WO2022147442A1 - Formulations of activating antigen carriers - Google Patents

Formulations of activating antigen carriers Download PDF

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Publication number
WO2022147442A1
WO2022147442A1 PCT/US2021/073142 US2021073142W WO2022147442A1 WO 2022147442 A1 WO2022147442 A1 WO 2022147442A1 US 2021073142 W US2021073142 W US 2021073142W WO 2022147442 A1 WO2022147442 A1 WO 2022147442A1
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Prior art keywords
aacs
formulation
antigen
adjuvant
formulation comprises
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PCT/US2021/073142
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English (en)
French (fr)
Inventor
Howard Bernstein
Defne YARAR
Katarina BLAGOVIC
Amritha RAMAKRISHNAN
Maisam DADGAR
Louise CLEAR
Jason Murray
Tarek ABDELJAWAD
Claire PAGE
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SQZ Biotechnologies Co
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SQZ Biotechnologies Co
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Priority to CN202180094095.6A priority Critical patent/CN116847880A/zh
Priority to CA3203709A priority patent/CA3203709A1/en
Priority to JP2023539809A priority patent/JP2024503277A/ja
Priority to EP21851939.5A priority patent/EP4271407A1/en
Publication of WO2022147442A1 publication Critical patent/WO2022147442A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6006—Cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates generally to formulations of activating antigen carriers (AACs) comprising at least one antigen and an adjuvant, and a cryopreservation medium. Also provided are methods of manufacturing such AACs comprising the at least one antigen and the adjuvant, methods of formulating the cryopreservable formulation, and methods of cryopreserving the formulation.
  • AACs activating antigen carriers
  • Papillomaviruses are small nonenveloped DNA viruses with a virion size of ⁇ 55 nm in diameter. More than 100 human papillomavirus (HPV) genotypes are completely characterized, and a higher number is presumed to exist. HPV is a known cause of cervical cancers, as well as some vulvar, vaginal, penile, oropharyngeal, anal, and rectal cancers. Although most HPV infections are asymptomatic and clear spontaneously, persistent infections with one of the oncogenic HPV types can progress to precancer or cancer.
  • HPV-associated diseases can include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermodysplasia, focal epithelial hyperplasia, mouth papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (SILs), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VEN) and vaginal intraepithelial neoplasia (VAIN).
  • SILs squamous intraepithelial lesions
  • CIN cervical intraepithelial neoplasia
  • VEN vulvar intraepithelial neoplasia
  • VAIN vaginal intraepithelial neoplasia
  • HPV 16 and 18 have benign lesions with a subset being oncogenic. Based on epidemiologic and phylogenetic relationships, HPV types are classified into fifteen “high-risk types” (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three “probable high-risk types” (HPV 26, 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 vulval, vaginal, penile, anal, and perianal cancer, as well as head and neck cancers. Recently, the association of high risk types HPV 16 and 18 with breast cancer was also described.
  • HPV 6 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 depressed and correspondingly, the antitumor response is significantly impaired. See Suresh and Burtness Am J Hematol Oncol 13(6):20-27 (2017).
  • Immunotherapy can be divided generally 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.
  • Several current active protocols include vaccination strategies with disease-associated peptides, lysates, or allogeneic whole cells, infusion of autologous dendritic cell (DCs) as vehicles for tumor antigen delivery, and infusion of immune checkpoint modulators. See Papaioannou, Nikos E., el al. Annals of translational medicine 4.14 (2016).
  • Adoptive immunotherapy can be employed to modulate the immune response, enhance antitumor activity, and achieve the goal of treating or preventing HPV-associated cancers.
  • CD8 + cytotoxic T lymphocytes (CTL) and CD4 + helper T (Th) cells stimulated by disease-associated antigens have the potential to target and destroy diseased cells; however, current methods for inducing endogenous T cell responses have faced challenges.
  • the formulations and vials described herein comprise AACs in cryopreservation medium that can be manufactured, stored and transported without loss in functionality, before administration to individuals in need thereof.
  • the methods described herein are used to efficiently generate AACs, which may be anucleate cells or anucleate cell-derived entities comprising at least one HPV antigen and an adjuvant, in a high throughput manner, which can be utilized in inducing a robust T cell response to the HPV antigens.
  • the disclosure herein also describes methods, treatments, doses and regimens for treating individuals with HPV-associated cancers using AACs comprising HPV antigens and adjuvants.
  • All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
  • the patent publications WO 2013/059343, WO 2015/023982, WO 2016/070136, W02017041050, W02017008063, WO 2017/192785, WO 2017/192786, WO 2019/178005, WO 2019/178006, WO 2020/072833, WO 2020/154696, and WO 2020/176789, US 20180142198, and US 20180201889 are hereby expressly incorporated by reference in their entirety.
  • the invention provides a pharmaceutical formulation comprising activating antigen carriers (AACs), the formulation comprising a) AACs wherein the AACs comprise at least one antigen and an adjuvant, and b) a cryopreservation medium.
  • AACs activating antigen carriers
  • the formulation comprises about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs.
  • the formulation comprises about 7x 10 9 AACs.
  • the formulation comprises about 7x 10 9 AACs prior to freezing.
  • the formulation comprises about 9 x 10 9 AACs after thawing.
  • the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing as measured by flow cytometry. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 0.7 x 10 9 AACs/mL after thawing as measured by Coulter counter.
  • At least about 70%, 80%, 90%, or 95% of AACs of a population of the AACs in the formulation are functional.
  • the AACs in the formulation maintain equal to or greater than about 70% functionality.
  • the formulation comprises about 1 x 10 8 functional AACs /mL to about 1 x 10 9 functional AACs/mL.
  • wherein at least about 70%, 80%, 90%, or 95% of AACs of a population of the AACs are positive for annexin staining.
  • the AACs in the formulation maintain equal to or greater than about 70% are positive for annexin staining.
  • the annexin is annexin V.
  • the cryopreservation medium in the formulation comprises dimethylsulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises about 0.5% to about 5% DMSO. In some embodiments, the cryopreservation medium comprises about 2% DMSO. In some embodiments, the cryopreservation medium is CryoStor® CS2.
  • the pH of the formulation is about 6.0 to about 8.5. In some embodiments, the pH of the formulation is about 7.6.
  • the invention provides a pharmaceutical formulation of AACs, the formulation comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • the formulation comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • formulation comprises about 7x 10 9 AACs prior to freezing.
  • the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x 10 9 cells after thawing. In some embodiments, the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing.
  • the formulation comprising about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the cryopreservation medium is CryoStor® CS2.
  • the formulation is sterile.
  • the formulation comprises less than about 2 EU/mL endotoxin.
  • the formulation is free of mycoplasma.
  • the AACs of the formulation comprises the at least one human papillomavirus (HPV) antigen.
  • the HPV antigen is a HPV-16 antigen or a HPV-18 antigen.
  • the antigen comprises a peptide derived from HPV E6 and/or E7. In some embodiments, wherein the antigen comprises a peptide derived from HPV E6 and a peptide from HPV E7.
  • the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25.
  • the AACs 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.
  • the AACs of the formulation comprise an adjuvant, wherein the adjuvant is a CpG oligodeoxynucleotide (ODN), LPS, IFN-a, STING agonists, RIG-I agonists, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR 9 agonist.
  • the adjuvant is a CpG 7909 oligodeoxynucleotide (ODN).
  • the formulation comprising AACs comprising the at least one antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed anucleate cells, thereby generating the AACs comprising the at least one antigen and the adjuvant.
  • the diameter of the constriction is about 1.6 pm to about 2.4 pm or about 1.8 pm to about 2.2 pm.
  • the invention provides a vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 1 x 10 9 AACs to about 1 x 10 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • the invention provides a vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 1 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • the formulation comprises about 7x 10 9 AACs prior to freezing.
  • the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x 10 9 cells after thawing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x 10 9 AACs after thawing as measured by flow cytometry. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 7 x IQ 9 AACs after thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL.
  • the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing as measured by flow cytometry. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 0.7 x 10 9 AACs/mL after thawing as measured by Coulter counter.
  • the invention provides a vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the AACs are in about 9.5 mL of the cryopreservation medium.
  • the pharmaceutical formulation comprises about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the formulation is sterile.
  • the invention provides a method of producing a pharmaceutical formulation of AACs, the method comprising adding a cryopreservation medium to the AACs wherein the AACs comprise at least one antigen and an adjuvant.
  • the invention provides a method of producing a pharmaceutical formulation of AACs, wherein the AACs comprise the at least one antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising a input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the
  • the diameter of the constriction is about 1.6 pm to about 2.4 pm or about 1.8 pm to about 2.2 pm.
  • the AACs are washed about 6 times.
  • the AACs are washed by centrifugation and resuspension or by centrifugation and filtration.
  • the centrifugation is at about 4000 rpm.
  • about 1 x 10 9 AACs to about 1 x 10 10 AACs are formulated in about 9 mL to about 10 mL of the cryopreservation medium.
  • the pharmaceutical formulation comprises about 7* 10 9 AACs prior to freezing.
  • the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 * 10 9 cells after thawing. In some embodiments, the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing. In some embodiments, the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing as measured by flow cytometry. .
  • the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 0.7 x 10 9 AACs/mL after thawing as measured by Coulter counter. In some embodiments, about 7 x io 9 AACs are formulated in about 10 mL of the cryopreservation medium. In some embodiments, the cryopreservation medium is CryoStor® CS2. In some embodiments, the input anucleate cell is a red blood cell.
  • the method further comprises freezing the formulation of AACs at about -170 °C.
  • the formulation of AACs are frozen by a process comprising: a) placing the formulation in a chamber b) reducing the temperature of the chamber to about -3 °C, c) reducing the temperature of the chamber to about -140°C at a rate of about - 20°C/minutes, d) reducing the temperature of the chamber to about -150°C at a rate of about 1.5°C/minutes, e) reducing the temperature of the chamber to about -170°C at a rate of about 1.0°C/minutes, and f) holding the temperature of the chamber at about -170°C for at least about 10 minutes.
  • Fig. 1 shows an assessment of platelet removal and RBC recovery was performed over the LOVO process taken from seven different healthy donors.
  • FIG. 2 shows average overall recovery of RBCs, platelets, and white blood cells of eleven runs over the RBC purification step encompassing cell washing and resuspension with delivery media on the LOVO and purification via the leukoreduction filter.
  • FIG. 3 A shows an increase in Annexin V+ population following passage of red blood cells through different chip configurations at 60 psi and 75 psi operating pressure.
  • FIG. 3B shows delivery of Ova647 following passage of red blood cells through different chip configurations at 60 psi and 75 psi operating pressure.
  • FIG. 4 shows delivery of Ova647 following passage of red blood cells through a chip a different cell concentrations.
  • FIG. 5 shows delivery of F AM-labeled E6 and E7 peptides following passage of red blood cells through a chip a different cell concentrations.
  • FIG. 6 shows delivery of F AM-labeled E7 peptides following passage of red blood cells through a chip at different pressures.
  • FIG. 7 shows a functional analysis as measured by IFNy levels when AACs are diluted or undiluted AACs during a 37 °C rest period.
  • FIG. 8 shows average recovery rates for LOVO processing using two different protocols.
  • FIG. 9 shows LOVO processing to achieve a target concentration of 7 x 10 8 AACs/mL.
  • FIG. 10 shows a representative load of 48 vials of SQZ-AAC-HPV being cryopreserved using the developed protocol.
  • FIG. 11 shows a representative load of 64 vials of SQZ-AAC-HPV being cryopreserved using a two-rack configuration.
  • FIG. 12 shows post thaw counts from five process development batches. The average AAC count across these five batches was 1.03 x io 9 AACs/mL.
  • the present invention provides a pharmaceutical formulation comprising AACs wherein the AACs comprise at least one antigen and an adjuvant, and a cryopreservation medium.
  • the present invention provides a pharmaceutical formulation of Activating Antigen Carriers (AACs), the formulation comprising about 0.5 x 10 9 AACs/mL to about 1 x 1O 10 AACs/mL in a cry opreservation medium, wherein the AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • the formulation comprises 0.7 x 10 9 AACs/mL in a cry opreservation medium.
  • the present invention provides a vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 0.5 x 10 9 AACs/mL to about 1 x IO 10 AACs/mL in a cryopreservation medium (such as but not limited to CryoStor® CS2), wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • a cryopreservation medium such as but not limited to CryoStor® CS2
  • the present invention provides a vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 0.7 x 10 9 AACs/mL in a cryopreservation medium (such as, but not limited to, CryoStor® CS2), wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • a cryopreservation medium such as, but not limited to, CryoStor® CS2
  • formulations and vials comprising AACs comprising at least one antigen and an adjuvant
  • the methods of preparing the formulation of AACs comprising the at least one antigen and adjuvant are prepared by a process comprising: a) passing a cell suspension comprising a population of input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed input anucleate cells; and b) incubating the population of perturbed input anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen to enter the perturbed input anucleate cells, thereby generating the AACs comprising the at least one antigen and the adjuvant
  • the antigen is a HPV antigen.
  • compositions for use in inducing an immune response to HPV antigens or for treating a HPV-associated cancer are also provided.
  • uses of the formulation comprising an effective amount of the AACs in the manufacture of a medicament for stimulating an immune response to a HPV antigen or for treating a HPV-associated cancer.
  • a pharmaceutical formulation of AACs wherein the AACs comprise the at least one antigen and an adjuvant
  • the method comprising: a) passing a cell suspension comprising an input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed anucleate cells, thereby generating the AACs comprising the at least one antigen and the adjuvant; c) washing the AACs; and d) formulating the AACs in a cryopreservation medium
  • anucleate cell refers to a cell lacking a nucleus. Such cells can include, but are not limited to, platelets, red blood cells (RBCs) such as erythrocytes and reticulocytes. Reticulocytes are immature (e.g., not yet biconcave) red blood cells, typically comprising about 1% of the red blood cells in the human body.
  • RBCs red blood cells
  • Reticulocytes are immature (e.g., not yet biconcave) red blood cells, typically comprising about 1% of the red blood cells in the human body.
  • Reticulocytes are also anucleate.
  • the systems and methods described herein are used the treatment and/or processing of enriched (e.g., comprising a greater percentage of the total cellular population than would be found in nature), purified, or isolated (e.g., from their natural environment, in substantially pure or homogeneous form) populations of anucleate cells (e.g., RBCs, reticulocytes, and/or platelets).
  • the systems and methods described herein are used for the treatment and/or processing of whole blood containing RBCs (e.g., erythrocytes or reticulocytes), platelets as well as other blood cells.
