Methods for Treating Cancer Using Activated Antigen Carriers

Abstract

The present application provides an activating antigen carrier (AAC) for treating HPV-associated cancer. The AAC is derived from anucleated cells into which at least one antigen and an adjuvant have been delivered intracellularly. In some embodiments, the AAC is administered in combination with a checkpoint inhibitor, such as a CTLA4 antagonist and / or a PD-1 / PD-L1 agonist. In some aspects, the present invention provides a method for treating human papillomavirus (HPV)-associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of the activating antigen carrier (AAC), wherein the effective amount is about 0.1×10 8 AAC / kg ~ approx. 1 x 10 9 AAC / kg, where the AAC comprises at least one HPV antigen delivered intracellularly and an adjuvant.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 131,506, filed December 29, 2020, the entire contents of which are incorporated herein by reference. Submission of sequence listing in ASCII text file

[0002] The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 750322003540SEQLIST.TXT, Recorded: December 23, 2021, Size: 13,033 bytes).

[0003] FIELD OF THEINVENTION The present disclosure generally relates to methods, dosages and regimens of using an activated antigen carrier (AAC) comprising an HPV antigen and an adjuvant to treat individuals with HPV-associated cancer. Methods of producing such an AAC comprising at least one HPV antigen and an adjuvant, as well as compositions thereof, are also disclosed. [Background technology]

[0004] 2. Background of the Invention Papillomaviruses are small, non-enveloped DNA viruses with a virion size of approximately 55 nm in diameter. Over 100 HPV genotypes have been fully characterized, and many more are estimated to exist. HPV is a known cause of cervical cancer, as well as some vulvar, vaginal, penile, oropharyngeal, anal and rectal cancers. Most HPV infections are asymptomatic and resolve spontaneously, but persistent infection with one of the oncogenic HPV types can progress to precancerous conditions or cancer. Other HPV-associated diseases include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermal dysplasia, focal epithelial thickening, oral papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (SIL), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VAIN).

[0005] Many of the known human papillomavirus (HPV) types cause benign lesions with a subset being tumorigenic. Based on epidemiological and phylogenetic relationships, HPV types are classified into 15 "high-risk types" (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and 3 "probable high-risk types" (HPV26, 53, and 66), which are known to appear together as low- and high-grade cervical changes and cancers, as well as other anogenital cancers such as vulvar, vaginal, penile, anal and perianal cancers, and head and neck cancers. Recently, the association of high-risk types HPV16 and 18 with breast cancer has also been described. Eleven HPV types classified as "low-risk types" (HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are known to manifest as benign low-grade cervical changes, genital warts, and recurrent respiratory papillomatosis. Cutaneous HPV types 5, 8, and 92 are associated with skin cancer. In some HPV-associated cancers, the immune system is suppressed and, accordingly, antitumor responses are significantly impaired. See Suresh and Burtness Am J Hematol Oncol 13(6):20-27 (2017).

[0006] Immunotherapy can be broadly divided into two main types of interventions, either passive or active. Passive protocols include administration of pre-activated and / or engineered cells (e.g., CAR T cells), disease-specific therapeutic antibodies, and / or cytokines. Active immunotherapy strategies are directed at stimulating immune system effector functions in vivo. Some current active protocols include vaccination strategies with disease-associated peptides, lysates, or whole allogeneic cells, injection of autologous dendritic cells (DCs) as a vehicle for tumor antigen delivery, and injection of immune checkpoint modulators. See Papaioannou, Nikos E., et al. Annals of translational medicine 4.14 (2016). Adoptive immunotherapy can be used to modulate immune responses, enhance antitumor activity, and achieve the goal of treating or preventing HPV-associated cancers. CD8 stimulated by disease-associated antigens + Cytotoxic T lymphocytes (CTL) and CD4 + Helper T (Th) cells have the potential to target and destroy diseased cells, but current methods for inducing endogenous T cell responses face challenges. Using the methods described herein, AAC can be anucleated cells or anucleated cell-derived entities that contain HPV antigens and / or adjuvants in a high-throughput manner, and can be utilized to induce robust T cell responses to HPV antigens. The methods described herein also describe methods, treatments, doses, and regimens for treating individuals with HPV-associated cancers using AAC that contain HPV antigens and adjuvants. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.Patent publications WO2013 / 059343, WO2015 / 023982, WO2016 / 070136, WO2017041050, WO2017008063, WO2017 / 192785, WO2017 / 192786, WO2019 / 178005, WO2019 / 178006, WO2020 / 072833, WO2020 / 154696, and WO2020 / 176789, US20180142198, and US20180201889 are expressly incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 059343 [Patent Document 2] International Publication No. 2015 / 023982 [Patent Document 3] International Publication No. 2016 / 070136 [Patent Document 4] International Publication No. 2017 / 041050 [Non-patent literature]

[0008] [Non-Patent Document 1] See Suresh and Burtness Am J Hematol Oncol 13(6):20-27 (2017). [Non-Patent Document 2] Papaioannou, Nikos E., et al. Annals of translational medicine 4.14 (2016) Summary of the Invention [Means for solving the problem]

[0009] BRIEF SUMMARY OF THE INVETION In some aspects, the present invention provides a method for treating a human papillomavirus (HPV) associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an activating antigen carrier (AAC), wherein the effective amount is about 0.1×10 8 AAC / kg ~ approx. 1 x 10 9 AAC / kg, the AAC comprising at least one HPV antigen delivered intracellularly and an adjuvant. In some aspects, the present invention provides a method for treating human papillomavirus (HPV)-associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an activating antigen carrier (AAC), the AAC comprising at least one HPV antigen delivered intracellularly and an adjuvant, and administering to the individual an effective amount of an antagonist of CTLA-4 and / or an antagonist of PD-1 / PD-L1. In some embodiments, the antagonist of CTLA4 is an antibody that binds to CTLA4. In some embodiments, 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 embodiments, an antibody that binds to CTLA-4 and an antibody that binds to PD-1 are administered to the individual. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab. In some embodiments, the antibody that binds to PD-1 is nivolumab. In some embodiments, the antibody that binds to PD-1 is pembrolizumab. In some embodiments, an antibody that binds to CTLA-4 is administered to an individual, and an antibody that binds to PD-L1 is administered to an individual. In some embodiments, the antibody that binds to PD-L1 is atezolizumab.

[0010] In some embodiments of the present invention, at least one HPV antigen is an HPV-16 antigen or an HPV-18 antigen. In some embodiments, at least one HPV antigen comprises a peptide derived from HPV E6 and / or E7. In some embodiments, at least one HPV antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the AAC comprises an antigen comprising the amino acid sequence of SEQ ID NO: 19 and an antigen comprising the amino acid sequence of SEQ ID NO: 23.

[0011] In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), LPS, IFN-α, STING agonist, RIG-I agonist, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist or TLR9 agonist.In some embodiments, the adjuvant is CpG 7909 oligodeoxynucleotide (ODN).

[0012] In some embodiments, the individual is a human. In some embodiments, the individual is an HLA-A * In some embodiments, the AAC is autologous or allogeneic to the individual. In some embodiments, the HPV-associated cancer is currently locally advanced or metastatic. In some embodiments, the HPV-associated cancer is head and neck cancer, cervical cancer, anal cancer, or esophageal cancer. In some embodiments, the composition comprising the AAC is administered intravenously. In some embodiments, the CTLA-4 antagonist and / or the PD-1 / PD-L1 antagonist are administered intravenously, orally, or subcutaneously. In some embodiments, the antibody that binds CTLA-4 and / or the antibody that binds PD-1 and / or the antibody that binds PD-L1 are administered intravenously. In some embodiments, the effective amount of the AAC comprising at least one HPV antigen and an adjuvant is about 0.5×10 8AAC / kg ~ approx. 1 x 10 9 In some embodiments, the effective amount of the AAC comprising at least one HPV antigen and an adjuvant is about 0.5×10 8 AAC / kg ~ approx. 1 x 10 9 In some embodiments, the effective amount of the AAC comprising at least one HPV antigen and an adjuvant is about 0.5×10 8 AAC / kg, approx. 2.5 × 10 8 AAC / kg, approx. 5 × 10 8 AAC / kg, or approximately 7.5 × 10 8 AAC / kg.

[0013] In some embodiments, the effective amount of ipilimumab is about 1 mg / kg to about 3 mg / kg. In some embodiments, the effective amount of nivolumab is about 360 mg. In some embodiments, the effective amount of atezolizumab is about 1200 mg.

[0014] In some embodiments, the composition comprising AAC is delivered on day 1 of a three-week cycle. In some embodiments, the composition comprising AAC is further administered on day 2 of the first three-week cycle. In some embodiments, about 0.5×10 8 Individual cells / kg ~ approx. 1×10 9 In some embodiments, about 0.5×10 cells / kg are administered on day 1 of each 3-week cycle. 8 Individual cells / kg, approximately 2.5×10 8 Individual cells / kg, approximately 5.0×10 8 cells / kg, or approximately 7.5 x 10 8 In some embodiments, about 0.5×10 cells / kg are administered on day 1 of each 3-week cycle. 8 Individual cells / kg ~ approx. 1×10 9 In some embodiments, about 0.5×10 cells / kg are administered on day 2 of each 3-week cycle. 8 Individual cells / kg, approximately 2.5×10 8 Individual cells / kg, approximately 5.0×10 8 cells / kg, or approximately 7.5 x 10 8Cells / kg will be administered on day 2 of the first 3-week cycle.

[0015] In some embodiments, the antibody that binds CTLA-4 and / or the antibody that binds PD-1 and / or the antibody that binds PD-L1 is administered once per three-week cycle. In some embodiments, the antibody that binds CTLA-4 is administered once per two three-week cycles. In some embodiments, the antibody that binds CTLA-4 is administered on day 1 of each three-week cycle. In some embodiments, the antibody that binds CTLA-4 is ipilimumab, and the ipilimumab is administered at a dose of about 3 mg / kg. In some embodiments, the antibody that binds PD-1 is administered on day 8 of the first three-week cycle and on day 1 of each subsequent cycle. In some embodiments, the antibody that binds PD-1 is nivolumab, and the nivolumab is administered at a dose of about 360 mg. In some embodiments, the antibody that binds CTLA-4 is ipilimumab, and ipilimumab is administered at a dose of about 1 mg / kg on day 1 of the first 3-week cycle of two 3-week cycles, and the antibody that binds PD-1 is administered at a dose of about 360 mg on day 8 of the first 3-week cycle and day 1 of each subsequent cycle. In some embodiments, the antibody that binds PD-L1 is administered on day 8 of the first 3-week cycle and day 1 of each subsequent cycle. In some embodiments, the antibody that binds PD-L1 is administered at a dose of about 1200 mg. In some embodiments, the composition comprising PBMCs is administered to the individual for at least about 3 months, 6 months, 9 months, or 1 year.

[0016] In some embodiments, a composition comprising AAC is present in a cryopreservation medium containing about 1×10 9 AAC ~ approx. 1×10 10 In some embodiments, the composition comprising AAC comprises about 7×10 9PBMCs. In some embodiments, the cryopreservation medium is Cryostor® CS2. In some embodiments, the AAC comprising at least one HPV antigen and an adjuvant is prepared by a process comprising: a) passing a cell suspension comprising a population of input anucleated material through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the at least one HPV antigen and the adjuvant to pass through, thereby forming perturbed input anucleated cells; and b) incubating the population of perturbed input anucleated cells with the at least one HPV antigen and the adjuvant for a time sufficient to allow the antigen to enter the perturbed input anucleated cells, thereby creating an AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the diameter of the constriction is about 1.6 μm to about 2.4 μm or about 1.8 μm to about 2.2 μm. In some embodiments, the input anucleated cells are red blood cells. In some embodiments, the at least one HPV antigen comprises a peptide derived from HPV E6 and a peptide derived from HPV E7. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the treatment regimen for Cohort 1.

[0018] [Diagram 2] FIG. 2 shows the treatment regimen for cohort 2a.

[0019] [Diagram 3] FIG. 3 shows the treatment regimen for cohort 2b.

[0020] [Figure 4] FIG. 4 shows the treatment regimen for cohort 2c.

[0021] [Diagram 5]5A and 5B show surface phosphatidylserine levels of AAC-treated versus untreated red blood cells containing HPV antigens, as measured by the number of Annexin V+ events using FACS. Cells were treated in PBS (FIG. 5A) or RPMI (FIG. 5B).

[0022] [Figure 6] Figure 6 shows the percentage of FAM+ events (gated on singlets). AAC-HPV and untreated RBCs are used as negative controls. Each point shown for each group is derived from data for an individual donor analyzed in a separate experiment represented by a circle. Values ​​shown for the third experiment (right circle for each condition) are the average of data from replicate samples.

[0023] [Figure 7] Figure 7 shows the percentage of Annexin V+ events (gated on singlets). Untreated RBCs are used as negative controls. Each point shown per group is derived from data for an individual donor analyzed in a separate experiment represented by a circle. Values ​​shown for the third experiment (right circle for each condition) are the average of data from replicate samples.

[0024] [Figure 8] Figure 8 shows representative images of AAC-HPV (F-E6, E7) from three separate donors as indicated (FAM is shown in green and PB is shown in blue). Graphs representing normalized (to minimum and maximum signals) FAM (green) and PB (blue) fluorescence intensities along with lines drawn across the length of AAC-HPV (F-E6, E7) are shown below each image.

[0025] [Figure 9]Figure 9 shows representative images of AAC-HPV (F-E6, E7) from three separate donors as indicated (FAM is shown in green and PB is shown in blue). Graphs representing normalized (to minimum and maximum signals) FAM (green) and PB (blue) fluorescence intensities along with lines drawn across the length of AAC-HPV (F-E6, E7) are shown below each image.

[0026] [Figure 10] Figure 10 shows representative images of AAC-HPV used as a negative control for FAM fluorescence from three separate RBC donors as indicated (FAM shown in green and PB shown in blue). Graphs representing normalized FAM (green) and PB (blue) fluorescence intensity with a line drawn across the length of the AAC-HPV are shown below each image. The display settings in A correspond to the display settings used for AAC-HPV (F-E6, E7) in Figure 8. The display settings in B correspond to the display settings used for AAC-HPV (E6, F-E7) in Figure 9.

[0027] [Figure 11] FIG. 11 shows PKH26 fluorescence (mean of biological replicates ± standard deviation of the mean) measured in CD11c+MODC / PKH26-labeled AAC-HPV co-cultures incubated at 37° C. (red) and 4° C. (blue) and in MODC / AAC-HPV co-cultures incubated at 37° C. (green). For presentation purposes, the condition without (PKH26-labeled) AAC-HPV is plotted at 0.2 on the x-axis. Each graph represents an independent experiment. Each experiment was performed with separate human blood and MODC donors.

[0028] [Figure 12]FIG. 12 presents summary data showing fold change in geometric mean fluorescence intensity (MFI) of surface levels of CD80, CD83, CD86, and MHC-II in MODCs after 46 hours of co-culture at 37° C. with C-vehicle or AAC-HPV compared to control vehicle alone. Data from individual MODC donors are shown as separate circles. *=P<0.05; **=P<0.01.

[0029] [Figure 13] FIG. 13 shows graphs depicting IFNγ values ​​secreted from E7-specific CD8+ T cells approximately 24 hours after co-culture with MODC and vehicle control, SQZ-AAC-HPV, or no E7 SLP. Each graph corresponds to co-culture with a separate batch of SQZ-AAC-HPV. MODC and CD8+ T cell co-cultures were incubated with vehicle control, SQZ-AAC-HPV, or no E7 peptide. Each individual data point (full circle) corresponds to an individual well of the co-culture sample. FP numbers represent batch numbers.

[0030] [Figure 14] FIG. 14 shows summary data from two independent experiments for CD86 geometric MFI of CD11chiMHC-IIhiCD8+ cells (CD8+ DC), CD11chiMHC-IIhiCD11b+ cells (CD11b+ DC) and F4 / 80+CD11blo / - cells (RPM) 14-16 hours after administration of M-AAC-HPV or MC vehicle. Each graph represents a separate experiment. Mice receiving M-AAC-HPV had higher CD86 geometric MFI compared to mice receiving MC vehicle. *=P<0.05.

[0031] [Figure 15]Figure 15 shows summary data of CD83 geometric MFI of CD11chiMHC-IIhiCD8+ cells (CD8+ DCs), CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) and F4 / 80+CD11blo / - (RPMs) 14-16 hours after administration of M-AAC-HPV or MC vehicle from two independent experiments. CD11chiMHC-IIhiCD8+ cells (CD8+ DCs) and CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) had higher CD83 geometric MFIs in mice receiving M-AAC-HPV compared to mice receiving MC vehicle. Of note, the CD83 geometric MFI of CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) in one experiment had negative data values ​​and therefore is not represented on the graph. As described by Bagwell BS and Park DR, et al, the spectral crossover comparison required for proper interpretation of multiparameter flow cytometry data is a subtractive process. As a result, after fluorescence compensation, data values ​​for cell populations that are essentially unstained or negative for a particular dye should be more or less distributed around a low value. Thus, data sets resulting from computer compensation typically (and properly) include populations whose distribution extends below zero. *=P<0.05.

[0032] [Figure 16]FIG. 16 shows summary data of CD40 geometric MFI of CD11chiMHC-IIhiCD8+ cells (CD8+ DCs), CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) and F4 / 80+CD11blo / - cells (RPM) 14-16 hours after administration of M-AAC-HPV or MC vehicle from two independent experiments. Each graph represents a separate experiment. CD11chiMHC-IIhiCD8+ cells (CD8+ DCs) and CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) had higher CD40 geometric MFI in mice that received M-AAC-HPV compared to mice that received MC vehicle. *=P<0.05.

[0033] [Figure 17] FIG. 17 shows summary data of CD80 geometric MFI of CD11chiMHC-IIhiCD8+ cells (CD8+ DCs), CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) and F4 / 80+CD11blo / - cells (RPM) 14-16 hours after administration of M-AAC-HPV or MC vehicle from two independent experiments. Each graph represents a separate experiment. CD11chiMHC-IIhiCD8+ cells (CD8+ DCs) and CD11chiMHC-IIhiCD11b+ cells (CD11b+ DCs) had higher CD80 geometric MFI in mice that received M-AAC-HPV compared to mice that received MC vehicle. *=P<0.05.

[0034] [Figure 18]FIG. 18 shows summary data of MHC-II geometric MFI of CD11chiMHC-IIhiCD8+ cells (CD8+ DC), CD11chiMHC-IIhiCD11b+ cells (CD11b+ DC) and F4 / 80+CD11blo / - cells (RPM) 14-16 hours after administration of M-AAC-HPV or MC vehicle from two independent experiments. Each graph represents a separate experiment. CD11chiMHC-IIhiCD8+ cells (CD8+ DC) and F4 / 80+CD11blo / - cells (RPM) had higher MHC-II geometric MFI in mice that received M-AAC-HPV compared to mice that received MC vehicle. NA stands for "not applicable." *=P<0.05.

[0035] [Figure 19] Figure 19 shows the requirement for antigen (E7 SLP) and adjuvant (poly I:C) in priming E7-specific CD8+ T cell responses. Percentages of IFNγ+ CD8+ T cells are shown. ****=P<0.0001.

[0036] [Figure 20] Figure 20 shows the effect of m-AAC-HPV dose on the magnitude of E7-specific CD8+ T cell responses. Percentage of IFNγ+ CD44+ CD8+ T cells is shown. B=109; M=106. ns=not statistically significant (P≧0.05); *=P<0.05; ***=P<0.001; ****=P<0.0001.

[0037] [Figure 21] Figure 21 shows the percentage of E7-tetramer+activated (CD44hi) CD8+ T cells measured in whole blood for each group. Data are presented for: PBS (day 0) to 8 days after last dose (study day 8); Prime alone (day 0) to 8 days after last dose (study day 8), Boost (day 2) to 8 days after last dose (study day 10); Boost (day 6) to 7 days after last dose (study day 13). **=P<0.01; ***=P<0.001.

[0038] [Figure 22] Figure 22 shows the percentage of E7-tetramer+activated (CD44hi) CD8+ T cells measured in whole blood for each group. Data are presented for: PBS (day 0) to 13 days after last dose (study day 13); Prime alone (day 0) to 13 days after last dose (study day 13); Boost (day 2) to 13 days after last dose (study day 15); Boost (day 6) to 14 days after last dose (study day 21). **=P<0.01; ****=P<0.0001.

[0039] [Diagram 23] Figure 23 shows the percentage of E7-tetramer+activated (CD44hi) CD8+ T cells measured in whole blood for each group. Data are presented for: PBS (day 0) to 21 days after the last dose (study day 21); Prime alone (day 0) to 21 days after the last dose (study day 21), Boost (day 2) to 21 days after the last dose (study day 23); **=P<0.01.

[0040] [Figure 24] Figures 24A and 24B show summary data of tumor volumes (mean ± standard error of the mean) shown for each experimental group over time in two experiments. The line ends when the median survival time was reached for that group. "M" stands for million; "B" stands for billion.

[0041] [Diagram 25] Figure 25 shows survival data for the two different experimental groups. Numbers in brackets indicate median survival time (days).

[0042] [Figure 26] Figure 26 shows summary data of tumor volume (mean ± standard error of the mean) for each experimental group over time. The line ends when the median survival time for that group is reached. "B" stands for billions and "M" stands for million. n=10 mice / group.

[0043] [Figure 27] Figure 27 shows survival data for the experimental groups. Numbers in parentheses indicate median survival times.

[0044] [Figure 28] FIG. 28 shows summary data of tumor volume (mean±standard error of the mean) for each experimental group over time in (A-B) Experiment 1 and (C-D) Experiment 2. The line ends when the median survival time was reached for that group. "M" stands for million. n=10 mice / group.

[0045] [Figure 29] Figure 29 shows survival data for the two experimental groups. Numbers in parentheses indicate median survival time (days). "M" stands for millions. n=10 mice / group.

[0046] [Diagram 30] Figures 30A-30F show the percentage of CD8+ T cells shown as a percentage of the live cell population. (Figure 30B, Figure 30E). The percentage of E7-specific (tetramer+) cells is shown as a percentage of the live cell population and (Figure 30C, Figure 30F) as a percentage of the CD8+ T cell population. (Figure 30A-30C) Data from experiment 1 and (Figure 30D-30F) from experiment 2 are shown. Statistical significance of M-AAC-HPV treated group compared to PBS treated group is shown (*p<0.05, ***p<0.001, ****p<0.0001).

[0047] [Diagram 31] Figures 31A-31D show graphs depicting the number of CD8+ T cells and E7-specific (tetramer+) CD8+ T cells, respectively, normalized to 100 mg of tumor. Statistical significance of M-AAC-HPV-treated group compared to PBS-treated group is shown (**p<0.01, ***p<0.001).

[0048] [Diagram 32]Figure 32 shows summary data of the mean tumor volume for mice included in each experimental group over time. Immunization with M-AAC-HPV was performed on day 14 (experiment 1) or day 13 (experiment 2) and is shown as a dotted line. Data (mean ± standard error of the mean) are shown up to the time the tumors were harvested (day 26 for experiment 1, day 25 for experiment 2). It should be noted that the 12 day post-immunization time point (day 25 or 26 after tumor implantation) was chosen to ensure sufficient tumor material was available for processing and analysis in the treatment groups.

[0049] [Diagram 33] FIG. 33 shows the study design for in vivo serum cytokine / chemokine analysis in mice following repeated intravenous administration of m-AAC-HPV as measured by Luminex analysis.

[0050] [Diagram 34] Figure 34 shows the percentage of PKH26-labeled cells in whole blood. *=P<0.05 (P values ​​were calculated by comparing the percentage of PKH26-labeled cells at the indicated time points in M-AAC-HPV with the corresponding time points in the PBS control). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Detailed Description of the Invention In some aspects, the present invention provides a method for treating a human papillomavirus (HPV) associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an activating antigen carrier (AAC), the AAC comprising an HPV antigen delivered intracellularly and an adjuvant.

[0052] In some aspects, the present invention provides a method for treating HPV-associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an AAC, the AAC comprising an intracellularly delivered HPV antigen and an adjuvant, and administering an effective amount of one or more immune checkpoint inhibitors. In some embodiments, the one or more immune checkpoint inhibitors comprise an antagonist of CTLA-4 (such as, but not limited to, ipilimumab), an antagonist of PD-1 (such as, but not limited to, nivolumab), and / or an antagonist of PD-L1 (such as, but not limited to, atezolizumab).

[0053] In some aspects, the invention provides a method for treating HPV-associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an AAC, the AAC comprising an HPV antigen delivered intracellularly and an adjuvant, and administering an effective amount of one or more of ipilimumab, nivolumab, or atezolizumab, the AAC comprising at least one HPV antigen and an adjuvant, and / or one or more immune checkpoint inhibitors are administered in a three week cycle, and the effective amount of the AAC is about 0.5×10 8 AAC / kg ~ approx. 1 x 10 9 AAC / kg, the effective amount of ipilimumab is about 1 mg / kg to about 3 mg / kg, the effective amount of nivolumab is about 360 mg / kg, and the effective amount of atezolizumab is about 1200 mg.

[0054] Also provided are compositions comprising AAC comprising at least one HPV antigen and adjuvant, and methods for preparing AAC comprising at least one HPV antigen and adjuvant.In some embodiments, AAC is prepared by a process comprising: a) passing a cell suspension comprising a population of input anucleated matter through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing perturbation of the input anucleated cells large enough for at least one HPV antigen and adjuvant to pass through, thereby forming perturbed input anucleated cells; and b) incubating the population of perturbed input anucleated cells with at least one HPV antigen and adjuvant for a sufficient time to allow antigen and adjuvant to enter the perturbed input anucleated cells, thereby producing AAC comprising at least one HPV antigen and adjuvant.Also provided are compositions for use in inducing immune response to HPV antigens or for treating HPV-related cancer. Also provided is the use of a composition comprising an effective amount of AAC in the manufacture of a medicament for stimulating an immune response to an HPV antigen or for treating an HPV-associated cancer.

[0055] general technique The techniques and procedures described or referenced herein are generally well understood and may be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012);Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003);the series Methods in Enzymology (Academic Press, Inc.);PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds., 1995);Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988);Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6 thed., J. Wiley and Sons, 2010);Oligonucleotide Synthesis (M.J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., Academic Press, 1998);Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, Plenum Press, 1998);Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., J. Wiley and Sons, 1993-8);Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds., 1996);Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (J.E. Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002);Immunobiology (C.A. Janeway et al., 2004);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C.Dean, eds., 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), which are commonly employed by those of skill in the art.

[0056] definition For purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0057] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0058] The terms "comprising," "having," "containing," and "including," as well as other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and are intended to be open-ended in that the item or items following any one of these words are not meant to be an inclusive list of such item or items, or to be limited to only the listed item or items. For example, an article "comprising" components A, B, and C may consist of (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, "comprises" and similar forms and their grammatical equivalents are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0059] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure subject to any specifically excluded limits in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0060] The term "about" as used herein refers to the normal error range for each value, which is readily known to those skilled in the art. Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0061] As used herein, "anucleated cells" refers to cells that lack a nucleus. Such cells include, but are not limited to, platelets, red blood cells (RBCs), e.g., red blood cells and reticulocytes. Reticulocytes are immature (e.g., not yet biconcave) red blood cells that typically constitute about 1% of the red blood cells in the human body. Reticulocytes are also anucleated. In certain embodiments, the systems and methods described herein are used to treat and / or process purified or isolated (e.g., in substantially pure or homogenous form from their natural environment) populations of enriched (e.g., constituting a higher percentage of the total cell population than found in nature) anucleated cells (e.g., RBCs, reticulocytes, and / or platelets). In certain embodiments, the systems and methods described herein are used for the treatment and / or processing of whole blood containing RBCs (e.g., red blood cells or reticulocytes), platelets, and other blood cells. Purification or enrichment of these cell types is achieved using known methods such as density gradient systems (e.g., Ficoll-Hypaque), fluorescence activated cell sorting (FACS), magnetic cell sorting, or in vitro differentiation of erythroblasts and erythroid progenitor cells.

[0062] The term "vesicle" as used herein refers to a structure that includes a liquid enclosed by a lipid bilayer. In some examples, the lipid bilayer is provided from a naturally occurring lipid composition. In some examples, the lipid bilayer can be provided from a cell membrane. In some examples, the vesicle can be derived from various types of entities, such as cells. In such examples, the vesicle can retain molecules (such as intracellular proteins or membrane components) from the entity of origin. For example, a vesicle derived from a red blood cell may contain any number of intracellular proteins that were present in the red blood cell and / or membrane components of the red blood cell. In some examples, the vesicle can contain any number of molecules intracellular in addition to the desired payload.

[0063] As used herein, "payload" refers to a material to be delivered, such as loaded into an AAC (e.g., an AAC). "Payload," "cargo," "delivery material," and "compound" are used interchangeably herein. In some embodiments, payload may refer to proteins, small molecules, nucleic acids (e.g., RNA and / or DNA), lipids, carbohydrates, macromolecules, vitamins, polymers, fluorescent dyes and fluorophores, carbon nanotubes, quantum dots, nanoparticles, and steroids. In some embodiments, payload may refer to proteins or small molecule drugs. In some embodiments, payload may include one or more compounds.

[0064] The term "heterologous" when referring to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence adjacent to a sequence that is not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutation events do not give rise to heterologous DNA as used herein.

[0065] The term "heterologous" when referring to amino acid sequences, such as peptide and polypeptide sequences, refers to sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a peptide sequence is a segment of amino acids within or attached to another amino acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a peptide construct may include an amino acid sequence of the peptide adjacent to a sequence that is not found in association with the amino acid sequence of the peptide in nature. Another example of a heterologous peptide sequence is a construct in which the peptide sequence itself is not found in nature (e.g., a synthetic sequence with different amino acids encoded from a native gene). Similarly, a cell transformed with a vector that expresses an amino acid construct that is not normally present in the cell would be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutation events do not give rise to heterologous peptides as used herein.

