These optional tests may include, but are not limited to, transcriptome analysis and T-cell receptor sequencing. No additional samples are required to complete this testing. If the patient provides consent, the test will be performed.

[0309] Cytokine assessment

[0310] Blood samples will be collected from all patients for cytokine testing as per the evaluation and procedure schedule. Patients with Grade 2, 3, or 4 CRS will have additional cytokine plasma levels measured during Grade 2, 3, or 4 CRS events. Blood should be collected at the time of diagnosis of CRS, when severity increases (e.g., when Grade 2 CRS progresses to Grade 3 CRS), at the onset of neurologic symptoms, and at discharge or remission.

[0311] Evaluation of the cytokine panel will include, but is not limited to, IFN gamma (IFNγ) and IL-6. Although CRS may have a delayed onset, it rarely develops more than 14 days after initiation of treatment. If a patient exhibits symptoms consistent with CRS occurring outside of this time frame, careful evaluation for alternative causes should be performed.

[0312] Cytokines will also be monitored for pharmacodynamic evaluation. Baseline and post-treatment serum samples will be collected to assess the anti-tumor immune response by measuring cytokines that can provide information about the inflammatory response to the drug.

[0313] Security Assessment

[0314] Safety was evaluated in this study by monitoring all SAEs and non-serious AEs and laboratory abnormalities, which were defined and graded according to the NCICTCAE version 5.0. General safety assessments included physical examinations and specific laboratory evaluations, including serum chemistry, coagulation, and blood cell counts (including differentiation). SAEs and ≥ grade 2 AESIs will be reported in a rapid manner for entry into the safety database.

[0315] During the study, all safety events observed (including CRS events that resolved to Grade 2) were reviewed and a decision was made as to whether a given event warranted the initiation of staged enrollment of patients following this event. Staggered enrollment of potential additional monotherapy cohorts (Part 1) or combination safety cohorts (Part 2) requires that all subsequent newly enrolled patients in one or more cohorts be staggered by 1 week. Semi-continuous enrollment of patients may continue in some cohorts, if applicable. Patients will undergo cytokine release assays during the study. Patients with Grade 2, 3, or 4 CRS will require additional blood samples for safety laboratory and cytokine panel evaluations.

[0316] Exposure to immune checkpoint inhibitors may increase the risk of irAEs, especially autoimmune diseases. Therefore, irAEs should be detected and treated as early as possible to avoid potential serious complications.

[0317] All patients returned to the clinic for safety follow-up within 15 to 45 days after the last dose of the study product. All AEs and SAEs were recorded until 6 weeks after the last dose of the study product (EOD6W) or 45 days after withdrawal from the study or initiation of another anticancer therapy, whichever occurred first. Only persistent SAEs determined by the investigator to be possibly, probably, or definitely related to the drug product monotherapy or combination therapy were followed up.

[0318] Physical examination and height and weight

[0319] The physical examination includes height (screening only), weight, and assessment of general appearance and evaluation of the following systems: integumentary system, head, eyes, ears, nose, mouth / throat / neck, thyroid, lymph nodes, respiratory system, cardiovascular system, gastrointestinal tract, extremities, musculoskeletal system, neurologic system, gynecologic, and genitourinary system (if indicated). It is particularly important to measure weight during the physical examination of patients within 24 hours after leukapheresis because patient dosing is determined by weight. Vital signs are collected, including measurement of systolic and diastolic blood pressure, heart rate, temperature, and respiratory rate with the patient in the seated position.

[0320] Physical status

[0321] Eastern Cooperative Oncology Group scales and criteria are used to assess a patient's performance status, evaluate the impact of the disease on a patient's ability to perform daily activities, and determine appropriate treatment and prognosis.

[0322] Vital signs

[0323] Vital signs were collected, including measurement of systolic and diastolic blood pressure, heart rate, temperature, and respiratory rate with the patient in a sitting position.

[0324] 12-lead ECG

[0325] A 12-lead ECG was performed by qualified on-site personnel using an ECG machine that determined heart rate, PR interval, QRS interval, RR interval, QT interval, and QTc interval collected by QTcB (QTc corrected by Bazett's formula) and QTcF (QTc corrected by Fridericia's formula). During the ECG collection, the patient should be in a resting position, in a quiet environment without distractions (e.g., television, cell phones) for at least 10 minutes before the ECG is collected. All ECGs must be evaluated by a qualified physician for abnormalities.

[0326] Echocardiography or multiple gated acquisition (MUGA) scanning was performed to measure LVEF at screening and as clinically indicated.

[0327] Laboratory evaluation

[0328] Samples will be collected for clinical laboratory evaluation. The clinical laboratory tests outlined in Table 3 were performed by the site. Samples for the laboratory tests outlined in Table 3 were collected into appropriate tubes and processed according to the site's standard procedures. Clinical laboratory variables are listed in Table 3.