  • Purification or enrichment of these cell types is accomplished 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 precursors.
  • density gradient systems e.g., Ficoll-Hypaque
  • FACS fluorescence activated cell sorting
  • magnetic cell sorting or in vitro differentiation of erythroblasts and erythroid precursors.
  • vesicle refers to a structure comprising liquid enclosed by a lipid bilayer.
  • the lipid bilayer is sourced from naturally existing lipid composition.
  • the lipid bilayer can be sourced from a cellular membrane.
  • vesicles can be derived from various kinds of entities, such as cells.
  • a vesicle can retain molecules (such as intracellular proteins or membrane components) from the originating entity.
  • a vesicle derived from a red blood cell may contain any number of intracellular proteins that were in the red blood cell and/or membrane components of the red blood cell.
  • a vesicle can contain any number of molecules intracellularly in addition to the desired payload.
  • a payload refers to the material that is being delivered into, such as loaded in, the anucleate cell-derived vesicle (e.g., an AAC).
  • a payload may refer to a protein, a small molecule, a nucleic acid (e.g., RNA and/or DNA), a lipid, a carbohydrate, a macromolecule, a vitamin, a polymer, fluorescent dyes and fluorophores, carbon nanotubes, quantum dots, nanoparticles, and steroids.
  • the payload may refer to a protein or small molecule drug.
  • the payload may comprise one or more compounds.
  • heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
  • a “heterologous” region of a nucleic acid construct or a 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.
  • a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention.
  • Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
  • heterologous as it relates to amino acid sequences such as peptide sequences and polypeptide sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
  • 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.
  • a heterologous region of a peptide construct could include the amino acid sequence of the peptide flanked by sequences not found in association with the amino acid sequence of the peptide in nature.
  • heterologous peptide sequence is a construct where the peptide sequence itself is not found in nature (e.g., synthetic sequences having amino acids different as coded from the native gene).
  • a cell transformed with a vector that expresses an amino acid construct which is not normally present in the cell would be considered heterologous for purposes of this invention.
  • Allelic variation or naturally occurring mutational events do not give rise to heterologous peptides, as used herein.
  • exogenous when used in reference to an agent, such as an antigen or an adjuvant, with relation to a cell or an AAC refers to an agent outside of the cell or an agent delivered into 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 has been delivered.
  • homologous refers to a molecule which is derived from the same organism. In some examples the term refers to a nucleic acid or protein which is normally found or expressed within the given organism.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of cancer (such as, for example, tumor volume).
  • the methods of the invention contemplate any one or more of these aspects of
  • prophylactic treatment refers to treatment, wherein an individual is known or suspected to have or be at risk for having a disorder but has displayed no symptoms or minimal symptoms of the disorder.
  • An individual undergoing prophylactic treatment may be treated prior to onset of symptoms.
  • an individual may be treated if they have a precancerous lesion, particularly a precancerous lesion associated with HPV infection.
  • “combination therapy” is meant that a first agent be administered in conjunction with another agent.
  • “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of a composition of nucleated cells as described herein in addition to administration of an immunoconjugate as described herein to the same individual.
  • “in conjunction with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual.
  • the term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes.
  • the first and second therapies may be contained in the same composition e.g., a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).
  • the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first therapy or the second therapy may be administered first.
  • the first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.
  • the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.
  • the term “treating” includes any or all of killing cancer cells, inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
  • modulate may refer to the act of changing, altering, varying, or otherwise modifying the presence, or an activity of, a particular target.
  • modulating an immune response may refer to any act leading to changing, altering, varying, or otherwise modifying an immune response.
  • modulate refers to enhancing the presence or activity of a particular target.
  • modulate refers to suppressing the presence or activity of a particular target.
  • modulating the expression of a nucleic acid may include, but not limited to a change in the transcription of a nucleic acid, a change in mRNA abundance (e.g., increasing mRNA transcription), a corresponding change in degradation of mRNA, a change in mRNA translation, and so forth.
  • the term “inhibit” may refer to the act of blocking, reducing, eliminating, or otherwise antagonizing the presence, or an activity of, a particular target. Inhibition may refer to partial inhibition or complete inhibition. For example, inhibiting an immune response may refer to any act leading to a blockade, reduction, elimination, or any other antagonism of an immune response.
  • inhibition of the expression of a nucleic acid may include, but not limited to reduction in the transcription of a nucleic acid, reduction of mRNA abundance (e.g., silencing mRNA transcription), degradation of mRNA, inhibition of mRNA translation, gene editing and so forth.
  • inhibition of the expression of a protein may include, but not be limited to, reduction in the transcription of a nucleic acid encoding the protein, reduction in the stability of mRNA encoding the protein, inhibition of translation of the protein, reduction in stability of the protein, and so forth.
  • inhibit may refer to the act of slowing or stopping growth; for example, retarding or preventing the growth of a tumor cell.
  • suppress may refer to the act of decreasing, reducing, prohibiting, limiting, lessening, or otherwise diminishing the presence, or an activity of, a particular target. Suppression may refer to partial suppression or complete suppression. For example, suppressing an immune response may refer to any act leading to decreasing, reducing, prohibiting, limiting, lessening, or otherwise diminishing an immune response. In other examples, suppression of the expression of a nucleic acid may include, but not limited to reduction in the transcription of a nucleic acid, reduction of mRNA abundance (e.g., silencing mRNA transcription), degradation of mRNA, inhibition of mRNA translation, and so forth.
  • mRNA abundance e.g., silencing mRNA transcription
  • suppression of the expression of a protein may include, but not be limited to, reduction in the transcription of a nucleic acid encoding the protein, reduction in the stability of mRNA encoding the protein, inhibition of translation of the protein, reduction in stability of the protein, and so forth.
  • the term “enhance” may refer to the act of improving, boosting, heightening, or otherwise increasing the presence, or an activity of, a particular target.
  • enhancing an immune response may refer to any act leading to improving, boosting, heightening, or otherwise increasing an immune response.
  • enhancing an immune response may refer to employing an antigen and/or adjuvant to improve, boost, heighten, or otherwise increase an immune response.
  • enhancing the expression of a nucleic acid may include, but not limited to increase in the transcription of a nucleic acid, increase in mRNA abundance (e.g., increasing mRNA transcription), decrease in degradation of mRNA, increase in mRNA translation, and so forth.
  • enhancing the expression of a protein may include, but not be limited to, increase in the transcription of a nucleic acid encoding the protein, increase in the stability of mRNA encoding the protein, increase in translation of the protein, increase in the stability of the protein, and so forth.
  • the term “induce” may refer to the act of initiating, prompting, stimulating, establishing, or otherwise producing a result.
  • inducing an immune response may refer to any act leading to initiating, prompting, stimulating, establishing, or otherwise producing a desired immune response.
  • inducing the expression of a nucleic acid may include, but not limited to initiation of the transcription of a nucleic acid, initiation of mRNA translation, and so forth.
  • inducing the expression of a protein may include, but not be limited to, increase in the transcription of a nucleic acid encoding the protein, increase in the stability of mRNA encoding the protein, increase in translation of the protein, increase in the stability of the protein, and so forth.
  • polynucleotide or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the backbone of the polynucleotide can comprise repeating units, such as N-(2-aminoethyl)- glycine, linked by peptide bonds (i.e., peptide nucleic acid).
  • the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and phorphorthi oates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphorothioate-phosphorodiester oligomer or a mixed phosphoramidate- phosphodiester oligomer.
  • P-NH2 oligodeoxynucleoside phosphoramidate
  • P-NH2 oligodeoxynucleoside phosphoramidate
  • P-NH2 oligodeoxynucleoside phosphoramidate
  • P-NH2 oligodeoxynucleoside phosphoramidate
  • mixed phosphorothioate-phosphorodiester oligomer or a mixed phosphoramidate- phosphodiester oligomer.
  • a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full- length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • the term “adjuvant” refers to a substance which modulates and/or engenders an immune response. Generally, the adjuvant is administered in conjunction with an antigen to effect enhancement of an immune response to the antigen as compared to antigen alone. Various adjuvants are described herein.
  • CpG oligodeoxynucleotide and “CpG ODN” herein refer to DNA molecules of 10 to 30 nucleotides in length containing a dinucleotide of cytosine and guanine separated by a phosphate (also referred to herein as a “CpG” dinucleotide, or “CpG”).
  • the CpG ODNs of the present disclosure contain at least one unmethylated CpG dinucleotide. That is, the cytosine in the CpG dinucleotide is not methylated (i.e., is not 5-methyl cytosine).
  • CpG ODNs may have a partial or complete phosphorothioate (PS) backbone.
  • PS phosphorothioate
  • pharmaceutically acceptable or “pharmacologically compatible” is meant 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.
  • Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • microfluidic systems refers to systems in which low volumes (e.g., mL, nL, pL, fL) of fluids are processed to achieve the discrete treatment of small volumes of liquids. Certain implementations described herein include multiplexing, automation, and high throughput screening.
  • the fluids e.g., a buffer, a solution, a payload-containing solution, or a cell suspension
  • the fluids can be moved, mixed, separated, or otherwise processed.
  • microfluidic systems are used to apply mechanical constriction to a cell suspended in a buffer, inducing perturbations in the cell (e.g., holes) that allow a payload or compound to enter the cytosol of the cell.
  • a “constriction” may refer to a portion of a microfluidic channel defined by an entrance portion, a centerpoint, and an exit portion, wherein the centerpoint is defined by a width, a length, and a depth.
  • a constriction may refer to a pore or may be a portion of a pore.
  • the pore may be contained on a surface (e.g., a filter and/or membrane).
  • a pharmaceutical formulation comprising AACs, the formulation comprising a) AACs wherein the AACs comprise at least one antigen and an adjuvant, and b) a cryopreservation medium.
  • the formulation comprises about 1 x 10 7 to about 1 x 10 12 AACs. In some embodiments, the formulation comprises about 1 x 10 9 to about 1 x 10 11 AACs in about 9 mL to aboutlO mb In some embodiments, the formulation comprises about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises any one of about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x 10 11 , about 0.5 x io 11 to about 1.0 x 10 11 , about 1.0 x io 11 to about 0.5 x io 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises about any one of 1 x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9 mL to about lOmL. In some embodiments, the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9.5 mL.
  • the formulation comprises about 7x 10 9 AACs in about 9 mL to about 10 mL. In some embodiments, the formulation comprises about 7x 10 9 AACs in about 9.5 mL. In some embodiments, the formulation comprises about 6.65 x 10 9 AACs in about 9.5 mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing as measured by Coulter counter.
  • an expected concentration post-thaw of 1.0 x io 9 AAC/mL as measured by a flow cytometry method is approximately equivalent to 7.0 x 10 8 AAC/mL postthaw as measured by a Coulter-based cell counter method based on the negative bias of the Coulter-based counter method due to a lower sensitivity.
  • the formulation comprises about 1 x 10 7 to about 1 x 10 12 AACs. In some embodiments, the formulation comprises about 1 x 10 9 to about 1 x 10 11 AACs.
  • the formulation comprises about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs.
  • the formulation comprises any one of about, about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x 10 11 , about 0.5 x io 11 to about 1.0 x 10 11 , about 1.0 x io 11 to about 0.5 x 10 12 AACs.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs. In some embodiments, the formulation comprises about 7* 10 9 AACs. In some embodiments, the formulation comprises about 6.65 * 10 9 AACs.
  • the volume of the formulation is about 2 mL to about 50 mL. In some embodiments, the volume of the formulation is about 5 mL to about 20 mL. In some embodiments, the volume of the formulation is about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more mL.
  • the volume of the formulation is any one of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10, about 10 to about 11, about 11 to about 12, about 12 to about 13, about 13to about 14, about 14 to about 15, about 15 to about 16, about 16 to about 17, about 17 to about 18, about 18 to about 19, or about 19 to about 20 mL. In some embodiments, the volume of the formulation is about 9.5 mL.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 AACs/mL. In some embodiments, the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x 10 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x 10 10 , about 1.0 x 1O 10 to about 0.5 x 10 11 AACs/mL.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL. In some embodiments, the formulation comprises about 7x 10 8 AACs/mL. In some embodiments, the formulation comprises about 6.65 x 10 8 AACs/mL.
  • the formulation is sterile. In some embodiments, the formulation comprises less than about 2 EU/mL endotoxin. In some embodiments, the formulation comprises less than any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mL endotoxin. In some embodiments, the formulation is free of mycoplasma. Formulations of AACs in cryopreservation media
  • the composition of AACs further comprises an agent that enhances the function of the AACs as compared to a corresponding composition of AACs that does not comprise the agent.
  • the composition of AACs further comprises an agent that enhances the function of the AAC upon freeze-thaw cycle as compared to a corresponding composition of AAC that does not comprise the agent.
  • the agent is a cryopreservation agent and/or a hypothermic preservation agent.
  • the cryopreservation agent nor the hypothermic preservation agent prevents more than 10% or 20% of cell death in a composition of AAC comprising the agent compared to a corresponding composition of AAC that does not comprise the agent before any freeze-thaw cycles.
  • freeze-thaw cycles of compositions of AACs comprising the cryopreservation agent and/or the hypothermic preservation agent causes not more than 10%, 20%, 30%, 40%, or 50% loss in function when compared to a corresponding composition of AACs before the freeze-thaw cycles.
  • freeze-thaw cycles of AAC compositions comprising the cryopreservation agent and/or the hypothermic preservation agent causes 10%, 20%, 30%, 40%, or 50% less loss of function when compared to freeze-thaw cycles of a corresponding composition of AACs without the cryopreservation agent and the hypothermic preservation agent.
  • the function or functionality of the AAC composition comprising at least one antigen is measured by the percentage of the AACs that are positive for annexin V staining. In some embodiments, the function or functionality of the AAC composition is measured by the percentage of the AACs that are positive for annexin V staining.
  • the function or functionality of the AAC composition is measured by the percentage of the AACs that are positive for CD235a staining. In some embodiments, the function or functionality of the AACs composition is measured by the percentage of the anucleate cell-derived vesicles that are positive CD235a and annexin V staining. In some embodiments, at least about 70%, about 80%, or about 90% of the AAC are functional after up to 1, 2, 3, 4, 5 freeze-thaw cycles.
  • the agent is a compound that enhances endocytosis, a stabilizing agent or a co-f actor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin.
  • the agent 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: a 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 one more of Mg2+, Zn2+ or Ca2+.
  • the agent is one or more of: sodium pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), dimethyl sulfoxide (DMSO), HEPES, glycerol, glutathione, inosine, dibasic sodium phosphate, monobasic sodium phosphate, sodium metal ions, potassium metal ions, magnesium metal ions, chloride, acetate, gluoconate, sucrose, potassium hydroxide, or sodium hydroxide.