[0066] The term "exogenous," when used in reference to an agent such as an antigen or adjuvant, with respect to a cell or cell-derived vesicle, 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.

[0067] The term "homologous" as used herein refers to molecules that are derived from the same organism. In some instances, the term refers to nucleic acids or proteins that are normally found or expressed in a given organism.

[0068] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a 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, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of the pathological consequences of cancer (e.g., tumor volume, etc.). The method of the present invention contemplates any one or more of these aspects of treatment.

[0069] As used herein, the term "prophylactic treatment" refers to treatment in which an individual is known to have a disorder, suspected to have a disorder, or at risk of having a disorder, but does not show symptoms of the disorder, or shows minimal symptoms of the disorder. The individual receiving prophylactic treatment can be treated before the onset of symptoms. In some embodiments, the individual can be treated when the individual has a precancerous lesion, particularly a precancerous lesion associated with HPV infection.

[0070] As used herein, "combination therapy" means that a first agent is administered in conjunction with another agent. "In conjunction with" refers to the administration of one treatment modality in addition to another treatment modality, for example, the administration of the composition of nucleated cells described herein in addition to the administration of the immunoconjugate described herein to the same individual. Thus, "in conjunction with" refers to the administration of one treatment modality before, during, or after the delivery of the other treatment modality to an individual.

[0071] The term "co-administration," as used herein, means that the first and second therapies in a combination therapy are administered no more than about 15 minutes apart, e.g., no more than about 10, 5, or 1 minute apart. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition that includes both the first and second therapies) or in separate compositions (e.g., the first therapy in one composition and the second therapy in another composition).

[0072] As used herein, the term "sequential administration" means that the first and second therapies in the combination therapy are administered at a time interval of more than about 15 minutes, for example, more than about 20, 30, 40, 50, 60 minutes, or longer.Either the first or second therapy can be administered first.The first and second therapies are contained in separate compositions, which can be contained in the same or different packages or kits.

[0073] As used herein, the term "concurrent administration" means that the administration of a first therapy and the administration of a second therapy in a combination therapy overlap with each other.

[0074] In the context of cancer, the term "treating" includes any or all of killing cancer cells, inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the total tumor burden, and ameliorating one or more symptoms associated with the disease.

[0075] As used herein, the term "modulate" may refer to the act of changing, altering, fluctuating, or otherwise modifying the presence or activity of a particular target. For example, modulating an immune response may refer to any act that results in changing, altering, fluctuating, or otherwise modifying an immune response. In some examples, "modulate" refers to enhancing the presence or activity of a particular target. In some examples, "modulate" refers to suppressing the presence or activity of a particular target. In other examples, modulating the expression of a nucleic acid may include, but is not limited to, a change in the transcription of the nucleic acid, a change in the abundance of mRNA (e.g., increasing mRNA transcription), a corresponding change in the degradation of mRNA, a change in the translation of mRNA, and the like.

[0076] As used herein, the term "inhibit" may refer to the action of blocking, reducing, eliminating, or otherwise antagonizing the presence or activity of a particular target. Inhibition may refer to partial or complete inhibition. For example, inhibiting an immune response may refer to any action that results in blocking, reducing, eliminating, or any other antagonism of an immune response. In other examples, inhibiting the expression of a nucleic acid may include, but is not limited to, reducing the transcription of a nucleic acid, reducing the abundance of an mRNA (e.g., silencing mRNA transcription), degrading an mRNA, inhibiting the translation of an mRNA, gene editing, and the like. In other examples, inhibiting the expression of a protein may include, but is not limited to, reducing the transcription of a nucleic acid encoding a protein, reducing the stability of an mRNA encoding a protein, inhibiting the translation of a protein, reducing the stability of a protein, and the like. In another example, inhibiting may refer to the action of slowing or stopping growth, e.g., impeding or preventing the growth of a tumor cell.

[0077] As used herein, the term "suppress" may refer to the action of decreasing, reducing, preventing, restricting, mitigating, or otherwise diminishing the presence or activity of a particular target. Suppression may refer to partial or complete suppression. For example, suppressing an immune response may refer to any action that results in decreasing, reducing, preventing, restricting, mitigating, or otherwise diminishing an immune response. In other examples, suppression of expression of a nucleic acid may include, but is not limited to, decreasing transcription of a nucleic acid, decreasing the abundance of an mRNA (e.g., silencing mRNA transcription), degrading an mRNA, inhibiting translation of an mRNA, and the like. In other examples, suppression of expression of a protein may include, but is not limited to, decreasing transcription of a nucleic acid encoding a protein, decreasing stability of an mRNA encoding a protein, inhibiting translation of a protein, decreasing stability of a protein, and the like.

[0078] As used herein, the term "enhancing" may refer to the action of improving, boosting, enhancing, or otherwise increasing the presence or activity of a particular target. For example, enhancing an immune response may refer to any action that results in improving, boosting, enhancing, or otherwise increasing an immune response. In an illustrative example, enhancing an immune response may refer to the use of an antigen and / or an adjuvant to improve, boost, enhance, or otherwise increase an immune response. In other examples, enhancing expression of a nucleic acid may include, but is not limited to, increasing transcription of a nucleic acid, increasing abundance of an mRNA (e.g., increasing mRNA transcription), decreasing degradation of an mRNA, increasing translation of an mRNA, and the like. In other examples, enhancing expression of a protein may include, but is not limited to, increasing transcription of a nucleic acid encoding a protein, increasing stability of an mRNA encoding a protein, increasing translation of a protein, increasing stability of a protein, and the like.

[0079] As used herein, the term "induce" may refer to the act of initiating, encouraging, stimulating, establishing, or otherwise producing a result. For example, inducing an immune response may refer to any act that results in initiating, encouraging, stimulating, establishing, or otherwise producing a desired immune response. In other examples, inducing expression of a nucleic acid may include, but is not limited to, initiating transcription of a nucleic acid, initiating translation of an mRNA, and the like. In other examples, inducing expression of a protein may include, but is not limited to, increasing transcription of a nucleic acid encoding a protein, increasing stability of an mRNA encoding a protein, increasing translation of a protein, increasing stability of a protein, and the like.

[0080] The term "polynucleotide" or "nucleic acid" as used herein refers to any length of polymeric form of nucleotides, including ribonucleotides and deoxyribonucleotides. Thus, the 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 and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of a polynucleotide can include sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide can include repeating units such as N-(2-aminoethyl)-glycine linked by peptide bonds (i.e., peptide nucleic acid). Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and phosphorothioates, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2), or a mixed phosphorothioate-phosphodiester oligomer or a mixed phosphoramidate-phosphodiester oligomer.In addition, a double-stranded polynucleotide can be obtained from a single-stranded polynucleotide product of chemical synthesis, either by synthesizing a complementary strand and annealing the strands under suitable conditions, or by synthesizing a complementary strand de novo using a DNA polymerase with a suitable primer.

[0081] 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, including, but not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or by errors due to PCR amplification.

[0082] As used herein, the term "adjuvant" refers to a substance that modulates and / or generates an immune response. Generally, an adjuvant is administered in conjunction with an antigen to result in an enhanced immune response to the antigen compared to the antigen alone. Various adjuvants are described herein.

[0083] The terms "CpG oligodeoxynucleotide" and "CpG ODN" as used herein refer to a DNA molecule of 10-30 nucleotides in length that contains a dinucleotide of cytosine and guanine separated by a phosphate (also referred to herein as a "CpG" dinucleotide 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., is not a 5-methylcytosine). The CpG ODN may have a partial or complete phosphorothioate (PS) backbone.

[0084] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharma-ceutically acceptable carrier or excipient preferably meets the necessary standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredient Guide produced by the U.S. Food and Drug Administration.

[0085] For any structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0086] As used herein, "microfluidic system" refers to a system in which low volumes (e.g., mL, nL, pL, fL) of fluids are processed to achieve discrete treatment of small volumes of fluid. Certain implementations described herein include multiplexing, automation, and high-throughput screening. Fluids (e.g., buffers, solutions, payload-containing solutions, or cell suspensions) can be moved, mixed, separated, or otherwise processed. In certain embodiments described herein, a microfluidic system is used to apply a mechanical constriction to cells suspended in a buffer to induce a disturbance (e.g., a pore) in the cell that allows the payload or compound to enter the cytosol of the cell.

[0087] 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, where the center point is defined by a width, a length, and a depth. In other examples, a constriction may refer to a pore or be a portion of a pore. A pore may be contained in a surface (e.g., a filter and / or a membrane).

[0088] For any structural and functional characteristics described herein, methods for determining these characteristics are known in the art. Treatment Method

[0089] In some aspects, a method of treating HPV-associated disease in an individual is provided, the method comprising administering to the individual a composition comprising an effective amount of an AAC, the AAC comprising an HPV antigen delivered intracellularly and an adjuvant.

[0090] In some aspects, a method of treating HPV-associated disease in an individual comprises administering to the individual a composition comprising an effective amount of AAC, the effective amount being about 0.5×10 7 AAC / kg ~ approx. 5 x 10 10 AAC / kg, where the AAC comprises an HPV antigen delivered intracellularly and an adjuvant.

[0091] In some embodiments, the HPV-related disease is HPV-related cancer.In some embodiments, the HPV-related cancer is cervical cancer, perianal cancer, anogenital cancer, oral cancer, salivary gland cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer or head and neck cancer.In some embodiments, the HPV-related disease is HPV-related infectious disease.

[0092] In some embodiments, an effective amount of AAC is about 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , 0.5×10 8 , 1.0×10 8 , 0.5×10 9 , 1.0×10 9 , 0.5×10 10 , 1.0×10 10 , 0.5×10 11 , and 1.0×10 11 AAC / kg. In some embodiments, the effective amount is about 0.5×10 6 ~Approx. 1.0×10 6, about 1.0×10 6 ~about 0.5×10 7 , about 0.5×10 7 ~Approx. 1.0×10 7 , about 1.0×10 7 ~about 0.5×10 8 AAC, approximately 0.5×10 8 ~Approx. 1.0×10 8 , about 1.0×10 8 ~about 0.5×10 9 AAC, approximately 0.5×10 9 ~Approx. 1.0×10 9 , about 1.0×10 9 ~about 0.5×10 10 AAC, approximately 0.5×10 10 ~Approx. 1.0×10 10 , about 1.0×10 10 ~about 0.5×10 11 , or approximately 0.5 × 10 11 ~Approx. 1.0×10 11 In some embodiments, a method of treating HPV-associated cancer in an individual comprises administering to the individual a composition comprising an effective amount of AAC, wherein the effective amount is about 0.5×10 8 ~Approx. 1×10 9 AAC / kg, where the AAC comprises an HPV antigen delivered intracellularly and an adjuvant.

[0093] In some embodiments, the method further comprises administering an effective amount of one or more immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors are, without limitation, antagonists of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is an antagonist of one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more of an antibody that binds PD-1, an antibody that binds PD-L1, an antibody that binds CTLA-4, an antibody that binds LAG3, or an antibody that binds TIM-3, an antibody that binds TIGIT, an antibody that binds VISTA, an antibody that binds TIM-1, an antibody that binds B7-H4, or an antibody that binds BTLA. In further embodiments, the antibody may be a full length antibody or any variant, such as, but not limited to, an antibody fragment, a single chain variable fragment (ScFv), or an antigen-binding fragment (Fab). In further embodiments, the antibody may be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemical compounds that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more peptides that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor targets PD-1. In some embodiments, the immune checkpoint inhibitor targets PD-L1. In some embodiments, the immune checkpoint inhibitor targets CTLA-4.

[0094] In some embodiments, a method of treating HPV-associated cancer in an individual comprises administering to the individual a composition comprising an effective amount of AAC, wherein the effective amount is about 0.5×10 8 ~Approx. 1×10 9 A method is provided, comprising administering an effective amount of one or more immune checkpoint inhibitors, the AAC comprising an intracellularly delivered HPV antigen and an adjuvant. In some embodiments, the immune checkpoint inhibitor is an antagonist of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antagonist of PD-1. In some embodiments, the immune checkpoint inhibitor is an antagonist of PD-L1. In some embodiments, the one or more immune checkpoint inhibitors comprise an antagonist of CTLA-4, an antagonist of PD-1, and / or an antagonist of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody that binds to CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody that binds to PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody that binds to PD-L1. In some embodiments, the one or more immune checkpoint inhibitors comprise an antibody that binds to CTLA-4, an antibody that binds to PD-1, and / or an antibody that binds to PD-L1.

[0095] In some aspects, a method of treating HPV-associated disease in an individual comprises administering to the individual a composition comprising an effective amount of AAC, the effective amount being about 0.5×10 8 ~Approx. 1×10 9 A method is provided that includes administering to an individual an effective amount of an antagonist of CTLA-4, an antagonist of PD-1, and / or an antagonist of PD-L1, wherein the AAC is an AAC comprising an HPV antigen and an adjuvant delivered intracellularly. In some embodiments, a method of treating HPV-associated disease in an individual is provided, comprising administering to the individual a composition comprising an effective amount of the AAC, wherein the effective amount is about 0.5×10 8 ~Approx. 1×109 A method is provided that includes administering an effective amount of an antibody that binds CTLA-4, an antibody that binds PD-1, and / or an antibody that binds PD-L1, wherein the AAC comprises an HPV antigen and an adjuvant delivered intracellularly. In some embodiments, the antibody that binds PD-1 is nivolumab. In some embodiments, the antibody that binds PD-1 is pembrolizumab. In some embodiments, the antibody that binds PD-L1 is atezolizumab. In some embodiments, the antibody that binds CTLA-4 is ipilimumab. In some embodiments, the antibody that binds CTLA-4 is administered to the individual. In some embodiments, the antibody that binds PD-L1 is administered to the individual. In some embodiments, the antibody that binds PD-1 is administered to the individual.

[0096] In some aspects, a method for stimulating an immune response to an HPV antigen in an individual is provided, comprising administering to the individual a composition (e.g., RBC-derived vesicles) comprising an effective amount of an AAC, wherein the AAC comprises an HPV antigen, and wherein at least one HPV antigen is delivered intracellularly to the AAC. In some embodiments, the AAC further comprises an adjuvant. In some embodiments, the method comprises administering an effective amount of any of the compositions described herein. In some embodiments, the individual has cancer.

[0097] In some aspects, a method for reducing tumor growth in an individual is provided, comprising administering to the individual an effective amount of a composition (e.g., RBC-derived vesicle) comprising an AAC, wherein the AAC comprises an HPV antigen, and wherein at least one HPV antigen is delivered intracellularly to the AAC. In some embodiments, the AAC further comprises an adjuvant. In some embodiments, the method comprises administering an effective amount of any of the compositions described herein. In some embodiments, the individual has cancer.

[0098] In some aspects, a method for vaccinating an individual in need thereof is provided, comprising administering to the individual a composition (e.g., RBC-derived vesicles) comprising an effective amount of an AAC, wherein the AAC comprises an HPV antigen, and wherein at least one HPV antigen is delivered intracellularly to the AAC. In some embodiments, the AAC further comprises an adjuvant. In some embodiments, the method comprises administering an effective amount of any of the compositions described herein. In some embodiments, the individual has cancer.

[0099] In some aspects, a method for treating cancer in an individual is provided, comprising administering to the individual a composition (e.g., RBC-derived vesicles) comprising an effective amount of an AAC, wherein the AAC comprises an HPV antigen, and wherein at least one HPV antigen is delivered intracellularly to the AAC. In some embodiments, the AAC further comprises an adjuvant. In some embodiments, the method comprises administering an effective amount of any of the compositions described herein.

[0100] In some aspects, a method is provided for stimulating an immune response to an HPV antigen in an individual, the method comprising: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow at least one HPV antigen and an adjuvant to pass through to form perturbed input anucleated cells; b) incubating the perturbed input anucleated cells with the at least one HPV antigen and an adjuvant for a time sufficient to allow the at least one HPV antigen and the adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and an adjuvant; and c) administering an effective amount of the AAC comprising the at least one HPV antigen and an adjuvant to the individual.

[0101] In some aspects, a method is provided for reducing tumor growth in an individual, the method comprising: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in suspension, thereby causing a perturbation of the input anucleated cells large enough to allow at least one HPV antigen and an adjuvant to pass through to form perturbed input anucleated cells; b) incubating the perturbed input anucleated cells with the at least one HPV antigen and an adjuvant for a time sufficient to allow the at least one HPV antigen and an adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and an adjuvant; and c) administering an effective amount of the AAC comprising the at least one HPV antigen and an adjuvant to the individual.

[0102] In some aspects, a method is provided for vaccinating an individual in need thereof, the method comprising: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow at least one HPV antigen and an adjuvant to pass through to form perturbed input anucleated cells; b) incubating the perturbed input anucleated cells with the at least one HPV antigen and an adjuvant for a time sufficient to allow the HPV antigen or the at least one HPV antigen and an adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and an adjuvant; and c) administering an effective amount of the AAC comprising the at least one HPV antigen and an adjuvant to the individual.

[0103] In some aspects, a method for treating cancer in an individual is provided, the method comprising: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the HPV antigen and adjuvant to pass through to form perturbed input anucleated cells; b) incubating the perturbed input anucleated cells with at least one HPV antigen and adjuvant for a time sufficient to allow the at least one HPV antigen and adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising at least one HPV antigen and adjuvant; and c) administering an effective amount of the AAC comprising the at least one HPV antigen and adjuvant to the individual.

[0104] In some embodiments according to any of the methods, uses, or compositions described herein, the method comprises: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in suspension, thereby causing a perturbation of the input anucleated cells large enough to allow HPV antigens to pass through to form perturbed input anucleated cells; b) incubating the perturbed input anucleated cells with at least one HPV antigen for a time sufficient to allow at least one HPV antigen to enter the perturbed input anucleated cells, thereby creating an AAC comprising at least one HPV antigen; and c) administering an effective amount of the AAC comprising at least one HPV antigen to an individual.

[0105] In some embodiments, a composition is provided for stimulating an immune response to HPV proteins in an individual, the composition comprising any one of the compositions comprising an effective amount of an AAC comprising an HPV antigen described herein. In some embodiments, a composition is provided for reducing tumor growth, the composition comprising any one of the compositions comprising an effective amount of an AAC comprising an HPV antigen described herein. In some embodiments, the individual has cancer. In some embodiments, a composition is provided for treating cancer in an individual, the composition comprising any one of the compositions comprising an effective amount of an AAC comprising an HPV antigen described herein. In some embodiments, the cancer is cervical cancer, perianal cancer, anogenital cancer, oral cancer, salivary gland cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer or head and neck cancer.

[0106] In some embodiments, a composition is provided for stimulating an immune response to HPV proteins in an individual, the composition comprising any one of the compositions comprising an AAC comprising an effective amount of an HPV antigen and an adjuvant as described herein. In some embodiments, a composition is provided for reducing tumor growth, the composition comprising any one of the compositions comprising an AAC comprising an effective amount of an HPV antigen and an adjuvant as described herein. In some embodiments, the individual has cancer. In some embodiments, a composition is provided for treating cancer in an individual, the composition comprising any one of the compositions comprising an AAC comprising an effective amount of an HPV antigen and an adjuvant as described herein.

[0107] In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for stimulating an immune response to an HPV antigen, the composition comprising any one of the compositions of AAC comprising an effective amount of an HPV antigen described herein. In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for reducing tumor growth in an individual, the composition comprising any one of the compositions comprising AAC comprising an effective amount of an HPV antigen described herein. In some embodiments, the individual has cancer. In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for treating cancer in an individual, the composition comprising any one of the compositions comprising AAC comprising an effective amount of an HPV antigen described herein.

[0108] In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for stimulating an immune response to HPV antigen protein, the composition comprising any one of the compositions of AAC comprising an effective amount of HPV antigen and an adjuvant described herein. In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for reducing tumor growth in an individual, the composition comprising any one of the compositions of AAC comprising an effective amount of HPV antigen and an adjuvant described herein. In some embodiments, the individual has cancer. In some embodiments, a use of a composition comprising an effective amount of AAC is provided in the manufacture of a medicament for treating cancer in an individual, the composition comprising any one of the compositions of AAC comprising an effective amount of HPV antigen and an adjuvant described herein.

[0109] In some embodiments of the methods, uses or compositions described herein, the individual has cancer. In some embodiments, the cancer is cervical cancer, perianal cancer, anogenital cancer, oral cancer, salivary gland cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer or head and neck cancer. In some embodiments, the cancer is HPV-associated cancer. In some embodiments, the cancer is localized cancer. In some embodiments, the cancer is localized cancer. In some embodiments, the cancer is locally advanced cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor.

[0110] In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is about 0.25 μm to about 4 μm, about 1 μm to about 4 μm, about 1.2 μm to about 3 μm, about 1.4 μm to about 2.6 μm, about 1.6 μm to about 2.4 μm, or about 1.8 μm to about 2.2 μm. In some embodiments, the width of the constriction is about 2.0 μm. In some embodiments, the width of the constriction is about 2.5 μm. In some embodiments, the width of the constriction is about 3.0 μm. In some embodiments, the width of the constriction is about any of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. or any one of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. In some embodiments, the cell suspension comprising the input anucleated cells is passed through a plurality of constrictions, wherein the plurality of constrictions are arranged in series and / or parallel.

[0111] In some embodiments, the anucleated cells are RBCs or platelets. In some embodiments, the anucleated cells are red blood cells or reticulocytes. In some embodiments, the AAC are anucleated cell-derived vesicles. In some embodiments, the AAC are RBC-derived vesicles or platelet-derived vesicles. In some embodiments, the AAC are red blood cell-derived vesicles or reticulocyte platelet-derived vesicles.

[0112] In some embodiments, the input anucleated cells are autologous to the individual receiving the composition. In some embodiments, the input anucleated cells are allogeneic to the individual receiving the composition. In some embodiments, the AAC is autologous to the individual receiving the composition. In some embodiments, the input AAC is allogeneic to the individual receiving the composition.

[0113] In some embodiments, when the AAC comprises an adjuvant, the adjuvant used for adjustment is CpG oligodeoxynucleotide (ODN), LPS, IFN-α, IFN-β, IFN-γ, alpha-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic acid-polycytidylic acid (poly I:C), R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist. In some embodiments, the adjuvant is polyinosinic acid-polycytidylic acid (poly I:C).

[0114] In some embodiments, at least one HPV antigen is a pool of multiple polypeptides that induce responses to the same and / or different HPV antigens. In some embodiments, at least one HPV antigen is a polypeptide that includes one or more antigenic HPV epitopes and one or more heterologous peptide sequences. In some embodiments, at least one HPV antigen is complexed with other antigens or with an adjuvant. In some embodiments, at least one HPV antigen can be processed into an MHC class I restricted peptide. In some embodiments, at least one HPV antigen can be processed into an MHC class II restricted peptide.

[0115] In some embodiments, the method includes multiple administrations of the AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the method includes about 3 to about 9 administrations of the AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the method includes any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 administrations of the AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the method optionally includes sequential administrations of the AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the time interval between two sequential administrations of the AAC comprising at least one HPV antigen and an adjuvant is about 1 day to about 30 days. In some embodiments, the time interval between two sequential administrations of the AAC comprising at least one HPV antigen and an adjuvant is about 21 days. In some embodiments, the time interval between two consecutive administrations of the AAC comprising at least one HPV antigen and an adjuvant is any one of about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 150 days. In some embodiments, the time interval between the first two consecutive administrations of the AAC comprising at least one HPV antigen and an adjuvant is 1 or 2 days. In some embodiments, the time interval between the first two consecutive administrations of the AAC comprising at least one HPV antigen and an adjuvant is one or two days, and the method includes more than two administrations of the AAC comprising at least one HPV antigen and an adjuvant (such as, but not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more administrations). In some embodiments, the AAC comprising at least one HPV antigen and an adjuvant is administered intravenously, intratumorally, orally, and / or subcutaneously. In some embodiments, the AAC comprising at least one HPV antigen is administered intravenously.

[0116] In some embodiments, the composition further comprises an adjuvant.In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), LPS, IFN-α, IFN-β, IFN-γ, alpha-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic-polycytidylic acid, R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.In some embodiments, the adjuvant is CpG oligodeoxynucleotide.In some embodiments, the adjuvant is poly I:C.

[0117] In some embodiments, the individual is HLA-A * 02. HLA-A * 01. HLA-A * 03. HLA-A * 24. HLA-A * 11. HLA-A * 26. HLA-A * 32. HLA-A * 31. HLA-A * 68, HLA-A * 29. HLA-A * 23. HLA-B * 07. HLA-B * 44. HLA-B * 08. HLA-B * 35. HLA-B * 15. HLA-B * 40, HLA-B * 27. HLA-B * 18. HLA-B * 51. HLA-B * 14. HLA-B * 13. HLA-B * 57, HLA-B * 38. HLA-C * 07. HLA-C * 04. HLA-C * 03. HLA-C * 06. HLA-C * 05. HLA-C * 12. HLA-C * 02. HLA-C * 01. HLA-C* 08, or HLA-C * Positive for expression of 16.

[0118] Immune checkpoints are regulators of the immune system that keep immune responses in check. Immune checkpoint inhibitors can be used to promote enhanced immune responses. In some embodiments, a composition comprising an AAC comprising at least one HPV antigen is administered in combination with administration of an immune checkpoint inhibitor. In some embodiments, a composition comprising an AAC comprising an HPV antigen and an immune checkpoint inhibitor are administered simultaneously. In some embodiments, a composition comprising an AAC comprising at least one HPV antigen and an immune checkpoint inhibitor are administered sequentially. In some embodiments, an immune checkpoint inhibitor and / or an AAC comprising at least one HPV antigen is administered intravenously, intratumorally, orally, and / or subcutaneously. In some embodiments, an AAC comprising at least one HPV antigen is administered intravenously. In some embodiments, an immune checkpoint inhibitor is administered intravenously, intratumorally, orally, and / or subcutaneously.

[0119] In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered after administration of the immune checkpoint inhibitor. For example, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour to about 1 week prior to administration of the immune checkpoint inhibitor. For example, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour to about 1 week prior to administration of the immune checkpoint inhibitor. For example, in some embodiments, a composition comprising an AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days prior to administration of an immune checkpoint inhibitor. In some embodiments, the composition comprising an AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days prior to administration of the immune checkpoint inhibitor.

[0120] In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 7 days, about 10 days, about 14 days, about 18 days, about 21 days, about 24 days, about 28 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days prior to administration of the immune checkpoint inhibitor. In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 7 to about 10 days, about 10 to about 14 days, about 14 to about 18 days, about 18 to about 21 days, about 21 to about 24 days, about 24 to about 28 days, about 28 to about 30 days, about 30 to about 35 days, about 35 to about 40 days, about 40 to about 45 days, or about 45 to about 50 days prior to administration of the immune checkpoint inhibitor.

[0121] In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered after administration of an immune checkpoint inhibitor. For example, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour to about 1 week after administration of an immune checkpoint inhibitor. For example, in some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of an immune checkpoint inhibitor. In some embodiments, the composition comprising an AAC comprising at least one HPV antigen and an adjuvant is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days after administration of the immune checkpoint inhibitor.

[0122] In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 7 days, about 10 days, about 14 days, about 18 days, about 21 days, about 24 days, about 28 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days after administration of the immune checkpoint inhibitor. In some embodiments, the composition comprising the AAC comprising at least one HPV antigen and an adjuvant is administered about 7 to about 10 days, about 10 to about 14 days, about 14 to about 18 days, about 18 to about 21 days, about 21 to about 24 days, about 24 to about 28 days, about 28 to about 30 days, about 30 to about 35 days, about 35 to about 40 days, about 40 to about 45 days, or about 45 to about 50 days after administration of the immune checkpoint inhibitor.

[0123] In some embodiments, the method includes multiple administrations of a composition comprising an AAC comprising at least one HPV antigen and an adjuvant, and / or multiple administrations of a checkpoint inhibitor. For example, in some embodiments, the method includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 administrations of a composition comprising an AAC comprising at least one HPV antigen and an adjuvant and / or a checkpoint inhibitor. For example, in some embodiments, the method includes less than 5 administrations, less than 10 administrations, less than 15 administrations, less than 20 administrations, less than 25 administrations, less than 30 administrations, less than 50 administrations, less than 75 administrations, less than 100 administrations, or less than 200 administrations of a composition comprising an AAC comprising at least one HPV antigen and an adjuvant and / or a checkpoint inhibitor.

[0124] Exemplary immune checkpoint inhibitors target, without limitation, PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more of an antibody that binds PD-1, an antibody that binds PD-L1, an antibody that binds CTLA-4, an antibody that binds LAG3, or an antibody that binds TIM-3, an antibody that binds TIGIT, an antibody that binds VISTA, an antibody that binds TIM-1, an antibody that binds B7-H4, or an antibody that binds BTLA. In further embodiments, the antibody may be a full-length antibody or any variant, including but not limited to, an antibody fragment, a single-chain variable fragment (ScFv), or an antigen-binding fragment (Fab). In further embodiments, the antibody may be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemical compounds that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more peptides that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor targets PD-1. In some embodiments, the immune checkpoint inhibitor targets PD-L1.

[0125] In some embodiments, there is provided a plurality of AACs (e.g., RBC-derived vesicles) comprising an HPV antigen and an adjuvant for use in a method of stimulating an immune response in an individual by any one of the methods described herein. Composition of AAC containing HPV antigens

[0126] In some embodiments, the AAC comprises an HPV antigen and an adjuvant delivered intracellularly. In some embodiments, the AAC is derived from input anucleated cells. In some embodiments, the AAC is derived from input red blood cells (RBCs). In some embodiments, the AAC is an AAC comprising at least one HPV antigen and an adjuvant. In some embodiments, the AAC is an RBC-derived vesicle comprising at least one HPV antigen and an adjuvant.