[0329] Table 3. Clinical laboratory evaluation

[0330]

[0331]

[0332] Abbreviations: β-hCG = β-human chorionic gonadotropin; CRS = cytokine release syndrome; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone.

[0333] a These laboratory tests need to be collected prior to or on the day of leukapheresis, with results available for review prior to leukapheresis.

[0334] b Coagulation parameter results should be available for review (by the investigator or designee) on the day of any tumor biopsy or the day before.

[0335] Adverse Events

[0336] Clinical sample AE is any adverse medical event that occurs in patients, but it does not necessarily have a causal relationship with the administered research product. Therefore, AE can be any adverse or unexpected signs (including abnormal laboratory findings), symptoms or diseases that are time-related to the use of the research product, whether or not it is related to the research product. Adverse events may be new events, or they may be pre-existing conditions that are aggravated or worsened in severity or frequency. Adverse events may be clinically significant changes compared to baseline in physical examinations, laboratory tests or other diagnostic studies. In this study, if the occurrence time of AE is within 6 weeks after the administration of the research product to the last dose of the research product, the AE is caused by treatment.

[0337] Serious Adverse Events (SAEs)

[0338] Any AE that results in any of the following is a SAE:

[0339] die

[0340] Immediately life-threatening

[0341] Requires hospitalization or prolongation of existing hospitalization

[0342] Causes persistent or severe disability or incapacity to work

[0343] Cause congenital anomalies or birth defects

[0344] Important medical events that could endanger the patient or require medical intervention to prevent 1 of the outcomes listed above

[0345] All SAEs, whether study related or not, occurring after any patient signs the ICF and before, during, or within 30 days of cessation of treatment must be recorded on the appropriate clinical procedure form.

[0346] Adverse Events of Special Interest

[0347] AESIs are AEs (serious or non-serious) that are specific to the scientific and medical issues of the investigational product and require ongoing monitoring by the investigator and immediate notification to the sponsor. Such AEs may require further investigation to describe and understand. During a study, adverse events of particular interest may be added or deleted by protocol amendment. The following AEs are considered AESIs:

[0348] Events suggestive of hypersensitivity reactions, cytokine release, systemic inflammatory response syndrome, or systemic inflammatory activation

[0349] Flu-like illness

[0350] Infusion reaction syndrome

[0351] irAEs associated with immunotherapy, such as myocarditis, neurologic irAEs, elevated aminotransferases of immune-related etiology, and nephritis

[0352] In addition, the following events were reported to the sponsor:

[0353] Liver test abnormalities met Hy's postulate criteria, i.e., AST or ALT laboratory values ​​≥3×ULN, and total bilirubin laboratory values ​​≥2×ULN, with alkaline phosphatase laboratory values ​​<2×ULN, as determined by protocol-specified or unplanned laboratory testing.

[0354] Severity Assessment

[0355] NCI CTCAE version 5.0 is used to assess and grade the severity of AEs and laboratory abnormalities. ASTCT consensus grading will be used for CRS and ICANS. Each AE term will be mapped to the latest version of the Medical Dictionary for Regulatory Activities (MedDRA) terms and codes. If the event is not covered by CTCAE version 5.0, the guidelines shown in Table 4 should be used to assess severity.

[0356] Table 4. Severity and toxicity grade of events not covered by CTCAE

[0357]

[0358]

[0359] Source: (NIAID, 2003)

[0360] Abbreviation: CTCAE = Common Terminology Criteria for Adverse Events

[0361] Causality Assessment

[0362] Investigators will provide an assessment of the causality and relationship of AEs and SAEs to study treatment. For patients receiving combination therapy (Parts 1B and 2), causality will be assessed separately for each protocol-assigned therapy. If the event is consistent with the pembrolizumab prescribing label, a reasonable suspicion of causality should be attributed solely to pembrolizumab.

[0363] Table 5. Causality of Adverse Events Assessed by Investigators

[0364]

[0365] Anticipation

[0366] AEs that were not listed in the applicable product information (e.g., Drug Product IB or approved labeling for atezolizumab, ipilimumab, or nivolumab), or that were inconsistent with the specificity or severity in the applicable product information, were considered unexpected.

[0367] Efficacy analysis

[0368] Progression-free survival (PFS) is defined as the time from Cycle 1 Day 1 to the first documentation of objective tumor progression (PD, radiological) according to RECIST 1.1 or death from any cause, whichever occurs first. For patients who do not have objective tumor progression and remain on study at the time of analysis, are receiving antitumor therapy other than investigational product, or have withdrawn from treatment follow-up before objective tumor progression is documented, progression-free survival data will be censored at the last tumor assessment date to document the absence of PD. Patients who do not have tumor assessments after enrollment and are unknown to have died will have PFS censored at Cycle 1 Day 1. PFS will be assessed according to both RECIST 1.1 and iRECIST criteria to accommodate different practices among participating sites.

[0369] Overall survival (OS) was defined as the time from Cycle 1 Day 1 to the date of death from any cause. If death was not confirmed, survival was censored at the last date the patient was known to be alive. Survival for patients with missing data beyond Cycle 1 Day 1 was censored at Cycle 1 Day 1.