  • 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®.
  • the process further comprises a step of incubating the composition of anucleate cells with an agent that enhances the function of the anucleate cells compared to corresponding anucleate cells prepared without the further incubation step.
  • the formulation comprises a cryopreservation medium. In some embodiments, the formulation comprises about 1 x 10 9 to about 1 x 10 11 AACs in about 9 mL to about 10 mL cryopreservation medium.
  • the formulation comprises about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs in about 9 mL to about 10 mL cryopreservation medium.
  • the formulation comprises any one of about 0.5 x 10 7 to about 1.0 x 10 7 , about 1.0 x 10 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9 mL to about lOmL cryopreservation medium.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9.5 mL cryopreservation medium.
  • the formulation comprises about 7x 10 9 AACs in about 9 mL to about 10 mL cryopreservation medium.
  • the formulation comprises about 7x 10 9 AACs in about 9.5 mL cryopreservation medium.
  • the formulation comprises about 6.65* 10 9 AACs in about 9.5 mL cryopreservation medium.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs in a cryopreservation medium.
  • the formulation comprises any one of about, about 0.5 x 10 7 to about 1.0 x 10 7 , about 1.0 x io 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACS, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x 10 11 , about 0.5 x 10 11 to about 1.0 x 10 11 AACs, about 1.0 x 10 11 to about 0.5 x 10 12 AACs in a cryopreservation medium.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in a cryopreservation medium.
  • the formulation comprises about 7x 10 9 AACs in a cryopreservation medium.
  • the formulation comprises about 6.65 x 10 9 AACs in a cryopreservation medium.
  • the formulation comprises about 0.7 x 10 9 AACs/mL in cryopreservation medium post-thawing.
  • the formulation comprises about 0.7 x 10 9 AACs/mL in cryopreservation medium post-thawing as measured by Coulter counter.
  • the cryopreservation medium comprises CryoStor® CS2. In some embodiments, the cryopreservation medium is CryoStor® CS2.
  • the composition comprising AACs comprise about 7 x 10 9 AACs in about 9 mL to about 10 mL of CryoStor® CS2. In some embodiments, the composition comprising AACs comprise about 7 x 10 9 AACs in about 9.5 mL of CryoStor® CS2. In some embodiments, the formulation comprises about 6.65 x 10 9 AACs in about 9.5 mL of CryoStor® CS2.
  • the AACs in the formulation maintain equal to or greater than about 50% functionality up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% functionality up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AACs in the formulation maintain equal to or greater than about 70% functionality following storage for at least 12 months at temperatures at or below -140°C. In some embodiments, the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% functionality following storage for at least 12 months at temperatures at or below -140°C.
  • the formulation maintain equal to or greater than about 70% functionality following storage for at least 6, 9, 12, 15, 18, 24, 30, or 36 months at temperatures at or below - 140°C. In some embodiments, the formulation maintain equal to or greater than about 70% functionality following storage for at least 12 months at temperatures 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.
  • the AACs in the formulation maintain equal to or greater than about 50% positive staining for annexin up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for annexin up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AACs in the formulation maintain equal to or greater than about 70% positive staining for annexin following storage for at least 12 months at temperatures at or below -140°C.
  • the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for annexin following storage for at least 12 months at temperatures at or below -140°C. In some embodiments, the formulation maintain equal to or greater than about 70% positive staining for annexin following storage for at least 6, 9, 12, 15, 18, 24, 30, or 36 months at temperatures at or below -140°C.
  • the formulation maintain equal to or greater than about 70% positive staining for annexin following storage for at least 12 months at temperatures 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.
  • the AACs in the formulation maintain equal to or greater than about 50% positive staining for annexin V and/or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for annexin V and/or CD235a up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the AACs in the formulation maintain equal to or greater than about 70% positive staining for annexin V and/or CD235a following storage for at least 12 months at temperatures at or below -140°C.
  • the formulation maintain equal to or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 99% positive staining for annexin V and/or CD235a following storage for at least 12 months at temperatures at or below -140°C. In some embodiments, the formulation maintain equal to or greater than about 70% positive staining for annexin V and/or CD235a following storage for at least 6, 9, 12, 15, 18, 24, 30, or 36 months at temperatures at or below -140°C.
  • the formulation maintain equal to or greater than about 70% positive staining for annexin V and/or CD235a following storage for at least 12 months at temperatures 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.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 10 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 functional AACs.
  • the formulation comprises any one of about, about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x 10 8 to about 0.5 x 10, about 0.5 x 10 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x 10 11 , about 0.5 x io 11 to about 1.0 x 10 11 , about 1.0 x io 11 to about 0.5 x io 12 functional AACs.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 functional AACs. In some embodiments, the formulation comprises about 7x 10 9 functional AACs. In some embodiments, the formulation comprises about l x 10 9 to about l x 10 10 functional AACs.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 functional AACs/mL. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 functional AACs/mL. In some embodiments, the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 functional AACs/mL.
  • the formulation comprises any one of about, about 0.5 x io 6 to about 1.0 x io 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x 1O 10 to about 1.0 x 1O 10 , about 1.0 x 1O 10 to about 0.5 x 10 11 functional AACs/mL.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 functional AACs/mL. In some embodiments, the formulation comprises about 7x 10 8 functional AACs/mL. In some embodiments, the formulation comprises about 1 x 10 8 to about 1 x 10 9 functional AACs/mL.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x IO 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 functional AACs post-thawing.
  • the formulation comprises any one of about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 , about 0.5 x io 11 to about 1.0 x 10 11 AACs, about 1.0 x io 11 to about 0.5 x 10 12 functional AACs post-thawing.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and lx IO 10 functional AACs post-thawing. In some embodiments, the formulation comprises about 7x 10 9 functional AACs post-thawing. In some embodiments, the formulation comprises about l x 10 9 to about l x 10 10 functional AACs post-thawing.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 functional AACs/mL post-thawing. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 10 10 functional AACs/mL post-thawing.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 functional AACs/mL post-thawing.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x 10 6 to about 0.5 x io 7 , about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x 10 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 10 , about 1.0 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x 10 11 functional AACs/m
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 functional AACs/mL post-thawing. In some embodiments, the formulation comprises about 7x 10 8 functional AACs/mL post-thawing. In some embodiments, the formulation comprises about 1 x 10 8 to about 1 x 10 9 functional AACs/mL post-thawing.
  • the cryopreservation medium comprises dimethylsulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises about 0.5% to about 25% DMSO. In some embodiments, the cryopreservation medium comprises about 0.5% to about 5% DMSO. In some embodiments, the cryopreservation medium comprises about any one of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20% and 25% DMSO. In some embodiments, the cryopreservation medium comprises any one of about 0.5% to about 5%, about 5% to about 10%, about 10% to about 20% DMSO. In some embodiments, the cryopreservation medium comprises about 2% DMSO.
  • DMSO dimethylsulfoxide
  • the pH of the formulation is about 5.0 to about 9.5. In some embodiments, the pH of the formulation is about 6.0 to about 8.5. In some embodiments, the pH of the formulation is about 7.6. In some embodiments, the pH of the formulation is any one of about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 10. In some embodiments, the pH of the formulation is any one of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH of the formulation is any one of about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10.
  • the pH of the formulation is any one of about 7 to about 7.1, about 7.1 to about 7.2, about 7.2 to about 7.3, about 7.3 to about 7.4, about 7.4 to about 7.5, about 7.5 to about 7.6, about 7.6 to about 7.7, about 7.7 to about 7.8, about 7.8 to about 7.9, or about 7.9 to about 8.0.
  • a pharmaceutical formulation of AACs comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • a pharmaceutical formulation of AACs the formulation comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing.
  • the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL prior to freezing. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 AACs/mL prior to freezing.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL prior to freezing.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x io 6 , about 1.0 x 10 6 to about 0.5 x 10 7 , about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x 10 9 to about 0.5 x 1Q 10 , about 0.5 x 1Q 10 to about 1.0 x 1Q 10 , about 1.0 x 1Q 10 to about 0.5 x 10 11 AACs/mL prior to freezing.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL prior to freezing. In some embodiments, the formulation comprises about 7x 10 8 AACs/mL prior to freezing. In some embodiments, the formulation comprises about 6.65 x 10 8 AACs/mL prior to freezing. In some embodiments, the concentration of AACs in the formulation is measured by Coulter counter.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 10 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x
  • the formulation comprises any one of about, about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0 x 10 7 to about 0.5 x 10 8 AACs, about 0.5 x 10 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x 10 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x 10 10 to about 0.5 x 10 11 , about 0.5 x 10 11 to about 1.0 x 10 11 AACs, about 1.0 x 10 11 to about 0.5 x 10 12 AACs prior to freezing.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs prior to freezing. In some embodiments, the formulation comprises about 7x 10 9 AACs prior to freezing. In some embodiments, the formulation comprises about 6.65 x 10 9 AACs prior to freezing.
  • the formulation comprises about 1 x io 6 to about 1 x io 11 AACs/mL post-thawing. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 AACs/mL post-thawing.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL post-thawing.
  • the formulation comprises any one of about, about 0.5 x io 6 to about 1.0 x io 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 AACs/mL post-thawing.
  • the formulation comprises about any one of lx 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , and 5x 10 9 AACs/mL post-thawing.
  • the formulation comprises about 7x 10 8 AACs/mL post-thawing.
  • the formulation comprises about 6.65 * 10 8 AACs/mL post-thawing.
  • the formulation comprises about l x 10 9 AACs/mL post-thawing.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x io 10 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL postthawing as measured by Coulter counter.
  • the formulation comprises any one of about, about 0.5 x io 6 to about 1.0 x io 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 7x 10 8 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 6.65 x 10 8 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL post-thawing as measured by flow cytometry. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x io 10 AACs/mL post-thawing as measured by flow cytometry.
  • the formulation comprises about any one of 0.5 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , and 1.0 x 10 11 AACs/mL post-thawing as measured by flow cytometry.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x 1O 10 to about 0.5 x 10 11 AACs/mL post-thawing as measured by flow cytometry.
  • the formulation comprises about any one of 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , l x 10 9 , 2* 10 9 , 3* 10 9 , 4* 10 9 , 5* 10 9 AACs/mL post-thawing as measured by flow cytometry. In some embodiments, the formulation comprises about l x 10 9 AACs/mL postthawing as measured by flow cytometry.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x IO 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs post-thawing.
  • the formulation comprises any one of about, about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 , about 0.5 x io 11 to about 1.0 x 10 11 AACs, about 1.0 x io 11 to about 0.5 x 10 12 AACs post-thawing.
  • the formulation comprises about any one of 1 x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x IO 10 AACs postthawing.
  • the formulation comprises about 9x 10 9 AACs post-thawing.
  • the formulation comprises about 7x 10 9 AACs post-thawing.
  • the formulation comprises about 6.65 x 10 9 AACs post-thawing.
  • the AACs are in about 2 mL to about 50 mL of cryopreservation medium. In some embodiments, the AACs are in about 5 mL to about 20 mL of cryopreservation medium. In some embodiments, the AACs are in about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more mL of cryopreservation medium.
  • the AACs are in any one of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10, about 10 to about 11, about 11 to about 12, about 12 to about 13, about 13to about 14, about 14 to about 15, about 15 to about 16, about 16 to about 17, about 17 to about 18, about 18 to about 19, or about 19 to about 20 mL of cryopreservation medium. In some embodiments, the AACs are in about 9.5 mL of cryopreservation medium.
  • a pharmaceutical formulation of AACs comprising about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the cryopreservation medium is CryoStor® CS2.
  • the formulation is sterile.
  • the formulation comprises less than about 2 EU/mL endotoxin.
  • the formulation comprises less than any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mL endotoxin.
  • the formulation is free of mycoplasma.
  • a vial comprising any one of the pharmaceutical formulations described herein.
  • a vial comprising a pharmaceutical formulation, wherein the pharmaceutical formulation comprises AACs, wherein the formulation comprising about 1 x 10 9 AACs to about 1 x 10 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • a pharmaceutical formulation of AACs the formulation comprising about 1 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • the AACs are in about 2 mL to about 50 mL of cryopreservation medium. In some embodiments, the AACs are in about 5 mL to about 20 mL of cryopreservation medium. In some embodiments, the AACs are in about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more mL of cryopreservation medium.
  • the AACs are in any one of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10, about 10 to about 11, about 11 to about 12, about 12 to about 13, about 13 to about 14, about 14 to about 15, about 15 to about 16, about 16 to about 17, about 17 to about 18, about 18 to about 19, or about 19 to about 20 mL of cry opreservation medium.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 AACs/mL. In some embodiments, the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x io 6 to about 0.5 x 10 7 , about 0.5 x io 7 to about 1.0 x 10 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x 10 8 , about 1.0 x io 8 to about 0.5 x 10 9 , about 0.5 x 10 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 1O 10 , about 0.5 x io 10 to about 1.0 x 10 10 , about 1.0 x 1O 10 to about 0.5 x 10 11 AACs/mL.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL. In some embodiments, the formulation comprises about 7x 10 8 AACs/mL. In some embodiments, the formulation comprises about 6.65 x 10 8 AACs/mL. In some embodiments, the cryopreservation medium is CryoStor® CS2. In some embodiments, the cryopreservation medium comprises DMSO. In some embodiments, the cryopreservation medium comprises about 0.5% to about 25% DMSO.
  • the cryopreservation medium comprises about 0.5% to about 5% DMSO. In some embodiments, the cryopreservation medium comprises about any one of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20% and 25% DMSO. In some embodiments, the cryopreservation medium comprises any one of about 0.5% to about 5%, about 5% to about 10%, about 10% to about 20% DMSO. In some embodiments, the cryopreservation medium comprises about 2% DMSO.
  • the pH of the formulation is about 5.0 to about 9.5. In some embodiments, the pH of the formulation is about 6.0 to about 8.5. In some embodiments, the pH of the formulation is about 7.6. In some embodiments, the pH of the formulation is any one of about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 10. In some embodiments, the pH of the formulation is any one of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH of the formulation is any one of about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10.
  • the pH of the formulation is any one of about 7 to about 7.1, about 7.1 to about 7.2, about 7.2 to about 7.3, about 7.3 to about 7.4, about 7.4 to about 7.5, about 7.5 to about 7.6, about 7.6 to about 7.7, about 7.7 to about 7.8, about 7.8 to about 7.9, or about 7.9 to about 8.0.