[0127] In some embodiments, the method includes administering an effective amount of an AAC comprising an HPV antigen and an adjuvant, wherein the AAC comprising at least one HPV antigen and an adjuvant is prepared by: a) passing a cell suspension comprising input anucleated cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the at least one HPV antigen and the adjuvant to pass through to form perturbed input anucleated cells; and b) incubating the perturbed input anucleated cells with the at least one HPV antigen and the adjuvant for a time sufficient to allow the at least one HPV antigen and the adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and the adjuvant. In some embodiments, the at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, at least one HPV antigen comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 18-25.

[0128] In some aspects, a composition of an AAC comprising an HPV antigen, or an HPV antigen and an adjuvant is provided, wherein the AAC comprising at least one HPV antigen, or at least one HPV antigen and an adjuvant, is prepared by: a) passing a cell suspension comprising input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the at least one HPV antigen and the adjuvant to pass through to form perturbed input anucleated cells; and b) incubating the perturbed input anucleated cells with the at least one HPV antigen for a time sufficient to allow the at least one HPV antigen and the adjuvant to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and the adjuvant. In some embodiments, the at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, at least one HPV antigen comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 18-25.

[0129] In some embodiments, the anucleated cells are RBCs or platelets. In some embodiments, the anucleated cells are red blood cells or reticulocytes. In some embodiments, the AAC are anucleated cell-derived vesicles. In some embodiments, the AAC are RBC-derived vesicles or platelet-derived vesicles. In some embodiments, the AAC are red blood cell-derived vesicles or reticulocyte platelet-derived vesicles.

[0130] In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is about 0.25 μm to about 4 μm, about 1 μm to about 4 μm, about 1.2 μm to about 3 μm, about 1.4 μm to about 2.6 μm, about 1.6 μm to about 2.4 μm, or about 1.8 μm to about 2.2 μm. In some embodiments, the width of the constriction is about 2.0 μm. In some embodiments, the width of the constriction is about 2.5 μm. In some embodiments, the width of the constriction is about 3.0 μm. In some embodiments, the width of the constriction is about any of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. or any one of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. In some embodiments, the cell suspension comprising the input anucleated cells is passed through a plurality of constrictions, wherein the plurality of constrictions are arranged in series and / or parallel.

[0131] In some embodiments, at least one HPV antigen is a pool of multiple polypeptides that induce responses to the same and / or different HPV antigens. In some embodiments, at least one HPV antigen is a polypeptide that includes one or more antigenic HPV epitopes and one or more heterologous peptide sequences. In some embodiments, at least one HPV antigen is complexed with other antigens or with an adjuvant. In some embodiments, at least one HPV antigen can be processed into an MHC class I restricted peptide. In some embodiments, at least one HPV antigen can be processed into an MHC class II restricted peptide.

[0132] In some embodiments, the composition further comprises an adjuvant.In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), LPS, IFN-α, IFN-β, IFN-γ, alpha-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic-polycytidylic acid (poly I:C), R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.In some embodiments, the adjuvant is polyinosinic-polycytidylic acid (poly I:C). Dosage and Regimen

[0133] In some embodiments, a method of treating HPV-associated disease in an individual comprises administering to the individual a composition comprising an effective amount of AAC, wherein the effective amount is about 0.5×10 8 ~Approx. 1×10 9 The method includes administering an effective amount of one or more immune checkpoint inhibitors to a patient, the AAC comprising an HPV antigen delivered intracellularly and an adjuvant.

[0134] In some embodiments according to any one of the methods described herein, the effective amount of the AAC comprising at least one HPV antigen and an adjuvant is about 0.5×10 8AAC / kg ~ approx. 1.0 x 10 9 AAC / kg. In some embodiments, the effective amount of AAC is about 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , 0.5×10 8 , 1.0×10 8 , 0.25×10 9 , 0.5×10 9 , 0.75×10 9 , 1.0×10 9 , 0.5×10 10 , 1.0×10 10 , 0.5×10 11 , and 1.0×10 11 In some embodiments, the effective amount of the AAC comprising at least one HPV antigen and an adjuvant is about 0.5×10 8 AAC / kg, approx. 2.5 × 10 8 AAC / kg, approx. 5 × 10 8 AAC / kg, or approximately 7.5 × 10 8 AAC / kg. In some embodiments, the effective amount of AAC is about 0.5×10 6 ~Approx. 1.0×10 6 , about 1.0×10 6 ~about 0.5×10 7 , about 0.5×10 7 ~Approx. 1.0×10 7 , about 1.0×10 7 ~about 0.5×10 8 AAC, approximately 0.5×10 8 ~Approx. 1.0×10 8 , about 1.0×10 8 ~about 0.5×10 9 AAC, approximately 0.5×10 9 ~Approx. 1.0×10 9 , about 1.0×10 9 ~about 0.5×10 10 AAC, approximately 0.5×10 10 ~Approx. 1.0×10 10 , about 1.0×10 10 ~about 0.5×10 10 AAC / kg.

[0135] In some embodiments, when the method further comprises administering an effective amount of an immune checkpoint inhibitor, the immune checkpoint inhibitor targets CTLA-4. In some embodiments, the immune checkpoint inhibitor is ipilimumab. In some embodiments, the effective amount of ipilimumab is about 0.1 mg / kg to about 30 mg / kg. In some embodiments, the effective amount of ipilimumab is any one of about 1 mg / kg to about 3 mg / kg. In some embodiments, the effective amount of ipilimumab is any one of about 0.1, 0.2, 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, or 30 mg / kg. In some embodiments, the effective amount of ipilimumab is any one of about 0.1-0.2, 0.2-0.5, 0.5-1.0, 1.0-1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2, 2.2-2.4, 2.4-2.6, 2.6-2.8, 2.8-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, or 25-30 mg / kg.

[0136] In some embodiments, when the method further comprises administering an effective amount of an immune checkpoint inhibitor, the immune checkpoint inhibitor targets PD-1. In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, the effective amount of nivolumab is about 30 mg to about 1000 mg. In some embodiments, the effective amount of nivolumab is about 300 mg to about 400 mg. In some embodiments, the effective amount of ipilimumab is about 360 mg. In some embodiments, the effective amount of ipilimumab is about 30, 50, 100, 150, 200, 250, 300, 320, 340, 360, 380, 400, 450, 500, 550, 600, 700, 800, 900, or 1000 mg. In some embodiments, the effective amount of ipilimumab is any one of about 30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-320, 320-340, 340-360, 360-380, 380-400, 400-450, 500-550, 550-600, 600-700, 700-800, 800-900, or 900-1000 mg.

[0137] In some embodiments, when the method further comprises administering an effective amount of an immune checkpoint inhibitor, the immune checkpoint inhibitor targets PD-L1. In some embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, the effective amount of atezolizumab is about 100 mg to about 2500 mg. In some embodiments, the effective amount of atezolizumab is any one of about 900 mg to about 1500 mg. In some embodiments, the effective amount of atezolizumab is any one of about 1200 mg. In some embodiments, the effective amount of atezolizumab is any one of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1150, 1200, 1250, 1300, 1400, 1500, 1600, 1800, 2000, 2200, or 2500 mg. In some embodiments, the effective amount of atezolizumab is any one of about 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1800, 1800-2000, 2000-2200, or 2200-2500 mg.

[0138] In some embodiments, the method of treatment includes multiple (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) cycles of administering any one of the AACs described herein to an individual. For example, in some embodiments, a method of vaccinating an individual against an antigen is provided by administering to the individual 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times an AAC comprising an antigen and / or adjuvant, the AAC being created by passing input anucleated cells through a constriction to form an AAC such that the antigen and / or adjuvant enters the AAC. In some embodiments, the time period between any two consecutive administrations of the AAC is at least about 1 day (including any range between these values, such as 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).

[0139] In some embodiments according to any one of the methods described herein, the composition comprising AAC is administered in any one of a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 week cycle. In some embodiments, the composition comprising AAC is administered in a 3 week cycle. In some embodiments, the composition comprising AAC is administered in a 6 week cycle. In some embodiments, the composition comprising AAC is administered on one or more of days 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. In some embodiments, the composition comprising AAC is administered on day 1 of the treatment cycle. In some embodiments, the composition comprising AAC is administered on day 2 of the treatment cycle. In some embodiments, the composition comprising AAC is administered on days 1 and 2 of the treatment cycle. In some embodiments, the composition comprising AAC is administered on days 1 and 3 of the treatment cycle. In some embodiments, the composition comprising AAC is administered on day 8 of the treatment cycle. In some embodiments, the composition comprising AAC is administered on day 1 of a three-week cycle. In some embodiments, the composition comprising AAC is further administered on day 2 of a three-week cycle. In some embodiments, the composition comprising AAC is administered in three-week cycles until the AAC composition supply is exhausted or for one year.

[0140] In some embodiments, about 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , 0.5×10 8 , 1.0×10 8 , 0.25×10 9 , 0.5×10 9 , 0.75×10 9 , 1.0×10 9 , 0.5×10 10 , 1.0×10 10 , 0.5×10 11 , and 1.0×10 11 AAC / kg is administered on day 1 of each 3-week cycle. In some embodiments, about 0.5×108 AAC / kg ~ approx. 1 x 10 9 AAC / kg is administered on day 1 of each 3-week cycle. In some embodiments, about 0.5×10 8 AAC / kg, approx. 2.5 × 10 8 AAC / kg, approx. 5.0 × 10 8 AAC / kg, or approximately 7.5 × 10 8 AAC / kg is administered on day 1 of each 3-week cycle. In some embodiments, about 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , 0.5×10 8 , 1.0×10 8 , 0.25×10 9 , 0.5×10 9 , 0.75×10 9 , 1.0×10 9 , 0.5×10 10 , 1.0×10 10 , 0.5×10 11 , and 1.0×10 11 AAC / kg is administered on day 2 of each three-week cycle. In some embodiments, about 0.5×10 8 AAC / kg ~ approx. 1 x 10 9 AAC / kg is administered on day 2 of each 3-week cycle. In some embodiments, about 0.5×10 8 AAC / kg, approx. 2.5 × 10 8 AAC / kg, approx. 5.0 × 10 8 AAC / kg, or approximately 7.5 × 10 8 AAC / kg is administered on day 2 of each 3-week cycle. In some embodiments, 0.5×10 8 AAC / kg is administered on day 1 of each 3-week cycle. In some embodiments, 0.5×10 8 AAC / kg was administered on day 1 of each 3-week cycle, with 0.5 × 10 8 AAC / kg is administered on day 2 of each 3-week cycle. In some embodiments, 0.5×10 8AAC / kg was administered on day 1 of each 3-week cycle, with 0.5 × 10 8 AAC / kg is administered on day 3 of each 3-week cycle. 8 AAC / kg is administered on day 1 of each 3-week cycle. In some embodiments, 2.5×10 8 AAC / kg was administered on day 1 of each 3-week cycle, with 2.5 × 10 8 AAC / kg is administered on day 2 of each 3-week cycle. 8 AAC / kg was administered on day 1 of each 3-week cycle, with 2.5 × 10 8 AAC / kg is administered on day 3 of each 3-week cycle. 8 AAC / kg is administered on day 1 of each 3-week cycle. 8 AAC / kg was administered on day 1 of each 3-week cycle, with 5 × 10 8 AAC / kg is administered on day 2 of each 3-week cycle. 8 AAC / kg was administered on day 1 of each 3-week cycle, with 5 × 10 8 AAC / kg is administered on day 3 of each 3-week cycle.

[0141] In some embodiments, when the method further comprises administering an effective amount of one or more immune checkpoint inhibitors, the immune checkpoint inhibitors target CTLA-4, PD-1, and / or PD-L1. In some embodiments, the antibody that binds CTLA-4, and / or the antibody that binds PD-1, and / or the antibody that binds PD-L1 are administered 1, 2, 3, 4, 5, 6 or more times in a cycle. In some embodiments, the antibody that binds CTLA-4, and / or the antibody that binds PD-1, and / or the antibody that binds PD-L1 are administered once per three-week cycle. In some embodiments, the antibody that binds CTLA-4 is administered once per three-week cycle. In some embodiments, the antibody that binds PD-1 is administered once per three-week cycle. In some embodiments, the antibody that binds PD-L1 is administered once per three-week cycle. In some embodiments, the antibody that binds CTLA-4, and / or the antibody that binds PD-1, and / or the antibody that binds PD-L1 are administered once per two three-week cycles. In some embodiments, the antibody that binds CTLA-4 is administered once every two three-week cycles. In some embodiments, the antibody that binds PD-1 is administered once every two three-week cycles. In some embodiments, the antibody that binds PD-L1 is administered once every two three-week cycles.

[0142] In some embodiments, the immune checkpoint inhibitor is administered one or more times on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of a treatment cycle. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 binding antibody, and the antibody that binds to CTLA-4 is administered on day 1 of each three-week cycle. In some embodiments, the antibody that binds to CTLA-4 is administered in up to four doses. In some embodiments, the effective amount of the antibody that binds to CTLA-4 is about 3 mg / kg. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab. In some embodiments, the ipilimumab is administered at a dose of about 3 mg / kg. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab, and the ipilimumab is administered at a dose of about 3 mg / kg on day 1 of every three-week cycle.

[0143] In some embodiments, the immune checkpoint inhibitor is a PD-1 binding antibody, and the antibody that binds to PD-1 is administered on day 8 of the first 3-week cycle and on day 1 of each subsequent 3-week cycle. In some embodiments, the antibody that binds to PD-1 is nivolumab. In some embodiments, nivolumab is administered at a dose of about 360 mg. In some embodiments, the antibody that binds to PD-1 is nivolumab, and nivolumab is administered at a dose of about 360 mg on day 8 of the first 3-week cycle and on day 1 of each subsequent cycle.

[0144] In some embodiments, the one or more immune checkpoint inhibitors include a CTLA-4 binding antibody and a PD-1 binding antibody, and the antibody that binds CTLA-4 is administered on day 1 of every alternate 3-week cycle (i.e., day 1 of every 6-week cycle), and the antibody that binds PD-1 is administered on day 8 of the first 3-week cycle and on day 1 of each subsequent 3-week cycle. In some embodiments, the antibody that binds CTLA-4 is ipilimumab, and the antibody that binds PD-1 is nivolumab. In some embodiments, ipilimumab is administered at a dose of about 1 mg / kg. In some embodiments, nivolumab is administered at a dose of about 360 mg. In some embodiments, the antibody that binds CTLA-4 is ipilimumab, which is administered at a dose of about 1 mg / kg on day 1 of all alternative 3 week cycles, and the antibody that binds PD-1 is nivolumab, which is administered at a dose of about 360 mg on day 8 of the first 3 week cycle and day 1 of each subsequent cycle.

[0145] In some embodiments, the immune checkpoint inhibitor is a PD-L1 binding antibody, and the antibody that binds to PD-L1 is administered on day 8 of the first 3-week cycle and on day 1 of each subsequent 3-week cycle. In some embodiments, the antibody that binds to PD-L1 is atezolizumab. In some embodiments, atezolizumab is administered at a dose of about 1200 mg. In some embodiments, the antibody that binds to PD-1 is atezolizumab, and atezolizumab is administered at a dose of about 360 mg on day 8 of the first 3-week cycle and on day 1 of each subsequent cycle. Methods for Producing Compositions of AAC Containing HPV Antigens

[0146] In some embodiments, a method is provided for making a composition comprising an AAC comprising an HPV antigen, wherein at least one HPV antigen is delivered intracellularly to the AAC. In some embodiments, a method is provided for making a composition comprising an AAC comprising an HPV antigen and an adjuvant, wherein at least one HPV antigen and an adjuvant are delivered intracellularly to the AAC.

[0147] In some embodiments, an AAC comprising at least one HPV antigen and an adjuvant is prepared by a process comprising: a) passing a cell suspension comprising a population of anucleated input cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the at least one HPV antigen and the adjuvant to pass through to form perturbed input anucleated cells; and b) incubating the population of perturbed input anucleated cells with the at least one HPV antigen and the adjuvant for a time sufficient to allow the antigen to enter the perturbed input anucleated cells, thereby creating an AAC comprising the at least one HPV antigen and the adjuvant.

[0148] In some embodiments, at least one HPV antigen comprises a peptide derived from HPV E6. In some embodiments, at least one HPV antigen comprises a peptide derived from HPV E7. In some embodiments, at least one HPV antigen comprises a peptide derived from HPV E6.

[0149] In some embodiments, the input non-nucleated cells are red blood cells (RBCs) or platelets. In some embodiments, the input non-nucleated cells are red blood cells or reticulocytes. In some embodiments, the AAC are non-nucleated cell-derived vesicles. In some embodiments, the AAC are RBC-derived vesicles or platelet-derived vesicles. In some embodiments, the AAC are red blood cell-derived vesicles or reticulocyte platelet-derived vesicles.

[0150] In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the input anuclear cells. In some embodiments, the width of the constriction is about 0.25 μm to about 4 μm, about 1 μm to about 4 μm, about 1.2 μm to about 3 μm, about 1.4 μm to about 2.6 μm, about 1.6 μm to about 2.4 μm, or about 1.8 μm to about 2.2 μm. In some embodiments, the width of the constriction is about 2.0 μm. In some embodiments, the width of the constriction is about 2.5 μm. In some embodiments, the width of the constriction is about 3.0 μm. In some embodiments, the width of the constriction is about any of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. or any one of 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. In some embodiments, the cell suspension comprising the input anucleated cells is passed through a plurality of constrictions, wherein the plurality of constrictions are arranged in series and / or parallel.

[0151] In some embodiments, the at least one HPV antigen is a pool of multiple polypeptides that induce responses to the same and / or different HPV antigens. In some embodiments, the at least one HPV antigen is a polypeptide that includes one or more antigenic HPV epitopes and one or more heterologous peptide sequences. In some embodiments, the at least one HPV antigen is delivered together with other antigens or with an adjuvant. In some embodiments, the at least one HPV antigen is a polypeptide that includes antigenic HPV epitopes and one or more heterologous peptide sequences. In some embodiments, the at least one HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, the at least one HPV is HPV-16 or HPV-18. In some embodiments, the at least one HPV antigen is composed of an HLA-A2 specific epitope. In some embodiments, the at least one HPV antigen is an HPV E6 antigen or an HPV E7 antigen. In some embodiments, the antigen includes peptides derived from HPV E6 and / or E7. In some embodiments, the antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7. In some embodiments, at least one HPV antigen can be processed into an MHC class I restricted peptide. In some embodiments, at least one HPV antigen can be processed into an MHC class II restricted peptide.

[0152] In some embodiments, the composition further comprises an adjuvant.In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), LPS, IFN-α, IFN-β, IFN-γ, alpha-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic-polycytidylic acid (poly I:C), R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.In some embodiments, the adjuvant is polyinosinic-polycytidylic acid (poly I:C). HPV antigen

[0153] In some embodiments according to the methods described herein, the exogenous antigen is a human papillomavirus (HPV) antigen. Papillomavirus is a small non-enveloped DNA virus with a virion size of about 55 nm in diameter. More than 100 HPV genotypes have been fully characterized, and many more are estimated to exist. HPV is a known cause of cervical cancer, as well as some vulvar, vaginal, penile, oropharyngeal, anal and rectal cancers. Most HPV infections are asymptomatic and disappear naturally, but persistent infection with one of the oncogenic HPV types can progress to pre-cancerous conditions or cancer. Other HPV-associated diseases include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermal dysplasia, focal epithelial hyperplasia, oral papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (SIL), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VAIN). Many of the known human papillomavirus (HPV) types cause benign lesions with a subset that are tumorigenic. Based on epidemiological and phylogenetic relationships, HPV types have been classified into 15 "high-risk types" (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "probable high-risk types" (HPV26, 53, and 66), which together are known to manifest as low- and high-grade cervical changes and cancers, as well as other anogenital cancers such as vulvar, vaginal, penile, anal and perianal cancers, and head and neck cancers. Recently, an association of high-risk types HPV16 and 18 with breast cancer has also been described. Eleven HPV types classified as "low-risk types" (HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are known to manifest as benign low-grade cervical changes, genital warts, and recurrent respiratory papillomatosis. Cutaneous HPV types 5, 8, and 92 are associated with skin cancer. In some HPV-associated cancers, the immune system is suppressed and, accordingly, antitumor responses are significantly impaired. See Suresh and Burtness, Am J Hematol Oncol 13(6):20-27 (2017).In some embodiments, the exogenous antigen is a pool of multiple polypeptides that induce responses against the same and / or different antigens. In some embodiments, the antigens in the pool of multiple antigens do not reduce immune responses directed against other antigens in the pool of multiple antigens. In some embodiments, at least one HPV antigen is a polypeptide that includes an antigenic HPV epitope and one or more heterologous peptide sequences. In some embodiments, at least one HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, at least one HPV is an HPV-16 antigen or an HPV-18 antigen. In some embodiments, at least one HPV antigen is composed of an HLA-A2 specific epitope. In some embodiments, at least one HPV antigen is an HPV E6 antigen or an HPV E7 antigen. In some embodiments, the antigen includes a peptide derived from HPV E6 and / or E7. In some embodiments, the antigen includes an HLA-A2 restricted peptide derived from HPV E6 and / or E7. In some embodiments, the antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7, the HLA-A2-restricted peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, at least one HPV antigen comprises an amino acid sequence having at least 90% similarity to any one of SEQ ID NOs: 18-25. In some embodiments, at least one HPV antigen comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 18. In some embodiments, at least one HPV antigen comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 19.In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, at least one HPV antigen comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, at least one HPV antigen is a plurality of antigens comprising at least one of the amino acid sequences of any one of SEQ ID NOs: 18-25. In some embodiments, the exogenous antigen is a plurality of antigens comprising 2, 3, 4, 5, 6, 7, or 8 of the amino acid sequences of any one of SEQ ID NOs: 18-25. In some embodiments, the exogenous antigen is a plurality of antigens comprising an amino acid sequence having at least 90% similarity to SEQ ID NO: 19 and an amino acid sequence having at least 90% similarity to SEQ ID NO: 23. In some embodiments, the exogenous antigen is a plurality of antigens comprising an amino acid sequence of SEQ ID NO: 19 and an amino acid sequence of SEQ ID NO: 23. In some embodiments, the plurality of antigens is contained within a pool of non-covalently linked peptides. In some embodiments, the plurality of antigens is contained within a pool of non-covalently linked peptides, each peptide comprising no more than one antigen. In some embodiments, the plurality of antigens is contained within a pool of non-covalently linked peptides, the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 25 are contained within separate peptides.

[0154] In some embodiments, at least one HPV antigen is in a pool of multiple polypeptides that elicit responses against the same and / or different HPV antigens. In some embodiments, an antigen in the pool of multiple antigens does not reduce immune responses directed against other antigens in the pool of multiple antigens. In some embodiments, at least one HPV antigen is a polypeptide that includes an antigenic HPV antigen and one or more heterologous peptide sequences. In some embodiments, at least one HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, at least one HPV antigen is comprised of an HLA-A2 specific epitope. In some embodiments, at least one HPV antigen is comprised of an HLA-A11 specific epitope. In some embodiments, at least one HPV antigen is comprised of an HLA-B7 specific epitope. In some embodiments, at least one HPV antigen is comprised of an HLA-C8 specific epitope. In some embodiments, at least one HPV antigen includes a portion or all of the N-terminal domain of a full-length HPV protein.

[0155] In some embodiments according to any one of the methods described herein, the AAC (e.g., RBC-derived vesicles) comprises multiple HPV antigens comprising multiple immunogenic epitopes. In further embodiments, following administration of the AAC comprising multiple antigens comprising multiple immunogenic epitopes to an individual, the multiple immunogenic epitopes do not reduce the immune response in the individual against any of the other immunogenic epitopes. In some embodiments, at least one HPV antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminally adjacent polypeptide and / or a C-terminally adjacent polypeptide. In some embodiments, at least one HPV antigen is a polypeptide comprising an immunogenic peptide epitope and one or more heterologous peptide sequences. In some embodiments, at least one HPV antigen is a polypeptide comprising an immunogenic peptide epitope flanked at its N-terminus and / or its C-terminus by a heterologous peptide sequence. In some embodiments, the flanking heterologous peptide sequence is derived from a disease-associated immunogenic peptide. In some embodiments, the flanking heterologous peptide sequence is a non-naturally occurring sequence. In some embodiments, the flanking heterologous peptide sequence is derived from an immunogenic synthetic long peptide (SLP). In some embodiments, at least one HPV antigen can be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. Adjuvants

[0156] As used herein, the term "adjuvant" may refer to a substance that modulates and / or generates an immune response, either directly or indirectly. In some embodiments of the present invention, the adjuvant is delivered intracellularly to a population of anuclear cells or AAC, such as a population of RBCs or RBC-derived vesicles (i.e., before administration to an individual, but before, during, and / or after stenosis treatment, incubation of the cells or vesicles with the adjuvant) to form an AAC containing the adjuvant. In some examples, the adjuvant is administered in conjunction with an HPV antigen to result in an enhanced immune response to at least one HPV antigen compared to the HPV antigen alone. Thus, the adjuvant can be used to boost the induction of immune cell responses (e.g., T cell responses) to HPV antigens. Exemplary adjuvants include, without limitation, stimulator of interferon genes (STING) agonists, and retinoic acid-inducible gene I (RIG-I) agonists, as well as agonists for TLR3, TLR4, TLR7, TLR8 and / or TLR9. Exemplary adjuvants include, without limitation, CpG ODN, interferon-α (IFN-α), polyinosinic:polycytidylic acid (poly I:C), imiquimod (R837), resiquimod (R848), or lipopolysaccharide (LPS). In some embodiments, the adjuvant is CpG ODN, LPS, IFN-α, IFN-β, IFN-γ, α-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic:polycytidylic acid (poly I:C), R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.In certain embodiments, the adjuvant is CpG ODN.In some embodiments, the adjuvant is CpG ODN.In some embodiments, the CpG ODN is class A CpG ODN, class B CpG ODN, or class C CpG ODN.In some embodiments, the CpG ODN adjuvant comprises one selected from the group of CpG ODN1018, CpG ODN1585, CpG ODN2216, CpG ODN2336, CpG ODN1668, CpG ODN1826, CPG ODN2006, CpG ODN2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN2395, CpG ODN M362, CpG ODN D-SL03. In some embodiments, the CpG ODN adjuvant is a CpG ODN1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN2006 (also known as CpG7909) (TCGTCGTTTTGTCGTTTTGTCGTT; SEQ ID NO: 31) oligonucleotide. In some embodiments, the adjuvant is CpG7909. In some embodiments, the RIG-I agonist comprises polyinosinic:polycytidylic acid (poly I:C). Multiple adjuvants can also be used in conjunction with HPV antigens to enhance induction of immune responses. In some embodiments, the AAC comprising at least one HPV antigen further comprises more than one adjuvant. In some embodiments, the AAC comprising at least one HPV antigen further comprises any combination of adjuvants CpG ODN, LPS, IFN-α, IFN-β, IFN-γ, alpha-galactosylceramide, STING agonist, cyclic dinucleotide (CDN), RIG-I agonist, polyinosinic:polycytidylic acid (poly I:C), R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist. Further Modifications of AAC Including HPV Antigens and Adjuvants

[0157] In some embodiments according to any one of the methods described herein, the AAC composition further comprises an agent that enhances the function of the AAC compared to a corresponding AAC composition without the agent. In some embodiments, the AAC composition further comprises an agent that enhances the function of the AAC upon freeze-thaw cycling compared to a corresponding AAC composition without the agent. In some embodiments, the agent is a cryopreservation agent and / or cryopreservation agent. In some embodiments, neither the cryopreservation agent nor the cryopreservation agent prevents more than 10% or 20% cell death in the AAC composition comprising the agent compared to a corresponding AAC composition without the agent prior to any freeze-thaw cycle. In some embodiments, freeze-thaw cycling of the non-nuclear cell-derived vesicle composition comprising a cryopreservation agent and / or cryopreservation agent causes no more than 10%, 20%, 30%, 40%, or 50% loss of function when compared to a corresponding non-nuclear cell-derived vesicle composition prior to the freeze-thaw cycle. In some embodiments, freeze-thaw cycling of an anucleated cell-derived vesicle composition comprising a cryopreservative and / or cryopreservative causes less than 10%, 20%, 30%, 40%, or 50% loss of function when compared to freeze-thaw cycling of a corresponding composition of anucleated cell-derived vesicles without a cryopreservative and / or cryopreservative. In some embodiments, the function or functionality of an anucleated cell-derived vesicle composition is measured by the percentage of anucleated cell-derived vesicles that are positive for Annexin V staining. In some embodiments, the function or functionality of an anucleated cell-derived vesicle composition is measured by the percentage of anucleated cell-derived vesicles that are positive for CD235a staining. In some embodiments, the function or functionality of an anucleated cell-derived vesicle composition is measured by the percentage of anucleated cell-derived vesicles that are positive for CD235a and Annexin V staining. In some embodiments, at least about 70%, about 80%, or about 90% of the AAC is functional after up to 1, 2, 3, 4, or 5 freeze-thaw cycles. In some embodiments, the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. In some embodiments, the agent is albumin.In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the albumin is one or more of mouse, bovine, or human albumin. In some embodiments, the agent is human albumin. In some embodiments, the agent is one or more of divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the divalent metal cation is Mg. 2+ , Zn 2+ or Ca 2+ In some embodiments, the agent is one or more of sodium pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), DMSO, HEPES, glycerol, glutathione, inosine, dibasic sodium phosphate, monobasic sodium phosphate, sodium metal ion, potassium metal ion, magnesium metal ion, chloride, acetate, gluconate, sucrose, potassium hydroxide, or sodium hydroxide. In some embodiments, the agent is one or more of sodium pyruvate, adenine, Rejuvesol®, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), PlasmaLyte®, DMSO, Cryostor® CS2, Cryostor® CS5, Cryostor® CS10, Cryostor® CS15, HEPES, glycerol, glutathione, HypoThermosol®.