[0370] Objective response rate (ORR) is defined as the proportion of patients who achieve a CR or PR according to RECIST 1.1. Objective response rate will be presented as unconfirmed and confirmed ORR. A confirmed response is one that persists on repeat imaging studies at least 28 days after the first documented response. Similarly, iORR by iRECIST will also be summarized and reported.

[0371] Duration of response is defined as the time in months from the start of study treatment to the first occurrence of CR or PR according to RECIST 1.1. This will be calculated only for patients who achieve a CR or PR.

[0372] Duration of response (DoR) is defined as the time from the first documented PR or CR to the first documented objective tumor progression or death due to any cause. For patients who have not had tumor progression at the time of analysis and remain on study, receive antitumor therapy other than the investigational product, or withdraw from study follow-up before documented objective tumor progression, the duration of response data will be reviewed on the day of the last tumor assessment to record the absence of PD and will be reviewed at the time of the last tumor assessment. Similarly, the iDoR by iRECIST will also be summarized and reported.

[0373] Once all tumor assessments from Cycle 1 Day 1 until disease progression or death are recorded, the best overall response (BOR) will be determined. Generally, this is the best response of all assessments; however, confirmation of CR, PR, and stable disease (SD) will also be used in determining the BOR. To confirm a CR or PR, changes in tumor measurements must be confirmed by repeat assessments no less than 4 weeks (28 days) after the response criteria were first met. To confirm an SD, it must occur at least 12 weeks from Cycle 1 Day 1; otherwise, the BOR will be based on subsequent assessments. The best overall response will be summarized as a percentage and use enrollment as the base date as the event time variable for the best response time. Similarly, the iBOR by iRECIST will also be summarized and reported.

[0374] The disease control rate (DCR) is the proportion of patients with a BOR of CR, PR, or SD as determined by RECIST 1.1 at a defined time point. All patients in the safety population who had measurable disease at baseline and were eligible for tumor assessment will be considered as the denominator for the DCR rates at 3, 6, and 12 months. Similarly, the iDCR by iRECIST will also be summarized and reported.

[0375] Stable disease 12 weeks is the proportion of patients with a BOR of CR, PR, or SD per RECIST 1.1 that is maintained for at least 12 weeks. All patients in the safety population with measurable disease at baseline and eligible for tumor assessment will be considered as the denominator for the 3-, 6-, and 12-month proportions. Similarly, SD 12 weeks by iRECIST will also be summarized and reported.

[0376] Efficacy analyses will be performed on the safety population. Best overall response will be reported as incidence and percentage. Kaplan-Meier analysis will be used to describe efficacy parameters, OS, PFS, response time, and DOR. Patients such as ORR, DCR, and proportions of patients with SD ≥ 12 weeks will be reported. When a meaningful sample size is available, 95% confidence intervals will also be reported. If the efficacy evaluable population is different from the safety population, the efficacy evaluable population will also be used for efficacy analysis. All assessments of response assessments using RECIST 1.1 or iRECIST will be analyzed using the investigator's review assessment.

[0377] Median PFS and two-sided 95% confidence intervals were estimated using the Kaplan-Meier method. Patients who died were considered to have experienced an event regardless of the cause of death, unless they received subsequent anticancer therapy before death. If subsequent therapy was received, the patient will be censored at the date of the last evaluable tumor assessment before the subsequent therapy. Patients who withdraw study consent will be considered censored at the last evaluable tumor assessment before the withdrawal of consent. Patients who are still alive at the clinical data cutoff date will be censored at the most recent evaluable tumor assessment. All patients lost to follow-up before the clinical data cutoff date will also be considered censored at the last evaluable tumor assessment before loss of follow-up.

[0378] Duration of response, best overall response, and overall survival will use the same methods as PFS. In addition, iPFS, iBOR, iDCR, and time to iBOR using iRECIST will be analyzed and reported using similar methods.

[0379] Objective response rate (ORR) is presented as a ratio with 95% confidence interval based on exact binomial distribution. Point estimates and two-sided 95% confidence intervals for ORR will be provided. DCR and SD sustained for at least 12 weeks will be reported as point estimates.

[0380] Security Analysis

[0381] All safety parameters were analyzed using the safety population. Safety parameters included: AEs, laboratory evaluations, vital signs, ECOG, exposure, ECG, ECHO / MUGA, and physical examination.

[0382] The primary endpoints for safety were the number of patients experiencing any AE and observed toxicity from drug product administration, with severity assessed using the NCI CTCAE version 5.0. All AEs occurring after the first administration of drug product were included in the analysis. Adverse events were collected from the time the informed consent was signed; however, the analysis focused on treatment-emergent AEs.

[0383] AEs were analyzed using descriptive statistics. For patients with multiple occurrences of a given AE, the highest severity was used.