  • the formulation comprises about 1 x 10 9 to about 1 x 10 11 AACs in about 9 mL to about 10 mL. In some embodiments, the formulation comprises about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises any one of about 0.5 x 10 7 to about 1.0 x 10 7 , about 1.0 x 10 7 to about 0.5 x 10 8 AACS, about 0.5 x io 8 to about 1.0 x 10 8 , about 1.0 x io 8 to about 0.5 x 10 9 , about 0.5 x 10 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 1O 10 , about 0.5 x io 10 to about 1.0 x 1O 10 , about 1.0 x 10 10 to about 0.5 x io 11 , about 0.5 x 10 11 to about 1.0 x io 11 , about 1.0 x 10 n to about 0.5 x 10 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9 mL to about lOmL. In some embodiments, the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9.5 mL.
  • the formulation comprises about 7x 10 9 AACs in about 9 mL to about 10 mL. In some embodiments, the formulation comprises about 7x 10 9 AACs in about 9.5 mL. In some embodiments, the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs. In some embodiments, the formulation comprises any one of about, about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0
  • the formulation comprises about any one of l x 10 9 , 2x
  • the formulation comprises about 7x 10 9 AACs in a cryopreservation medium. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL in cryopreservation medium post-thawing. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL in cryopreservation medium post-thawing as measured by Coulter counter. In some embodiments, the cryopreservation medium comprises CryoStor® CS2.
  • the cryopreservation medium is CryoStor® CS2.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL post-thawing. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x 1O 10 AACs/mL post-thawing.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 10 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL post-thawing.
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x io 6 to about 0.5 x 1Q 7 , about 0.5 x 1Q 7 to about 1.0 x 1Q 7 , about 1.0 x 1Q 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x 10 8 , about 1.0 x io 8 to about 0.5 x 10 9 , about 0.5 x 10 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 1O 10 , about 0.5 x io 10 to about 1.0 x
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL post-thawing.
  • the formulation comprises about 7x 10 8 AACs/mL post-thawing.
  • the formulation comprises about 6.65 x 10 8 AACs/mL post-thawing.
  • the formulation comprises about l x 10 9 AACs/mL post-thawing.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x io 10 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about any one of 0.5 x 10 6 , 0.7 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , and 1.0 x 10 11 AACs/mL postthawing as measured by Coulter counter.
  • the formulation comprises any one of about, about 0.5 x io 6 to about 1.0 x io 6 , about 1.0 x io 6 to about 0.5 x io 7 , about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x io 8 , about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x io 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about any one of l x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , and l x 10 9 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 7x 10 8 AACs/mL post-thawing as measured by Coulter counter. In some embodiments, the formulation comprises about 6.65 x 10 8 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about 1 x 10 6 to about 1 x 10 11 AACs/mL post-thawing as measured by flow cytometry. In some embodiments, the formulation comprises about 1 x 10 7 to about 1 x io 10 AACs/mL post-thawing as measured by flow cytometry. In some embodiments, the formulation comprises about any one of 0.5 x 10 6 , 1.0 x 10 6 , 0.5 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 1.0 x 1O 10 , 0.5 x
  • the formulation comprises any one of about, about 0.5 x 10 6 to about 1.0 x 10 6 , about 1.0 x io 6 to about 0.5 x 10 7 , about 0.5 x io 7 to about 1.0 x 10 7 , about 1.0 x io 7 to about 0.5 x 10 8 , about 0.5 x io 8 to about 1.0 x 10 8 , about 1.0 x io 8 to about 0.5 x 10 9 , about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 1O 10 , about 0.5 x io 10 to about 1.0 x 1O 10 , about 1.0 x 1O 10 to about 0.5 x 10 11 AACs/mL post-thawing as measured by flow cytometry.
  • the formulation comprises about any one of 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 AACs/mL post-thawing as measured by flow cytometry. In some embodiments, the formulation comprises about l x 10 9 AACs/mL postthawing as measured by flow cytometry.
  • the formulation comprising AACs comprise about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 10 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs post-thawing.
  • the formulation comprises any one of about, about 0.5 x io 7 to about 1.0 x io 7 , about 1.0 x io 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x io 8 , about 1.0 x io 8 to about 0.5 x 10 9 AACs, about 0.5 x io 9 to about 1.0 x io 9 , about 1.0 x io 9 to about 0.5 x 10 10 AACs, about 0.5 x io 10 to about 1.0 x io 10 , about 1.0 x io 10 to about 0.5 x io 11 , about 0.5 x io 11 to about 1.0 x 10 11 AACs, about 1.0 x io 11 to about 0.5 x 10 12 AACs post-thawing.
  • the formulation comprises about any one of 1 x 10 9 , 2 x 10 9 , 3 x 10 9 , 4 x 10 9 , 5 x 10 9 , 6 x 10 9 , 7 x 10 9 , 8 x 10 9 , 9 x 10 9 , and 1 x 10 10 AACs post-thawing.
  • the formulation comprises about 9 x 10 9 AACs postthawing.
  • the formulation comprises about 7x 10 9 AACs post-thawing.
  • the formulation comprises about 6.65 x 10 9 AACs post-thawing.
  • a pharmaceutical formulation of activated AACs comprising about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the cryopreservation medium is CryoStor® CS2.
  • the formulation is sterile. In some embodiments, the formulation comprises less than about 2 EU/mL endotoxin. In some embodiments, the formulation comprises less than any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mL endotoxin. In some embodiments, the formulation is free of mycoplasma. Compositions of AACs comprising antigens and adjuvants
  • the AACs comprise an antigen and an adjuvant delivered intracellularly. In some embodiments, the AACs comprise an HPV antigen and an adjuvant delivered intracellularly. In some embodiments, the AACs are derived from input anucleate cells. In some embodiments, the AACs are derived from input erythrocytes. In some embodiments, the AACs are derived from input reticulocytes. In some embodiments, the AACs are derived from input red blood cells (RBCs). In some embodiments, the AACs are anucleate cell-derived vesicles comprising the HPV antigen and the adjuvant. In some embodiments, the AACs are RBC-derived vesicles comprising the HPV antigen and the adjuvant.
  • the AACs comprising the at least one antigen and the adjuvant are prepared by: a) passing a cell suspension comprising input anucleate cells through a celldeforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed input anucleate cells; and b) incubating the perturbed input anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed input anucleate cells; thereby generating AACs comprising the at least one antigen and the adjuvant.
  • the AACs comprising the HPV antigen and the adjuvant are prepared by: a) passing a cell suspension comprising input anucleate cells through a celldeforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the HPV antigen and the adjuvant to pass through to form perturbed input anucleate cells; and b) incubating the perturbed input anucleate cells with the HPV antigen and the adjuvant for a sufficient time to allow the HPV antigen and the adjuvant to enter the perturbed input anucleate cells; thereby generating AACs comprising the HPV antigen and the adjuvant.
  • the HPV antigen comprises the amino acid sequence of any one of SEQ ID Nos: 1-4 and 18-25. In some embodiments, the HPV antigen comprises an amino acid sequence with at least 90% identity to any one of SEQ ID Nos: 1-4 and 18-25.
  • the anucleate cell is an RBC or a platelet. In some embodiments, the anucleate cell is an erythrocyte or a reticulocyte. In some embodiments, the AAC is an anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is an RBC-derived vesicle or a platelet-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is an erythrocyte-derived vesicle or a reticulocyte-derived vesicle.
  • the AACs comprising the at least one antigen and the adjuvant are prepared by: a) passing a cell suspension comprising input red blood cells through a celldeforming constriction, wherein a diameter of the constriction is a function of a diameter of the input red blood cells in the suspension, thereby causing perturbations of the input red blood cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed input red blood cells; and b) incubating the perturbed input red blood cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed input red blood cells; thereby generating AACs comprising the at least one antigen and the adjuvant.
  • the AACs comprising the HPV antigen and the adjuvant are prepared by: a) passing a cell suspension comprising input red blood cells through a celldeforming constriction, wherein a diameter of the constriction is a function of a diameter of the input red blood cells in the suspension, thereby causing perturbations of the input red blood cells large enough for the HPV antigen and the adjuvant to pass through to form perturbed input red blood cells; and b) incubating the perturbed input red blood cells with the HPV antigen and the adjuvant for a sufficient time to allow the HPV antigen and the adjuvant to enter the perturbed input red blood cells; thereby generating AACs comprising the HPV antigen and the adjuvant.
  • the HPV antigen comprises the amino acid sequence of any one of SEQ ID Nos: 1-4, and 18-25. In some embodiments, the HPV antigen comprises an amino acid sequence with at least 90% identity to any one of SEQ ID Nos: 1-4, and 18-25.
  • the width of the constriction is about 10% to about 99% of the mean diameter of the input anucleate cells (e.g., red blood cells).
  • 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 mean diameter of the input anucleate cells.
  • the width of the constriction about 0.25 pm to about 4 pm, about 1 pm to about 4 pm, about 1.2 pm to about 3 pm, about 1.4 pm to about 2.6 pm, about 1.6 pm to about 2.4 pm, or about 1.8 pm to about 2.2 pm. In some embodiments, the width of the constriction is about 2.0 pm. In some embodiments, the width of the constriction is about 2.5 pm. In some embodiments, the width of the constriction is about 3.0 pm.
  • the width of the constriction is about or less than any one of 0.25 pm, 0.5 pm, 1.0 pm, 1.2 pm, 1.4 pm, 1.6 pm, 1.8 pm, 2.0 pm, 2.2 pm, 2.4 pm, 2.6 pm, 2.8 pm, 3.0 pm, 3.2 pm, 3.4 pm, 3.6 pm, 3.8 pm, 4.0 pm, 4.2 pm, 4.4 pm, 4.6 pm, 4.8 pm, 5.0 pm, 5.2 pm, 5.4 pm, 5.6 pm, 5.8 pm, 6.0 pm.
  • the cell suspension comprising the input anucleate cells are passed through multiple constrictions wherein the multiple constrictions are arranged in series and/or in parallel.
  • the HPV antigen is a pool of multiple polypeptides that elicit a response against the same and or different HPV antigens.
  • the HPV antigen is a polypeptide comprising one or more antigenic HPV epitope and one or more heterologous peptide sequences.
  • the HPV antigen complexes with other antigens or with an adjuvant.
  • the HPV antigen is capable of being processed into an MHC class I-restricted peptide.
  • the HPV antigen is capable of being processed into an MHC class Il-restricted peptide.
  • the composition further comprises an adjuvant.
  • the adjuvant is a CpG oligodeoxynucleotide (ODN), LPS, IFN-a, IFN-P, IFN-y, alpha-Galactosyl Ceramide, STING agonists, cyclic dinucleotides (CDN), RIG-I agonists, polyinosinic-polycytidylic acid (poly I:C), R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR9 agonist.
  • the adjuvant is polyinosinic-polycytidylic acid (poly I:C).
  • the AACs comprised within the pharmaceutical formulations are anucleate cell-derived vesicles.
  • the AACs comprising at least one antigen and adjuvant are prepared by a process comprising: a) passing a cell suspension comprising a population of input anucleate through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and adjuvant to pass through to form perturbed input anucleate cells; and b) incubating the population of perturbed input anucleate cells with the at least one antigen and adjuvant for a sufficient time to allow the at least one antigen to enter the perturbed input anucleate cells, thereby generating the AACs comprising the at least one antigen and adjuvant.
  • the AACs comprising the HPV antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising a population of input anucleate through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the HPV antigen and the adjuvant to pass through to form perturbed input anucleate cells; and b) incubating the population of perturbed input anucleate cells with the HPV antigen and the adjuvant for a sufficient time to allow the at least one antigen to enter the perturbed input anucleate cells, thereby generating the AACs comprising the HPV antigen and the adjuvant.
  • the HPV antigen comprises a peptide derived from HPV E6. In some embodiments, the HPV antigen comprises a peptide derived from HPV E7. In some embodiments, the HPV antigen comprises a peptide derived from HPV E6 and a peptide derived from HPV E7.
  • the input anucleate cell is a red blood cell (RBC) or a platelet.
  • the input anucleate cell is an erythrocyte or a reticulocyte.
  • the AAC is an anucleate cell-derived vesicle.
  • the anucleate cell-derived vesicle is a RBC-derived vesicle or a platelet-derived vesicle.
  • the anucleate cell-derived vesicle is an erythrocyte-derived vesicle or a reticulocyte-derived vesicle.
  • the input anucleate cell is autologous to the individual who will receive the composition.
  • the anucleate cell is allogeneic to the individual who will receive the composition.
  • the width of the constriction is about 10% to about 99% of the mean diameter of the input anucleate 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 mean diameter of the input anucleate cells.
  • the width of the constriction about 0.25 pm to about 4 pm, about 1 pm to about 4 pm, about 1.2 pm to about 3 pm, about 1.4 pm to about 2.6 pm, about 1.6 pm to about 2.4 pm, or about 1.8 pm to about 2.2 pm. In some embodiments, the width of the constriction is about 2.0 pm. In some embodiments, the width of the constriction is about 2.5 pm. In some embodiments, the width of the constriction is about 3.0 pm.
  • the width of the constriction is about or less than any one of 0.25 pm, 0.5 pm, 1.0 pm, 1.2 pm, 1.4 pm, 1.6 pm, 1.8 pm, 2.0 pm, 2.2 pm, 2.4 pm, 2.6 pm, 2.8 pm, 3.0 pm, 3.2 pm, 3.4 pm, 3.6 pm, 3.8 pm, 4.0 pm, 4.2 pm, 4.4 pm, 4.6 pm, 4.8 pm, 5.0 pm, 5.2 pm, 5.4 pm, 5.6 pm, 5.8 pm, 6.0 pm.
  • the cell suspension comprising the input anucleate cells are passed through multiple constrictions wherein the multiple constrictions are arranged in series and/or in parallel.
  • the HPV antigen is a pool of multiple polypeptides that elicit a response against the same and or different HPV antigens.
  • the HPV antigen is a polypeptide comprising one or more antigenic HPV epitope and one or more heterologous peptide sequences.
  • the HPV antigen is delivered with other antigens or with an adjuvant.
  • the HPV antigen is a polypeptide comprising an antigenic HPV epitope and one or more heterologous peptide sequences.
  • the HPV antigen complexes with itself, with other antigens, or with the adjuvant.
  • the HPV is HPV-16 or HPV-18.
  • the HPV antigen is comprised of an HLA-A2-specific epitope.
  • the HPV antigen is an HPV E6 antigen or an HPV E7 antigen.
  • the at least one antigen comprises a peptide derived from HPV E6 and/or E7.
  • the at least one antigen comprises an HLA-A2 -restricted peptide derived from HPV E6 and/or E7.
  • the HPV antigen is capable of being processed into an MHC class I-restricted peptide.