[0158] In some embodiments according to any one of the methods described herein, the process further comprises a step of incubating the AAC composition with an agent that enhances the function of the AAC compared to a corresponding AAC prepared without an additional incubation step.

[0159] In some embodiments, the formulation comprises a cryopreservation medium. In some embodiments, the formulation comprises about 1×10 9 ~Approx. 1×10 11 In some embodiments, the formulation comprises about 0.5×10 AAC in about 9 mL to about 10 mL of cryopreservation medium. 7 , 0.7×10 7 , 1.0×10 7 , 0.5×10 8 , 0.7×10 8 , 1.0×10 8 , 0.5×10 9 , 0.7×10 9 , 1.0×10 9 , 0.5×10 10 , 0.7×10 10 , 1.0×10 10 , 0.5×10 11 , 0.7×10 11 , 1.0×10 11 , 0.5×10 12 , 0.7×10 12 , and 1.0×10 12 AAC. In some embodiments, the formulation comprises about 0.5×10 in about 9 mL to about 10 mL of cryopreservation medium. 7 ~Approx. 1.0×10 7 , about 1.0×10 7 ~about 0.5×10 8 AAC, approximately 0.5×10 8 ~Approx. 1.0×10 8 , about 1.0×10 8 ~about 0.5×10 9 AAC, approximately 0.5×10 9 ~Approx. 1.0×10 9 , about 1.0×10 9 ~about 0.5×10 10 , about 0.5×10 10 ~Approx. 1.0×10 10 , about 1.0×10 10 ~about 0.5×10 11 , about 0.5×10 11 ~Approx. 1.0×10 11 AAC, approximately 1.0×10 11 ~about 0.5×10 12AAC. In some embodiments, the formulation comprises about 1 x 10 in about 9 mL to about 10 mL of cryopreservation medium. 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , and 1 × 10 10 AAC. In some embodiments, the formulation comprises about 1 x 10 in about 9.5 mL of cryopreservation medium. 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , and 1 × 10 10 AAC. In some embodiments, the formulation comprises about 7×10 IgG in about 9 mL to about 10 mL of cryopreservation medium. 9 In some embodiments, the formulation comprises about 7×10 AAC in about 9.5 mL of cryopreservation medium. 9 In some embodiments, the formulation comprises about 6.65×10 AAC in about 9.5 mL of cryopreservation medium. 9 In some embodiments, the formulation comprising AAC comprises about 0.5×10 7 , 0.7×10 7 , 1.0×10 7 , 0.5×10 8 , 0.7×10 8 , 1.0×10 8 , 0.5×10 9 , 0.7×10 9 , 1.0×10 9 , 0.5×10 10 , 0.7×10 10 , 1.0×10 10 , 0.5×10 11 , 0.7×10 11 , 1.0×10 11 , 0.5×10 12, 0.7×10 12 , and 1.0×10 12 AAC. In some embodiments, the formulation comprises about 0.5×10 7 ~Approx. 1.0×10 7 , about 1.0×10 7 ~about 0.5×10 8 AAC, approximately 0.5×10 8 ~Approx. 1.0×10 8 , about 1.0×10 8 ~about 0.5×10 9 AAC, approximately 0.5×10 9 ~Approx. 1.0×10 9 , about 1.0×10 9 ~about 0.5×10 10 , about 0.5×10 10 ~Approx. 1.0×10 10 , about 1.0×10 10 ~about 0.5×10 11 , about 0.5×10 11 ~Approx. 1.0×10 11 AAC, approximately 1.0×10 11 ~about 0.5×10 12 AAC. In some embodiments, the formulation comprises about 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , and 1 × 10 10 AAC. In some embodiments, the formulation comprises about 7×10 9 In some embodiments, the formulation comprises about 6.65×10 AAC in a cryopreservation medium. 9 In some embodiments, the formulation comprises about 0.7×10 AAC in frozen storage medium after thawing. 9 In some embodiments, the formulation contains about 0.7×10 AAC / mL in the cryopreservation medium after thawing, as measured by Coulter Counter. 9AAC / mL. In some embodiments, the cryopreservation medium comprises CryoStor® CS2. In some embodiments, the cryopreservation medium is CryoStor® CS2.

[0160] In some embodiments, a composition comprising AAC is about 7×10 in about 9 mL to about 10 mL of CryoStor® CS2. 9 In some embodiments, the composition comprising AAC comprises about 7×10 9 In some embodiments, the formulation comprises about 6.65×10 AAC in about 9.5 mL of CryoStor® CS2. 9 Includes AAC.

[0161] In some embodiments, the AAC in the formulation maintains functionality equal to or greater than about 50% up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintains functionality equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AAC in the formulation maintains functionality equal to or greater than about 70% after storage at or below −140° C. for at least 12 months. In some embodiments, the formulation maintains functionality equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% after storage at or below −140° C. for at least 12 months. In some embodiments, the formulation maintains about 70% or greater functionality after at least 6, 9, 12, 15, 18, 24, 30, or 36 months of storage at or below −140° C. In some embodiments, the formulation maintains about 70% or greater functionality after at least 12 months of storage at or below −100° C., −110° C., −120° C., −130° C., −140° C., −150° C., −160° C., −170° C., −180° C., −190° C., or −200° C.

[0162] In some embodiments, the AAC in the formulation maintains about 50% or greater positive staining for Annexin V and / or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintains about 50%, 60%, 70%, 80%, 90%, 95%, or 99% or greater positive staining for Annexin V and / or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AAC in the formulation maintains about 70% or greater positive staining for Annexin V and / or CD235a after storage at or below -140°C for at least 12 months. In some embodiments, the formulation maintains greater than or equal to about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for Annexin V and / or CD235a after at least 12 months of storage at or below −140° C. In some embodiments, the formulation maintains greater than or equal to about 70% positive staining for Annexin V and / or CD235a after at least 6, 9, 12, 15, 18, 24, 30, or 36 months of storage at or below −140° C. In some embodiments, the formulation maintains greater than or equal to about 70% positive staining for Annexin V and / or CD235a after storage for at least 12 months at or below temperatures of -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, or -200°C.

[0163] In some embodiments, the AAC in the formulation maintains about 50% or greater positive staining for Annexin V and / or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintains about 50%, 60%, 70%, 80%, 90%, 95%, or 99% or greater positive staining for Annexin V and / or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AAC in the formulation maintains about 70% or greater positive staining for Annexin V and / or CD235a after storage at or below -140°C for at least 12 months. In some embodiments, the formulation maintains greater than or equal to about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for Annexin V and / or CD235a after at least 12 months of storage at or below −140° C. In some embodiments, the formulation maintains greater than or equal to about 70% positive staining for Annexin V and / or CD235a after at least 6, 9, 12, 15, 18, 24, 30, or 36 months of storage at or below −140° C. In some embodiments, the formulation maintains greater than or equal to about 70% positive staining for Annexin V and / or CD235a after storage for at least 12 months at or below temperatures of -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, or -200°C. Stenosis used in the production of HPV antigen-containing AAC compositions

[0164] In some embodiments, the present invention provides a composition of AAC comprising HPV antigen for stimulating immune response. In some embodiments, the anucleated cell is RBC or platelet. In some embodiments, the anucleated cell is red blood cell or reticulocyte. In some embodiments, at least one HPV antigen is delivered intracellularly to anucleated cell. Methods of introducing payload into anucleated cell are known in the art.

[0165] In some embodiments, at least one HPV antigen is introduced into anucleated cells by passing the cells through a constriction such that a transient pore is introduced into the membrane of the cell, thereby allowing the at least one HPV antigen to enter the cell. Examples of constriction-based delivery of compounds to cells are provided by WO2013 / 059343, WO2015 / 023982, WO2016 / 070136, WO2017041050, WO2017008063, WO2017 / 192785, WO2017 / 192786, WO2019 / 178005, WO2019 / 178006, WO2020 / 072833, WO2020 / 154696, and WO2020 / 176789, US20180142198, and US20180201889.

[0166] In some embodiments, at least one HPV antigen and adjuvant are delivered to anucleated cells (e.g., RBCs) by passing a cell suspension containing the anucleated cells through a constriction to produce the AAC of the present invention, where the constriction deforms the cells, thereby causing perturbation of the cells such that the HPV antigen and adjuvant enter the cells. In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, multiple constrictions can be arranged in parallel and / or in series within the microfluidic channel.

[0167] In some embodiments, the constriction in the microfluidic channel includes an inlet portion, a center point, and an outlet portion. In some embodiments, the length, depth, and width of the constriction in the microfluidic channel can be varied. In some embodiments, the width of the constriction in the microfluidic channel is a function of the diameter of the anucleated cell. Methods for determining the diameter of anucleated cells are known in the art, such as high content imaging, cell counters, or flow cytometry.

[0168] In some embodiments of constriction-based delivery of HPV antigens to the AAC, the width of the constriction is about 0.5 μm to about 10 μm. In some embodiments, the width of the constriction is about 1 μm to about 4 μm. In some embodiments, the width of the constriction is about 1 μm to about 3 μm. In some embodiments, the width of the constriction is about 1.5 μm to about 2.5 μm. In some embodiments, the width of the constriction is about 1.2 μm to about 2.8 μm. In some embodiments, the width of the constriction is about 0.5 μm to about 5 μm. In some embodiments, the width of the constriction is about 2 μm to about 2.5 μm. In some embodiments, the width of the constriction is about 1.5 μm to about 2 μm. In some embodiments, the width of the constriction is about 0.5 μm to about 3.5 μm. In some embodiments, the width of the constriction is about 3.2 μm to about 3.8 μm. In some embodiments, the width of the constriction is between about 3.8 μm and about 4.3 μm. In some embodiments, the width of the constriction is between about 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, and 6.0 μm. or any one of less than 0.25 μm, 0.5 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.2 μm, 5.4 μm, 5.6 μm, 5.8 μm, 6.0 μm. In some embodiments, the width of the constriction is about 2 μm. In some embodiments, the width of the constriction is about 2.2 μm. In some embodiments, the width of the constriction is about 2.5 μm. In some embodiments, the width of the constriction is about 3 μm.

[0169] Examples of parameters that may affect the delivery of compounds to the AAC include, but are not limited to, the dimensions of the constriction, the angle of the entrance of the constriction, the nature of the surface of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), the operating flux (e.g., cell passage time through the constriction), the concentration of cells, the concentration of the compound in the cell suspension, the amount of buffer in the cell suspension and the time to recover or incubate the AAC after passing through the constriction, which may affect the passage of compounds delivered to the AAC. Additional parameters that affect the delivery of compounds to the AAC may include the velocity of the input anucleated cells at the constriction, the shear rate at the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time at the constriction. In addition, multiple chips containing channels in series and / or in parallel may affect the delivery to the AAC. Multiple chips in parallel may be useful to enhance throughput. Such parameters may be designed to control the delivery of compounds. In some embodiments, the concentration of cells may range from about 10 to at least about 10 12 In some embodiments, the concentration of the delivery compound may range from about 10 ng / mL to about 1 g / mL, or any concentration or range of concentrations therebetween. In some embodiments, the concentration of the delivery compound may range from about 1 pM to at least about 2M, or any concentration or range of concentrations therebetween.

[0170] In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or anucleated cell-derived vesicles is about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is any of about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is higher than about 10 mM. In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is any of about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is about 0.1 μM to about 1 mM. In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is about 0.1 μM to about 10 μM. In some embodiments, the concentration of the HPV antigen incubated with anucleated cells or AAC is 1 μM.

[0171] In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is any of about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is greater than about 10 mM. In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is any of about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is about 0.1 μM to about 1 mM. In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is about 0.1 μM to about 10 μM. In some embodiments, the concentration of the antigen incubated with the perturbed input anucleated cells is 1 μM.

[0172] In some embodiments, the molar ratio of antigen to adjuvant incubated with the perturbed input anucleated cells is anywhere from about 10000:1 to about 1:10000. For example, in some embodiments, the molar ratio of antigen to adjuvant incubated with the perturbed input anucleated cells is anywhere from about 10000:1, about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant incubated with the perturbed input anucleated cells is about 10000:1 to about 1000:1, about 1000:1 to about 100:1, about 100:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:100, about 1:100 to about 1:1000, about 1:1000 to about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant incubated with the perturbed input anucleated cells is about 200:1. In some embodiments, the molar ratio of antigen to adjuvant incubated with the perturbed input anucleated cells is about 20:1.

[0173] In some embodiments, the AAC includes an adjuvant at a concentration of about 1 nM to about 1 mM. For example, in some embodiments, the AAC includes an adjuvant at a concentration of about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the AAC includes an adjuvant at a concentration of greater than about 10 mM. In some embodiments, the AAC includes an adjuvant at a concentration of about 1 nM to about 10 nM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the AAC includes an adjuvant at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the AAC includes an adjuvant at a concentration of about 1 μM.

[0174] In some embodiments, the AAC contains an antigen at a concentration of about 1 nM to about 1 mM. For example, in some embodiments, the AAC contains an antigen at a concentration of about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the AAC contains an antigen at a concentration of more than about 10 mM. In some embodiments, the AAC contains an antigen at a concentration of about 1 nM to about 10 nM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the AAC contains an antigen at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the AAC contains an antigen at a concentration of about 1 μM.

[0175] In some embodiments, the molar ratio of antigen to adjuvant in the AAC is anywhere from about 10000:1 to about 1:10000. For example, in some embodiments, the molar ratio of antigen to adjuvant in the modified PBMC is anywhere from about 10000:1, about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant in the AAC is about 10000:1 to about 1000:1, about 1000:1 to about 100:1, about 100:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:100, about 1:100 to about 1:1000, or about 1:1000 to about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant in the AAC is about 200:1. In some embodiments, the molar ratio of antigen to adjuvant in the AAC is about 20:1. Characteristics of AAC and its internalization by antigen-presenting cells

[0176] In an embodiment according to any one of the methods, uses, or compositions described herein, the method includes: a) passing a cell suspension containing input anucleated cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the HPV antigen and adjuvant to pass through, forming a perturbed input anucleated cell; b) incubating the perturbed input anucleated cells with at least one HPV antigen and adjuvant for a time sufficient to allow the at least one HPV antigen and adjuvant to enter the perturbed input anucleated cell, thereby creating an AAC containing at least one HPV antigen and adjuvant. In some embodiments, the AAC containing a payload (such as an HPV antigen and an adjuvant) exhibits different characteristics compared to the input anucleated cells. In some embodiments, AAC containing a payload (such as an HPV antigen and an adjuvant) exhibit distinct characteristics compared to anucleated cells containing a payload introduced by other delivery methods (such as hemolytic loading or electroporation).

[0177] In some embodiments, the half-life of the AAC after administration to a mammal is decreased compared to the half-life of the input anucleated cells after administration to a mammal. In some embodiments, the hemoglobin content of the AAC is decreased compared to the hemoglobin content of the input anucleated cells. In some embodiments, the ATP production of the AAC is decreased compared to the ATP production of the input anucleated cells. In some embodiments, the AAC exhibits a globular morphology. In some embodiments, the AAC is an erythrocyte, and the AAC has a reduced biconcave shape compared to the input anucleated cells. In some embodiments, the AAC is an erythrocyte cell ghost. In some embodiments, the AAC prepared by the process has about 1.5 times more phosphatidylserine on its surface compared to the input anucleated cells. In some embodiments, the population profile of the AAC prepared by the process exhibits a higher average phosphatidylserine level on its surface compared to the input anucleated cells. In some embodiments, at least 50% of the population profile of the AAC prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleated cells. In some embodiments, the AAC exhibits preferential uptake in tissues or cells compared to the input anucleated cells. In some embodiments, the AAC exhibits preferential uptake in phagocytes and / or antigen presenting cells compared to the input anucleated cells. In some embodiments, the AAC is modified to enhance uptake in tissues or cells compared to the input anucleated cells. In some embodiments, the AAC is modified to enhance uptake in phagocytes and / or antigen presenting cells compared to unmodified AAC. In some embodiments, the phagocytes and / or antigen presenting cells comprise one or more of dendritic cells or macrophages. In some embodiments, the tissue or cells comprise one or more of liver or spleen. In some embodiments, the AAC comprises CD47 on its surface.

[0178] In some embodiments of the above methods for creating an AAC, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel includes multiple constrictions. In some embodiments, the multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple micropillars, multiple micropillars arranged in an array, or between one or more movable plates. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the size of the constriction is a function of the diameter of the input anucleated cells in suspension. In some embodiments, the size of the constriction is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the diameter of the input anucleated cells in suspension. In some embodiments, the constriction has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction has a width of about 2.2 μm. In some embodiments, the input anucleated cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi. In some embodiments, the cell suspension is contacted with an antigen prior to, concurrently with, or after passing through the constriction.

[0179] In some embodiments, when an AAC comprising a payload (e.g., an HPV antigen, an HPV antigen and an adjuvant) is prepared from input anucleated cells, the AAC has one or more of the following properties: (a) decreased circulating half-life in a mammal compared to the input anucleated cells, (b) decreased hemoglobin levels compared to the input anucleated cells, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the input anucleated cells, or (e) decreased ATP production compared to the input anucleated cells.

[0180] In some embodiments, the input non-nucleated cells are mammalian cells. In some embodiments, the input non-nucleated cells are human cells. In some embodiments, the input non-nucleated cells are red blood cells or platelets. In some embodiments, the red blood cells are red blood cells or reticulocytes.

[0181] In some embodiments, the circulating half-life of the AAC in a mammal is reduced compared to the input anucleated cells. In some embodiments, the circulating half-life in a mammal is reduced by greater than about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to the input anucleated cells.

[0182] In some embodiments, the input non-nuclear cells are human cells and the circulating half-life of the AAC is less than about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 25 days, about 50 days, about 75 days, about 100 days, or about 120 days.

[0183] In some embodiments, the input non-nucleated cells are red blood cells, and the hemoglobin level in the AAC is reduced compared to the input non-nucleated cells. In some embodiments, the hemoglobin level in the AAC is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 100% compared to the input non-nucleated cells. In some embodiments, the hemoglobin level in the AAC is about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the level of hemoglobin in the input non-nucleated cells.

[0184] In some embodiments, the input non-nucleated cells are red blood cells and the AAC have a spherical morphology. In some embodiments, the input non-nucleated cells are red blood cells and the AAC have a reduced biconcave shape compared to the input non-nucleated cells.

[0185] In some embodiments, the input anucleated cells are red blood cells or red blood cells and the AAC are red blood cell ghosts (RBC ghosts).

[0186] In some embodiments, the AAC comprises CD47 on its surface.

[0187] In some embodiments, the AAC has increased surface phosphatidylserine levels compared to the input anucleated cells. In some embodiments, the AAC prepared by the process has about 1.5 times more phosphatidylserine on its surface compared to the input anucleated cells. In some embodiments, the AAC has about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, about 100%, or more than about 100% more phosphatidylserine on its surface compared to the input anucleated cells. In some embodiments, the level of phosphatidylserine on the surface of the AAC is determined by measuring the level of annexin staining (e.g., annexin V staining) on ​​the surface of the AAC.

[0188] In some embodiments, the AAC has reduced ATP production compared to the input anucleated cells. In some embodiments, the AAC produces ATP at about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the level of ATP produced by the input anucleated cells. In some embodiments, the AAC does not produce ATP.

[0189] In some embodiments, the AAC exhibits enhanced uptake in tissues or cells compared to the input anucleated cells. In some embodiments, the AAC exhibits preferential uptake in the liver or spleen, or by phagocytes or antigen-presenting cells, compared to the uptake of the input anucleated cells.

[0190] In some embodiments, the AAC is further modified to enhance uptake in tissues or cells compared to the input anucleated cells, In some embodiments, the AAC is further modified to enhance uptake in the liver or spleen, or by phagocytes or antigen presenting cells compared to the input anucleated cells.

[0191] In some embodiments, when the AAC shows enhanced uptake in the liver or spleen or by phagocytes and / or antigen-presenting cells, internalization of the AAC results in increased expression of maturation markers of phagocytes or antigen-presenting cells. In some embodiments, the phagocytes and / or antigen-presenting cells are monocyte-derived dendritic cells (MODCs). In some embodiments, the maturation markers are one or more of CD80, CD86, CD83, and MHC-II. In some embodiments, the expression of one or more of CD80, CD86, CD83, and MHC-II is increased by at least about any one of 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, or more in phagocytes and / or antigen-presenting cells contacted with AAC comprising HPV antigens, compared to phagocytes and / or antigen-presenting cells not contacted with AAC comprising HPV antigens. In some embodiments, expression of one or more of CD80, CD86, CD83, and MHC-II is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or more in phagocytes and / or antigen presenting cells contacted with AAC comprising HPV antigens compared to phagocytes and / or antigen presenting cells contacted with input anucleated cells.

[0192] In some embodiments, if the AAC comprising the HPV antigen or the HPV antigen and an adjuvant exhibits enhanced uptake in the liver or spleen or by phagocytes and / or antigen presenting cells, internalization of the AAC results in increased presentation of at least one HPV antigen contained within the AAC. In some embodiments, presentation of at least one HPV antigen is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold or more in phagocytes and / or antigen presenting cells contacted with the AAC comprising the HPV antigen, compared to phagocytes and / or antigen presenting cells contacted with corresponding anucleated cells comprising the same HPV antigen introduced by other delivery methods (such as, but not limited to, hemolytic loading).

[0193] In some embodiments, when the AAC comprising the HPV antigen or HPV antigen and an adjuvant exhibits enhanced uptake in the liver or spleen or by phagocytes and / or antigen-presenting cells, internalization of the AAC results in an increased ability of the phagocytes and / or antigen-presenting cells to induce an antigen-specific immune response. In some embodiments, the antigen-specific immune response mediated by the phagocytes and / or antigen-presenting cells contacted with the AAC comprising at least one HPV antigen and an adjuvant is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold or more compared to the phagocytes and / or antigen-presenting cells contacted with the input anucleated cells. In some embodiments, the antigen-specific immune response mediated by phagocytes and / or antigen-presenting cells contacted with AAC comprising at least one HPV antigen and an adjuvant is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold or more compared to phagocytes and / or antigen-presenting cells contacted with anucleated cells comprising the same HPV antigen introduced by other delivery methods (such as, but not limited to, hemolytic loading). In some embodiments, the antigen-specific immune response is an antigen-specific CD4+ T cell response. In some embodiments, the antigen-specific immune response is an antigen-specific CD8+ T cell response.

[0194] In some embodiments, the individual is HLA-A * 02. HLA-A * 01. HLA-A * 03. HLA-A * 24. HLA-A * 11. HLA-A * 26. HLA-A * 32. HLA-A * 31. HLA-A * 68, HLA-A * 29. HLA-A * 23. HLA-B * 07. HLA-B *44. HLA-B * 08. HLA-B * 35. HLA-B * 15. HLA-B * 40, HLA-B * 27. HLA-B * 18. HLA-B * 51. HLA-B * 14. HLA-B * 13. HLA-B * 57, HLA-B * 38. HLA-C * 07. HLA-C * 04. HLA-C * 03. HLA-C * 06. HLA-C * 05. HLA-C * 12. HLA-C * 02. HLA-C * 01. HLA-C * 08, and / or HLA-C * Tested positive for 16.

[0195] In some embodiments of any one of the methods, compositions, or uses described herein, the phagocyte is HLA-A * 02. HLA-A * 01. HLA-A * 03. HLA-A * 24. HLA-A * 11. HLA-A * 26. HLA-A * 32. HLA-A * 31. HLA-A * 68, HLA-A * 29. HLA-A * 23. HLA-B * 07. HLA-B * 44. HLA-B * 08. HLA-B * 35. HLA-B * 15. HLA-B * 40, HLA-B * 27. HLA-B * 18. HLA-B * 51. HLA-B * 14. HLA-B *13. HLA-B * 57, HLA-B * 38. HLA-C * 07. HLA-C * 04. HLA-C * 03. HLA-C * 06. HLA-C * 05. HLA-C * 12. HLA-C * 02. HLA-C * 01. HLA-C * 08, and / or HLA-C * In some embodiments, the antigen presenting cells are human cells having a haplotype of HLA-A 16. * 02. HLA-A * 11. HLA-B * 07, or HLA-C * 08 haplotype. In some embodiments, the HPV antigens presented to the phagocytes and / or antigen presenting cells described herein are comprised of HLA-A2 specific epitopes. In some embodiments, the HPV antigens presented to the phagocytes and / or antigen presenting cells described herein are comprised of HLA-A11 specific epitopes. In some embodiments, the HPV antigens presented to the phagocytes and / or antigen presenting cells described herein are comprised of HLA-B7 specific epitopes. In some embodiments, the HPV antigens presented to the phagocytes and / or antigen presenting cells described herein are comprised of HLA-C8 specific epitopes.

[0196] In some embodiments, the method includes administering to an individual an AAC comprising at least one HPV antigen and an adjuvant, wherein the AAC is internalized by a phagocyte and / or an antigen-presenting cell. In some embodiments, when the AAC is internalized by a phagocyte and / or an antigen-presenting cell, the internalization of the AAC results in increased expression of a maturation marker of the phagocyte or antigen-presenting cell. In some embodiments, the phagocyte and / or antigen-presenting cell is a monocyte-derived dendritic cell (MODC). In some embodiments, the maturation marker is one or more of CD80, CD86, CD83, and MHC-II. In some embodiments, expression of one or more of CD80, CD86, CD83, and MHC-II is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or more in phagocytes and / or antigen presenting cells contacted with AAC comprising an HPV antigen compared to phagocytes and / or antigen presenting cells not contacted with AAC comprising an HPV antigen. In some embodiments, expression of one or more of CD80, CD86, CD83, and MHC-II is increased by at least any one of about 10%, 20%, 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or more in phagocytes and / or antigen presenting cells contacted with AAC comprising HPV antigens compared to phagocytes and / or antigen presenting cells contacted with input anucleated cells.

[0197] In some embodiments, the input anucleated cells were not (a) heat treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during preparation of the AAC. In some embodiments, osmolarity was maintained during preparation of the AAC from the input anucleated cells. In some embodiments, osmolarity was maintained at about 200 mOsm to about 600 mOsm. In some embodiments, osmolarity was maintained at about 200 mOsm to about 400 mOsm. Systems and Kits

[0198] In some aspects, the present invention provides a system comprising one or more of the constriction, anucleated cell suspension, HPV antigen, or adjuvant for use in the methods disclosed herein. The system can include any of the embodiments described in the methods disclosed above, including a surface having a microfluidic channel or pores, providing a cell deformation constriction, a cell suspension, a cell perturbation, delivery parameters, compounds, and / or application, etc. The system can include any of the embodiments described in the compositions and methods of the subject matter disclosed herein, including those disclosed in the section above entitled "Microfluidic Systems and Components Thereof." In some embodiments, the cell deformation constriction is sized for delivery to anucleated cells. In some embodiments, the delivery parameters, such as the operating flux, cell and compound concentrations, the velocity of cells in the constriction, and the composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.), are optimized for maximum response of the compound to suppress immune response or induce tolerance.

[0199] Also provided are kits or articles of manufacture for use in treating individuals with HPV-associated cancer. In some embodiments, the kits include AACs that include mature antigens in cells and adjuvants in cells. In some embodiments, the kits include one or more of HPV antigens or adjuvants for use in making AACs for use in treating individuals with HPV-associated diseases, such as stenosis, anuclear cell suspensions, and cancer. In some embodiments, the kits include compositions (e.g., microfluidic channels or surfaces containing pores, cell suspensions, and / or compounds) described herein in suitable packaging. Suitable packaging materials are known in the art, and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging materials (e.g., sealed Mylar or plastic bags), and the like. These products can be further sterilized and / or sealed.

[0200] The present invention also provides kits that include components of the methods described herein, and may further include instructions for performing the method of treating an individual with HPV-associated cancer, and / or instructions for introducing HPV antigens and / or adjuvants into anucleated cells. The kits described herein may further include other materials, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein; for example, instructions for treating an individual with HPV-associated cancer, or instructions for creating an AAC that contains HPV antigens in cells and adjuvants in cells. Exemplary embodiments

[0201] Embodiment 1. A method for treating a human papillomavirus (HPV) associated cancer in an individual, comprising administering to the individual a composition comprising an effective amount of an activating antigen carrier (AAC), wherein the effective amount is about 0.5×10 8 AAC / kg ~ approx. 1 x 10 9 AAC / kg, said AAC comprising at least one HPV antigen delivered intracellularly and an adjuvant.

[0202] Embodiment 2. A method for treating a human papillomavirus (HPV) associated cancer in an individual, comprising: administering to the individual a composition comprising an effective amount of an activating antigen carrier (AAC), the AAC comprising at least one HPV antigen to be delivered intracellularly and an adjuvant; and administering to said individual an effective amount of an antagonist of CTLA-4 and / or an antagonist of PD-1 / PD-L1. A method comprising:

[0203] Embodiment 3. The method of embodiment 2, wherein the CTLA4 antagonist is an antibody that binds to CTLA4.

[0204] Embodiment 4 The method of embodiment 2 or 3, wherein the PD-1 / PD-L1 antagonist is an antibody that binds to PD-1 or an antibody that binds to PD-L1.

[0205] Embodiment 5. The method of embodiment 3 or 4, wherein an antibody that binds CTLA-4 and an antibody that binds PD-1 are administered to the individual.

[0206] Embodiment 6. The method of any one of embodiments 3 to 5, wherein the antibody that binds to CTLA-4 is ipilimumab.

[0207] Embodiment 7. The method of any one of embodiments 4 to 6, wherein the antibody that binds to PD-1 is nivolumab.

[0208] Embodiment 8. The method of any one of embodiments 4 to 6, wherein the antibody that binds to PD-1 is pembrolizumab.

[0209] Embodiment 9. The method of any one of embodiments 4-6, wherein an antibody that binds CTLA-4 is administered to the individual and an antibody that binds PD-L1 is administered to the individual.