[0384] Adverse Events

[0385] Adverse events will be collected from the time each patient signs the initial ICF until EODW6. AEs will be coded using the MedDRA dictionary. Adverse events that the investigator considers to be possibly, probably, or definitely related to the study product will be classified as related for ease of summary. All AEs are provided in the patient listing.

[0386] Clinical laboratory evaluation

[0387] Baseline is defined as the last nonmissing value prior to first exposure to study product. This is usually pre-dose on Cycle Day 1, but can be earlier. Actual values ​​and changes from baseline clinical laboratory tests are summarized by study visit.

[0388] Laboratory test results were classified according to NCI CTCAE version 5.0 and clinical significance was determined by the researchers. If multiple laboratory results were reported for each parameter at each study visit, the result with the most severe classification was selected for analysis. The change table was created to show the maximum change in the graded laboratory parameters relative to baseline. All laboratory assessments are provided in a list.

[0389] Patients with clinically significant abnormal laboratory test results are listed. This list includes all abnormal and clinically significant laboratory parameter results for the patient as determined by the investigator during the study visit.

[0390] Vital signs

[0391] Vital sign values ​​were categorized according to clinical significance as determined by the investigators. The number of patients with nonmissing results, the number and percentage of patients with clinically insignificant results, and the number and percentage of patients with clinically significant results were summarized by study visit and study time point. If more than one vital sign result was reported for each parameter per study visit and study time point, the result that produced the most severe categorization was selected for analysis.

[0392] Patients with clinically significant vital sign values ​​are listed. This list includes all vital sign parameter results determined by the investigator to be clinically significant for the patient at the study time point.

[0393] Physical examination

[0394] Abnormal physical examination findings are listed.

[0395] ECG results are presented in a change table (normal, not clinically significant abnormal, abnormal, clinically significant) to show the maximum change from baseline. All ECG results are displayed in the patient list.

[0396] All safety data are provided in the table.

[0397] ECOG PS was collected at each scheduled visit and the change in ECOG PS from baseline was summarized. The change in ECOG PS from baseline was summarized as a continuous variable and a categorical variable. A decrease of ≥1 point from baseline was classified as an "improvement" from baseline. An increase of ≥1 point from baseline was classified as a "worse" from baseline. At each post-enrollment time point when ECOG PS was assessed, improvement, worsening, and no change in ECOG PS from baseline were summarized by treatment status as a categorical variable.

[0398] Pharmacodynamic analysis

[0399] Biomarker analyses in this study are exploratory and will be summarized for each time point for change from baseline and percent change from baseline. Descriptive statistics and graphical methods can be used to explore the association between pharmacodynamic markers and drug product dose. Descriptive statistics (mean, standard deviation, median, minimum, maximum, and geometric mean (if applicable)) will be reported for each biomarker. Graphs of individual values ​​over time according to dose group will be presented.

[0400] Dose Manufacturing Feasibility

[0401] The feasibility of dose manufacturing will be assessed based on individual patient batch yields, product failures that contraindicate use, and any additional information deemed relevant from leukapheresis to drug product production.

[0402] Example 2. Preliminary efficacy

[0403] Two patients in the low-dose cohort had significant increases in CD8+ T-cell tumor infiltration and stable disease. One of these 2 patients, who had a low tumor burden, has been on study for more than 10 months and appears to have achieved clinical benefit. Of the 5 evaluable patients in the highest-dose cohort (which has been determined to be RP2D), 1 patient with checkpoint-refractory head and neck cancer had a clinical response and symptomatic improvement. The target lesion demonstrated a complete response on both radiologic assessments. At the most recent assessment, the predominant oropharyngeal lesions showed continued improvement on physical examination; however, new skin lesions were noted. This patient had the greatest increase in the number of tumor-infiltrating CD8+ T cells, along with a decrease in the number of cells expressing E6 and E7 (not shown). Increased PD-L1 expression also suggests potential synergy with combination therapies, such as Figure 3 shown.

[0404] It should be understood that the detailed description section, rather than the summary and abstract sections, is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventors, and therefore, are not intended to limit the present disclosure and the appended claims in any way.

[0405] The present disclosure has been described above with the aid of functional building blocks that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be defined as long as the specific functions and their relationships are appropriately performed.

[0406] The foregoing description of specific aspects will fully reveal the general nature of the present disclosure so that others can easily modify and / or adapt such specific aspects for various applications by applying knowledge within the technical scope of the art without departing from the general concept of the present disclosure. Therefore, based on the teaching content and guidance content presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is intended to illustrate rather than limit, so the technical staff will interpret the terms or wording of this specification based on the teaching content and guidance content.

[0407] The breadth and scope of the present disclosure should not be limited to the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0408] The claims in this application are different from those in the parent application or other related applications. Applicant therefore withdraws any disclaimer regarding the scope of the claims made in the parent application or any prior application related to this application. Examiners are therefore advised to revisit any such prior disclaimers and the references made for avoidance. Examiners are also reminded that any disclaimer made in this application should not be construed as or against the parent application.