  • the HPV antigen is capable of being processed into an MHC class Il-restricted peptide.
  • the composition further comprises an adjuvant.
  • the adjuvant is a CpG oligodeoxynucleotide (ODN), LPS, IFN-a, IFN-P, IFN-y, alpha-Galactosyl Ceramide, STING agonists, cyclic dinucleotides (CDN), RIG-I agonists, polyinosinic-polycytidylic acid (poly I:C), R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR9 agonist.
  • the adjuvant is polyinosinic-polycytidylic acid (poly I:C).
  • the method comprises: a) passing a cell suspension comprising a input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed anucleate cells, thereby generating the AACs comprising the at least one
  • the AACs are washed for about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the AACs are washed for about 6 times. In some embodiments, the AACs are washed in PBS. In some embodiments, the AACs are washed in medium. In some embodiments, the medium is substantially the same as medium for the input anucleate cells. In some embodiments, the AACs are washed in medium comprising one or more stabilizing agents. In some embodiments, the AACs are washed in medium comprising one or more cryopreservation agents. In some embodiments, the AACs are washed by centrifugation and resuspension.
  • the AACs are washed by centrifugation and filtration. In some embodiments, the AACs are washed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps of centrifugation and resuspension. In some embodiments, the AACs are washed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps of centrifugation and filtration. In some embodiments, the one or more centrifugation steps are conducted at about any one of lOOxg, 150xg, 200xg, 250xg, 300xg, 350xg, 400xg, 450xg, 500xg, 550xg, 600xg, 650xg, 700xg, 750xg, and 800xg. In some embodiments, the one or more centrifugation steps are conducted at about any one of lOOOrpm, 1500rpm, 2000rpm, 2500rpm, 3000rpm, 3500rpm, 4000rpm, or 4500 rpm.
  • the formulation comprises about 1 x 10 7 to about 1 x 10 12 AACs. In some embodiments, the formulation comprises about 1 x 10 9 to about 1 x 10 11 AACs in about 9mL to aboutlO mL.
  • the formulation comprises about any one of 0.5 x 10 7 , 0.7 x 10 7 , 1.0 x 10 7 , 0.5 x 10 8 , 0.7 x 10 8 , 1.0 x 10 8 , 0.5 x 10 9 , 0.7 x 10 9 , 1.0 x 10 9 , 0.5 x 1O 10 , 0.7 x 1O 10 , 1.0 x 1O 10 , 0.5 x 10 11 , 0.7 x 10 11 , 1.0 x 10 11 , 0.5 x 10 12 , 0.7 x 10 12 , and 1.0 x 10 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises any one of about 0.5 x 10 7 to about 1.0 x io 7 , about 1.0 * 10 7 to about 0.5 x 10 8 AACs, about 0.5 x io 8 to about 1.0 x 10 8 , about 1.0 x io 8 to about 0.5 x io 9 AACs, about 0.5 x io 9 to about 1.0 x 10 9 , about 1.0 x io 9 to about 0.5 x io 10 , about 0.5 x io 10 to about 1.0 x 1O 10 , about 1.0 x io 10 to about 0.5 x io 11 , about 0.5 x io 11 to about 1.0 x io 11 AACs, about 1.0 x io 11 to about 0.5 x io 12 AACs in about 9 mL to about 10 mL.
  • the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9 mL to about lOmL. In some embodiments, the formulation comprises about any one of l x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , and l x 10 10 AACs in about 9.5 mL.
  • the formulation comprises about 7x 10 9 AACs in about 9 mL to about 10 mL. In some embodiments, the formulation comprises about 7x 10 9 AACs in about 9.5 mL. In some embodiments, the formulation comprises about 6.65 x 10 9 AACs in about 9.5 mL. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing. In some embodiments, the formulation comprises about 0.7 x 10 9 AACs/mL post-thawing as measured by Coulter counter.
  • the formulation comprises about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • the cryopreservation medium is CryoStor® CS2.
  • the method further comprises cryopreservation (such as freezing) of the formulation of AACs at between about -80 °C to about -250 °C. In some embodiments, the method further comprises cryopreservation (such as freezing) of the formulation of AACs at about -170 °C.
  • the method comprises freezing the formulation of AACs at about any one of -80 °C, -90 °C, -100 °C, -110 °C, -120 °C, -130 °C, -140 °C, -150 °C, -160 °C, -170 °C, -180 °C, -190 °C, -200 °C, -210 °C, -220 °C, -230 °C, -240 °C or -250 °C.
  • the method comprises freezing the formulation of AACs at any one of about -80 °C to about -90 °C, about -90 °C to about -100 °C, about -100 °C to about -110 °C, about -110 °C to about -120 °C, about -120 °C to about -130 °C, about -130 °C to about -140 °C, about -140 °C to about -150 °C, about -150 °C to about -160 °C, about -160 °C to about -170 °C, about -170 °C to about -180 °C, about -180 °C to about -190 °C, about -190 °C to about -200 °C, about -200 °C to about -210 °C, about -210 °C to about -220 °C, about -220 °C to about -230
  • the formulation are cryopreserved (such as frozen) by a process comprising: a) placing the formulation in a chamber; b) a first step reducing the temperature of the chamber to between about 0°C to about -20°C; c) a second step of reducing the temperature of the chamber to between about -130°C to about -150°C at a rate of between about - 10°C/minute to about -30°C/minute; d) a third step of reducing the temperature of the chamber to between about -140°C to about -160°C at a rate of between about -0.5°C/minute to about - 5°C/minute; e) a forth step of reducing the temperature of the chamber to between about -150°C to about -200°C at a rate of between about -0.
  • the chamber is a cryopreservation chamber, such as but not limited to a cryopreservation chamber that facilitates stable rate of temperature decrease, such as MR. FROSTYTM (Thermo ScientificTM).
  • different cryopreservation chambers are used for one or more of the steps described above.
  • the formulations are subsequently removed from the chamber and moved to permanent storage (such as but not limited to liquid nitrogen tank, and such as but not limited to liquid phase, liquid-vapor transition phase or vapor phase of liquid nitrogen).
  • the first step of reducing the temperature of the chamber of the chamber comprises reducing the temperature to any one of about 0 °C, -1 °C, -2 °C, -3 °C, or -4 °C, or any temperature or range therebetween.
  • the second step of reducing the temperature of the chamber comprises reducing the temperature of the chamber to any one of about -130 °C, -131 °C, -132 °C, -133 °C, -134 °C, -135 °C, -136 °C, -137 °C, -138 °C, -139 °C, -140 °C, -141 °C, -142 °C, -143 °C, -144 °C, -145 °C, -146 °C, -147 °C, -148 °C, - 149 °C, -150 °C or any temperature or range therebetween.
  • the second step of reducing the temperature of the chamber comprises reducing the temperature at a rate of any one of about -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20 °C per minute, or any rate or range therebetween.
  • the third step of reducing the temperature of the chamber comprises reducing the temperature of the chamber to any one of about -140 °C, -141 °C, -142 °C, -143 °C, -144 °C, -145 °C, -146 °C, -147 °C, -148 °C, -149 °C, -150 °C, -151 °C, -152 °C, -153 °C, -154 °C, -155 °C, -156 °C, -157 °C, -158 °C, - 159 °C, -160 °C or any temperature or range therebetween.
  • the third step of reducing the temperature of the chamber comprises reducing the temperature at a rate of any one of about -0.5, -1, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5 °C per minute, or any rate or range therebetween.
  • the fourth step of reducing the temperature of the chamber comprises reducing the temperature of the chamber to any one of about -150 °C, -152 °C, -154 °C, -156 °C, -158 °C, -160 °C, -162 °C, -164 °C, -166 °C, -168 °C, -170 °C, -172 °C, -174 °C, - 176 °C, -178 °C, -180 °C, -182 °C, -184 °C, -186 °C, -188 °C, -190 °C or any temperature or range therebetween.
  • the third step of reducing the temperature of the chamber comprises reducing the temperature at a rate of any one of about -0.1, -0.2, -0.3, -0.4, - 0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, -1.2, -1.3, -1.4, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5 °C per minute.
  • the method comprises holding the temperature of the chamber at about -150 °C, -152 °C, -154 °C, -156 °C, -158 °C, -160 °C, -162 °C, -164 °C, -166 °C, -168 °C, -170 °C, -172 °C, -174 °C, -176 °C, -178 °C, -180 °C, -182 °C, -184 °C, -186 °C, -188 °C, -190 °C or any temperature or range therebetween for at least any one of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or any time-intervals or range therebetween.
  • the formulation are cryopreserved (such as frozen) by a process comprising: a) placing the formulation in a chamber; b) reducing the temperature of the chamber to about -3 °C; c) reducing the temperature of the chamber to about -140°C at a rate of about -20°C/minutes; d) reducing the temperature of the chamber to about -150°C at a rate of about -1.5°C/minutes; e) reducing the temperature of the chamber to about -170°C at a rate of about -1.0°C/minutes; and f) holding the temperature of the chamber at about -170°C for at least about 10 minutes.
  • the at least one antigen is a exogenous antigen.
  • the exogenous antigen is a HPV antigen.
  • Papillomaviruses are small nonenveloped DNA viruses with a virion size of ⁇ 55 nm in diameter. More than 100 HPV genotypes are completely characterized, and a higher number is presumed to exist. HPV is a known cause of cervical cancers, as well as some vulvar, vaginal, penile, oropharyngeal, anal, and rectal cancers.
  • HPV-associated diseases can include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermodysplasia, focal epithelial hyperplasia, mouth papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (SILs), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VAIN).
  • SILs squamous intraepithelial lesions
  • CIN cervical intraepithelial neoplasia
  • VIN vulvar intraepithelial neoplasia
  • VAIN vaginal intraepithelial neoplasia
  • HPV types cause benign lesions with a subset being oncogenic. Based on epidemiologic and phylogenetic relationships, HPV types are classified into fifteen “high-risk types” (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three “probable high-risk types” (HPV 26, 53, and 66), which together are known to manifest as low and high grade cervical changes and cancers, as well as other anogential cancers such as vulval, vaginal, penile, anal, and perianal cancer, as well as head and neck cancers. Recently, the association of high-risk types HPV 16 and 18 with breast cancer was also described.
  • HPV 6, 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.
  • the immune system is depressed and correspondingly, the antitumor response is significantly impaired. See Suresh and Burtness, Am J Hematol Oncol 13(6):20-27 (2017).
  • the exogenous antigen is a pool of multiple polypeptides that elicit a response against the same and or different antigens.
  • an antigen in the pool of multiple antigens does not decrease the immune response directed toward other antigens in the pool of multiple antigens.
  • the HPV antigen is a polypeptide comprising an antigenic HPV epitope and one or more heterologous peptide sequences.
  • the HPV antigen complexes with itself, with other antigens, or with the adjuvant.
  • the HPV is HPV-16 or HPV-18.
  • the HPV antigen is comprised of an HLA-A2-specific epitope.
  • the HPV antigen is an HPV E6 antigen or an HPV E7 antigen.
  • the at least one antigen comprises a peptide derived from HPV E6 and/or E7. In some embodiments, the at least one 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, the HLA-A2- restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the HPV antigen comprises an amino acid sequence with at least 90% similarity to any one of SEQ ID NOs: 18-25.
  • the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 1. In some embodiments, the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO:2. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO:4. In some embodiment, the HPV antigen consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the HPV antigen consists of the amino acid sequence of SEQ ID NO:20.
  • the HPV antigen consists of the amino acid sequence of SEQ ID NO:21. In some embodiments, the HPV antigen consists of the amino acid sequence of SEQ ID NO:22. In some embodiments, the HPV antigen consists of the amino acid sequence of SEQ ID NO:23. In some embodiments, the HPV antigen consists of the amino acid sequence of SEQ ID NO:24. In some embodiments, the HPV antigen consists of the amino acid sequence of SEQ ID NO:25. In some embodiments, the HPV antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25.
  • the 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.
  • 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.
  • the exogenous antigen is a plurality of antigens comprising an amino acid sequence with at least 90% similarity to SEQ ID NO: 19 and an amino acid sequence with at least 90% similarity to SEQ ID NO:23.
  • the exogenous antigen is a plurality of antigens comprising the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:23.
  • the plurality of antigens is contained within a pool of non-covalently linked peptides.
  • the plurality of antigens is contained within a pool of non- covalently linked peptides, wherein each peptide comprises no more than one antigen.
  • the plurality of antigens is contained within a pool of non-covalently linked peptides, wherein the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:23 are contained within separate peptides.
  • the HPV antigen is within a pool of multiple polypeptides that elicit a response against the same and or different HPV antigens. In some embodiments, an antigen in the pool of multiple antigens does not decrease the immune response directed toward other antigens in the pool of multiple antigens. In some embodiments, the HPV antigen is a polypeptide comprising an antigenic HPV antigen and one or more heterologous peptide sequences. In some embodiments, the HPV antigen complexes with itself, with other antigens, or with the adjuvant. In some embodiments, the HPV antigen is comprised of an HLA-A2- specific epitope.
  • the HPV antigen is comprised of an HLA-A11 -specific epitope. In some embodiments, HPV antigen is comprised of an HLA-B7-specific epitope. In some embodiments, the HPV antigen is comprised of an HLA-C8-specific epitope. In some embodiments, the HPV antigen comprises part or all of the N-terminal domain of a full-length HPV protein.
  • the AACs comprise a plurality of HPV antigens that comprise a plurality of immunogenic epitopes.
  • the HPV antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope.
  • the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and/or a C-terminal flanking polypeptide.
  • the HPV antigen is a polypeptide comprising an immunogenic peptide epitope and one or more heterologous peptide sequences.
  • the HPV antigen is a polypeptide comprising an immunogenic peptide epitope that is flanked on the N-terminus and/or the C-terminus by heterologous peptide sequences.
  • the flanking heterologous peptide sequences are derived from disease- associated immunogenic peptides.
  • flanking heterologous peptide sequences are non-naturally occurring sequence.
  • the flanking heterologous peptide sequences are derived from an immunogenic synthetic long peptide (SLP).
  • the HPV antigen is capable of being processed into an MHC class I- restricted peptide and/or an MHC class Il-restricted peptide.
  • an adjuvant can refer to a substance which either directly or indirectly modulates and/or engenders an immune response.
  • an adjuvant is delivered intracellularly to a population of anucleate cells or anucleate- derived vesicles such as a population of RBCs or RBC-derived vesicles (z.e, incubation of cells or vesicles with an adjuvant before, during and/or after constriction processing, but prior to administration to an individual) to form AACscomprising the adjuvant.