[0210] Embodiment 10 The method of any one of embodiments 4 and 9, wherein the antibody that binds PD-L1 is atezolizumab.

[0211] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the at least one HPV antigen is an HPV-16 antigen or an HPV-18 antigen.

[0212] Embodiment 12. The method of any one of embodiments 1-11, wherein the at least one HPV antigen comprises a peptide derived from HPV E6 and / or E7.

[0213] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the at least one HPV antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7.

[0214] Embodiment 14. The method of embodiment 13, wherein the HLA-A2 restricted peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 4.

[0215] Embodiment 15. The method of any one of embodiments 1 to 12, wherein the at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18 to 25.

[0216] Embodiment 16. The method of any one of embodiments 1 to 12, wherein the AAC comprises an antigen comprising the amino acid sequence of SEQ ID NO: 19 and an antigen comprising the amino acid sequence of SEQ ID NO: 23.

[0217] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the adjuvant is 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 TLR9 agonist.

[0218] Embodiment 18. The method of embodiment 17, wherein the adjuvant is a CpG 7909 oligodeoxynucleotide (ODN).

[0219] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the individual is a human.

[0220] Embodiment 20. The individual has HLA-A * The method of any one of the preceding claims, wherein the test is positive for 02.

[0221] Embodiment 21. The method of any one of embodiments 1-20, wherein the AAC is autologous or allogeneic to the individual.

[0222] Embodiment 22. The method of any one of embodiments 1-21, wherein the HPV-associated cancer is currently a locally advanced or metastatic cancer.

[0223] Embodiment 23. The method of any one of embodiments 1-22, wherein the HPV-associated cancer is head and neck cancer, cervical cancer, anal cancer, or esophageal cancer.

[0224] Embodiment 24 The method of any one of embodiments 1 to 23, wherein the composition comprising AAC is administered intravenously.

[0225] Embodiment 25. The method of any one of embodiments 2-24, wherein the CTLA-4 antagonist and / or the PD-1 / PD-L1 antagonist is administered intravenously, orally, or subcutaneously.

[0226] Embodiment 26. The method of any one of embodiments 3 to 25, wherein the antibody that binds CTLA-4 and / or the antibody that binds PD-1 and / or the antibody that binds PD-L1 is administered intravenously.

[0227] Embodiment 27. The effective amount of AAC comprising the at least one HPV antigen and the adjuvant is about 0.5×10 8 AAC / kg ~ approx. 7.5 x 10 8 The method according to any one of the preceding claims, wherein the AAC is / kg.

[0228] Embodiment 28. The effective amount of AAC comprising the at least one HPV antigen and the adjuvant is about 0.5×10 8 AAC / kg ~ approx. 1 x 10 9 The method according to any one of the preceding claims, wherein the AAC is / kg.

[0229] Embodiment 29. The effective amount of AAC comprising the at least one HPV antigen and the adjuvant is about 0.5×10 8AAC / kg, approx. 2.5 × 10 8 AAC / kg, approx. 5 × 10 8 AAC / kg, or approximately 7.5 × 10 8 The method according to any one of the preceding claims, wherein the AAC is / kg.

[0230] Embodiment 30. The method of any one of embodiments 6 to 29, wherein the effective amount of ipilimumab is about 1 mg / kg to about 3 mg / kg.

[0231] Embodiment 31. The method of any one of embodiments 7 and 11-30, wherein the effective amount of nivolumab is about 360 mg.

[0232] Embodiment 32 The method of any one of embodiments 10 to 30, wherein the effective amount of atezolizumab is about 1200 mg.

[0233] Embodiment 33 The method of any one of embodiments 1 to 32, wherein the composition comprising the AAC is delivered on day 1 of a three-week cycle.

[0234] Embodiment 34 The method of any one of embodiments 1 to 33, wherein the composition comprising the AAC is further administered on day 2 of an initial 3-week cycle.

[0235] Embodiment 35. Approximately 0.5×10 8 Individual cells / kg ~ approx. 1×10 9 The method of embodiment 33 or 34, wherein cells / kg are administered on day 1 of each 3 week cycle.

[0236] Embodiment 36. Approximately 0.5×10 8 Individual cells / kg, approximately 2.5×10 8 Individual cells / kg, approximately 5.0×10 8 cells / kg, or approximately 7.5 x 10 8 The method of any one of embodiments 33-35, wherein cells / kg are administered on day 1 of each three-week cycle.

[0237] Embodiment 37. Approximately 0.5×10 8 Individual cells / kg ~ approx. 1×10 9 The method of any one of embodiments 33-36, wherein cells / kg are administered on day 2 of each three-week cycle.

[0238] Embodiment 38. Approximately 0.5×10 8 Individual cells / kg, approximately 2.5×10 8 Individual cells / kg, approximately 5.0×10 8 cells / kg, or approximately 7.5 x 10 8 The method of any one of embodiments 33-37, wherein cells / kg are administered on day 2 of the first 3 week cycle.

[0239] Embodiment 39. The method of any one of embodiments 33-38, wherein the antibody that binds CTLA-4 and / or the antibody that binds PD-1 and / or the antibody that binds PD-L1 is administered once per three week cycle.

[0240] Embodiment 40 The method of any one of embodiments 33-39, wherein the antibody that binds CTLA-4 is administered once per two three-week cycles.

[0241] Embodiment 41 The method of any one of embodiments 33-40, wherein the antibody that binds CTLA-4 is administered on day 1 of each three-week cycle.

[0242] Embodiment 42. The method of any one of embodiments 39-41, wherein the antibody that binds CTLA-4 is ipilimumab, and the ipilimumab is administered at a dose of about 3 mg / kg.

[0243] Embodiment 43 The method of any one of embodiments 39-42, wherein the antibody that binds PD-1 is administered on day 8 of the first 3 week cycle and on day 1 of each subsequent cycle.

[0244] Embodiment 44. The method of embodiment 43, wherein the antibody that binds PD-1 is nivolumab, and the nivolumab is administered at a dose of about 360 mg.

[0245] Embodiment 45. The method of any one of embodiments 39-44, wherein the antibody that binds CTLA-4 is ipilimumab, and the ipilimumab is administered at a dose of about 1 mg / kg on day 1 of the first 3 week cycle of two 3 week cycles, and the antibody that binds PD-1 is administered at a dose of about 360 mg on day 8 of the first 3 week cycle and on day 1 of each subsequent cycle.

[0246] Embodiment 46 The method of any one of embodiments 33-39, wherein the antibody that binds PD-L1 is administered on day 8 of the first 3 week cycle and on day 1 of each subsequent cycle.

[0247] Embodiment 47. The method of embodiment 46, wherein the antibody that binds PD-L1 is atezolizumab, and the atezolizumab is administered at a dose of about 1200 mg.

[0248] Embodiment 48 The method of any one of embodiments 1 to 47, wherein the composition comprising PBMCs is administered to the individual for at least about 3 months, 6 months, 9 months, or 1 year.

[0249] Embodiment 49. The composition comprising AAC comprises about 1×10 9 AAC ~ approx. 1×10 10 The method of any one of embodiments 1 to 48, comprising AAC.

[0250] Embodiment 50. The composition comprising AAC comprises about 7×10 9 The method of any one of embodiments 1 to 49, comprising PBMCs.

[0251] Embodiment 51 The method of embodiment 49 or 50, wherein the cryopreservation medium is Cryostor® CS2.

[0252] Embodiment 52. The AAC comprising at least one HPV antigen and an adjuvant comprises: a) passing a cell suspension including a population of input anucleated material through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells sufficiently large to allow the at least one HPV antigen and the adjuvant to pass therethrough to form perturbed input anucleated cells; and b) incubating said population of perturbed input anucleated cells with said at least one HPV antigen and said adjuvant for a time sufficient to allow said antigen to enter said perturbed input anucleated cells, thereby generating said AAC comprising said at least one HPV antigen and said adjuvant. 52. The method of any one of the preceding embodiments, wherein the compound is prepared by a process comprising:

[0253] Embodiment 53. The method of embodiment 52, wherein the diameter of the stenosis is from about 1.6 μm to about 2.4 μm or from about 1.8 μm to about 2.2 μm.

[0254] Embodiment 54 The method of embodiment 52 or 53, wherein the input anucleated cells are red blood cells.

[0255] Embodiment 55. The method of any one of embodiments 52-54, wherein the at least one HPV antigen comprises a peptide derived from HPV E6 and a peptide derived from HPV E7.

[0256] Embodiment 56. The method of any one of embodiments 52-55, wherein the at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0257] Embodiment 57. The method of any one of embodiments 52-55, wherein the at least one HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0258] Embodiment 58. The method of any one of embodiments 52 to 55, wherein the AAC comprises an antigen comprising the amino acid sequence of SEQ ID NO: 19 and an antigen comprising the amino acid sequence of SEQ ID NO: 23.

[0259] Embodiment 59. The method of any one of embodiments 52 to 58, wherein the adjuvant is 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 TLR9 agonist.

[0260] Embodiment 60. The method of embodiment 59, wherein the adjuvant is a CpG 7909 oligodeoxynucleotide (ODN). EXAMPLES

[0261] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. Example 1 A Phase I Study of the Safety and Tolerability of SQZ-AAC-HPV

[0262] HLA A with recurrent, locally advanced or metastatic HPV16+ solid tumors * Conduct a Phase 1, open-label, multicenter study of the safety and tolerability, antitumor activity, and immunogenic and pharmacodynamic effects of SQZ-AAC-HPV as monotherapy and in combination with (1) ipilimumab, (2) nivolumab, and (3) nivolumab and ipilimumab in 02+ patients.

[0263] SQZ-AAC-HPV is HLA-A serogroup positive (HLA-A * SQZ-AAC-HPV is a red blood cell (RBC)-derived product of activating antigen carrier (AAC) used as a treatment for human papillomavirus (HPV) strain 16-positive (HPV16+) cancers in human leukocyte antigen (HLA) serotype 02+ (HPV16+) patients. * It consists of autologous RBCs treated with 02-restricted E6 and E7 epitopes and the adjuvant polyinosinic-polycytidylic acid (poly I:C), which is delivered to the cytosol during manufacture. E6 SLP: QLCTELQTTIHDIILECVYCKQQLL (SEQ ID NO: 19) E7 SLP: QLCTELQTYMLDLQPETTYCKQQLL (SEQ ID NO: 23)

[0264] The process begins with a particular patient at the clinical site where whole blood is collected and then transported to a manufacturing site. At the manufacturing site, platelets and white blood cells are removed and E6 and E7 epitopes along with poly I:C adjuvant are delivered to the cells using Cell Squeeze technology. The Cell Squeeze technology results in increased phosphatidylserine on the surface of the AAC compared to the starting RBCs. The resulting cells are the AAC-HPV bulk. The AAC-HPV bulk is washed with cryopreservation medium and then formulated into SQZ-AAC-HPV homemade agent and cryopreserved. The SQZ-PBMC-HPV bulk contains the HPV16 HLA-A antigen delivered to the cytosol during the manufacturing process. * 02 consists of autologous PBMCs with synthetic long peptides (SLPs) containing restricted E6 and E7 epitopes. overview

[0265] The study population consisted of HLA-A resistant pediatric patients with advanced stage HPV16+ solid tumors (head and neck cancer, cervical cancer, and other tumor types). * Patients who are HLA A 02+ *HPV 02+ status and HPV16+ tumor status must be confirmed by clinical laboratory reports and all eligibility criteria must be met prior to patient blood collection for the manufacture of autologous blood products. Patients with locally confirmed HPV16+ status may have central confirmation from a fresh tumor biopsy taken at screening if documentation of clinical laboratory evaluation is deemed insufficient by the sponsor.

[0266] Eligible patients will undergo a single blood draw at the study site for the manufacture of the home-grown agent. At least 200 mL of whole blood will be taken for this purpose. This blood draw will be sent to a contract manufacturer for the manufacture of each patient's personalized autologous cell therapy. Frozen vials of SQZ AAC HPV will then be sent to the study site for administration.

[0267] The study will be conducted in two parts, part 1 will consist of dose escalation to determine the safety profile, preliminary efficacy, and RP2D of SQZ-AAC-HPV monotherapy. Part 2 of the study will evaluate the safety and preliminary efficacy of SQZ-AAC-HPV when combined with immune checkpoint inhibitors in a combination safety phase.

[0268] In all cohorts, SQZ AAC-HPV will be administered every 3 weeks for up to one year or until the SQZ-AAC-HPV supply is exhausted or treatment discontinuation criteria are met, whichever occurs first.

[0269] All patients in Part 1 and Part 2 will be observed for at least 4 hours after each dose of SQZ AAC HPV. In addition, the first two patients in each cohort will be observed for a minimum of 23 hours after their first dose of SQZ AAC-HPV.

[0270] Tumor assessments will be performed throughout the study by RECIST 1.1 and iRECIST until disease progression, unacceptable toxicity, withdrawal of consent, death, or 2 years from the date of first dose of SQZ AAC HPV, whichever occurs first. Patients who experience disease progression by RECIST 1.1 may continue on medication if considered in their best interest by the treating investigator to allow confirmation of disease progression: iCPD by iRECIST (Seymour et al., 2017).

[0271] After the final dose of investigational product, there will be follow-up visits to monitor safety and tolerability, as well as to assess overall survival. Part 1: Escalation Phase (SQZ-AAC-HPV Monotherapy)

[0272] The planned dose cohorts for the escalation phase are shown in Table 1. The traditional 3+3 design is intended to assess safety and tolerability, but it may be prudent to treat up to a total of 12 additional patients in the cohort to further examine safety and tolerability, immunogenicity effects, and antitumor activity. In this modified 3+3 design, there are a maximum of 12 patients per cohort. [Table 1] a. SQZ-AAC-HPV dosing will continue every 3 weeks until treatment discontinuation criteria are met, the SQZ-AAC-HPV supply is exhausted, or for a maximum of 1 year, whichever occurs first. b. In Cycle 1, patients will receive SQZ-AAC-HPV on days 1 and 2. c. If no safety signals are observed, the higher dose level will be 5 x 10 8 If grade 2 non-PD-related toxicity is observed in 1 of 3 patients or 2 of 6 patients during the DLT period, the high dose level is 2.5×10 8 AAC / kg. d.2.5×10 8If a high dose of AAC / kg is chosen, 2.5 × 10 8 Once the DLT evaluation of AAC / kg is completed, 8 A third cohort with AAC / kg may be released.

[0273] At least two monotherapy dose levels will be tested. The low dose of SQZ-AAC-HPV is 0.5 × 10 8 AAC / kg. To ensure patients in cohort 2 are exposed to the highest immunogenic cell dose possible, the high dose level of SQZ-AAC-HPV is based on safety findings in cohort 1. If no safety signal is observed (i.e., no treatment-related toxicity of ≥ grade 2), the high dose level will be 5×10 8 If grade 2 for non-PD-related toxicity (or DLT) is observed in 1 of 3 patients or 2 of 6 patients during the DLT period, the high dose level is 2.5×10 8 AAC / kg. 2.5×10 8 If a high dose of AAC / kg is chosen, 2.5 × 10 8 Once the DLT evaluation of AAC / kg is completed, 8 A third cohort with AAC / kg may be released. After review of available safety, efficacy, and pharmacodynamic data from patients in a given cohort, the SSC will determine whether investigation of additional higher or lower single or double antigen loading dose levels is warranted. In this case, the magnitude of dose escalation or de-escalation will be determined by the SSC based on the type and severity of TEAEs observed.

[0274] Patients will receive SQZ-AAC-HPV on days 1 and 2 of cycle 1 and on day 1 of each subsequent 21-day cycle. In part 1, the DLT observation period is 28 days (Figure 1).

[0275] Patients will be enrolled in a coordinated manner across study sites, meaning that no more than one patient in a cohort will receive their first dose of SQZ AAC-HPV within one week. Dosing of SQZ AAC-HPV in subsequent cohorts will not begin until the SSC has reviewed the available safety data and determined that dose escalation is warranted. Dose escalation and RP2D determination

[0276] Although the traditional 3+3 design is intended to assess safety and tolerability, it may be prudent to treat up to 6 additional patients in a cohort to further examine safety and tolerability, immunogenicity effects, and antitumor activity. In this modified 3+3 design, there are a maximum of 12 patients per cohort.

[0277] Dose escalation, or an increase in cohort size from 6 to 12 patients, will be considered after the first 3 patients at a given dose level have completed the DLT observation period and are found to be evaluable for safety upon review of safety data conducted by the SSC. The DLT observation period is defined as 28 days for Part 1.

[0278] If no DLT is observed in any of the first three enrolled patients at a given dose level over the DLT observation period, then a cohort at a higher dose level may be opened for enrollment. If one of the first three patients experiences a DLT, three additional patients are enrolled (for a total of six evaluable patients at the same dose level). If ≧1 of the first three patients or ≧2 of the first six patients experience a DLT, no further dose escalation is considered, and this becomes the maximum administered dose (MAD). The RP2D may be a lower dose level previously evaluated, or an alternative intermediate dose level may be selected for further evaluation. The RP2D determination is made by the SSC based on safety data from at least six patients. The RP2D is further evaluated in part 2 (combination safety phase) of the study. Alternatively, the RP2D may be declared based on a pharmacodynamic evaluation that determines that the maximum biological effect has been achieved and that the patient would not benefit from further dose escalation.

[0279] Patients are considered inevaluable if they are unable to complete the DLT observation period for any reason other than safety or if the pharmacodynamic assessments are insufficient to define the biological effect of the study treatment. Part 1 patients who are considered inevaluable may be replaced following discussion between the investigator and sponsor.

[0280] Adverse events occurring after any administered dose will resolve to ≤ Grade 2 at the time of the subsequent dose. Similarly, adverse events of special interest (AESI) occurring after any administered dose will resolve to < Grade 2 at the time of the subsequent dose. If these retreatment criteria are met after the first dose of cycle 1, the second SQZ AAC-HPV dose will be given during the ≥ 23 hour observation period (i.e., 16-24 hours after the first dose). Patients will be observed for a minimum of 4 hours after the administration of the second antigen load. The minimum interval between the two doses is 16 hours.

[0281] Patients will be monitored for the emergence of DLTs for 28 days after the first dose of SQZ AAC HPV in the monotherapy cohort. According to the modified 3+3 rule, the minimum number of patients required to confirm safety and a cohort with respect to DLTs is 0 DLTs in 3 patients, ≤1 DLT in 6 patients, ≤2 DLTs in 9 patients, or ≤3 DLTs in 12 patients.

[0282] DLT evaluation in all cohorts must be completed for the decision on the monotherapy RP2D regimen. The RP2D regimen will be selected based on review of all available safety, tolerability, immunogenicity, and other pharmacodynamic and antitumor data. The SSC will review the data and make a recommendation to the DSMB, which will be responsible for approval of the RP2D.

[0283] Once the RP2D regimen is defined, part 2 (combination safety phase) may begin. Part 2: Combination safety phase (SQZ-AAC-HPV + checkpoint inhibitors)

[0284] The SQZ-AAC-HPV dose to be evaluated during the combination safety study will be selected based on a review of all available safety, tolerability, immunogenicity, and other pharmacodynamic and antitumor data. The DSMB will decide whether to select SQZ-AAC-HPV monotherapy RP2D for the combination safety phase or to start at a lower dose.

[0285] Cohorts are defined by SQZ AAC HPV RP2D and combination partner. SQZ AAC-HPV is administered at the RP2D in cohorts 2a, 2b, and 2c. Cohort 2a: SQZ-AAC-HPV (RP2D) plus ipilimumab (3 mg / kg every 3 weeks for up to 4 doses if tolerated) Cohort 2b: SQZ-AAC-HPV (RP2D) and nivolumab (360 mg every 3 weeks) Cohort 2c (subject to safety assessment of 6 patients treated in cohorts 2a and 2b, respectively): SQZ-AAC-HPV (RP2D) plus nivolumab (360 mg every 3 weeks) and ipilimumab (1 mg / kg every 6 weeks)

[0286] Enrollment in part 2 will begin with cohorts 2a and 2b. Cohorts 2a and 2b will each have six patients enrolled and successfully complete the 42-day DLT evaluation period; i.e., when <33% of patients experience a DLT, cohort 2c will open for enrollment. Based on available safety data from both cohorts, the SSC will decide whether to select the SQZ-AAC-HPV dose regimen selected for cohorts 2a and 2b for cohort 2c, or to start with a lower dose regimen. If the SSC recommends starting cohort 2c with a lower dose of SQZ-AAC-HPV, six patients will be enrolled initially, with at least four patients observed for 42 days. If the SSC deems the combination safe with <33% of patients experiencing DLT, the dose of SQZ-AAC-HPV may be escalated to the full monotherapy RP2D and enrollment may continue until a maximum of 12 patients are enrolled, if warranted.

[0287] Patients in the combination safety cohort in part 2 will receive SQZ-AAC-HPV on days 1 and 2 of cycle 1 and on day 1 of each subsequent 21-day cycle. After the first two patients in each cohort have completed day 8 of cycle 1, additional patients in the cohort may be treated in that cohort.

[0288] All patients will be evaluated for safety and tolerability, as well as preliminary evidence of anti-tumor response.

[0289] Cohort 2a - SQZ-AAC-HPV and ipilimumab

[0290] In cycle 1, SQZ-AAC-HPV is administered IV according to the RP2D determined in part 1, i.e., either as a double antigen loading dose on days 1 and 2, or as a single antigen loading dose on day 1. Ipilimumab 3 mg / kg is administered IV over 90 minutes prior to SQZ AAC HPV on day 1. In cycles 2, 3, and 4, ipilimumab is given on day 1 after administration of SQZ-AAC-HPV. Ipilimumab is administered for up to 4 cycles. SQZ-AAC-HPV is given in 3-week cycles until discontinuation criteria are met, SQZ-AAC-HPV supply is exhausted, or for up to 1 year, whichever occurs first (Figure 2).

[0291] Cohort 2b – SQZ-AAC-HPV and nivolumab

[0292] In cycle 1, SQZ-AAC-HPV is administered IV according to the RP2D determined in part 1, i.e., either as a double antigen loading dose on days 1 and 2, or as a single antigen loading dose on day 1. On day 8 of cycle 1, nivolumab is administered IV at a dose of 360 mg over 30 minutes immediately following the end of the SQZ-AAC-HPV infusion. In subsequent cycles, SQZ AAC-HPV followed by nivolumab is administered on day 1 every 3 weeks. Nivolumab may be given every 3 weeks for up to 2 years or until discontinuation criteria are met. SQZ-AAC-HPV is administered in 3-week cycles until discontinuation criteria are met, SQZ-AAC-HPV supply is exhausted, or for up to 1 year, whichever occurs first (Figure 3).

[0293] Cohort 2c - SQZ-AAC-HPV and nivolumab and ipilimumab

[0294] In cycle 1, SQZ-AAC-HPV is administered IV according to the RP2D based on findings in cohorts 2a and 2b. Of note, the SSC may decide to advise a procedure at a dose below that selected for cohorts 2a and 2b, or with a modified dose regimen (e.g., as a single antigen loading dose on day 1 only). Ipilimumab is administered IV at a dose of 1 mg / kg over 30 minutes on day 1 prior to SQZ-AAC-HPV. On day 8 of cycle 1, nivolumab 360 mg IV is administered over 30 minutes. Nivolumab is given on day 1 of the subsequent 3-week cycle after administration of SQZ-AAC-HPV. Ipilimumab is administered every 6 weeks after administration of SQZ-AAC-HPV and nivolumab in subsequent cycles (Figure 4). Nivolumab and ipilimumab may be given for 2 years from day 1 of cycle 1 until one of the criteria for discontinuation of treatment is met. SQZ-AAC-HPV will be administered in 3-week cycles until a discontinuation criterion is met, the SQZ-AAC-HPV supply is exhausted, or for up to 1 year, whichever occurs first.

[0295] If a patient meets the criteria for discontinuation of checkpoint inhibitors due to an immune-mediated AE (per Appendix E) and the investigator is unable to determine whether the event is related to nivolumab or ipilimumab, the patient may discontinue both drugs and continue SQZ-AAC-HPV.

[0296] For all cohorts in Part 2, the second SQZ-AAC-HPV dose on day 2 of cycle 1 will be given during a ≥23 hour observation period. Adverse events occurring after any administered dose will resolve to ≤ grade 2 at the time of the subsequent dose. Similarly, AESIs occurring after any administered dose will resolve to < grade 2 at the time of the subsequent dose. If these retreatment criteria are met, the second SQZ AAC HPV dose will be given during a ≥23 hour observation period (i.e., 16-24 hours after the first dose). Patients will be observed for a minimum of 4 hours after administration of the second antigen load. The minimum interval between the two doses is 16 hours. After the first two patients in each cohort have completed day 14 of cycle 1, additional patients in the cohort will be treated.

[0297] Patients will be monitored for the emergence of DLTs for 42 days after the first dose of SQZ AAC-HPV in the combination therapy cohort.

[0298] In the event of DLT or other significant toxicity in an individual patient, a de-escalation to a lower SQZ AAC-HPV dose will be performed. After review of available safety, efficacy, and pharmacodynamic data from patients in the individual combination safety cohorts, the SSC may determine that dual antigen loading is not advisable for one or more dose combinations. In this case, the SSC may recommend lowering the SQZ-AAC-HPV dose for the second dose (day 2 of cycle 1). Alternatively, the SSC may determine that a lower dose level for SQZ-AAC-HPV may be investigated (dose de-escalation). For example, if DLT is observed in ≥ 33% ​​of patients in the individual combination safety cohorts, a cohort evaluating a lower SQZ-AAC-HPV level will be released and investigated. The higher dose cohorts will be designated 2c, 2d, etc., and the lower dose cohorts will be designated 2a-1, 2b-1, etc. Dosing schedule and study duration

[0299] All patients will undergo a single blood draw to be used for the manufacture of autologous blood products prior to the start of treatment. Patients will undergo this blood draw at the study site, which typically occurs approximately 1-2 weeks prior to the first dose of SQZ AAC HPV (Day 1 of Cycle 1). The schedule for the first dose of SQZ AAC HPV will take into account the location of the site and transportation logistics.

[0300] A cycle is defined as a 21 day treatment period.

[0301] Patients will receive SQZ AAC HPV every 3 weeks for up to 1 year until the investigational product is depleted or treatment discontinuation criteria are met, whichever occurs first.

[0302] Accumulating clinical evidence indicates that some subjects treated with immune system stimulants may reveal signs of disease progression (by traditional response criteria) before demonstrating clinical objective responses and / or stable disease. Two hypotheses have been put forward to explain this phenomenon. First, enhanced inflammation within the tumor leads to an increase in tumor size, which manifests as enlarged index lesions and as new visible small non-index lesions. Over time, both the noxious and inflammatory nature of the mass then diminishes, leading to overt signs of clinical improvement (Wolchok et al, 2009). Alternatively, in some individuals, the dynamics of tumor growth may initially outweigh antitumor immune activity. With sufficient time, antitumor activity dominates and becomes clinically evident. Therefore, it is important to evaluate RECIST 1.1 and iRECIST in parallel at each time point.

[0303] Patients may continue on study therapy after initial RECIST 1.1-defined progression, thus allowing for confirmation of disease progression according to iRECIST (Seymour et al., 2017) if the following criteria are met: 1. Investigator-assessed clinical benefit and lack of rapid disease progression 2. Tolerability of the investigational drug as defined by the investigator 3. Stable general condition 4. Treatment beyond progression does not delay urgent intervention to prevent serious outcomes from rapidly progressive disease 5. Absence of complications of disease progression (e.g., CNS metastases)

[0304] Assessment of clinical benefit takes into account whether a patient is clinically deteriorating and is unlikely to receive further benefit from continued treatment. Dose-limiting toxicity

[0305] A patient is considered evaluable for DLT assessment if: 1) they experience a DLT during the DLT assessment period, regardless of the cell dose received; or 2) they do not experience a DLT during the DLT assessment period after receiving at least 70% of the intended dose of SQZ-AAC-HPV during the DLT assessment period. Patients who do not experience a DLT during the DLT assessment period and have not yet received less than 70% of the intended SQZ-AAC-HPV dose are not considered evaluable for DLT and are replaced.

[0306] Patients experiencing a DLT that is not an IRR will be discontinued from the study. If, in the opinion of the investigator and sponsor, it is in the patient's best interest to continue treatment on the investigational product, subsequent treatment will be determined by the investigator in consultation with the sponsor. Because of an IRR, pre-dosing or dosing rate may be adjusted to allow the patient to remain in the study.

[0307] DLTs were defined as any AE or clinically significant abnormal laboratory value assessed by the principal investigator, occurring within either the first 28 days of treatment with monotherapy or within the first 42 days of treatment with combination therapy, related to SQZ-AAC-HPV (either alone or in combination) but unrelated to disease, disease progression, intercurrent illness, concomitant medications / procedures, or environmental factors, and confirmed by the SSC, that met any of the predefined criteria listed below using the National Cancer Institute's (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. CRS and neurotoxicity were graded using the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading, see Sections 6.1.4 and 6.1.5, respectively. Non-hematological toxicity Grade 4 or Grade 5. Grade 3 toxicity, ≤ Grade 1 or not resolving to baseline within 7 days despite optimal supportive care, except for Grade 3 CRS or neurotoxicity not resolving to ≤ Grade 2 within 24 hours. Grade 3 laboratory values ​​lasting >7 days and requiring medical intervention. Hepatotoxicity >Grade 3 lasting >48 hours, except for the following: For patients with baseline Grade 2 aspartate aminotransferase (AST), alanine aminotransferase (ALT), and / or alkaline phosphatase abnormalities, only an increase to >8× upper limit of normal (ULN) lasting >48 hours is considered a DLT. Abnormal liver tests meeting Hy's law criteria. hematological toxicity Any grade 5 toxicity. Any grade 4 anemia. Any ≥ grade 3 febrile neutropenia. ≥ Grade 4 neutropenia (absolute neutrophil count <500 / μL) lasting >7 days. ≥ Grade 4 thrombocytopenia (<25,000 / μL). ≥ Grade 3 thrombocytopenia (<50,000 / μL) lasting >7 days associated with clinically significant bleeding. TEAEs at least possibly related to SQZ-AAC-HPV (either alone or in combination) resulting in permanent interruption or >14-day delay of scheduled SQZ AAC HPV administration on Day 1 of Cycle 2. Any other event that, in the discretion of the investigator and sponsor, is considered to be a DLT.