[0409] Table B. Exemplary sequences:

[0410]

[0411]

[0412]

[0413]

[0414]

Claims

1. A method for treating a human papillomavirus (HPV)-associated cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner.

2. The method of claim 1, further comprising administering an immune checkpoint inhibitor to the subject.

3. A method for treating a human papillomavirus (HPV)-associated cancer in a subject in need thereof, the method comprising administering to the subject (i) a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner, and (ii) an immune checkpoint inhibitor.

4. The method of any one of claims 1 to 3, wherein the enhanced APCs comprise T cells, B cells, NK cells, monocytes, or a combination thereof.

5. The method of any one of claims 1 to 4, wherein the HPV antigen comprises an HPV-16 antigen or an HPV-18 antigen.

6. The method of any one of claims 1 to 5, wherein the HPV antigen comprises a peptide derived from HPV E6 and / or HPV E7.

7. The method of claim 6, wherein the HPV antigen comprises a peptide derived from HPV E6 and a peptide derived from HPV E7.

8. The method of claim 6 or 7, wherein the peptide derived from HPV E6 and / or HPV E7 is full-length E6 and / or E7.

9. The method of any one of claims 1 to 8, wherein the HPV antigen comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-17.

10. The method according to any one of claims 1 to 8, wherein the HPV antigen comprises a first antigen comprising the amino acid sequence shown in SEQ ID NO: 14 and a second antigen comprising the amino acid sequence shown in SEQ ID NO:

15.

11. The method according to any one of claims 1 to 10, wherein the HPV antigen comprises a first antigen comprising the amino acid sequence shown in SEQ ID NO: 16 and a second antigen comprising the amino acid sequence shown in SEQ ID NO:

17.

12. The method of any one of claims 1 to 11, wherein the enhanced APC exhibits increased expression of a co-stimulatory molecule compared to a corresponding non-enhanced APC ("reference APC").

13. The method of claim 12, wherein the co-stimulatory molecule comprises CD86.

14. The method of any one of claims 1 to 13, wherein the enhanced APC exhibits increased expression of a cytokine compared to a corresponding unmodified APC ("reference APC").

15. The method of claim 14, wherein the cytokine comprises a membrane-bound cytokine.

16. The method of claim 14 or 15, wherein the cytokine comprises IL-2, IL-12, or both.

17. The method of any one of claims 1 to 16, wherein the enhanced APC has been prepared by passing a cell suspension containing the input APC through a cell deformation constriction, thereby causing a disturbance in the APC, so that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine enters the APC through the disturbance when in contact with the APC, thereby producing the enhanced APC.

18. The method of claim 17, wherein the cell suspension containing the input APC has been cultured in combination with the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine, so that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine are in contact with the input APC.

19. The method of claim 17 or 18, wherein the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine is mRNA.

20. The method of any one of claims 17 to 19, wherein the diameter of the cell deformation constriction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

21. The method of any one of claims 1 to 20, wherein the enhanced APCs have been conditioned in a culture medium comprising an adjuvant.

22. The method of claim 21, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 2 hours to about 10 hours.

23. The method of claim 21 or 22, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 3 hours to about 6 hours.

24. The method of any one of claims 21 to 23, wherein the enhanced APCs have been conditioned in medium comprising an adjuvant for about 4 hours.

25. The method of any one of claims 21 to 24, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant at about 37°C.

26. The method of any one of claims 21 to 25, wherein the adjuvant comprises a CpG oligodeoxynucleotide (ODN), LPS, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist, or a TLR 9 agonist.

27. The method of claim 26, wherein the adjuvant is ODN.

28. The method of any one of claims 2 to 27, wherein the immune checkpoint inhibitor comprises an antagonist of CTLA-4 and / or an antagonist of PD-1 / PD-L1.

29. The method of claim 28, wherein the immune checkpoint inhibitor is an antagonist of PD-1 / PD-L1.

30. The method of claim 28 or 29, wherein the antagonist of PD-1 / PD-L1 is an antibody that binds to PD-1 or an antibody that binds to PD-L1.

31. The method of any one of claims 28 to 30, wherein the antagonist of CTLA-4 is an antibody that binds to CTLA-4.

32. The method of claim 30 or 31, wherein the antibody that binds to PD-1 is pembrolizumab.

33. The method of claim 30 or 31, wherein the antibody that binds to PD-1 is nivolumab.

34. The method of claim 30 or 31, wherein the antibody that binds PD-L1 is atezolizumab.

35. The method of any one of claims 31 to 34, wherein the antibody that binds CTLA-4 is ipilimumab.

36. A method for treating HPV in a subject in need thereof + A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering to the subject a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV antigens and are capable of activating T cells in an HLA-agnostic manner.

37. The method of claim 36, wherein the enhanced APCs comprise T cells, B cells, NK cells, monocytes, or a combination thereof.