  • the adjuvant is administered in conjunction with a HPV antigen to effect enhancement of an immune response to the HPV antigen as compared to HPV antigen alone. Therefore, adjuvants can be used to boost elicitation of an immune cell response (e.g. T cell response) to a HPV antigen.
  • exemplary adjuvants include, without limitation, stimulator of interferon genes (STING) agonists, retinoic acid-inducible gene I (RIG-I) agonists, and agonists for TLR3, TLR4, TLR7, TLR8 and/or TLR9.
  • Exemplary adjuvants include, without limitation, CpG ODN, interferon-a (IFN-a), polyinosinic:polycytidylic acid (polyLC), imiquimod (R837), resiquimod (R848), or lipopolysaccharide (LPS).
  • IFN-a interferon-a
  • polyLC polyinosinic:polycytidylic acid
  • imiquimod R837)
  • resiquimod R848
  • lipopolysaccharide LPS
  • the adjuvant is CpG ODN, LPS, IFN-a, IFN- P, IFN-y, alpha-Galactosyl Ceramide, STING agonists, cyclic dinucleotides (CDN), RIG-I agonists, polyinosinic:polycytidylic acid (polyLC), R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR9 agonist.
  • the adjuvant is a CpG ODN.
  • the adjuvant is a CpG ODN.
  • the CpG ODN is a Class A CpG ODN, a Class B CpG ODN, or a Class C CpG ODN.
  • the CpG ODN adjuvant comprise of a selection from the group of CpG ODN 1018, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CPG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362, CpG ODN D-SL03.
  • the CpG ODN adjuvant is CpG ODN 1826 (TCCATGACGTTCCTGACGTT (SEQ ID NO:30)) or CpG ODN 2006 (also known as CpG 7909) (TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO:31)) oligonucleotide.
  • the adjuvant is CpG 7909.
  • the RIG-I agonist comprises polyinosinic:polycytidylic acid (polyLC). Multiple adjuvants can also be used in conjunction with HPV antigens to enhance the elicitation of immune response.
  • the AACscomprising the HPV antigen further comprise more than one adjuvant. Multiple adjuvants can also be used in conjunction with HPV antigens to enhance the elicitation of immune response. In some embodiments, the AACs comprising the HPV antigen further comprise more than one adjuvant.
  • the AACs comprising the HPV antigen further comprise any combination of the adjuvants CpG ODN, LPS, IFN-a, IFN-P, IFN-y, alpha- Galactosyl Ceramide, STING agonists, cyclic dinucleotides (CDN), RIG-I agonists, polyinosinic:polycytidylic acid (polyLC), R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR9 agonist.
  • the adjuvants CpG ODN, LPS, IFN-a, IFN-P, IFN-y, alpha- Galactosyl Ceramide, STING agonists, cyclic dinucleotides (CDN), RIG-I agonists, polyinosinic:polycytidylic acid (polyLC), R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR
  • compositions of AACs comprising an antigen and an adjuvant
  • the invention provides formulations comprising AACs comprising at least one antigen and an adjuvant.
  • the anucleate cell is an RBC or a platelet.
  • the anucleate cell is an erythrocyte or a reticulocyte.
  • the anucleate cell is autologous to the individual who will receive the composition of AACs.
  • the anucleate cell is autologous to the individual who will receive the composition of AACs.
  • the at least one HPV antigen is delivered to the anucleate cells intracellularly.
  • the adjuvant is delivered to the anucleate cells intracellularly.
  • the HPV antigen is introduced into the anucleate cells by passing the cell through a constriction such that transient pores are introduced to the membrane of the cell thereby allowing the HPV antigen to enter the cell.
  • constriction-based delivery of compounds into a cell are provided by WO 2013/059343, WO 2015/023982, WO 2016/070136, W02017041050, W02017008063, WO 2017/192785, WO 2017/192786, WO 2019/178005, WO 2019/178006, WO 2020/072833, WO 2020/154696, and WO 2020/176789, US 20180142198, and US 20180201889.
  • the HPV antigen and adjuvant are delivered into the anucleate cells to produce the AACs of the invention by passing a cell suspension comprising the anucleate cells (e.g., RBCs) through a constriction, wherein the constriction deforms the cells thereby causing a perturbation of the cells such that a HPV antigen and an adjuvant enter the cells.
  • the constriction is contained within a microfluidic channel.
  • multiple constrictions can be placed in parallel and/or in series within the microfluidic channel.
  • the constriction within the microfluidic channel includes an entrance portion, a center point, and an exit portion.
  • the length, depth, and width of the constriction within the microfluidic channel can vary.
  • the width of the constriction within the microfluidic channel is a function of the diameter of the anucleate cells. Methods to determine the diameter of anucleate cells are known in the art; for example, high-content imaging, cell counters or flow cytometry.
  • the width of the constriction is about 0.5 pm to about 10 pm. In some embodiments, the width of the constriction is about 1 pm to about 4 pm. In some embodiments, the width of the constriction is about 1 pm to about 3 pm. In some embodiments, the width of the constriction is about 1.5 pm to about 2.5 pm. In some embodiments, the width of the constriction is about 1.2 pm to about 2.8 pm. In some embodiments, the width of the constriction is about 0.5 pm to about 5 pm. In some embodiments, the width of the constriction is about 2 pm to about 2.5 pm.
  • the width of the constriction is about 1.5 pm to about 2 pm. In some embodiments, the width of the constriction is about 0.5 pm to about 3.5 pm. In some embodiments, the width of the constriction is about 3.2 pm to about 3.8 pm. In some embodiments, the width of the constriction is about 3.8 pm to about 4.3 pm.
  • the width of the constriction is about or less than any one of 0.25 pm, 0.5 pm, 1.0 pm, 1.2 pm, 1.4 pm, 1.6 pm, 1.8 pm, 2.0 pm, 2.2 pm, 2.4 pm, 2.6 pm, 2.8 pm, 3.0 pm, 3.2 pm, 3.4 pm, 3.6 pm, 3.8 pm, 4.0 pm, 4.2 pm, 4.4 pm, 4.6 pm, 4.8 pm, 5.0 pm, 5.2 pm, 5.4 pm, 5.6 pm, 5.8 pm, 6.0 pm.
  • the width of the constriction is about 2 pm.
  • the width of the constriction is about 2.2 pm.
  • the width of the constriction is about 2.5 pm.
  • the width of the constriction is about 3 pm.
  • parameters that may influence the delivery of the compound into the AAC include, but are not limited to, the dimensions of the constriction, the entrance angle of the constriction, the surface properties of the constrictions (e.g., roughness, chemical modification, hydrophilic, hydrophobic, etc.), the operating flow speeds (e.g., cell transit time through the constriction), the cell concentration, the concentration of the compound in the cell suspension, buffer in the cell suspension, and the amount of time that the AACs recover or incubate after passing through the constrictions can affect the passage of the delivered compound into the anucleate-derived vesicles.
  • the dimensions of the constriction the entrance angle of the constriction
  • the surface properties of the constrictions e.g., roughness, chemical modification, hydrophilic, hydrophobic, etc.
  • the operating flow speeds e.g., cell transit time through the constriction
  • the cell concentration e.g., the concentration of the compound in the cell suspension, buffer in the cell suspension
  • Additional parameters influencing the delivery of the compound into the AACs can include the velocity of the input anucleate cells in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component that is perpendicular to flow velocity, and time in the constriction.
  • multiple chips comprising channels in series and/or in parallel may impact delivery to anucleate-derived vesicles. Multiple chips in parallel may be useful to enhance throughput.
  • Such parameters can be designed to control delivery of the compound.
  • the cell concentration ranges from about 10 to at least about 10 12 cells/mL or any concentration or range of concentrations therebetween.
  • delivery compound concentrations can range from about 10 ng/mL to about 1 g/mL or any concentration or range of concentrations therebetween. In some embodiments, delivery compound concentrations can range from about 1 pM to at least about 2 M or any concentration or range of concentrations therebetween. [0142] In some embodiments, the concentration of HPV antigen incubated with the anucleate cells or AACs is between about 0.01 pM and about 10 mM.
  • the concentration of HPV antigen incubated with the anucleate cells or AACs is any of less than about 0.01 pM, about 0.1 pM, about 1 pM, about 10 pM, about 100 pM, about 1 mM or about 10 mM. In some embodiments, the concentration of HPV antigen incubated with the anucleate cells or AACs is greater than about 10 mM.
  • the concentration of HPV antigen incubated with the anucleate cells or AACs is any of between about 0.01 pM and about 0.1 pM, between about 0.1 pM and about 1 pM, between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the concentration of HPV antigen incubated with the anucleate cells or AACs is between about 0.1 pM and about 1 mM. In some embodiments, the concentration of HPV antigen incubated with the anucleate cells or AACs is between about 0.1 pM and about 10 pM. In some embodiments, the concentration of HPV antigen incubated with the anucleate cells or AACs is 1 pM.
  • the concentration of antigen incubated with the perturbed input anucleate cell is between about 0.01 pM and about 10 mM.
  • the concentration of antigen incubated with the perturbed input anucleate cell is any of less than about 0.01 pM, about 0.1 pM, about 1 pM, about 10 pM, about 100 pM, about 1 mM or about 10 mM.
  • the concentration of antigen incubated with the perturbed input anucleate cell is greater than about 10 mM.
  • the concentration of antigen incubated with the perturbed input anucleate cell is any of between about 0.01 pM and about 0.1 pM, between about 0.1 pM and about 1 pM, between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the concentration of antigen incubated with the perturbed input anucleate cell is between about 0.1 pM and about 1 mM. In some embodiments, the concentration of antigen incubated with the perturbed input anucleate cell is between about 0.1 pM and about 10 pM. In some embodiments, the concentration of antigen incubated with the perturbed input anucleate cell is 1 pM.
  • the molar ratio of antigen to adjuvant incubated with the perturbed input anucleate cell is any of between about 10000: 1 to about 1 : 10000.
  • the molar ratio of antigen to adjuvant incubated with the perturbed input anucleate cell is about any of 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.
  • the molar ratio of antigen to adjuvant incubated with the perturbed input anucleate cell is any of between about 10000: 1 and about 1000: 1, between about 1000:1 and about 100: 1, between about 100:1 and about 10: 1, between about 10: 1 and about 1 : 1, between about 1 :1 and about 1 : 10, between about 1 : 10 and about 1 : 100, between about 1 : 100 and about 1 : 1000, between about 1 : 1000 and about 1 : 10000.
  • the molar ratio of antigen to adjuvant incubated with the perturbed input anucleate cell is about 200: 1.
  • the molar ratio of antigen to adjuvant incubated with the perturbed input anucleate cell is about 20: 1.
  • the AACs comprise the adjuvant at a concentration between about 1 nM and about 1 mM.
  • the AACs comprise the adjuvant at a concentration of any of less than about 0.01 pM, about 0.1 pM, about 1 pM, about 10 pM, about 100 pM, about 1 mM or about 10 mM.
  • the AACs comprise the adjuvant at a concentration of greater than about 10 mM.
  • the AACs comprise the adjuvant at a concentration of any of between about 1 nM to about 10 nM, about 0.1 pM and about 1 pM, between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the AACs comprise the adjuvant at a concentration between about 0.1 pM and about 1 mM. In some embodiments, the AACs comprise the adjuvant at a concentration of about 1 pM.
  • the AACs comprise the at least one antigen at a concentration between about 1 nM and about 1 mM.
  • the AACs comprises the at least one antigen at a concentration of any of less than about 0.01 pM, about 0.1 pM, about 1 pM, about 10 pM, about 100 pM, about 1 mM or about 10 mM.
  • the AACs comprise the at least one antigen at a concentration of greater than about any of 10 mM.
  • the AACs comprise the at least one antigen at a concentration of any of between about 1 nM to about 10 nM, about 0.1 pM and about 1 pM, between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the AACs comprise the at least one antigen at a concentration between about 0.1 pM and about 1 mM. In some embodiments, the AACs comprise the at least one antigen at a concentration of about 1 pM.
  • the molar ratio of antigen to adjuvant in the AACs is any of between about 10000: 1 to about 1 : 10000.
  • the molar ratio of antigen to adjuvant in the AACs is about any of 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.
  • the molar ratio of antigen to adjuvant in the modified PBMCs is any of between about 10000: 1 and about 1000: 1, between about 1000: 1 and about 100:1, between about 100: 1 and about 10: 1, between about 10: 1 and about 1 : 1, between about 1 : 1 and about 1 : 10, between about 1 : 10 and about 1 : 100, between about 1 : 100 and about 1 : 1000, between about 1 :1000 and about 1 : 10000.
  • the molar ratio of antigen to adjuvant in the AACs is about 200: 1. In some embodiments, the molar ratio of antigen to adjuvant in the AACs is about 20: 1.
  • the AACs are generated in a process comprising: a) passing a cell suspension comprising input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for an antigen and an adjuvant to pass through to form perturbed input anucleate cells; b) incubating the perturbed input anucleate cells with the at least one antigen and adjuvant for a sufficient time to allow the at least one antigen and adjuvant to enter the perturbed input anucleate cells; thereby generating AACs comprising the at least one antigen and adjuvant.
  • the AACs comprising the payload displays different characteristics compared to an input anucleate cell.
  • the anucleate cell-derived vesicle comprising the payload displays different characteristics compared to an anucleate cell comprising a payload introduced by other delivery methods (such as hemolytic loading or electroporation).
  • the half-life of the AACs following administration to a mammal is decreased compared to a half-life of the input anucleate cell following administration to the mammal.
  • the hemoglobin content of the AACs is decreased compared to the hemoglobin content of the input anucleate cell.
  • ATP production of the AACs is decreased compared to ATP production of the input anucleate cell.
  • the AACs exhibits a spherical morphology.
  • the input anucleate cell is an erythrocyte and wherein the AACs have a reduced biconcave shape compared to the input erythrocyte.
  • the AAC is a red blood cell ghost.
  • the AACs prepared by the process have greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell.
  • a population profile of AACs prepared by the process exhibits higher average phosphatidyl serine levels on the surface compared to the input anucleate cells.
  • at least 50% of the population profile of AACs prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleate cells.
  • the AACs exhibit preferential uptake in a tissue or cell compared to the input anucleate cell.
  • the AACs exhibit preferential uptake in phagocytic cells and/or antigen presenting cells compared to the input anucleate cell. In some embodiments, the AACs are modified to enhance uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the AACs are modified to enhance uptake in phagocytic cells and/or antigen presenting cells compared to an unmodified anucleate cell-derived vesicle. In some embodiments, the phagocytic cells and/or antigen presenting cells comprise one or more of a dendritic cell or macrophage. In some embodiments, the tissue or cell comprises one or more of liver or spleen. In some embodiments, the anucleate cell-derived vesicle comprises CD47 on its surface.