[0308] The following events are not considered DLTs: ≥ grade 3 amylase level or isolated grade 3 lipase level not associated with clinical or radiographic evidence of pancreatitis. Grade 3 CRS improving to ≤ grade 2 within 24 hours with or without symptomatic treatment. Grade 3 skin rash that resolves to ≤ Grade 2 within 7 days with or without appropriate supportive care. An immediate allergic reaction occurring within 2 hours of cell product administration that reverts to ≦Grade 2 in 24 hours. Grade 1 or grade 2 electrolyte abnormalities corrected within 72 hours without clinical sequelae. Alopecia.

[0309] Grade 3 IRRs that can be adequately managed with the addition of premedication or modification of infusion rate are not considered DLTs unless these changes are deemed applicable to all subsequent patients enrolled in the study based on SSC recommendations. If the modifications are applicable to all subsequent patients, the cohort will reopen for DLT assessment. Patients who experience a grade 3 infusion reaction may continue in the study with modifications to their premedication or infusion rate.

[0310] If the maximum tolerated dose (MTD) is not reached in any cohort, additional cell dose levels or regimens may be tested. In the event of an AE not related to SQZ-AAC-HPV but encompassed by the definition of a DLT, the finding will be discussed by the SSC. Stopping criteria for cohorts, and cessation of dose escalation or progression into cohorts, and termination of the study

[0311] The modified 3+3 rule defines the final decision to declare a cohort safe. The minimum number of patients required to confirm a cohort as safe is 3 patients with 0 DLT, which can be increased up to 12 patients to confirm the cohort as safe (i.e., <33% of patients with DLT; e.g., 6 patients with <2 DLT, 9 patients with <3 DLT, or 12 patients with <4 DLT to confirm the safety of the cohort). If the cohort does not demonstrate that the MTD has been reached, additional cell dose levels or regimens may be tested. In the event of an AE not related to SQZ-AAC-HPV but encompassed by the definition of DLT, the findings will be discussed by the SSC.

[0312] AEs that meet the definition of a DLT and occur outside the DLT window will not be counted as DLTs, but will instead be considered in the overall safety assessment of a given cohort and in the selection of the RP2D regimen.

[0313] The stopping rule for a cohort is the occurrence of ≧3 DLTs (≧33%) in up to 12 patients receiving the investigational product within the same dose cohort. When a stopping rule is triggered, the SSC may make one of the following recommendations: The previous tolerated dose level is declared the MTD. The maximum administered dose (MAD) level without observed DLT will be declared as the dose level. Therefore, the RP2D is not the MTD. Studies at intermediate dose levels are recommended. We recommend protocol modifications to increase patient safety. Discontinue enrollment and / or study.

[0314] Following review by the SSC, a patient's medication may be suspended in the interest of patient safety based on these general safety criteria: Any serious adverse event (SAE) that is deemed to be potentially life-threatening and is assessed by the medical monitor as related to the investigational product. Any other clinically significant change that indicates a major tolerability concern to the investigator or sponsor. Study population

[0315] The study population consisted of HLA-A resistant pediatric patients with advanced stage HPV16+ solid tumors (head and neck cancer, cervical cancer, and other tumor types). * Includes patients who are 02+.

[0316] Patients may have received prior therapy with a PD-1, CTLA-4 inhibitor, or other immune checkpoint inhibitor.

[0317] Number of patients

[0318] The number of patients will depend on safety and immunogenic effects observed. It is expected that approximately 9-36 DLT-evaluable patients may be enrolled in monotherapy part 1 (escalation phase). If the planned cohorts do not demonstrate that the MTD has been reached, additional cell dose levels or regimens may be tested. A maximum of approximately 36 total evaluable patients may be enrolled in part 2 (n=12 per cohort). Depending on the need to rotate patients within cohorts, it is expected that approximately 72 evaluable patients will be treated in the study. Inclusion criteria 1. HLA-A confirmed by genotyping assay from blood * Male or female patients aged ≥ 18 years who are 02+. 2. Histologically confirmed incurable or metastatic solid tumors (including but not limited to cervical and head and neck tumors) that are HPV16+. 3. For cervical cancer not amenable to curative treatment with surgery, radiation therapy, and / or radiochemotherapy, the cancer must have progressed after prior systemic chemotherapy treatment with a platinum-based regimen in the adjuvant or recurrent setting. Patients must have had progressive disease during or after completion of their most recent prior treatment. For patients who cannot tolerate or refuse platinum-based systemic chemotherapy treatment for recurrent disease, the reasons must be documented. 4. For recurrent and metastatic head and neck cancer not amenable to curative treatment with surgery, radiation therapy, and / or radiochemotherapy, the cancer must have progressed after at least one prior platinum-based chemotherapy, primarily in the adjuvant or recurrent setting, and have been offered checkpoint immunotherapy. Patients who relapse after platinum-containing definitive chemoradiotherapy or after adjuvant chemoradiotherapy are eligible if platinum rechallenge is not seen to be beneficial at the time of relapse. For patients who cannot tolerate or refuse platinum-based systemic chemotherapy treatment for recurrent disease, the reasons must be documented. 5. Patients with incurable or metastatic HPV16+ cancer other than cervical or head and neck cancer must have progressed after at least one available standard therapy for incurable disease OR patients cannot tolerate or refuse standard therapy or have a tumor for which no standard therapy exists. Titration Phase (Part 1) and Combination Safety Phase (Part 2) 6. Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-1. 7. Patients must consent to venous access for blood collection for the manufacture of autologous blood products and, if venous access is an issue, must be receptive to insertion of a central line. 8. Patients with unresectable or metastatic solid tumors must have disease amenable to biopsy at acceptable clinical risk and must consent to a new biopsy at Screening and Day 8 (± 2 days) of Cycle 2. a. Lesions in previously irradiated areas may be biopsied so long as there is objective evidence of disease progression prior to study enrollment. 9. At least one measurable lesion per RECIST 1.1. a. Lesions in previously irradiated areas are eligible to be considered as measurable disease if there is objective evidence of disease progression prior to study enrollment. 10. Adequate organ function and bone marrow reserve as demonstrated by the following laboratory assessments performed within 14 days prior to blood collection for the manufacture of autologous blood products: Bone marrow function: absolute neutrophil count ≥ 1000 / μL; hemoglobin ≥ 9 g / dL; platelet count ≥ 75,000 / μL. Note: Transfusions may be available in stable patients with hemoglobin levels < 9 g / dL to meet inclusion criteria. b. Liver function: total serum bilirubin, ≤ 1.5 × ULN; serum AST / ALT, ≤ 2.5 × ULN (≤ 5 × ULN in the presence of liver metastases); alkaline phosphatase < 2.5 × ULN except in patients with liver and bone involvement: alkaline phosphatase ≤ 5 × ULN. i. Patients with inherited disorders of bilirubin metabolism should be discussed with the sponsor. c. Renal function: serum creatinine ≤ 2.5 x ULN or creatinine clearance ≥ 30 mL / min based on either urine collection or Cockcroft-Gault estimate. d. Coagulation profile: Prothrombin time (PT), International Normalized Ratio (INR) / Partial Thromboplastin Time (PTT) ≦1.5×ULN. Patients on a stable maintenance regimen of anticoagulant therapy for at least 30 days prior to blood collection for the manufacture of autologous blood products may have a PT / INR measurement >1.5×ULN if in the opinion of the investigator the patient is suitable for the study. Appropriate justification must be provided to the sponsor prior to enrollment. 11. Patients with immune-mediated endocrine abnormalities following treatment with immune checkpoint inhibitors requiring hormone replacement therapy are eligible. a. Patients who require prednisone as part of their hormone replacement therapy are eligible if the daily dose does not exceed 10 mg. 12. 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 time of informed consent until at least 5 months after the final dose of immune checkpoint inhibitor or SQZ-AAC-HPV (CTFG, 2020). Examples of highly effective contraceptive methods include: Combined hormonal contraceptives (containing both estrogen and progesterone) that are associated with the inhibition of ovulation; can be oral, intravaginal or transdermal Intrauterine device (IUD) Intrauterine hormone releasing system (IUS) Bilateral tubal obstruction Partner who has had a vasectomy ·abstinence 13. Non-vasectomized male patients must accept condom use from the time of informed consent until at least 5 months after the last dose of immune checkpoint inhibitor or SQZ-AAC-HPV. 14. Patient understands and is able to follow the protocol and has signed the required informed consent form (ICF). The appropriate ICF must be signed before any relevant study procedures are performed. If applicable, the female partner of the male patient understands and signs the pregnant partner's ICF. Exclusion criteria 1. Treatment with anti-cancer therapy, including investigational therapy, within 2 weeks prior to blood collection for autologous blood product manufacturing. For prior therapies with half-lives longer than 3 days, the timing of therapy interruption must be discussed with the sponsor. 2. Patients with > Grade 1 adverse event (AE) (excluding Grade 2 anemia) according to NCI CTCAE version 5.0 related to previous treatment with anticancer or investigational therapy that has not resolved (i.e., ≤ Grade 1 or above) at least 2 weeks prior to blood collection for autologous blood product manufacturing. 3. History of any grade 4 irAE from prior immunotherapy (patients with endocrine abnormalities or asymptomatic elevations of serum amylase or lipase controlled with replacement therapy are eligible), any irAE resulting in permanent discontinuation of prior immunotherapy, or any grade 3 irAE occurring ≤6 months prior to blood collection for the manufacture of autologous blood products. 4. Patients treated with non-corticosteroid immunosuppressants within the last 6 months may not be eligible and must be discussed with the sponsor. 5. Patients with active, known, or suspected autoimmune disease may not be eligible and must be discussed with the sponsor. 6.Patients who have undergone splenectomy. 7. Patients who have received or are expected to require a blood transfusion within 4 weeks prior to blood collection for in-house investigational product manufacturing. Note: Patients may receive transfusions following blood collection as clinically indicated. 8. Patients with prior allogeneic bone marrow or solid organ transplants may not be eligible and must be discussed with the sponsor. 9. Receipt of live virus vaccination within 4 weeks prior to blood collection for the manufacture of autologous blood products. 10. Systemic treatment with either corticosteroids (>10 mg prednisone or equivalent per day) or other immunosuppressants within 14 days prior to blood collection for the manufacture of autologous blood products. Note: Inhaled, intranasal, intra-articular and topical (including ophthalmic) steroids are permitted. The use of steroid replacement for patients with adrenal insufficiency is permitted. The use of fludrocortisone for mineralocorticoid replacement in patients with adrenal insufficiency is permitted. 11. Known active central nervous system metastases and / or carcinomatous meningitis. Patients with previously treated brain metastases may participate provided they are stable (no evidence of progression by imaging for at least 4 weeks prior to the first dose of investigational product and any neurological symptoms have returned to baseline), have no evidence of new or growing brain metastases, and have not used steroids for at least 7 days prior to blood collection for the manufacture of autologous blood products. This exception does not include carcinomatous meningitis, which is excluded regardless of clinical status. 12. History of interstitial lung disease, idiopathic pulmonary fibrosis, pneumonia (including drug-induced), or organizing pneumonia (e.g., bronchiolitis obliterans, cryptopathic organizing pneumonia) requiring steroids. a. Patients with asymptomatic pneumonia who are not requiring steroid therapy for their pneumonia are eligible. 13. Clinically significant cardiac disease, including unstable angina, acute myocardial infarction within 6 months prior to blood collection for the manufacture of autologous blood products, New York Heart Association class III or IV congestive heart failure, and arrhythmias requiring therapy. 14. Systemic arterial thrombotic or embolic events, such as cerebrovascular accident (including ischemic stroke), within 1 month prior to blood collection for the manufacture of autologous blood products. 15. Systemic venous thrombotic event (e.g., deep vein thrombosis) or pulmonary artery event (e.g., pulmonary embolism) within 1 month prior to blood collection for the manufacture of autologous blood products. a. Patients with a venous thrombotic event prior to blood collection for the manufacture of autologous blood products for stable anticoagulant therapy are eligible. 16. History or presence of clinically significant electrocardiogram abnormalities (ECG) in the opinion of the investigator. 17. Left ventricular ejection fraction (LVEF) <50%. 18. Major surgery within 2 weeks of blood collection for the production of autologous blood products; after major surgery >2 weeks prior to blood collection for the production of autologous blood products, all surgical wounds must be healed and without infection or dehiscence. 19. Any other clinically significant comorbidity, e.g. active infection, known psychiatric or neurological disorder, or any other condition that, in the investigator's judgment, impairs adherence to the protocol, interferes with interpretation of the study results, or renders the patient a safety risk. 20. Known active hepatitis B or C, or active mycobacterium tuberculosis infection. 21. The patient has a history of alcohol and / or illicit drug abuse within 12 months of entry. 22. Female patients who are breastfeeding or have a positive serum pregnancy test at the screening visit. 23. The patient has a history of allergy or hypersensitivity to any component of SQZ-AAC-HPV. 24. History of severe allergic anaphylactic reaction to chimeric, human, or humanized antibodies, or infused proteins (combination cohort only). 25. Known hypersensitivity to ipilimumab, nivolumab, Chinese hamster ovary cell products, or any components of the ipilimumab or nivolumab formulations (combination cohorts only). 26. Enrollment of HIV+ patients must be discussed with the sponsor. Blood collection for the production of autologous blood products

[0319] The blood collection goal for home production is approximately 500 × 10 to support a long treatment period. 9 The objective of the present invention is to provide a yield of RBCs for each patient in individual cells. For this purpose, at least 200 mL of whole blood (± 10%) is required to obtain at least 500 × 10 9 A sample is taken to collect RBCs. According to local procedures, an RBC or complete blood count is performed during the blood draw so that the volume of processed blood can be increased. In the event that an RBC or complete blood count cannot be performed during the blood draw, a sample is taken at the end of the blood draw, if possible, to determine the RBC count at the RBC draw. Results must be processed as soon as possible and provided to the sponsor in real time. Tumor Response Assessment and Schedule

[0320] Tumor assessments will be performed at screening (baseline) and tumor response will be assessed by the investigator every 9 weeks (± 7 days) for 1 year after the first dose of SQZ AAC HPV, and then every 12 weeks (± 7 days) thereafter until disease progression as confirmed by RECIST and iRECIST, unacceptable toxicity, withdrawal of consent, death, or 2 years from the date of the first dose of SQZ AAC HPV, whichever occurs first.

[0321] For patients who achieve a partial response (PR) or complete response (CR), tumor assessment is repeated after 4 weeks to confirm response.

[0322] Disease will be assessed via radiological imaging, either CT scan or MRI, with radiological modalities remaining consistent throughout the study. Patients experiencing disease progression by RECIST 1.1 may continue on medication if considered in their best interest by the treating investigator to allow confirmation of disease progression: iCPD by iRECIST (Seymour et al., 2017).

[0323] If a patient discontinues investigational product for reasons other than progression, the patient will continue imaging according to the schedule outlined above. If a patient discontinues treatment due to clinical deterioration, TEAEs related to clinical progression will be recorded on the AE page. Radiographic assessments must be obtained and recorded.

[0324] At screening and all subsequent time points, cervical, anal / rectal, vulvar / vaginal, and penile cancers will require computed tomography (CT) of the torso (chest, abdomen, and pelvis) and all known sites of disease; oropharyngeal cancer will require CT of the head, neck, and chest, as well as other areas of known involvement. If, for any discernible reason, a CT scan cannot be used or adequate tumor assessment is not possible, magnetic resonance imaging (MRI) will be permitted and the sponsor will be informed during screening. The same radiographic procedures used to assess sites of disease at screening will be used throughout the study. For all other advanced solid tumor types, the investigator will image all known sites of disease using the imaging modality the investigator believes is best for that tumor type.

[0325] Magnetic resonance imaging of the brain is required at screening in all patients with a history of brain metastases and may be repeated at subsequent time points in any patient with a history of brain metastases and / or who develops symptoms suggestive of brain metastases. If a patient cannot tolerate or has a contraindication to MRI, a CT scan is used.

[0326] When possible, the same assessor will perform the assessments to ensure internal consistency across visits. At the investigator's discretion, CT scans will be repeated at any time if progressive disease (PD) is suspected. For patients who achieve a partial response (PR) or complete response (CR), tumor assessments will be repeated after 4 weeks to confirm response. Pharmacodynamic evaluation including immunogenicity measurements Sampling Schedule

[0327] Blood and tumor biopsy samples will be collected to assess the effect of SQZ-AAC-HPV on pharmacodynamics, including immunogenicity measures. Tumor biopsy

[0328] Prior to blood collection for the manufacture of autologous blood products, patients undergo a screening tumor biopsy (primary tumor or metastasis), which may be from a previously irradiated site with active tumor growth. All patients are required to undergo a repeat tumor biopsy of the same primary tumor or metastasis on day 8 (±2 days) of cycle 2. If possible, an additional repeat tumor biopsy is obtained (pre-dose) on day 1 (+2 days) of cycle 5, and this sample is as needed. If preliminary data suggests that altering the on-treatment tumor biopsy time point is more appropriate, alternative on-treatment tumor biopsy time points may be considered.

[0329] Tumor tissue must be of good quality based on total and viable tumor content. A new tumor biopsy will be taken at screening from the primary tumor or metastatic site, and subsequent biopsies will be from the same primary tumor or metastasis biopsied at screening. Anatomical location (organ and area within organ) must be noted in the CRF. Pharmacodynamic evaluation

[0330] Whenever possible, baseline samples will be used for longitudinal evaluation of cellular correlates including, but not limited to, immunophenotyping by flow cytometry including tetramer staining, assessment of T cell cytokine production after co-culture with HPV peptides (IFNγ and granzyme B enzyme-linked immunospot [ELISPOT]), and circulating cell-free HPV16 DNA (cfHPV DNA). Baseline tumor biopsies and selected blood samples will be used only for comparison with post-treatment samples (Table 2). [Table 2] Abbreviations: DNA = deoxyribonucleic acid; HPV16 = human papillomavirus strain 16; IFNγ = interferon gamma

[0331] Assessment of the development of endogenous immune responses via ELISPOT, T cell receptor sequencing, and epitope spreading can be performed. Information about endogenous immune responses detected via ELISPOT informs immunohistochemical analysis of tumor biopsies. Cytokine evaluation

[0332] Patients with grade 2, 3, or 4 CRS will have additional cytokine plasma levels taken during a grade 2, 3, or 4 CRS event. Blood draws will be obtained pre-dose on cycle 1 day 1, as well as at the time of diagnosis of CRS, increasing severity (e.g., when grade 2 CRS progresses to grade 3 CRS), onset of neurological symptoms, and discharge or resolution.

[0333] Evaluation of a cytokine panel includes, but is not limited to, IFN gamma (IFNγ) and IL6. CRS may be delayed in onset but is rarely present beyond 14 days after initiation of therapy. Patients presenting with symptoms consistent with CRS that are outside of this window are carefully evaluated for other causes.

[0334] Cytokines are also monitored for pharmacodynamic evaluation. Baseline and post-treatment serum samples are collected to evaluate the anti-tumor immune response by measuring cytokines that provide information about the inflammatory response of the drug.

[0335] Blood samples will be taken in cycle 1 and cycle 2 to assess the kinetics of SQZ-AAC-HPV clearance from the bloodstream following its IV administration. Safety assessment

[0336] Eligibility criteria for this study were established to ensure the safety of participating patients. Safety will be assessed in this study through monitoring of all SAEs and nonserious AEs, as well as laboratory abnormalities, and will be defined and graded according to NCI CTCAE version 5.0. General safety assessments will include physical examination and special laboratory evaluations, including blood chemistry, coagulation, and blood counts with differential. SAEs and ≥ grade 2 AESIs will be reported in an expedited format for entry into the safety database.

[0337] During study conduct, the totality of observed safety events will be reviewed (including CRS events that resolved to grade 2) to determine if a given event requires the initiation of coordinated enrollment of patients following this event. Coordinated enrollment in potential additional monotherapy cohorts (part 1) or combination safety cohorts (part 2) will require one-week coordination of all subsequent newly enrolled patients in one or more cohorts. Parallel enrollment of patients may continue in some cohorts, if applicable. Patients with grade 2, 3, or 4 CRS will have additional blood samples taken for safety testing and cytokine panel evaluation.

[0338] Exposure to immune checkpoint inhibitors may increase the risk of irAEs, especially autoimmune conditions, so irAEs must be recognized early and treated promptly to avoid potential major complications.

[0339] All patients will return to the clinic for a safety follow-up visit within 15 to 45 days after the last dose of investigational product. All AEs and SAEs will be recorded 6 weeks after the last dose of investigational product (EOD6W) or up to 45 days after dropout or initiation of another anticancer therapy, whichever occurs first. Only ongoing SAEs determined by the investigator to be possibly, probably, or definitely related to SQZ-AAC-HPV monotherapy or combination therapy will be followed up. Physical examination and height and weight

[0340] The physical examination will include an assessment of height (screening only), weight, and general appearance, as well as an assessment of the following systems: skin, head, eyes, ears, nose, mouth / throat / neck, thyroid, lymph nodes, respiratory system, cardiovascular system, gastrointestinal system, extremities, musculoskeletal system, neurological system, and gynecological and genitourinary systems, as indicated. It is particularly important to capture weight during the patient's physical examination within 24 hours of blood collection for the manufacture of autologous blood products, as patient medications are determined by weight. General condition

[0341] Eastern Cooperative Oncology Group scales and criteria are used to assess the patient's performance status, evaluate how the disease affects the patient's ability to live daily life, and determine appropriate treatment and prognosis. 12-lead electrocardiogram

[0342] A 12-lead ECG is performed by qualified on-site personnel at scheduled times using ECG equipment that determines heart rate, PR interval, QRS interval, RR interval, and QT interval. QTcB (QTc corrected by Bazett's formula) and / or QTcF (QTc corrected by Fridericia's formula) are calculated based on the QT and RR intervals. During ECG collection, the patient should be in a motionless position in a quiet environment free of distractions (e.g., no television, cell phones) for at least 10 minutes prior to ECG collection.

[0343] All ECGs are evaluated by a qualified physician for the presence of abnormalities. Echocardiogram

[0344] Echocardiograms or multiple gated acquisition (MUGA) scans will be performed to measure LVEF at screening and when clinically indicated. Clinical Laboratory Evaluation

[0345] Samples for clinical laboratory evaluations will be collected at the time points. Clinical laboratory tests outlined in Table 3 will be performed at the site. Samples for the clinical tests outlined in Table 3 will be collected in appropriate tubes and handled according to the site's standard procedures.

[0346] Laboratory test variables are listed in Table 3. For safety monitoring purposes, the investigator must review, sign, and date all out-of-range laboratory test results. Laboratory test results must be documented. [Table 3] a. Results for these laboratory tests should be collected prior to or on the day of blood collection for autologous blood product production, with the results available prior to blood collection for autologous blood product production. b. Coagulation parameter results are required on the day of any tumor biopsy or the day after. Abbreviations: CRS = cytokine release syndrome; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone. Adverse events Adverse events

[0347] An AE is any untoward medical occurrence in a patient that does not necessarily have a causal relationship with the administered investigational product. Thus, an AE can be any untoward or unintended sign (including abnormal laboratory findings), symptom, or disease that is temporally related to the use of an investigational product, whether or not related to the investigational product. An adverse event can be a new event, or a pre-existing condition that has become serious or worse in severity or frequency.

[0348] An adverse event may be a clinically significant change from baseline in a physical examination, clinical laboratory test, or other diagnostic investigation.

[0349] In this study, AEs were treatment-emergent if their onset occurred after administration of the investigational product and up to 6 weeks after the last dose of study treatment (SQZ-AAC-HPV or immune checkpoint inhibitor). Serious Adverse Events

[0350] An SAE is any AE that results in any of the following: death. Immediately life threatening. Requires hospitalization of the patient or an extension of an existing hospital stay. Causes lasting or significant disability or incapacity. Causing congenital abnormalities or birth defects. A significant medical event that may endanger the patient or require medical intervention to prevent one of the outcomes listed above.

[0351] After any patient signs the ICF, all SAEs occurring before, during, or within 30 days after cessation of treatment, whether or not they are study-related, must be reported on the appropriate clinical procedure form. Adverse events of particular interest

[0352] AESIs are AEs (serious or not) of scientific and medical interest specific to the investigational product that require ongoing monitoring and immediate notification by the investigator to the sponsor. Such AEs may require further investigation to characterize and understand them. Adverse events of particular interest may be added or removed during the study by protocol amendment.

[0353] The following AEs are considered AESIs: Hypersensitivity, cytokine release, systemic inflammatory response syndrome, events suggestive of systemic inflammatory activation. Influenza-like illness. Infusion reaction syndrome. Immunotherapy-associated irAEs, e.g., myocarditis, neurological irAEs, transaminopathy of immune-related etiology, and nephritis. All AESIs of grade 2 or higher will be reported to the sponsor within 24 hours of recognition. Events deemed by the investigator to be irAEs or suspected to be immune-related must be discussed immediately with the sponsor.

[0354] In addition, the following events will be reported to the Sponsor: Suspected SQZ-AAC-HPV overdose. Abnormalities in liver tests fulfilling Hy's law criteria, i.e., AST or ALT laboratory values ​​≥ 3 × ULN and total bilirubin laboratory value ≥ 2 × ULN, and concomitant alkaline phosphatase laboratory value < 2 × ULN, as determined by specific protocol or unscheduled laboratory testing. General

[0355] Adverse events, including SAEs, will be collected for each patient from the date the ICF was first signed up to a maximum of 45 days after EOD6W or dropout, or until the start of another anticancer therapy, whichever occurs first. All SAEs and grade ≥ 2 AESIs occurring within the reporting period must be reported by the investigator to the sponsor or designee within 24 hours of the investigator becoming aware of the SAE or AESI, regardless of causality. Only ongoing SAEs determined by the investigator to be possibly, probably, or definitely related to SQZ AAC HPV monotherapy or combination therapy will be followed.

[0356] AE terms will be reported in standard medical terminology when possible. For each AE, the investigator will assess and report onset (date and time), resolution (date and time), severity, causality, actions taken, whether serious, and whether the event caused the patient to discontinue study or result in an alteration or delay in investigational product administration.

[0357] All AEs (both expected and unexpected) spontaneously reported by patients, or in response to open-ended questions from study personnel, or revealed by observation will be documented in the appropriate study-specific clinical procedure form at the study site and during the study. Clinical outcomes or symptoms related to PD will be reported as SAEs and / or deaths if they meet the SAE and / or death criteria and occur within 6 weeks of the last investigational product administration. They will be reported according to the diagnosis or symptoms of the event and will not be reported by the term "progressive disease."

[0358] Any laboratory values ​​outside the normal range will be alerted to the Investigator or designee's attention at that location. The Investigator or designee will review for clinical significance. If a clinically significant abnormality is found in a sample taken after dosing, during the study, and / or within 6 weeks after cessation of investigational product, it must be reported as an AE and the patient must be followed until tests normalize or stabilize, at the Investigator's discretion. Abnormal laboratory values ​​that constitute an SAE or result in the discontinuation of investigational product administration will be reported and recorded on the AE page of the Case Report Form (CRF).

[0359] SAEs and AESIs will be tracked until resolved, stabilized, or the investigator and sponsor agree that follow-up is no longer necessary. If the event has not resolved at the end of the study reporting period, it must be documented as ongoing. All SAEs and non-serious ≥ Grade 2 AESIs will be reported to the Global Pharmacovigilance Processing Group within 24 hours of learning of the event. Rating severity

[0360] The severity of AEs and of laboratory abnormalities is assessed and graded using NCI CTCAE version 5.0. The ASTCT consensus grading is used for CRS and ICANS. Each AE term is mapped to the latest version of the Medical Dictionary for Clinical Practice (MedDRA) terminology and codes.

[0361] If an event is not covered by CTCAE Version 5.0, the guidelines shown in Table 4 are for rating severity. [Table 4] Origin: (NIAID, 2003) Abbreviation: CTCAE = Common Terminology Criteria for Adverse Events Causal assessment

[0362] Relationships to the investigational product will be assessed by the investigator. Thus, the AE and SAE reporting form includes options to attribute a causal relationship to SQZ-AAC-HPV, ipilimumab, nivolumab, or the combination. For patients receiving concomitant therapy with SQZ AAC-HPV and an immune checkpoint inhibitor, causality will be assessed individually for each protocol-specified therapy. Reasonably suspected causal relationships will be attributed solely to the immune checkpoint inhibitor if the event is consistent with the immune checkpoint inhibitor label.

[0363] The relationship between AEs and investigational products (i.e., SQZ-AAC-HPV, ipilimumab, nivolumab, or combination) will be documented as follows: Unequivocal: The AE is clearly related to the investigational product. Probable: The AE is possibly related to the investigational product. Possible: The AE may be related to the investigational product. Unlikely: The AE is unlikely to be related to the investigational product. Unrelated: The AE is not clearly related to the investigational product.

[0364] A drug-qualified investigator will make the investigational product relationship determination for each AE. The investigator will determine whether, in his or her medical judgment, there is a reasonable possibility that the event may be caused by the investigational product. If no valid reason exists to suggest a relationship, the AE will be classified as "unrelated." If any valid reason exists, even if uncertain, for suspecting a possible causal relationship between the investigational product and the occurrence of the AE, the AE will be considered "related."