38. The method of claim 36 or 37, wherein the HPV antigen comprises an HPV-16 antigen or an HPV-18 antigen.

39. The method of any one of claims 36 to 38, wherein the HPV antigen comprises a peptide derived from HPV E6 and / or HPV E7.

40. The method of any one of claims 36 to 39, wherein the HPV antigens comprise a peptide derived from HPV E6 and a peptide derived from HPV E7.

41. The method of claim 39 or 40, wherein the peptide derived from HPV E6 and / or HPV E7 is full-length E6 and / or E7.

42. The method of any one of claims 36 to 41, wherein the HPV antigen comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-17.

43. The method of any one of claims 36 to 41, wherein the HPV antigen comprises a first antigen comprising the amino acid sequence shown in SEQ ID NO: 14 and a second antigen comprising the amino acid sequence shown in SEQ ID NO:

15.

44. The method of claim 43, wherein the HPV antigen comprises a first antigen comprising the amino acid sequence shown in SEQ ID NO:16 and a second antigen comprising the amino acid sequence shown in SEQ ID NO:

17.

45. The method of any one of claims 36 to 44, wherein the enhanced APC exhibits increased expression of a co-stimulatory molecule compared to a corresponding unmodified APC ("reference APC").

46. ​​The method of claim 45, wherein the co-stimulatory molecule comprises CD86.

47. The method of any one of claims 36 to 46, wherein the enhanced APC exhibits increased expression of a cytokine compared to a corresponding unmodified APC ("reference APC").

48. The method of claim 47, wherein the cytokine comprises a membrane-bound cytokine.

49. The method of claim 47 or 48, wherein the cytokine comprises IL-2, IL-12, or both.

50. The method of any one of claims 36 to 49, wherein the enhanced APC has been prepared by passing a cell suspension containing the input APC through a cell deformation constriction, thereby causing a perturbation in the APC, such that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule, and / or the nucleic acid encoding the cytokine enters the APC through the perturbation when in contact with the APC, thereby producing the enhanced APC.

51. A method as claimed in claim 50, wherein the cell suspension containing the input APC has been cultured in combination with the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine, so that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine are in contact with the input APC.

52. The method of claim 50 or 51, wherein the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and / or the nucleic acid encoding the cytokine is mRNA.

53. The method of any one of claims 50 to 52, wherein the diameter of the cell deformation constriction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

54. The method of any one of claims 36 to 53, wherein the enhanced APCs have been conditioned in a culture medium comprising an adjuvant.

55. The method of claim 54, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 2 hours to about 10 hours.

56. The method of claim 54 or 55, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 3 hours to about 6 hours.

57. The method of any one of claims 54 to 56, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 4 hours.

58. The method of any one of claims 54 to 57, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant at about 37°C.

59. The method of any one of claims 54 to 58, wherein the adjuvant comprises a CpG oligodeoxynucleotide (ODN), LPS, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist, or a TLR 9 agonist.

60. The method of claim 59, wherein the adjuvant is ODN.

61. The method of any one of claims 36 to 60, wherein the composition comprising enhanced APCs is administered in combination with an immune checkpoint inhibitor.

62. The method of claim 61, wherein the one or more immune checkpoint inhibitors comprise an antagonist of CTLA-4 and / or an antagonist of PD-1 / PD-L1.

63. The method of claim 62, wherein the immune checkpoint inhibitor is an antagonist of PD-1 / PD-L1.

64. The method of claim 62 or 63, wherein the antagonist of PD-1 / PD-L1 is an antibody that binds to PD-1 or an antibody that binds to PD-L1.

65. The method of any one of claims 62 to 64, wherein the antagonist of CTLA-4 is an antibody that binds to CTLA-4.

66. The method of claim 64 or 65, wherein the antibody that binds to PD-1 is pembrolizumab.

67. The method of claim 64 or 65, wherein the antibody that binds to PD-1 is nivolumab.

68. The method of claim 64 or 65, wherein the antibody that binds PD-L1 is atezolizumab.

69. The method of any one of claims 64 or 65, wherein the antibody that binds CTLA-4 is ipilimumab.

70. The method of any one of claims 1 to 69, wherein the subject is a human.

71. The method of any one of claims 1 to 70, wherein the subject is infected with human papillomavirus type 16 (HPV16 + ) was positive.

72. The method of any one of claims 1 to 71, wherein the HPV-related cancer and / or HPV + Cancers include cancer of the cervix, anus, vulva, vagina, penis, or oropharynx.

73. The method of any one of claims 1 to 72, wherein the composition comprising the enhanced APCs is present at about 0.25×10 6 cells / kg to about 7.50×10 6 cells / kg.

74. The method of claim 73, wherein the composition comprising the enhanced APC is present in an amount of about 0.5×10 6 cells / kg to about 5.0×10 6 cells / kg.

75. The method of claim 73, wherein the composition comprising the enhanced APC is present in an amount of about 0.5×10 6 cells / kg.