  • the constriction is contained within a microfluidic channel.
  • the microfluidic channel comprises a plurality of constrictions.
  • the plurality of constrictions is arranged in series and/or in parallel.
  • the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates.
  • the constriction is a pore or contained within a pore.
  • the pore is contained in a surface.
  • the surface is a filter.
  • the surface is a membrane.
  • the constriction size is a function of the diameter of the input anucleate cell in suspension. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the diameter of the input anucleate cell in suspension. In some embodiments, the constriction has a width of about 0.25 pm to about 4 pm. In some embodiments, the constriction has a width of about 4 pm, 3.5 pm, about 3 pm, about 2.5 pm, about 2 pm, about 1.5 pm , about 1 pm, about 0.5 pm, or about 0.25 pm. In some embodiments, the constriction has a width of about 2.2 pm.
  • the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi.
  • said cell suspension is contacted with the at least one antigen before, concurrently, or after passing through the constriction.
  • an AAC comprising the payload e.g. at least one HPV antigen and an adjuvant
  • the AAC having one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the input anucleate cell, (b) decreased hemoglobin levels compared to the input anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the input anucleate cell, or (e) reduced ATP production compared to the input anucleate cell.
  • the input anucleate cell is a mammalian cell. In some embodiments, the input anucleate cell is human cell. In some embodiments, the input anucleate cell is a red blood cell or a platelet. In some embodiments, the red blood cell is an erythrocyte or a reticulocyte. In some embodiments, the anucleate cell is autologous to the individual who will receive the composition. In some embodiments, the anucleate cell is autologous to the individual who will receive the composition.
  • the circulating half-life of the AAC in a mammal is decreased compared to the input anucleate cell. In some embodiments, the circulating half-life in the mammal is decreased by more 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 anucleate cell.
  • the input anucleate cell is a human cell and wherein 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, about 120 days.
  • the input anucleate cell is a red blood cell, wherein the hemoglobin levels in the AAC are decreased compared to the input anucleate cell.
  • the hemoglobin levels in the AAC are decreased 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 anucleate cell.
  • the hemoglobin levels in the AAC are about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the level of hemoglobin in the input anucleate cell.
  • the input anucleate cell is an erythrocyte and wherein the AAC is spherical in morphology. In some embodiments, the input anucleate cell is an erythrocyte and wherein the AAC has a reduced biconcave shape compared to the input anucleate cell.
  • the input anucleate cell is a red blood cell or an erythrocyte and wherein the AAC is a red blood cell ghost (RBC ghost).
  • RBC ghost red blood cell ghost
  • the AAC comprises CD47 on its surface.
  • the AAC has increased surface phosphatidylserine levels compared to the input anucleate cell. In some embodiments, the AACs prepared by the process has greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell. 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 anucleate cell.
  • the AAC has reduced ATP production compared to the input anucleate cell. In some embodiments, the AAC produces ATP at less than about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the level of ATP produced by the input anucleate cell. In some embodiments, the AAC does not produce ATP.
  • the AAC exhibits enhanced uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the AAC exhibits preferential uptake in liver or spleen or by a phagocytic cell or an antigen-presenting cell compared to the uptake of the input anucleate cell.
  • the AAC is further modified to enhance uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the AAC is further modified to enhance uptake in liver or spleen or by a phagocytic cell or an antigen-presenting cell compared to the uptake of the input anucleate cell.
  • the AAC exhibits enhanced uptake in liver or spleen or by a phagocytic cell and/or an antigen-presenting cell
  • internalization of the AAC results in increased expression of maturation markers of the phagocytic cell or the antigen-presenting cell.
  • the phagocytic cell and/or the antigen-presenting cell is a monocyte- derived dendritic cell (MODC).
  • the maturation marker is one or more of CD80, CD86, CD83, and MHC-II.
  • the expression of one or more of CD80, CD86, CD83, and MHC-II is increased in the phagocytic cell and/or the antigen- presenting cell contacted with an AAC comprising a HPV antigen 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 compared to a phagocytic cell and/or an antigen-presenting cell not contacted with a AAC comprising a HPV antigen.
  • the expression of one or more of CD80, CD86, CD83, and MHC-II is increased in the phagocytic cell and/or the antigen-presenting cell contacted with an AAC comprising a HPV antigen 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 compared to a phagocytic cell and/or an antigen-presenting cell contacted with the input anucleate cell.
  • the AAC comprising an HPV antigen, or a HPV antigen and adjuvant exhibits enhanced uptake in liver or spleen or by a phagocytic cell and/or an antigen-presenting cell
  • internalization of the AAC results in increased presentation of the HPV antigen comprised within the anucleate cell-derived vesicle.
  • the presentation of the HPV antigen is increased in the phagocytic cell and/or the antigen-presenting cell contacted with a AAC comprising a HPV antigen 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 compared to a phagocytic cell and/or an antigen-presenting cell contacted with corresponding anucleate cells comprising the same HPV antigen introduced by other delivery methods (such as but not limited to hemolytic loading).
  • the AAC comprising an HPV antigen, or a HPV antigen and adjuvant exhibits enhanced uptake in liver or spleen or by a phagocytic cell and/or an antigen-presenting cell
  • internalization of the AAC results in increased ability of the phagocytic cell and/or the antigen-presenting cell to induce an anti gen- specific immune response.
  • the antigen-specific immune response mediated by the phagocytic cell and/or the antigen-presenting cell contacted with a AAC comprising the HPV antigen or the HPV antigen and adjuvant 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 compared to a phagocytic cell and/or an antigen-presenting cell contacted with the input anucleate cells.
  • the antigen-specific immune response mediated by the phagocytic cell and/or the antigen-presenting cell contacted with a AAC comprising the HPV antigen or the HPV antigen and adjuvant 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 compared to a phagocytic cell and/or an antigen-presenting cell contacted with the anucleate cells comprising the same HPV antigen introduced by other delivery methods (such as but not limited to hemolytic loading).
  • the antigen-specific immune response is an antigen-specific CD4+ T cell response.
  • the antigenspecific immune response is an antigen-specific CD8+ T cell response.
  • the individual is positive for 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* 16.
  • the phagocytes are human cells with a haplotype of 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*
  • the antigen presenting cells are human cells with a haplotype of HLA-A*02, HLA-A*11, HLA-B*07, or HLA-C*08.
  • HPV antigens presented by the phagocytes and/or antigen presenting cells described herein are comprised of an HLA-A2-specific epitope.
  • HPV antigens presented by the phagocytes and/or antigen presenting cells described herein are comprised of an HLA-A11 -specific epitope.
  • HPV antigens presented by the phagocytes and/or antigen presenting cells described herein are comprised of an HLA-B7- specific epitope.
  • HPV antigens presented by the phagocytes and/or antigen presenting cells described herein are comprised of an HLA-C8-specific epitope.
  • the method comprises administering AACs comprising the HPV antigen and adjuvant to the individual, wherein the AACs are internalized by phagocytic cells and/or antigen-presenting cell.
  • AACs are internalized by phagocytic cells and/or antigen-presenting cell
  • internalization of the AAC results in increased expression of maturation markers of the phagocytic cell or the antigen-presenting cell.
  • the phagocytic cell and/or the antigen-presenting cell is a monocyte-derived dendritic cell (MODC).
  • the maturation marker is one or more of CD80, CD86, CD83, and MHC-II.
  • the expression of one or more of CD80, CD86, CD83, and MHC-II is increased in the phagocytic cell and/or the antigen-presenting cell contacted with a AAC comprising a HPV antigen 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 compared to an phagocytic cell and/or an antigen-presenting cell not contacted with a AAC comprising a HPV antigen.
  • the expression of one or more of CD80, CD86, CD83, and MHC-II is increased in the phagocytic cell and/or the antigen-presenting cell contacted with a AAC comprising a HPV antigen 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 compared to a phagocytic cell and/or an antigen-presenting cell contacted with the input anucleate cell.
  • the input anucleate cell was not (a) heat processed, (b) chemically treated, and/or (c) subjected to hypotonic or hypertonic conditions during the preparation of the anucleate cell-derived vesicles.
  • osmolarity was maintained during preparation of the AAC from the input anucleate cell. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 600 mOsm. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 400 mOsm.
  • the invention provides a system comprising one or more of the constriction, an anucleate cell suspension, antigens or adjuvants for use in the methods disclosed herein.
  • the system can include any embodiment described for the methods disclosed above, including microfluidic channels or a surface having pores to provide cell-deforming constrictions, cell suspensions, cell perturbations, delivery parameters, compounds, and/or applications etc.
  • the cell-deforming constrictions are sized for delivery to anucleate cells.
  • the delivery parameters such as operating flow speeds, cell and compound concentration, velocity of the cell in the constriction, and the composition of the cell suspension (e.g., osmolarity, salt concentration, serum content, cell concentration, pH, etc.) are optimized for maximum response of a compound for suppressing an immune response or inducing tolerance.
  • the kit comprises an AAC comprising intracellularly an antigen and intracellularly an adjuvant.
  • the kit comprises one or more of the constriction, an anucleate cell suspension, HPV antigens or adjuvants for use in generating AACs for use in treating an individual with a disease associated with HPV, such as cancer.
  • the kits comprise the compositions described herein (e.g. a microfluidic channel or surface containing pores, cell suspensions, and/or compounds) in suitable packaging.
  • suitable packaging materials are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.
  • kits comprising components of the methods described herein and may further comprise instructions for performing said methods treat an individual with a cancer associated with HPV and/or instructions for introducing a HPV antigen and an adjuvant into an anucleate cell.
  • the kits described herein may further include other materials, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein; e.g., instructions for treating an individual with a cancer associated with HPV or instructions for generating AACs to contain intracellularly a HPV antigen and intracellularly an adjuvant.
  • Embodiment 1 A pharmaceutical formulation comprising activating antigen carriers (AACs), the formulation comprising a) AACs wherein the AACs comprise at least one antigen and an adjuvant, and b) a cryopreservation medium.
  • AACs activating antigen carriers
  • Embodiment 2 The pharmaceutical formulation of embodiment 1, wherein the formulation comprises about 0.5 x io 9 AACs to about 1 x io 10 AACs.
  • Embodiment 3 The pharmaceutical formulation of embodiment 1 or 2, wherein the formulation comprises about 7x 10 9 AACs.
  • Embodiment 4 The pharmaceutical formulation of any one of embodiments 1-3, wherein the formulation comprises about 7x 10 9 AACs prior to freezing.
  • Embodiment 5. The pharmaceutical formulation of any one of embodiments 1-4, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 * 10 9 AACs after thawing.
  • Embodiment 6 The pharmaceutical formulation of any one of embodiments 1-5, wherein the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL.
  • Embodiment 7 The pharmaceutical formulation of any one of embodiments 1-6, wherein the formulation comprises about 0.7 x 10 9 AACs/mL.
  • Embodiment 8 The pharmaceutical formulation of any one of embodiments 1-7, wherein the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing.
  • Embodiment 9 The pharmaceutical formulation of any one of embodiments 1-8, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing.
  • Embodiment 10 The pharmaceutical formulation of any one of embodiments 1-9, wherein at least about 70%, 80%, 90%, or 95% of AACs of a population of the AACs are functional.
  • Embodiment 11 The pharmaceutical formulation of any one of embodiments 1-10, wherein the AACs in the formulation maintain equal to or greater than about 70% functionality.
  • Embodiment 12 The pharmaceutical formulation of any one of embodiments 1 or 2, wherein the formulation comprises about 1 x 10 8 functional AACs /mL to about 1 x 10 9 functional AACs/mL.
  • Embodiment 13 The pharmaceutical formulation of any one of embodiments 1-12, wherein at least about 70%, 80%, 90%, or 95% of AACs of a population of the AACs are positive for annexin staining.
  • Embodiment 14 The pharmaceutical formulation of any one of embodiments 1-13, wherein the AACs in the formulation maintain equal to or greater than about 70% are positive for annexin staining.
  • Embodiment 15 The pharmaceutical formulation of embodiment 13 or 14, wherein the annexin is annexin V.
  • Embodiment 16 The pharmaceutical formulation of any one of embodiments 1-15, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Embodiment 17 The pharmaceutical formulation of any one of embodiments 1-16, wherein the cryopreservation medium comprises about 0.5% to about 5% DMSO.
  • Embodiment 18 The pharmaceutical formulation of any one of embodiments 1-17, wherein the cryopreservation medium comprises about 2% DMSO.
  • Embodiment 19 The pharmaceutical formulation of any one of embodiments 1-18, wherein the cryopreservation medium is CryoStor® CS2.
  • Embodiment 20 The pharmaceutical formulation of any one of embodiments 1-19, wherein the pH of the formulation is about 6.0 to about 8.5.
  • Embodiment 21 The pharmaceutical formulation of any one of embodiments 1-20, wherein the pH of the formulation is about 7.6.
  • Embodiment 22 A pharmaceutical formulation of AACs, the formulation comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • Embodiment 23 A pharmaceutical formulation of AACs, the formulation comprising about 0.5 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • Embodiment 24 The pharmaceutical formulation of embodiment 22 or 23 wherein the formulation comprises about 7x 10 9 AACs prior to freezing.
  • Embodiment 25 The pharmaceutical formulation of any one of embodiments 22- 25, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x 10 9 cells after thawing.
  • Embodiment 26 The pharmaceutical formulation of any one of embodiments 22-
  • the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL.
  • Embodiment 27 The pharmaceutical formulation of any one of embodiments 22-
  • the formulation comprises about 0.7 x 10 9 AACs/mL.
  • Embodiment 28 The pharmaceutical formulation of any one of embodiments 22-
  • the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing.
  • Embodiment 29 The pharmaceutical formulation of any one of embodiments 22-
  • the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing.
  • Embodiment 30 A pharmaceutical formulation of AACs, the formulation comprising about 7 x 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • Embodiment 31 The pharmaceutical formulation of any one of embodiments 22- 30, wherein the cryopreservation medium is CryoStor® CS2.
  • Embodiment 32 The pharmaceutical formulation of any one of embodiments 1-31, wherein the formulation is sterile.
  • Embodiment 33 The pharmaceutical formulation of any one of embodiments 1-32, wherein the formulation comprises less than about 2 EU/mL endotoxin.
  • Embodiment 34 The formulation of any one of embodiments 1-33, wherein the formulation is free of mycoplasma.