[0365] If the relationship between the AE / SAE and the investigational product is determined to be "definite," "probable," or "possible," the event will be considered related to the investigational product for purposes of expedited regulatory reporting. Predictable

[0366] AEs that are not listed in, or whose specificity or severity is inconsistent with, the applicable product information (e.g., IB for SQZ-AAC-HPV, or approved labeling for ipilimumab or nivolumab) are considered unexpected. Efficacy analysis definition

[0367] Progression-free survival (PFS) is defined as the time from day 1 of cycle 1 to the first documentation of an objective tumor progression (PD, radiological) according to RECIST 1.1 or death from any cause, whichever occurs first. Progression-free survival data is censored at the date of the last tumor assessment documenting the absence of PD for patients who have not had objective tumor progression and are still on study at the time of analysis, or are given antitumor treatment other than the investigational product, or are removed from treatment follow-up prior to documentation of objective tumor progression. Patients who have no tumor assessment after enrollment who are not known to have died have PFS censored at day 1 of cycle 1. PFS is assessed by both RECIST 1.1 and iRECIST criteria to accommodate different implementations across participating sites.

[0368] Overall survival (OS) is defined as the time from the date of cycle 1 day 1 to the date of death from any cause. In the absence of confirmed death, survival is censored at the last date the patient is known to be alive. Patients missing dates beyond cycle 1 day 1 have their survival censored at cycle 1 day 1.

[0369] Objective response rate (ORR) is defined as the proportion of patients with CR or PR by RECIST 1.1. Objective response rates are provided as unconfirmed and confirmed ORR. A confirmed response is one that is sustained on repeat imaging studies at least 28 days after initial documentation of response. Similarly, iORR by iRECIST is also summarized and reported.

[0370] Duration of response (DoR) is defined as the time from the first documentation of PR or CR to the first documentation of objective tumor progression or death due to any cause. Duration of response data is censored at the date of the last tumor assessment documenting the absence of PD for patients who: 1) do not have tumor progression and are still on study at the time of analysis; 2) are given antitumor treatment other than the investigational product; or 3) are removed from study follow-up prior to the documentation of objective tumor progression. Similarly, iDoR by iRECIST is also summarized and reported.

[0371] Best overall response (BOR) is determined once all tumor assessments from cycle 1 day 1 to disease progression or death have been recorded. Generally, this is the best response across all assessments, however, confirmation of CR, PR, and stable disease (SD) is used in BOR determination. To confirm CR or PR, the change in tumor measurement is confirmed by repeat assessment, which must be 4 weeks (28 days) or more after the first meeting of the criteria for the reason. To confirm SD, this must occur at least 12 weeks from cycle 1 day 1, otherwise BOR depends on the subsequent evaluation. Best overall response is summarized by percentage and as a variable time to event for time to best response, using enrollment as a fixed date. Similarly, iBOR by iRECIST is summarized and reported.

[0372] Disease control rate (DCR) is the proportion of patients with a BOR determined as CR, PR, or SD by RECIST 1.1 at a defined time point. All patients in the safety analysis population with measurable disease at baseline and eligible for tumor assessment at 3, 6, and 12 months are considered in the denominator of the DCR rate. Similarly, iDCR by iRECIST will be summarized and reported. analysis

[0373] Efficacy analyses will be performed in the safety analysis population. Antitumor activity (ORR, PFS, OS) will also be described for patients with documented HLA class I expression. If the per-protocol population is different from the safety analysis population, efficacy analyses will also be performed using the PP population.

[0374] All assessments using response assessment by RECIST 1.1 or iRECIST will be analyzed using the investigator review assessment.

[0375] The Kaplan-Meier method is used to estimate median PFS, with two-sided 95% confidence intervals. Patients who die are considered to have had an event, regardless of cause of death, as long as they received subsequent anticancer therapy before death. If they receive subsequent therapy, they are censored at the date of their last evaluable tumor assessment before the subsequent therapy. Patients who withdraw consent for the study are considered censored at the time of their last evaluable tumor assessment before withdrawing consent. Patients who are still alive at the time of the clinical data cut-off date are censored at their most recent evaluable tumor assessment. All patients with no follow-up before the clinical data cut-off date are also considered censored at the time of their last evaluable tumor assessment before loss of follow-up.

[0376] Duration of response, time to best overall response, and overall survival will use the same censoring algorithm as PFS. In addition, iPFS using iRECIST, iBOR, iDCR, and time to iBOR will be analyzed and reported using similar methods.

[0377] Objective response rates (ORR) and DCR are expressed as percentages with 95% two-sided confidence intervals based on exact binomial distribution. SD lasting at least 12 weeks is reported as point estimates. Safety analysis

[0378] All safety parameters will be analyzed using the safety analysis population, including AEs, clinical laboratory assessments, vital signs, ECOG, exposure, ECG, ECHO / MUGA, and physical examination.

[0379] The primary safety endpoint is the number of patients with any AEs and observed toxicities to SQZ-AAC-HPV administration, where severity is assessed using NCI CTCAE version 5.0. All AEs beginning after the first dose of SQZ-AAC-HPV are included in the analysis. Adverse events are initially collected at the time of signing of informed consent, however, the analysis focuses on AEs that occur during treatment.

[0380] AEs will be analyzed using descriptive statistics. For patients with multiple indicators of a given AE, the highest severity will be used. Adverse events

[0381] AEs will be coded using the most current version of the MedDRA coding dictionary.

[0382] An AE is treatment-emergent if its onset occurs between Day 1 of Cycle 1 and 6 weeks after the last dose of investigational product. For AEs with a partial onset time, the non-missing date portion is used to determine if the AE was treatment-emergent. If it is not possible to determine when the AE occurred in relation to investigational product administration, the AE is classified as treatment-emergent. Treatment-emergent AEs also include any AE that was present before the first dose of investigational product and worsened in toxicity after administration.

[0383] The analysis described in this section is based on TEAEs, simply referred to as AEs in this section.

[0384] Adverse events deemed by the investigator to be possibly, with high certainty, or definitely related to the investigational product will be classified as related for summary purposes.

[0385] The numbers and percentages of patients with any AE, any related AE, any SAE, any related SAE, any grade 3 or higher AE, any related grade 3 or higher AE, and the total number of events for each category will be summarized. Deaths due to AE, hospitalizations due to AE, and treatment discontinuations due to AE, as well as the number of DLTs and AESIs will be summarized.

[0386] The numbers and percentages of patients with AEs, and the total number of AEs, will be summarized by system organ class and preferred term. This tabulation will be repeated for related AEs, AESIs, SAEs, related SAEs, and AEs > Grade 3 and AEs > Grade 3.

[0387] All AEs, including non-TEAEs, will be provided to a patient listing. A listing of patients will be generated for AEs leading to discontinuation of investigational product, AEs leading to death, SAEs, related AEs, AESIs, DLTs, and AEs ≥ Grade 3. Clinical Laboratory Evaluation

[0388] Baseline is defined as the last nonmissing value before first exposure to investigational product. This is typically pre-dose on day 1 of the cycle, but may be earlier. Actual values, and changes from baseline laboratory tests, will be summarized by study visit.

[0389] Laboratory test results are classified according to NCI CTCAE version 5.0 and clinical significance as determined by the investigator. If more than one laboratory test result is reported per study visit per parameter, the result giving the most severe classification is selected for analysis. A shift table is generated to show the maximum change from baseline for the graded laboratory parameters.

[0390] All clinical laboratory evaluations are provided in the listing.

[0391] Patients with clinically significant abnormal laboratory results will be listed. This list will include all results of laboratory parameters that were determined by the investigator to be abnormal and clinically significant for the patient across study visits. Vital signs

[0392] Baseline is defined as the last non-missing value before first exposure to investigational product. Actual values ​​and changes from baseline in vital signs will be summarized by study visit and study time point. All vital sign data will be presented in a patient listing.

[0393] Vital sign values ​​are classified according to clinical significance as determined by the investigator. The number of patients with non-missing results, the number and percentage of patients with clinically insignificant results, and clinically significant results are summarized by study visit and study time point. If more than one vital sign result is reported per parameter per study visit and study time point, the result giving the most severe classification is selected for analysis.

[0394] Patients with clinically significant vital sign values ​​will be listed. This listing will include all results of vital sign parameters determined by the investigator to be clinically significant for the patient across study time points. Physical examination

[0395] Abnormal physical examination findings are listed. 12 lead ECG

[0396] The ECG results are presented in a shift table (normal, clinically insignificant abnormal, abnormal, clinically significant) showing the maximum change from baseline. All ECG results are presented in a patient list. Other Safety Variables

[0397] All safety data is provided in the listing.

[0398] ECOG PS and change from baseline in ECOG PS will be summarized at each scheduled visit collected. Change from baseline in ECOG PS will be summarized as a continuous variable and as a categorical variable. A decrease of ≥ 1 point from baseline will be classified as "improvement" from baseline. An increase of ≥ 1 point from baseline will be classified as "worsening" from baseline. Improvement, worsening, and unchanged ECOG PS from baseline will be summarized as a categorical variable by treatment at each post-enrollment time point at which ECOG PS is assessed. Pharmacodynamic analysis

[0399] Biomarkers will be summarized for change from baseline and % change from baseline for each time point. Correlations between pharmacodynamic markers and SQZ-AAC-HPV will be explored in a descriptive and graphical manner.

[0400] Descriptive statistics (mean, standard deviation, median, minimum, maximum, and geometric mean) for each marker will be reported. Graphs of individual values ​​over time by dose group will be presented. Feasibility of dose production

[0401] The feasibility of dose manufacturing will be evaluated based on individual patient batch yields, product failures that would prevent use, and any additional information from blood collection for autologous blood product manufacturing to production of SQZ AAC HPV product that may be relevant. Example 2 Characterization of M-AAC-HPV production and surface phosphatidylserine on Annexin V+ cells measured by flow cytometry

[0402] The aim of these studies was to characterize surface phosphatidylserine (PS) levels via annexin V staining and flow cytometric analysis of M-AAC-HPV. Description of the process used to generate M-AAC-HPV

[0403] To generate M-AAC-HPV, mouse RBCs are SQZ-treated with E7 synthetic long peptide (SLP) and the adjuvant polyinosinic-polycytidylic acid (poly I:C). The mouse E7 SLP, shown below in bold and underlined, contains the mouse E7 antigen epitope presented in C57BL / 6J class I MHC H2-Kb. This sequence is contained within the same HPV16 E7 protein from which the human E7 SLP is derived. Note that for C57BL / 6J mice, E7 is the immunodominant antigen, and immunization against E6 provides little therapeutic benefit in the HPV16 TC-1 tumor model (Oosterhuis 2011; Peng 2016, Li 2010). Therefore, M-AAC-HPV contains only mouse E7 SLP. Below is a comparison of the structures of mouse E7 SLP and human E7 SLP. Mouse E7 SLP: GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25) Human E7 SLP: QLCTELQTYMLDLQPETTYCKQQLL (SEQ ID NO: 23)

[0404] The adjuvant used in the production of M-AAC-HPV is poly I:C, the same adjuvant used in the human formulation SQZ-AAC-HPV.

[0405] In addition to facilitating delivery of E7 SLP and poly I:C to the interior of M-AAC-HPV, the SQZ process increases the levels of PS exposed on the M-AAC-HPV membrane. This surface PS is hypothesized to serve as a ligand recognized by receptors on antigen-presenting cells that internalize M-AAC-HPV after intravenous administration. A fluorescent derivative of annexin V, a phospholipid-binding protein that binds PS with high affinity (Koopman 1994), is used to stain cells for cell surface PS and detected using flow cytometry.

[0406] Whole blood was collected from mice and mouse RBCs were isolated. Since these studies are being compared to the use of PBS or RPMI in this process, mouse RBCs were then transferred to a solution containing antigen (mouse E7 SLP; 100M) and adjuvant (Poly I:C; 1mg / mL) in either PBS (phosphate buffered saline) or RPMI (Roswell Park Memorial Institute (culture medium)) at 1x10 9 The M-AAC-HPV suspension was then washed with PBS using centrifugation and finally diluted with PBS at 2 × 10 cells / mL. The resulting cell suspension was transferred to the syringe of a small-scale SQZ device and then subjected to SQZ treatment. After SQZ treatment, the resulting suspension of AAC was incubated at room temperature for 20-60 min. The M-AAC-HPV suspension was then washed with PBS using centrifugation and finally diluted with PBS at 2 × 10 cells / mL. 9 The cells were resuspended at 100 cells / mL. Surface PS levels on M-AAC-HPV

[0407] Surface PS levels were characterized via analysis of data acquired by Annexin V staining and flow cytometry. Summary data displaying the percentage of M-AAC-HPV that were Annexin V+ are shown in Figures 5A and 5B).

[0408] The average percentage of Annexin V+ M-AAC-HPV from six independently prepared batches (three batches SQZ-treated in PBS and three batches SQZ-treated in RPMI) was 94.8±5.3% (mean±standard deviation), and the average percentage of Annexin V+ untreated RBCs was 2.0±1.3%. The average ratio of Annexin V MFI (geometric mean fluorescence intensity) in M-AAC-HPV to that in untreated RBCs from six independently prepared batches was 99±59 (mean±standard deviation). In addition, the ratio of Annexin V MFI of M-AAC-HPV to Annexin V MFI in untreated RBCs was not significantly different when cells were treated in PBS or RPMI.

[0409] These studies demonstrate that surface levels of PS are elevated under the conditions used for SQZ treatment of RBCs. The percentage of Annexin V+AAC for M-AAC-HPV is comparable to that of the human product SQZ-AAC-HPV, which is at least 95.8%. Example 3 Cellular characterization of AAC-HPV by flow cytometry

[0410] The objectives of this study were to quantify the delivery of FAM (5-carboxy-fluorescein)-labeled SLPs (synthetic long-chain peptides), FAM-E6 and FAM-E7 SLPs, to AAC and to characterize surface phosphatidylserine (PS) levels on AAC. Description of the small-scale process used to produce human AAC

[0411] Whole blood was collected the day before the study and RBCs were isolated on the day of the study. The RBCs were then diluted at 2×10 9 The cells were suspended at 2 × 10 cells / mL. The resulting cell suspension was incubated on ice for 10 min, transferred to the syringe of a small-scale SQZ device, and then subjected to SQZ treatment. After SQZ treatment, the resulting suspension of AAC was incubated at 2-8 °C for 20 min and then at 37 °C for 60 min. The AAC suspension was then washed with PBS using centrifugation and finally diluted to 2 × 10 cells / mL with PBS. 9 The cells were resuspended at 100 cells / mL. Delivery of FAM-E6 and FAM-E7 SLPs to the AAC

[0412] RBCs isolated from three healthy donors in three separate experiments were: A) used as is (no SQZ treatment) as a control, B) SQZ-treated with unlabeled E6 SLP, unlabeled E7 SLP, and poly I:C to generate AAC-HPV, C) SQZ-treated with 5-carboxy-fluorescein (FAM)-labeled E6 SLP, unlabeled E7 SLP, and poly I:C to generate AAC-HPV(F-E6, E7), or D) SQZ-treated with FAM-labeled E7 SLP, unlabeled E6 SLP, and poly I:C to generate AAC-HPV(F-E7, E6). Table 3 describes these experimental groups. Three batches of human RBCs, each from a different donor, were SQZ-treated to generate AAC-HPV, AAC-HPV(F-E6, E7) and AAC-HPV(E6, F-E7). SQZ-treated cells were stained with AF647-Annexin V and analyzed by flow cytometry to quantify incorporation of fluorescently labeled SLPs and to assess surface PS levels (based on Annexin V; results described in 2.4.3). Untreated RBCs and AAC-HPV served as negative controls (no FAM labeling). [Table 5] NA stands for "not applicable."

[0413] Summary data displaying the percentages of FAM-E6 SLP+ and FAM-E7 SLP+ samples are shown in Figure 6. FAM fluorescence was detected in 0.1% and 0.0% of AAC-HPV and untreated RBCs, respectively (negative control). The majority of AAC were positive for FAM-E6 or FAM-E7 SLP; on average, 97.8% of AAC-HPV (F-E6, E7) and 95.0% of AAC-HPV (E6, F-E7) were positive for FAM-E6 and FAM-E7 SLP, respectively.

[0414] Untreated RBCs and SQZ-treated cells were stained with AF647-Annexin V and analyzed by flow cytometry to quantify surface PS levels (based on Annexin V). Summary data displaying the percentage of Annexin V+ samples are shown in Figure 7. The average percentage of untreated RBCs that were Annexin V+ was 1.0%, while at least 95.8% of AAC-HPV, AAC-HPV(F-E6, E7) or AAC-HPV(E6, F-E7) were positive for Annexin V, demonstrating that the SQZ process increases PS on the cell membrane. Example 4 Imaging of SQZ-treated human RBCs with fluorescently labeled SLPs

[0415] This study demonstrates intracellular delivery of fluorescently labeled HPV E6 and E7 SLPs into AAC following SQZ treatment.

[0416] Table 6 describes the experimental groups used in these studies. In each of three independent experiments, RBCs from healthy donors were isolated from whole blood and A) SQZ-treated with 5-carboxy-fluorescein (FAM)-labeled E6 SLP, unlabeled E7 SLP, and poly I:C to generate AAC-HPV (F-E6, E7), B) SQZ-treated with FAM-labeled E7 SLP, unlabeled E6 SLP, and poly I:C to generate AAC-HPV (E6, F-E7), or C) SQZ-treated with unlabeled E6 SLP, unlabeled E7 SLP, and poly I:C to generate AAC-HPV. SQZ-treated samples were stained with Pacific Blue (PB)-conjugated anti-CD235a antibody and imaged with an epifluorescence microscope. Images for each sample were subjected to line scan analysis to determine whether the localization of FAM-labeled SLPs was luminal (inside the AAC). [Table 6]

[0417] Representative epifluorescence images and their corresponding line scan traces for AAC-HPV(F-E6, E7), AAC-HPV(E6, F-E7), and AAC-HPV generated by SQZ treatment of RBCs from three individual donors (one donor per experiment) are shown in Figure 8, Figure 9, and Figure 10, respectively. The mean and range of percentages of FAM+AAC-HPV(F-E6, E7), AAC-HPV(E6, F-E7), and AAC-HPV are shown in Table 7. [Table 7] Table notes: N = 3 donors / group.

[0418] The intracellular delivery of fluorescently labeled E6 and E7 SLPs (FAM-E6 and FAM-E7) into human AAC by the SQZ process was visualized by fluorescence microscopy. AAC-HPV(F-E6, E7), AAC-HPV(E6, F-E7), and AAC-HPV were stained with PB-conjugated anti-CD235a antibody to define the cell membrane. The localization of FAM-E6 or FAM-E7 SLPs was then visualized by fluorescence microscopy. AAC-HPV SQZ-treated with unlabeled SLPs served as a negative control.

[0419] Line scans performed on the fluorescent images confirmed the intra-AAC localization of FAM-E6 and FAM-E7 after SQZ treatment. Specifically, intra-AAC FAM was observed in all (100%) of the analyzed AAC-HPVs (F-E6, E7) and in the majority (95.0% on average) of the analyzed AAC-HPVs (E6, F-E7).

[0420] This imaging study confirms the delivery of fluorescently labeled E6 or E7 SLPs to the majority of human AAC-HPV(F-E6, E7) and AAC-HPV(E6, F-E7), respectively, as a result of SQZ treatment. Example 5 In vitro uptake of AAC-HPV by human antigen-presenting cells (APCs) measured by flow cytometry

[0421] The aim of the study was to evaluate the in vitro uptake of AAC-HPV by human antigen-presenting cells (APCs).

[0422] 5-day GM-CSF / IL-4 differentiation of CD14+ monocytes resulted in HLA-A * Monocyte-derived dendritic cells (MODCs) generated from 02+ donors were used as an in vitro model of human APCs.

[0423] Red blood cells (RBCs) from three healthy human donors were labeled with the lipid-soluble fluorescent membrane dye PKH26. Unlabeled and PKH26-labeled RBCs were SQZ-treated with E6 SLP, E7 SLP, and poly I:C using the process described in report number SQZ-AAC-0124 to generate unlabeled and PKH26-labeled AAC-HPV, respectively.

[0424] In vitro uptake of AAC-HPV by MODCs was characterized as the increase in MODC (CD11c+ cells) PKH26 fluorescence after overnight co-culture with PKH26-labeled AAC-HPV at 37° C., as measured by flow cytometry. Co-culture of MODCs with PKH26-labeled AAC-HPV or co-culture of MODCs with unlabeled AAC-HPV at 4° C. was used as negative controls. Summary graphs from three independent experiments are shown in FIG. 11. Summary data showing the fold increase in PKH26 fluorescence of MODCs cultured at 37° C. relative to MODCs co-cultured at 4° C. are shown in Table 8. [Table 8] N / A indicates not applicable. In ELN1084, the highest tested dose of PKH26-labeled AAC-HPV or AAC-HPV was 200 × 10 per well. 6 It was an individual cell.

[0425] The PKH26 MFI of MODCs co-cultured with PKH26-labeled AAC-HPV at 37°C showed an increase (2.8-31.2-fold) relative to the PKH26 MFI of MODCs co-cultured at 4°C, a temperature at which uptake is inhibited (Albert 1998), ranging from 2 to 600 × 10 in all studies (three of three). 6 Fluorescence was observed for AAC-HPV doses ranging from 0.01 to 0.01. No fluorescence was observed in co-cultures containing unlabeled AAC-HPV, demonstrating that the increase in PKH26 MFI of MODCs was dependent on PKH26-labeled AAC-HPV. Therefore, CD11c+ MODCs internalize PKH26-labeled AAC-HPV in a dose- and temperature-dependent manner.

[0426] This study demonstrates that MODC takes up PKH26-labeled AAC-HPV in a dose- and temperature-dependent manner. Example 6 In vitro maturation of APCs following AAC-HPV uptake measured by flow cytometry

[0427] The aim of this study was to evaluate the in vitro upregulation of maturation markers in MODCs (monocyte-derived dendritic cells), a human model APC, approximately 2 days after co-culture with AAC-HPV.

[0428] 5 HLA-A * Monocytes from each of the 02+ donors were incubated with GM-CSF / IL-4 for 4 days to generate 5 lots of MODCs. MODCs were phenotyped, frozen, and stored at <140°C until thawed for use.

[0429] Human RBCs were SQZ-treated with E6 SLPs, E7 SLPs and poly I:C to generate AAC-HPV using the process described in report number SQZ-AAC-0124. Similarly, human RBCs were SQZ-treated with vehicle in the absence of antigen (E6 and E7 SLPs) and adjuvant (poly I:C) to generate C vehicle.

[0430] MODCs from five different donors were co-cultured with AAC-HPV for approximately 2 days.

[0431] Upregulation of maturation markers was determined by measuring the geometric mean fluorescence intensity (MFI) of CD86, CD80, CD83, MHC-II, and CD40 staining by flow cytometry and comparing it to maturation marker levels in MODCs cultured with C-medium or control medium alone.

[0432] Summary graphs for CD86, CD80, CD83 and MHC-II are shown in Figure 12. AAC-HPV co-cultured with MODC did not result in increased CD40 expression in MODC compared to culture with control vehicle, therefore CD40 is not represented on the graph.

[0433] A statistically significant increase in upregulation of maturation markers on the MODC surface was observed for CD80, CD86, and MHC-II. Although a statistically significant increase in upregulation of maturation marker CD83 was not observed, three of five MODC donors showed upregulation of CD83 after coculture with AAC-HPV compared to C-medium. In addition, statistical analysis performed on the raw (non-normalized) data of control medium, C-medium, and AAC-HPV showed no differences between control medium and C-medium, confirming that SQZ-treated RBCs without adjuvant (and antigen) do not upregulate maturation markers in MODCs.

[0434] This study demonstrates that MODCs cocultured in vitro with AAC-HPV significantly upregulate multiple maturation markers, including CD80, CD86, and MHC-II, on the surface of MODCs. A non-significant upregulation of CD83 is observed in three of the five donors used to generate MODCs. Example 7 In vitro activity of SQZ-AAC-HPV measured as IFN secretion by E711-20-reactive CD8+ T cells after coculture with MODCs

[0435] The aim of the study was to demonstrate the functional response of human model APCs, MODCs (monocyte-derived dendritic cells), to SQZ-AAC-HPV co-cultured with E711-20-specific CD8+ T cells.

[0436] Seven different batches of SQZ-AAC-HPV were generated by SQZ treatment of fresh healthy donor blood with E6 and E7 SLPs and poly I:C and formulated into preparations. SQZ-AAC-HPV was expressed as HLA-A by 5-day stimulation of CD14+ monocytes with GM-CSF and IL-4. * 02+ donor-derived MODCs. Media from the resulting co-cultures was analyzed for IFN secretion from E711-20-specific CD8+ T cells by ELISA.

[0437] Summary data from seven different lots showing SQZ-AAC-HPV-induced antigen-specific IFN responses from E7-specific CD8+ T cells co-cultured with MODC as measured by ELISA are shown in Figure 13. Summary data showing the magnitude of IFN secretion expressed as the fold increase between IFN measured in SQZ-AAC-HPV-containing co-cultures and vehicle control co-cultures are shown in Table 9.

[0438] Coculture of MODC and CD8+ T cells with all seven batches of SQZ-AAC-HPV resulted in at least a six-fold increase in secreted IFNγ compared with coculture of MODC and CD8+ T cells with vehicle control.

[0439] This study demonstrated that SQZ-AAC-HPV inhibits HLA-A * We demonstrate that in vitro co-culture with 02+ MODC and E7-specific CD8+ T cells induces secretion of IFNγ by E7-specific CD8+ T cells that recognize the E711-20 minimal epitope. [Table 9] a Fold increases are calculated for SQZ-AAC-HPV and vehicle control conditions. Example 8 In vivo maturation of endogenous APCs in mice following intravenous administration of M-AAC-HPV as measured by flow cytometry

[0440] The aim of study SQZ-AAC-0127 was to evaluate the in vivo upregulation of maturation markers in various endogenous splenic APCs (antigen presenting cells) following immunization of mice with the murine prototype M-AAC-HPV.

[0441] Table 10 shows the design of the study to evaluate the activation of splenic APCs by M-AAC-HPV in female C57BL / 6J mice in vivo. MC vehicle (SQZ-treated mouse RBCs in vehicle (in the absence of antigen or adjuvant)) was used as a control. The day of animal sacrifice is the day immunophenotyping was performed.

[0442] Splenic APCs evaluated were CD11chiMHC-IIhiCD8+ cells (CD8+ dendritic cells or CD8+ DCs), CD11chiMHC-IIhiCD11b+ cells (CD11b+ dendritic cells or CD11b+ DCs), and F4 / 80+CD11blo / - (RPM; red pulp macrophages).

[0443] Upregulation of APC maturation markers was demonstrated by measuring the geometric mean fluorescence intensity (MFI) of CD40, CD86, CD80, CD83 and MHC-II staining by flow cytometry. Flow cytometric analysis of spleens was performed 14–16 h after administration of M-AAC-HPV or MC vehicle, allowing accumulation of maturation markers on the cell surface. [Table 10] M-AAC-HPV = mouse prototype of AAC-HPV (mouse RBCs SQZ-treated with mouse E7 SLP and poly I:C); MC-vehicle = mouse RBCs SQZ-treated with vehicle (in the absence of antigen or adjuvant); RO: retro-orbital (route of administration)

[0444] Summary graphs for markers in splenic APCs are shown in Figure 14 for CD86 geometric MFI, Figure 15 for CD83 geometric MFI, Figure 16 for CD40 geometric MFI, Figure 17 for CD80 geometric MFI, and Figure 18 for MHC-II geometric MFI.

[0445] Results from two independent experiments demonstrated a statistically significant increase in CD86 geometric MFI in all three splenic APC populations (CD8+ DC, CD11b+ DC, RPM) in mice receiving M-AAC-HPV compared to mice receiving MC vehicle. Results from two independent experiments demonstrated a statistically significant increase in selective CD83, CD40, and CD80 geometric MFI in splenic dendritic cells, i.e., CD8+ DC and CD11b+ DC, in mice receiving M-AAC-HPV compared to mice receiving MC vehicle. Results from two independent experiments demonstrated a statistically significant increase in selective MHC-II geometric MFI in splenic CD8+ DC and RPM in mice receiving M-AAC-HPV compared to mice receiving MC vehicle.

[0446] This study demonstrated that immunization of mice with the murine prototype M-AAC-HPV activated splenic APCs, including CD8+ DCs, CD11b+ DCs, and RPMs in vivo. Upregulation of costimulatory markers, markers for maturation (CD86, CD83, CD40, CD80, and MHC-II), was observed in various APC populations 14-16 hours after intravenous (IV) administration of M-AAC-HPV, but not in mice that received SQZ-treated mouse RBCs (MC vehicle) without any antigen or adjuvant. Example 9 In vivo immunization of mice with M-AAC-HPV to measure CD8+ T cell-intrinsic responses assessed by flow cytometric ICS - Effect of antigen and adjuvant

[0447] The requirement for antigen and adjuvant in priming E7-specific CD8+ T cell responses in mice was examined using intracellular cytokine staining (ICS) for IFNγ.

[0448] Collection of mouse blood and isolation of RBC were performed as described above.RBC were resuspended and SQZ-treated with PBS solution containing either E7 SLP alone (to generate M-AC) or poly I:C alone (to generate MC-poly I:C) or solution containing both E7 SLP and poly I:C (to generate M-AAC-HPV) using the SQZ-treatment conditions described in report SQZ-AAC-0126.