76. The method of claim 73, wherein the composition comprising the enhanced APCs is present in an amount of about 2.5×10 6 cells / kg.

77. The method of claim 73, wherein the composition comprising the enhanced APC is present at about 5.0×10 6 cells / kg.

78. The method of claim 73, wherein the composition comprising the enhanced APC is present in an amount of about 0.25×10 6 cells / kg.

79. The method of claim 73, wherein the composition comprising the enhanced APC is present at about 1.25×10 6 cells / kg.

80. The method of claim 73, wherein the composition comprising the enhanced APCs is present in an amount of about 3.25×10 6 cells / kg.

81. The method of claim 73, wherein the composition comprising the enhanced APCs is present at about 7.5×10 6 cells / kg.

82. The method of any one of claims 1 to 81, wherein the composition comprising enhanced APCs is administered intravenously.

83. The method of any one of claims 2 to 35 and 61 to 82, wherein the immune checkpoint inhibitor is administered intravenously, orally, or subcutaneously.

84. The method of claim 83, wherein the immune checkpoint inhibitor is administered intravenously.

85. The method of any one of claims 32, 35, 66, and 68, wherein pembrolizumab is administered in an amount of about 10 mg to about 400 mg.

86. The method of claim 85, wherein pembrolizumab is administered in an amount of about 200 mg to about 400 mg.

87. The method of claim 85 or 86, wherein pembrolizumab is administered in an amount of about 200 mg.

88. The method of any one of claims 33, 35, 67, and 69, wherein nivolumab is administered in an amount of about 15 mg to about 500 mg.

89. The method of claim 88, wherein nivolumab is administered in an amount of about 240 mg to about 480 mg.

90. The method of claim 88 or 89, wherein nivolumab is administered in an amount of about 360 mg.

91. The method of any one of claims 34, 35, 38, and 69, wherein atezolizumab is administered in an amount of about 75 mg to about 1700 mg.

92. The method of claim 91, wherein atezolizumab is administered in an amount of about 840 mg to about 1680 mg.

93. The method of claim 91 or 92, wherein atezolizumab is administered in an amount of about 1200 mg.

94. The method of claim 35 or 69, wherein ipilimumab is administered in an amount of about 1 mg / kg to about 10 mg / kg.

95. The method of claim 94, wherein ipilimumab is administered in an amount of about 1 mg / kg to about 3 mg / kg.

96. The method of any one of claims 1 to 95, wherein the composition comprising enhanced APCs is administered on day 1 of a three-week cycle.

97. The method of any one of claims 1 to 96, wherein the composition comprising enhanced APCs is further administered on day 2 of the first three-week cycle.

98. The method of claim 95 or 96, wherein about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg or about 7.50×10 6 cells / kg.

99. The method of claim 97 or 98, wherein about 0.25×10 6 cells / kg, about 0.5×10 6 cells / kg, about 1.25×10 6 cells / kg, about 2.5×10 6 cells / kg, about 3.25×10 6 cells / kg, about 5.0×10 6 cells / kg or about 7.50×10 6 cells / kg.

100. The method of any one of claims 30 to 35 and 64 to 99, wherein the antibody that binds to PD-1, the antibody that binds to PD-L1, and / or the antibody that binds to CTLA-4 is administered once every three-week cycle.

101. The method of claim 100, wherein the antibody that binds PD-1 is administered on day 1 of a three-week cycle.

102. The method of claim 100, wherein the antibody that binds PD-1 is administered on day 8 of the first three-week cycle.

103. The method of claim 100, wherein the antibody that binds to PD-1 is administered on Day 8 of the first three-week cycle and Day 1 of each subsequent three-week cycle.

104. The method of claim 100, wherein the antibody that binds PD-1 is administered on day 1 of the third three-week cycle.

105. The method of claim 104, wherein the antibody that binds to PD-1 is administered on Day 1 of the third three-week cycle and Day 1 of each subsequent three-week cycle.

106. The method of any one of claims 96 to 105, wherein the antibody that binds to PD-1 is pembrolizumab, wherein the pembrolizumab is administered in an amount of about 200 mg.

107. The method of any one of claims 96 to 105, wherein the antibody that binds to PD-1 is nivolumab, wherein the nivolumab is administered in an amount of about 360 mg.

108. The method of any one of claims 96 to 100, wherein the antibody that binds CTLA-4 is administered on day 1 of each three-week cycle.

109. The method of any one of claims 96 to 100, wherein the antibody that binds CTLA-4 is administered once every two three-week cycles.

110. The method of any one of claims 96 to 100, 108, and 109, wherein the antibody that binds CTLA-4 is ipilimumab, wherein the ipilimumab is administered at a dose of about 3 mg / kg.

111. The method of any one of claims 96 to 100, wherein the antibody that binds PD-L1 is administered on Day 8 of the first three-week cycle and Day 1 of each subsequent cycle.

112. The method of any one of claims 96 to 100 and 111, wherein the antibody that binds to PD-L1 is atezolizumab, wherein the atezolizumab is administered at a dose of about 1200 mg.

113. The method of any one of claims 1 to 112, wherein the composition comprising enhanced APCs is administered to the subject for at least about 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months.

114. The method of any one of claims 1 to 113, wherein the composition comprising enhanced APCs comprises: (a) About 5×10 6 Enhanced APCs to about 1x 10 9 Enhanced APC, (b) a cryopreservation medium at a concentration of about 40% to about 95% (weight / weight), (c) a cryopreservation medium at a concentration of about 25% to about 35% (weight / weight), and (d) a human serum albumin solution having a concentration of 15% to about 25% (weight / weight), wherein the pH of the composition is from about pH 6.0 to about pH 8.

5.

115. The method of any one of claims 1 to 114, wherein the composition comprising enhanced APCs comprises: (a) About 8.1×10 7 Enhanced APC, (b) a cryopreservation medium at a concentration of about 50% (weight / weight), (c) a cryopreservation medium at a concentration of about 30% (weight / weight), and (d) a human serum albumin solution having a concentration of 20% (weight / weight), wherein the pH of the composition is from about pH 7.0 to about pH 7.

9.

116. The method of any one of claims 1 to 114, wherein the composition comprising enhanced APCs comprises: (a) About 1.05×10 8 Enhanced APC, (b) a cryopreservation medium at a concentration of about 50% (weight / weight), (c) a cryopreservation medium at a concentration of about 30% (weight / weight), and (d) a human serum albumin solution having a concentration of 20% (weight / weight), wherein the pH of the composition is from about pH 7.0 to about pH 7.

9.

117. The method of any one of claims 1 to 116, wherein the composition comprising enhanced APCs comprises about 1×10 6 Enhanced APC / mL to about 1x 10 8 Enhanced APC / mL.

118. The method of any one of claims 1 to 117, wherein the composition comprising enhanced APCs comprises about 8.5×10 6 Enhanced APC / mL.

119. The method of any one of claims 1 to 117, wherein the composition comprising enhanced APCs comprises about 1.1×10 7 Enhanced APC / mL.

120. The method of any one of claims 106 to 119, wherein the cryopreservation medium is CS10.

121. The method of any one of claims 106 to 120, wherein the cryopreservation medium is FRS.

122. A method for treating a human papillomavirus (HPV)-associated cancer in a subject in need thereof, the method comprising: The subject is administered: (i) a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner, and (ii) an antagonist of PD-1.

123. A method for treating HPV in a subject in need thereof + A method for treating recurrent, locally advanced or metastatic tumors, the method comprising administering a composition comprising modified antigen presenting cells ("enhanced APCs"), wherein the enhanced APCs comprise HPV E6 and / or HPV E7 antigens, CD86, membrane-bound interleukin-2 ("mbIL-2"), and membrane-bound interleukin-12 ("mbIL-12"), and wherein the enhanced APCs are capable of activating T cells in an HLA-agnostic manner.

124. The method of claim 123, wherein the composition comprising enhanced APCs is administered in combination with an antagonist of PD-1.

125. The method of claim 122 or 124, wherein the antagonist of PD-1 is an antibody that binds to PD-1.

126. The method of claim 125, wherein the antibody that binds PD-1 is pembrolizumab.

127. The method of any one of claims 122 to 126, wherein the enhanced APC has been prepared by passing a cell suspension containing the input APC through a cell deformation constriction, thereby causing a disturbance in the APC, so that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule, and the nucleic acid encoding the cytokine enter the APC through the disturbance when in contact with the APC, thereby producing the enhanced APC.

128. A method as claimed in claim 127, wherein the cell suspension containing the input APC has been cultured in combination with the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and the nucleic acid encoding the cytokine, so that the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule and the nucleic acid encoding the cytokine are in contact with the input APC.

129. The method of claim 127 or 128, wherein the nucleic acid encoding the antigen, the nucleic acid encoding the co-stimulatory molecule, and the nucleic acid encoding the cytokine are mRNA.

130. The method of any one of claims 127 to 129, wherein the diameter of the cell deformation constriction is about 4.2 μm to about 6 μm or about 4.2 μm to about 4.8 μm.

131. The method of any one of claims 127 to 130, wherein the enhanced APCs have been conditioned in a culture medium comprising an adjuvant.

132. The method of claim 131, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 2 hours to about 10 hours.

133. The method of claim 131 or 132, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 3 hours to about 6 hours.

134. The method of any one of claims 131 to 133, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant for about 4 hours.

135. The method of any one of claims 131 to 134, wherein the enhanced APCs have been conditioned in a medium comprising an adjuvant at about 37°C.

136. The method of any one of claims 131 to 135, wherein the adjuvant comprises a CpG oligodeoxynucleotide (ODN), LPS, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist, or a TLR 9 agonist.

137. The method of claim 136, wherein the adjuvant is ODN.

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