[0449] Table 11 shows the study design to evaluate the requirement for antigen (E7 SLP) and adjuvant (Poly I:C) in SQZ-treated red blood cells (RBCs) to induce E7-specific CD8+ T cell responses in vivo in female C57BL / 6J mice. The day of animal sacrifice is the day that intracellular cytokine staining (ICS) was performed on splenocytes. [Table 11] ICS = intracellular cytokine staining; M-AAC-HPV = mouse prototype of AAC-HPV (mouse RBCs SQZ-treated with mouse E7 SLP and poly I:C); M-AC = mouse RBCs SQZ-treated with antigen alone (absence of antigen); MC-Poly I:C = mouse RBCs SQZ-treated with adjuvant alone (absence of antigen); NA = not applicable; PBS = phosphate buffered saline; RO = retro-orbital (method of IV administration)

[0450] On the days indicated in Table 11 above for ICS, mice were sacrificed, spleens were harvested, and cells were isolated for analysis. E7-specific CD8+ T cell responses are measured by assessing the percentage of CD8+ T cells that produce IFNγ when restimulated with E7 minimal epitope peptide.

[0451] The magnitude of the E7-specific CD8+ T cell response is shown in Figure 19. The percentage of IFNγ-producing CD8+ T cells is greatest when the SQZ-treated cells used for immunization contain both adjuvant (poly I:C) and antigen (E7 SLP). The percentage of CD8+ T cells producing IFNγ when restimulated with E7 minimal epitope peptide in animals receiving M-AAC-HPV was significantly greater (p<0.0001) than in animals receiving SQZ-treated RBCs prepared with adjuvant alone (MC-poly I:C) or antigen alone (M-AC) (average 0.60% for M-AAC-HPV compared to 0.02% for MC-poly I:C and 0.03% for M-AC).

[0452] Mice treated with M-AAC-HPV elicited significant E7-specific CD8+ T cell responses that were dependent on the presence of antigen (E7) and adjuvant (poly I:C). Example 10 Assessment of CD8+ T cell-intrinsic responses in mice following M AAC-HPV challenge - dose response

[0453] The aim of this study was to evaluate the effect of increasing doses (5 × 10 7 , 1×10 8 , 2.5×10 8 , 5×10 8 , and 1 × 10 9 The purpose of this study was to investigate the efficacy of M-AAC (HPV / mouse).

[0454] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0455] Table 12 shows the design of the study to evaluate different doses of M-AAC-HPV in vivo. Female C57BL / 6J mice were used for the study. The day of animal sacrifice is the day intracellular cytokine staining (ICS) was performed on splenocytes. [Table 12] M stands for million; B stands for billion; NA stands for "not applicable"; RO: retro-orbital (method of IV administration); ICS = intracellular cytokine staining

[0456] On the days indicated in the table above for ICS, mice were sacrificed, spleens were harvested, and cells were isolated for analysis. E7-specific CD8+ T cell responses are measured by assessing the percentage of CD8+ T cells that produce IFNγ when restimulated with E7 minimal epitope peptide. The magnitude of the E7-specific CD8+ T cell responses shown in Figure 20 demonstrates the effect of a range of doses of M-AAC-HPV.

[0457] As can be seen in Figure 20, the magnitude of the E7-specific CD8+ T cell IFN response is dependent on the dose of M-AAC-HPV. As the dose of M-AAC-HPV increases, the response increases, as does the percentage of CD8+ T cells that produce IFNγ when restimulated with the E7 short peptide, increasing by 5x10 compared to the mean value for the 0.02% PBS control. 7 The mean dose of M-AAC-HPV / mouse was 0.10% to 1 × 10 9 The effect of HPV infection on the immune system increased to a mean value of 0.63% for M-AAC-HPV / mouse at doses of 100 mg / mouse. 7 M-AAC-HPV / mouse ~1 × 10 9 M-AAC-HPV / mouse), the response was 2.5 × 10 8A plateau appeared in M-AAC-HPV / mouse.

[0458] Mice treated with M-AAC-HPV elicited significant CD8+ T cell IFNγ responses, the magnitude of which was dependent on the M-AAC-HPV dose. Example 11 Effect of in vivo immunization-boosting schedules in mice using M-AAC-HPV to measure E7-specific CD8+ cells in blood as assessed by tetramer staining

[0459] The objective was to determine the effect of boosting mice with M-AAC-HPV on the magnitude of the endogenous E7-specific CD8+ T cell response as measured in blood using tetramer staining.

[0460] Table 13 shows the design of a study to evaluate the effect of administering an additional booster dose of M-AAC-HPV to female C57BL / 6J mice on the magnitude of E7-specific CD8+ T cell responses. E7-specific CD8+ T cells were measured by staining cells in whole blood with MHC class I tetramers that bind to the T cell receptor (TCR) specific for the E7 immunodominant epitope in mice (E749-57 - RAHYNIVTF) and assessing the percentage of E7 tetramer+ CD8+ T cells in whole blood by flow cytometry. [Table 13] NA stands for "not applicable"; RO: retroorbital (method of IV administration)

[0461] The magnitude of the E7-specific CD8+ T cell response at various time points after the final immunization is shown in Figures 21, 22, and 23.

[0462] In this study, we analyzed the CD8+ T cell response to E7 over time using total CD8 + Compared with T cells, activated (CD44 hi )E7-tetramer positive CD8 +As can be seen in Figures 21-23, administration of a single dose of M-AAC-HPV significantly reduced E7 tetramer counts in whole blood in the prime-only group compared to the PBS control (range 0.02%-0.07%, average 0.03%). + CD8 + As evidenced by an increase in E7-specific CD8 T cells (range 0.22%-0.44%, mean 0.28%) + Although the E7 tetramer primed T cell responses, this difference did not reach statistical significance. + CD8 + The percentage of T cells was significantly increased by the boost given either 2 or 6 days after the prime compared to animals that received only the priming dose.

[0463] Maximum E7 tetramers for all groups + CD8 + T cell responses were observed approximately one week after the final immunization. E7-specific CD8 + The percentages of T cells ranged from 0.02% to 0.07% (mean 0.03%) for the PBS control group, 0.22% to 0.44% (mean 0.28%) for animals in the prime alone group, 0.47% to 1.27% (mean 0.79%) for animals boosted on day 2, and between 0.68% to 1.30% (mean 0.98%) for animals boosted on day 6. By approximately 2 weeks after the final immunization, E7-specific CD8+ T cell responses in animals that received a single priming dose (range 0.03% to 0.11%) were not significantly different from PBS controls (range 0.00% to 0.03%). In contrast, E7-specific CD8+ T cell responses in animals boosted on either day 2 (range: 0.21% to 0.6%) or day 6 (range 0.76% to 1.08%) were significantly different from PBS controls (range 0.00% to 0.03%). + T cell responses remained elevated and were significantly greater than those in the prime alone animals. Furthermore, animals boosted on day 6 also harbored significantly more E7-specific CD8 + had T cells.

[0464] The studies herein demonstrate that immunization with M-AAC-HPV primes E7-specific CD8+ T cell responses in the blood in vivo. 6 Mice were administered 250 × 10 6 The booster dose of M-AAC-HPV resulted in a significant and sustained increase in E7-specific CD8+ T cell responses compared to both the PBS control group and the prime-only group. Example 12 In vivo determination of efficacy in mice following therapeutic immunization with intravenously administered M-AAC-HPV in the TC-1 tumor model - antigen requirements

[0465] The objective of this study was to compare the efficacy of a single intravenous administration of the same dose of MC-Poly I:C (mouse RBCs (no antigen) SQZ-treated with Poly I:C) at 250 × 10 per mouse administered intravenously compared with intravenous PBS administration. 6 M-AAC-HPV or 1 × 10 9 The aim of the study was to evaluate the requirement for an antigen that inhibits tumor growth and extends median survival in a therapeutic TC-1 tumor model following a single vaccination with M-AAC-HPV.

[0466] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described in report number SQZ-AAC-0126. MC-poly I:C was generated equivalently using SQZ treatment of mouse RBCs with poly I:C.

[0467] The following tables show the study design for evaluating the effect of antigen in tumor studies (Table 14 and Table 15). Mice (female C57BL / 6J) were injected subcutaneously (SC) with TC-1 tumor cells (50,000 cells) on day 0. Mice were treated with test substances by retro-orbital administration on the days listed in Table 14 and Table 15. Survival was monitored daily and tumor growth was measured twice a week. [Table 14] NA stands for "not applicable." "M" stands for one million. [Table 15] NA stands for "not applicable." "B" stands for billions.

[0468] Summary tumor growth data from two independent experiments (ELN103212 and ELN1416) are shown in FIG. 23, and summary survival data are shown in FIG. 25 and Table 16. [Table 16]

[0469] As shown in Figures 24A and 24B, 6 or 1×10 9 A dose of M-AAC-HPV administered intravenously significantly slowed the rate of tumor growth in both studies. Administration of an equal dose of the antigen-free adjuvant carrier MC-Poly I:C did not slow tumor growth compared to the PBS control.

[0470] Mice treated with either dose of M-AAC-HPV showed statistically significant increased survival compared to control-treated mice, as seen in Figure 25 and Table 16. Survival was 250 x 10 6 38 to 53 days in mice treated with M-AAC-HPV (study ELN103212), and 1 × 10 9 The range was 38–48 days in mice treated with M-AAC-HPV (study ELN1416). 6 In mice treated with MC-Poly I:C, survival ranged from 26 to 45 days and 26 to 34 days, respectively (ELN103212). In a second study (ELN1416), survival was significantly longer than in mice treated with PBS and 1 × 10 9 For MC-Poly I:C treated mice, it ranged from 27 to 34 days, and from 27 to 41 days.

[0471] 250×10 6 or 1×10 9 Treatment of mice with intravenously administered doses of M-AAC-HPV resulted in significantly delayed tumor growth compared to control-treated mice. In addition, M-AAC-HPV-treated mice showed statistically significant extended survival at both doses. In contrast, mice treated with MC-Poly I:C did not show improved tumor growth delay or extended survival compared to controls, regardless of MC-Poly I:C dose.

[0472] These data support the requirement for the presence of antigen for efficacy in the therapeutic TC-1 model. Example 13 In vivo determination of therapeutic efficacy in mice following therapeutic immunization with intravenously administered M-AAC-HPV in the TC-1 tumor model - dose response

[0473] The purpose of this study was to evaluate the efficacy and safety of escalating doses (50 × 10) of M-AAC-HPV administered intravenously in the TC-1 mouse tumor model. 6 , 100×10 6 , 250×10 6 , and 1 × 10 9 The objective of this study was to evaluate the antitumor activity of M-AAC-HPV / mouse.

[0474] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0475] The following tables show the study design for evaluating different doses of M-AAC-HPV in tumor studies (Tables 17-20). Mice (female C57BL / 6J) were injected subcutaneously (SC) with TC-1 tumor cells (50,000 cells) on day 0. On day 10 of the study, mice were treated with test substances by retro-orbital administration. Survival was monitored daily and tumor growth was measured twice weekly. [Table 17] NA stands for "not applicable" [Table 18] NA stands for "not applicable" [Table 19] NA stands for "not applicable" [Table 20] NA stands for "not applicable"

[0476] Summary tumor growth data from four independent experiments is shown in FIG. 26, and summary survival data is shown in FIG. 27 and Table 21. [Table 21-1] NA stands for "not applicable." Group was not included in the study. Statistical significance of M-AAC-HPV group at a given dose compared to PBS group is shown, * represents p<0.05, *** represents p<0.001, **** represents p<0.0001.

[0477] These studies demonstrate that intravenous immunization of mice with M-AAC-HPV therapeutically inhibits tumor growth and prolongs survival in the HPV-16 E6 and E7 expressing TC-1 mouse tumor model. The ability of M-AAC-HPV to inhibit TC-1 tumor growth and prolong survival of tumor-bearing mice is dependent on the dose of M-AAC-HPV. Specifically, at doses of 1 × 10 per mouse, 9 or 250×10 6 Mice treated with intravenously administered M-AAC-HPV doses of 100 × 10 showed delayed tumor growth compared to mice receiving PBS in all studies (4 out of 4) using the TC-1 model. 6Mice receiving the M-AAC-HPV dose showed delayed tumor growth compared to mice receiving PBS in most studies (2 of 3). Furthermore, there was a significant increase in median survival time of 1×10 9 Four of four studies by dose, 250 × 10 6 Three of the four studies by dose, and 100 × 10 6 In one of three dose studies, inhibition of tumor growth and extension of median survival were both observed at doses of 50 × 10 6 was not observed in mice receiving doses (0 of 2 studies). Example 14 In vivo determination of efficacy in mice following therapeutic immunization with intravenously administered M-AAC-HPV in the TC-1 tumor model - effect of boosting regimens

[0478] The objective of this study was to compare the efficacy and safety of 100 × 10 per mouse with that of a single dose (prime only) at the same dose in the TC-1 tumor model. 6 or 250×10 6 The objective of this study was to evaluate the antitumor efficacy of two doses (prime and boost) of M-AAC-HPV administered intravenously.

[0479] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0480] The following tables show the study design for evaluating different doses of M-AAC-HPV in tumor studies (Table 22 and Table 23). Mice (female C57BL / 6J) were injected subcutaneously (SC) with TC-1 tumor cells (50,000 cells) on day 0. Mice were treated with test articles by retro-orbital administration according to the schedules described in Table 22 and Table 23. [Table 21-2] NA stands for "not applicable" [Table 22] NA stands for "not applicable"

[0481] Summary tumor growth data from two independent experiments are shown in Figure 28. Summary survival data are shown in Figure 29 and Table 24. [Table 24-1]

[0482] These studies demonstrate that two intravenous administrations of M-AAC-HPV (prime and boost) can result in slower tumor growth in the TC-1 tumor model compared with administration of prime alone, without an effect on median survival. Mice treated with prime and boost on day 2 had a 100 × 10 6 M-AAC-HPV at a dose of AAC / mouse showed statistically significantly slower tumor growth in one of two studies and a possible trend toward slower growth in the second study. There was no statistical difference at this dose level in median survival observed in two of two studies for prime and boost compared to prime alone. 6 Mice treated with a prime and day 2 boost of M-AAC-HPV at a dose of AAC / mouse showed statistically significantly slower tumor growth when compared to prime alone in one of two studies, with a possible trend toward slower growth in the second study. There was no statistical difference in median survival data at this dose level in two of two studies for prime and boost compared to prime alone. Finally, at 100 × 10 per mouse 6 or 250×10 6 Treatment of mice with prime or prime and boost of M-AAC-HPV can delay tumor growth and extend median survival compared to control PBS-treated mice. Example 15 In vivo immunization of TC-1 tumor-bearing mice with M-AAC-HPV to measure recruitment of E7-specific CD8+ TILs

[0483] The aim of this study was to quantify E7-specific CD8+ T cells in the tumor microenvironment of TC-1 tumors 12 days after intravenous immunization with M AAC-HPV.

[0484] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0485] Table 25 shows the design of the study used to quantify E7-specific CD8+ T cells in TC-1 tumors after M-AAC-HPV administration. Mice (female C57BL / 6J; 5 per group) were subcutaneously injected with TC-1 tumor cells (50,000 cells) on day 0. On day 14 (ELN68 study) or day 13 (ELN221 study) of the study, mice were immunized with the test substances listed in Table 25. Tumor volume and survival were monitored until the day before sacrifice (day 24 or 25). Mice were sacrificed 12 days after test substance administration (day 26 for ELN68 and day 25 for ELN221), and tumors were removed for enzymatic processing into single cell suspension. Tetramer staining was performed on the cell suspension to determine the percentage of infiltrating CD8+ T cells specific for E7 by flow cytometry. [Table 24-2] NA stands for "not applicable"

[0486] Figure 30 shows the CD8 + Intratumoral percentage of T cells, E7 tetramers among total viable cells + Percentage of cells, and CD8 + E7 tetramers in T cell populations + Percentage of cells expressed as total CD8 normalized to tumor mass +T cells and E7-specific CD8 + T cells are shown in Figure 31. Tumor growth summary data for mice sacrificed for tumor harvest is shown in Figure 32.

[0487] As seen in FIG. 30, mice immunized with M-AAC-HPV had significantly lower CD8+ in tumors compared to PBS-treated controls 12 days after immunization in ELN68 and ELN221, respectively. + There was a 12.8-fold and 20.8-fold increase in the mean percentage of CD8+ T cells in the M-AAC-HPV-treated animals. In the M-AAC-HPV-treated animals, the majority of these CD8+ T cells were specific for the E7 antigen as determined by tetramer staining (76.6±12.6% in ELN68 and 86.2±7.9% in ELN221 of the CD8+ T cell population). These data suggest that immunization with M-AAC-HPV significantly increased the number of E7-specific CD8+ T cells in the tumor microenvironment 12 days after immunization compared to PBS treatment. + The results demonstrate that the mean percentage of E7-specific CD8 T cells was significantly increased (209.3-fold increase in ELN68 and 71.2-fold increase in ELN221). + This increase in T cells was also noted when converted to cell numbers normalized to tumor mass. + The increase in T cells suggests that M-AAC-HPV reduces tumor burden by expanding E7-specific effector CD8+ T cells.

[0488] These studies demonstrated that intravenous immunization with M-AAC-HPV in the TC-1 mouse tumor model increased tumor-infiltrating E7-specific CD8 + We demonstrated that M-AAC-HPV resulted in a significant increase in T cells, an observation consistent with the proposed mechanism of action for M-AAC-HPV. (Example 16) In vivo serum cytokine / chemokine analysis in mice after repeated intravenous administration of M-AAC-HPV as measured by Luminex analysis

[0489] The objective of this study was to measure serum cytokines / chemokines in mice at various time points following intravenous immunization with 1, 2, 3, 4 or 5 doses of M-AAC-HPV compared to control PBS-injected mice.

[0490] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0491] Table 27 shows the design of a study to evaluate serum cytokine / chemokine concentrations in C57BL / 6J female mice immunized with up to five doses of M-AAC-HPV. Analysis of cytokine / chemokine concentrations using the Milliplex assay was performed after serum collection at all time points shown in FIG. [Table 27-1] B represents boost; all intravenous administration was by retro-orbital (RO) route. a Serum was obtained via puncture of the submandibular vein. b Serum was obtained by terminal cardiac puncture.

[0492] Analytes included in the Milliplex assay are: G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, MIP-2, RANTES and TNF-α. Table 28 is a summary table listing key findings for any cytokine / chemokine that showed a statistical difference in at least one time point compared to the corresponding PBS time point.

[0493] Four chemokines, IP-10, MIP-1β, MCP-1 and RANTES, demonstrated a transient but significant consistent increase in serum concentrations 1 day after the final immunization in all groups with one exception. The fold change of MIP-1β relative to pre-immunization values ​​in one of the groups (P / B3) was not significantly different between M-AAC-HPV and PBS controls at any time point throughout the course of the study (p=0.46). IP-10 concentrations remained elevated in all groups until 4 days after immunization. By day 7, the fold change (relative to pre-immunization values) of all cytokines / chemokines in the M-AAC-HPV group was no longer significantly elevated compared to the changes observed in the PBS control, with the exception of IL-12p70. The fold change in IL-12p70 remained significantly higher in one of the five groups that received M-AAC-HPV (the prime alone group) than that observed in the corresponding PBS group at day 7. However, the change in IL-12p70 relative to pre-immune values ​​in mice that received M-AAC-HPV in the prime alone group (range 0.43-2.05) was within the range observed in the corresponding PBS group throughout the study (range 0.43-2.37). Concentrations of other cytokines / chemokines that were elevated were not consistently significantly elevated, although sporadic statistically significant increases were observed for some other cytokines / chemokines. These included GM-CSF, IL-7, IL-12p40, IL-12p70, IL-13, KC, and MIP-1α.

[0494] Adjuvants such as poly I:C have previously been shown to be activators of the innate immune system leading to the secretion of chemokines including IP-10, MIP-1β, MCP-1 and RANTES by various cell types (Longhi 2015; De Waele 2018). These particular chemokines have also been shown to be important for the migration of CD4+ and CD8+ T cells to antigen-presenting cell (APC)-rich areas of secondary lymphoid organs such as the spleen (reviewed in Sokol 2015). Furthermore, they have been shown to promote clustering and the formation of stable contacts between T cells and APCs, thereby facilitating productive activation and differentiation of naive T cells into effector T cells. Thus, the early and transient increase in serum levels of IP-10, MIP-1β, MCP-1 and RANTES likely indicates early activation of innate immune cells by M-AAC-HPV.

[0495] Other analytes measured in this study, including G-CSF, IFN-γ, IL-1β, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-15, IL-17, MIP-2, and TNF-α, showed no significant changes in all M-AAC-HPV groups compared to PBS-treated subjects at all time points.

[0496] This study demonstrates that intravenous administration of M-AAC-HPV generally resulted in a transient but significant increase in serum concentrations of IP-10, MIP-1β, MCP-1, and RANTES compared with the respective PBS controls, beginning 1 day after the final immunization. Changes in concentrations of all analytes relative to pre-immunization values ​​were comparable between mice receiving M-AAC-HPV and mice receiving PBS until day 7 after the final vaccination. [Table 27-2] [Table 27-3] (Example 17) In vivo determination of RBC clearance in mice following repeated IV administration of M-AAC-HPV

[0497] The purpose of this study was to determine whether five repeated doses of M-AAC-HPV would result in an immune response against components of RBCs, thereby resulting in accelerated clearance of subsequent doses of intact RBCs. This study was conducted because previous studies in mice have shown that adjuvants such as polyinosinic-polycytidylic acid (Poly I:C) administered intravenously at the time of intravenous blood administration can induce or enhance immune responses against surface RBC antigens (Gibb 2017). Such responses have been shown to result in accelerated clearance of RBCs upon subsequent intravenous administration (Stowell SR 2014).

[0498] Methods for isolating mouse RBCs and SQZ treatment of RBCs with E7 SLP and poly I:C to generate the mouse prototype of AAC-HPV (M-AAC-HPV) are described above.

[0499] Table 29 shows the design of a study to evaluate the circulatory dynamics of intravenously administered untreated (not SQZ treated) syngeneic RBCs (labeled with PKH26) in female C57BL / 6J mice immunized with 5 doses of M-AAC-HPV. Analysis of circulating RBCs was performed immediately after blood collection at the time points indicated in Table 29 by flow cytometry for PKH26 labeled RBCs. [Table 29] All intravenous administration was by the retro-orbital (RO) route. M-AAC-HPV = murine prototype of AAC-HPV (mouse E7 SLPs and mouse RBCs SQZ-treated with poly I:C); PBS = phosphate-buffered saline

[0500] The percentage of labeled RBCs in whole blood at various time points after intravenous administration is shown in FIG.

[0501] This study evaluated the effect of repeated dosing of M-AAC-HPV on the induction of an immune response to syngeneic RBCs by measuring the clearance of intravenously administered labeled RBCs. Labeled RBCs were administered 1 week after the fifth and final immunization of M-AAC-HPV to allow sufficient time for the development of an immune response. After intravenous administration, labeled RBCs were monitored for a period of 48 hours. There were no statistical differences between the two groups at all time points, except at the 2 hour time point. At the 2 hour time point, the value of the percentage of PKH26-labeled RBCs in whole blood for M-AAC-HPV was 6.05% (range: 5.86-6.21%) compared to 6.41% (range: 5.87-7.11%) for PBS, a difference that is within expected inter-animal variation and not physiologically significant.

[0502] This study demonstrates that intravenous administration of five doses of M-AAC-HPV does not result in an immune response capable of causing accelerated clearance of syngeneic RBCs, as evidenced by comparable circulation of intravenously administered syngeneic labeled RBCs up to 48 hours post-dose when compared to five doses of PBS control. [Table 30-1] [Table 30-2] [Table 30-3] [Table 30-4]

Claims

1. 1. A composition for treating a human papillomavirus (HPV)-associated cancer in an individual, the composition comprising an activating antigen carrier (AAC), wherein an effective amount of the composition is administered to the individual, the effective amount being about 0.1×10 ８ AAC / kg to about 1 x 10 ９ AAC / kg, said AAC comprising at least one HPV antigen to be delivered intracellularly and an adjuvant.

2. A composition for treating a human papillomavirus (HPV) associated cancer in an individual, the composition comprising an activating antigen carrier (AAC), wherein an effective amount of the composition is administered to the individual, the AAC comprising at least one HPV antigen delivered intracellularly and an adjuvant; The composition is administered to the individual in combination with an effective amount of a CTLA-4 antagonist and / or a PD-1 / PD-L1 antagonist. composition. (a) the CTLA4 antagonist is an antibody that binds to CTLA4; and / or (b) the PD-1 / PD-L1 antagonist is an antibody that binds to PD-1 or an antibody that binds to PD-L1; The composition of claim 2. (a) the antibody that binds to CTLA-4 is ipilimumab, and / or (b) the antibody that binds to PD-1 is nivolumab or pembrolizumab, and / or (c) the antibody that binds to PD-L1 is atezolizumab; The composition of claim 3.

5. The adjuvant is (a) 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 TLR9 agonist; or (b) CpG 7909 oligodeoxynucleotide (ODN) The composition according to any one of claims 1 to 4, (a) the individual is a human; and / or (b) the individual is positive for HLA-A*02, and / or (c) the AAC is autologous or allogeneic to the individual; The composition according to any one of claims 1 to 5.

7. The composition of any one of claims 1 to 6, wherein the HPV-associated cancer is a recurrent, locally advanced or metastatic cancer.

8. The composition according to any one of claims 1 to 7, characterized in that the composition comprising AAC is administered intravenously.

9. The composition according to any one of claims 2 to 8, characterized in that the CTLA-4 antagonist and / or the PD-1 / PD-L1 antagonist are administered intravenously, orally or subcutaneously.

10. The composition according to any one of claims 3 to 9, wherein the antibody that binds to CTLA-4 and / or the antibody that binds to PD-1 and / or the antibody that binds to PD-L1 is administered intravenously. (a) the effective amount of the AAC comprising the at least one HPV antigen and the adjuvant is about 0.5×10 ８ AAC / kg to about 1 x 10 ９ AAC / kg, (b) the effective amount of AAC comprising the at least one HPV antigen and the adjuvant is from about 0.5×10 8 AAC / kg to about 7.5×10 8 AAC / kg; (c) the effective amount of AAC comprising the at least one HPV antigen and the adjuvant is about 0.5×10 8 AAC / kg, about 2.5×10 8 AAC / kg, about 5×10 8 AAC / kg, or about 7.5×10 8 AAC / kg; The composition according to any one of claims 1 to 10.

12. (a) the effective amount of ipilimumab is from about 1 mg / kg to about 3 mg / kg, and / or (b) the effective amount of nivolumab is about 360 mg; and / or (c) the effective amount of atezolizumab is about 1200 mg; The composition according to any one of claims 4 to 11.

13. The composition comprising the AAC, (a) delivered on day 1 of a three-week cycle; and / or (b) further administered on day 2 of the first 3-week cycle; and / or (c) administered to the individual for at least about three months, six months, nine months, or one year. The composition according to any one of claims 1 to 12. Claim 14: (a) (i) Approximately 0.5×10 ８ Individual cells / kg ~ approx. 1 x 10 ９ (ii) about 0.5×10 8 cells / kg, about 2.5×10 8 cells / kg, about 5.0×10 8 cells / kg, or about 7.5×10 8 cells / kg are administered on day 1 of each three-week cycle; and / or (b) (i) about 0.5×10 8 cells / kg to about 1×10 9 cells / kg are administered on day 2 of each three-week cycle; or (ii) about 0.5×10 8 cells / kg, about 2.5×10 8 cells / kg, about 5.0×10 8 cells / kg, or about 7.5×10 8 cells / kg are administered on day 2 of said first three-week cycle. The composition of claim 13.

15. The composition according to claim 13 or 14, characterized in that the antibody that binds to CTLA-4 and / or the antibody that binds to PD-1 and / or the antibody that binds to PD-L1 is administered once per three-week cycle.

16. An antibody that binds to CTLA-4, (a) administered once per two three-week cycles; and / or (b) administered on day 1 of each three-week cycle or on day 1 of two three-week cycles; and / or (c) ipilimumab, wherein the ipilimumab is administered at a dose of about 3 mg / kg.

15. The composition according to claim 13 or 14.

17. An antibody that binds to PD-1, (a) administered on day 8 of an initial 3-week cycle and on day 1 of each subsequent cycle; and / or (b) nivolumab, wherein the nivolumab is administered at a dose of about 360 mg. The composition according to any one of claims 13 to 16.

18. The composition of any one of claims 15 to 17, wherein the antibody that binds CTLA-4 is ipilimumab, and wherein the ipilimumab is administered at a dose of about 1 mg / kg on day 1 of a first 3 week cycle of two 3 week cycles, and the antibody that binds PD-1 is administered at a dose of about 360 mg on day 8 of the first 3 week cycle and on day 1 of each subsequent cycle.

19. An antibody that binds to PD-L1, (a) administered on day 8 of an initial 3-week cycle and on day 1 of each subsequent cycle; and / or (b) atezolizumab, wherein the atezolizumab is administered at a dose of about 1200 mg. The composition according to any one of claims 13 to 15.

20. The composition comprising AAC comprises: (i) about 1×10 ９ AAC ~ approx. 1×10 １０ 20. The composition of any one of claims 1 to 19, comprising AAC; or (ii) about 7 x 10 9 PBMCs in about 10 mL of cryopreservation medium, optionally wherein the cryopreservation medium is Cryostor® CS2.

21. The AAC comprising the at least one HPV antigen and an adjuvant, a) passing a cell suspension containing a population of input anucleated material through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input anucleated cells in the suspension, thereby causing a perturbation of the input anucleated cells large enough to allow the at least one HPV antigen and the adjuvant to pass through to form perturbed input anucleated cells; and b) incubating said population of perturbed input anucleated cells with said at least one HPV antigen and said adjuvant for a time sufficient to allow said antigen to enter said perturbed input anucleated cells, thereby generating said AAC comprising said at least one HPV antigen and said adjuvant. The composition of any one of claims 1 to 20, prepared by a process comprising:

22. 22. The composition of claim 21, wherein the diameter of the constriction is from about 1.6 μm to about 2.4 μm or from about 1.8 μm to about 2.2 μm.

23. 23. The composition of claim 21 or 22, wherein the input anucleated cells are red blood cells.

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