  • Embodiment 35 The formulation of any one of embodiments 1-34, wherein the at least one antigen is a human papillomavirus (HPV) antigen.
  • HPV human papillomavirus
  • Embodiment 36 The formulation of embodiment 35, wherein the HPV is HPV-16 or HPV-18.
  • Embodiment 37 The formulation of embodiment 35 or 36, wherein the antigen comprises a peptide derived from HPV E6 and/or E7.
  • Embodiment 38 The formulation of any one of embodiments 35-37, wherein the antigen comprises a peptide derived from HPV E6 and a peptide from HPV E7.
  • Embodiment 39 The formulation of any one of embodiments 35-38, wherein the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1-4.
  • Embodiment 40 The formulation of any one of embodiments 35-39, wherein the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25.
  • Embodiment 41 The formulation of any one of embodiments 35-40, wherein the
  • AACs 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.
  • Embodiment 42 The formulation of any one of embodiments 1-41, wherein the adjuvant is a CpG oligodeoxynucleotide (ODN), LPS, IFN-a, STING agonists, RIG-I agonists, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR 9 agonist.
  • ODN CpG oligodeoxynucleotide
  • LPS LPS
  • IFN-a STING agonists
  • RIG-I agonists poly I:C
  • R837, R848 a TLR3 agonist
  • TLR4 agonist a TLR4 agonist
  • Embodiment 43 The formulation of embodiment 42, wherein the adjuvant is a CpG 7909 oligodeoxynucleotide (ODN).
  • ODN oligodeoxynucleotide
  • Embodiment 44 The formulation of any one of embodiments 1-43, wherein the
  • AACs comprising the at least one antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed anucleate cells, thereby generating the AACs comprising the at least one antigen and the adjuvant.
  • Embodiment 45 The method of embodiment 44, wherein the diameter of the constriction is about 1.6 pm to about 2.4 pm or about 1.8 pm to about 2.2 pm.
  • Embodiment 46 The method of embodiment 44 or 45, wherein the input anucleate cell is a red blood cell.
  • Embodiment 47 A vial comprising the pharmaceutical formulation of any one of embodiments 1-46.
  • Embodiment 48 A vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 1 x 10 9 AACs to about 1 x IO 10 AACs in cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 6.0 to about pH 8.5.
  • Embodiment 49 A vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 1 x 10 9 AACs to about 1 x 1O 10 AACs in a cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, wherein the pH of the formulation is about pH 7.6.
  • Embodiment 50 The vial of embodiment 48 or 49 wherein the formulation comprises about 7x 10 9 AACs prior to freezing.
  • Embodiment 51 The vial of any one of embodiments 48-50, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x io 9 cells after thawing.
  • Embodiment 52 The vial of any one of embodiments 48-51, wherein the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL.
  • Embodiment 53 The vial of any one of embodiments 48-52, wherein the formulation comprises about 0.7 x 10 9 AACs/mL.
  • Embodiment 54 The vial of any one of embodiments 48-53, wherein the formulation comprises about 0.7 * 10 9 AACs/mL prior to freezing.
  • Embodiment 55 The vial of any one of embodiments 48-54, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing.
  • Embodiment 56 A vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 7 * 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • Embodiment 57 The vial of any one of embodiments 48-56 or 39, wherein the AACs are in about 9.5 mL of the cryopreservation medium.
  • Embodiment 58 A vial comprising a pharmaceutical formulation; the pharmaceutical formulation comprising about 7 * 10 9 AACs in about 9.5 mL of cryopreservation medium, wherein AACs comprise at least one antigen and an adjuvant, and wherein the pH of the formulation is about pH 7.6.
  • Embodiment 59 The vial of any one of embodiments 47-58, wherein the formulation is sterile.
  • Embodiment 60 A method of producing a pharmaceutical formulation of AACs, the method comprising adding a cryopreservation medium to the AACs wherein the AACs comprise at least one antigen and an adjuvant.
  • Embodiment 61 A method of producing a pharmaceutical formulation of AACs, wherein the AACs comprise the at least one antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising a input anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cells in the suspension, thereby causing perturbations of the input anucleate cells large enough for the at least one antigen and the adjuvant to pass through to form perturbed anucleate cells; and b) incubating the perturbed anucleate cells with the at least one antigen and the adjuvant for a sufficient time to allow the at least one antigen and the adjuvant to enter the perturbed anucleate cells, thereby generating the AACs comprising the at least one antigen and the adjuvant; c) washing the AACs; and d) formulating the AACs in a cryopreserv
  • Embodiment 62 The method of embodiment 61, wherein the diameter of the constriction is about 1.6 pm to about 2.4 pm or about 1.8 pm to about 2.2 pm.
  • Embodiment 63 The method of embodiment 61 or 62, wherein the AACs are washed about 6 times.
  • Embodiment 64 The method of any one of embodiments 61-63, wherein the AACs are washed by centrifugation and resuspension or by centrifugation and filtration.
  • Embodiment 65 The method of embodiment 64, wherein the centrifugation is at about 4000 rpm.
  • Embodiment 66 The method of any one of embodiments 61-65, wherein about 1 x 10 9 AACs to about 1 x 10 10 AACs are formulated in about 9 mL to about 10 mL of the cry opreservation medium.
  • Embodiment 67 The method of embodiment 66, wherein the pharmaceutical formulation comprises about 7x 10 9 AACs prior to freezing.
  • Embodiment 68 The method of embodiment 66 or 67, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 9 x 10 9 cells after thawing.
  • Embodiment 69 The method of any one of embodiments 66-68, wherein the formulation comprises about 0.5 x 10 9 AACs/mL to about 1 x 10 9 AACs/mL.
  • Embodiment 70 The method of any one of embodiments 66-69, wherein the formulation comprises about 0.7 x 10 9 AACs/mL.
  • Embodiment 71 The method of any one of embodiments 66-70, wherein the formulation comprises about 0.7 x 10 9 AACs/mL prior to freezing.
  • Embodiment 72 The method of any one of embodiments 66-71, wherein the formulation comprises AACs that were previously frozen, wherein the formulation comprises about 1 x 10 9 AACs/mL after thawing.
  • Embodiment 73 The method of any one of embodiments 66-72, wherein about 7 x 10 9 AACs are formulated in about 10 mL of the cryopreservation medium.
  • Embodiment 74 The method of any one of embodiments 66-73, wherein the cryopreservation medium is CryoStor® CS2.
  • Embodiment 75 The method of any one of embodiments 61-74, wherein the input anucleate cell is a red blood cell. [0248] Embodiment 75. The method of any one or embodiments 60-75, wherein the method further comprises freezing the formulation of AACs at about -170 °C.
  • Embodiment 76 The method of embodiment 75, where the formulation of AACs are frozen by a process comprising: a) placing the formulation in a chamber b) reducing the temperature of the chamber to about -3°C c) reducing the temperature of the chamber to about -140°C at a rate of about - 20°C/minutes d) reducing the temperature of the chamber to about -150°C at a rate of about 1.5°C/minutes. e) reducing the temperature of the chamber to about -170°C at a rate of about 1.0°C/minutes, and f) holding the temperature of the chamber at about -170°C for at least about 10 minutes.
  • the starting material information was verified against the manufacturing batch record.
  • a sample was aseptically withdrawn from the blood bag and a complete blood count (CBC) measurement is obtained via a hematology analyzer for information only, as well as an RBC count using a Coulter-based cell counter where the tentative criterion for total number of RBC s is >5 x IQ 11 total RBCs.
  • CBC complete blood count
  • the starting whole blood was diluted with RPMI + 5% DMSO (dilution media) to reduce the cell concentration for processing on a LOVO cell washing system.
  • the dilution media contains 5% DMSO to be similar to the media required for dissolution of the SLPs & adjuvant used in the delivery process downstream of this unit operation.
  • dilution ratios from 1 :3 to 1 :9 all show similar RBC recoveries over the LOVO process.
  • the expected patient starting material whole blood including anticoagulant
  • the expected patient starting material is 260 to 270 mL
  • a set dilution media volume of IL was implemented to simplify the manufacturing process.
  • the final parameters for the whole blood dilution step are shown in Table 2.
  • the intermediate product is gravity filtered through a sterile single-use 40 pm blood filter to remove any potential cell aggregates.
  • a sample was aseptically withdrawn from the diluted blood bag and an RBC count is taken.
  • the diluted blood is then processed on the LOVO cell washing system where the cells are washed for platelet removal with a sterile filtered mixture of 50 pM E6 SLP, 250 pM E7 SLP and 1.5 mg/mL poly I:C in RPMI 1640 Medium with 5% DMSO (referred to as the delivery media).
  • the delivery media a sterile filtered mixture of 50 pM E6 SLP, 250 pM E7 SLP and 1.5 mg/mL poly I:C in RPMI 1640 Medium with 5% DMSO (referred to as the delivery media).
  • the delivery media After platelet removal, the cells were resuspended with the delivery media using the LOVO system.
  • the in-process material was aseptically processed through a leukoreduction filter where WBCs are removed, resulting in a purified RBC suspension in delivery media.
  • the purified cell suspension was then assessed for R
  • the LOVO system utilizes a single-use LOVO kit which consists of a 4 pm spinning membrane, allowing for removal of platelets while maximizing recovery of RBCs. Using variable inlet and outlet flow rates, the LOVO system allows for washing and concentration of cells. Processing parameters used on the LOVO system for washing and resuspending RBCs with delivery media were provided by the equipment vendor (Fresenius Kabi). The recommended parameters provided by the vendor were evaluated during the development activities.
  • the LOVO processing parameters for platelet removal are provided in Table 3.
  • the intermediate product is filtered through a sterile single-use leukoreduction-filtration assembly at ambient conditions.
  • the filtration assembly consists of a leukoreduction filter (LRF), a 40 pm blood filter and an 18 pm blood filter assembled in line.
  • LRF leukoreduction filter
  • the leukoreduction-filtration assembly allows for removal of WBCs, and any potential cell aggregates. It has been noted that the leukoreduction filter also removes platelets in a non-specific manner.
  • Initial trial runs with the leukoreduction filtration step attempted to leukoreduce the incoming donor whole blood prior to other handling, however the filtration time was observed to be prohibitively long in duration. Ultimately it was determined that the leukoreduction step was best suited after the initial RBC purification on the LOVO had occurred, reducing the filtration time to ⁇ 5 minutes.
  • the data demonstrates suitable RBC purification in preparation for peptide & adjuvant delivery in the subsequent processing steps.
  • a microfluidic chip with a 2.2 pm constriction was used for the development of the RBC delivery process.
  • the microfluidic chip was selected to align with the chip constriction width used during the pre-clinical research studies. It is noted that the small number of residual WBCs remaining (see FIG. 2) in-process during manufacture of AAC-HPV prior to the delivery step do not clog the chip.
  • Ovalbumin 647 fluorescently labeled Ovalbumin
  • FAM-E6 and FAM-E7 fluorescently labeled E6 & E7 SLPs
  • Poly I:C was also used as a delivery material but was not measured in these studies as fluorescently labeled poly I:C was not available.
  • E6 SLP QLCTELQTTIHDIILECVYCKQQLL (SEQ ID NO: 19)
  • RBC delivery using Ovalbumin 647 was analyzed using flow cytometry to measure cell delivery of Ovalbumin 647 and Annexin V+ phenotype (a measure of phosphatidylserine on the membrane outer leaflet) for each study.
  • a study of operating pressure & microfluidic chip configuration was performed to evaluate Ovalbumin 647 delivery and Annexin V positivity at different operating pressures and utilizing a different number of 2.2 pm constriction chips arranged in parallel.
  • the study evaluated three discrete chip configurations of the same chip type (1 chip, 2x chips in parallel, 4x chips in parallel) and two operating pressures (60 psi, 75 psi).
  • FIG. 3 shows an increase in Annexin V+ population in the study arm performed at 75 psi operating pressure compared to the study arm performed at 60 psi operating pressure. Additionally, the population delivered with Ovalbumin 647 at the 75 psi operating pressure showed most consistent results (> 90% delivery) across all three chip configurations tested. As a result of this evaluation, the operating pressure of 75 psi and the 4x chip configuration was selected to maximize intracellular delivery and throughput of the delivery process.
  • FIG. 4 results show that delivery using the previously selected operating parameters is consistent across cell concentrations ranging from 0.5 x 10 9 to 4.0 x 10 9 RBCs/mL.
  • the operating range for RBC delivery with SLPs & adjuvant was established for the cell concentrations tested.
  • the cell concentration in the SQZ-AAC-HPV manufacturing process is expected to fall within the range tested.
  • Delivery Evaluations with Labeled Peptides (FAM-E6 and FAM-E7 SLPs) & Poly I:C Content [0268] Following the preliminary studies using Ovalbumin 647, confirmatory studies were performed using FAM labeled E6 & E7 SLPs and non-labeled poly I:C. Studies were performed using the delivery parameters (4x chip configuration, 75 psi, 2-8 °C).
  • FIG. 5 shows the % of population to which FAM-E6 SLP and FAM-E7 SLP were delivered to be > 88% and > 96% respectively.
  • Poly I:C content was measured from SQZ-AAC-HPV drug product vials after the development of delivery process parameters were completed. Results from representative batches are shown in Table 5.
  • the AACs are diluted, and then further rested by placing on a rocker inside an incubator at 37 °C for 2 hours. To determine these parameters, evaluations of rest time and pre-rest dilution volume were performed.
  • Whole blood hold time is defined as the end time of whole blood collection (z.e., end of patient blood draw) to the start of the whole blood dilution (start of manufacturing).
  • the initial evaluation of the whole blood hold time was performed with hold times of 36 and 50 hours at 2- 8 °C.
  • the studies consisted of splitting one healthy donor blood unit and using one half to produce SQZ-AAC-HPV following a 1-day hold (approximately 24 hours) for the control arm, and the other half following a 36- or 50-hour hold.
  • the resultant SQZ-AAC-HPV drug products were analyzed for functional response.
  • the drug product, SQZ-AAC-HPV is prepared by formulating the AAC-HPV drug substance in CryoStor® CS2, to target a concentration of 7.0 * 10 8 AACs/mL (as analyzed using a Coulter-based cell counter (Moxi GO II)), accounting for AAC losses during the formulation step, and filling into vials.
  • the vials are cryopreserved using a controlled-rate freezer with a chamber temperature of ⁇ -170 °C, with the vials reaching and maintaining a temperature of ⁇ -140 °C during production, storage and shipment.
  • Post-thaw AAC recoveries confirm that this cryopreservation media is suitable for the SQZ-AAC-HPV drug product.
  • Protocol 2 the LOVO processing parameters were adjusted to target a lower maximum outlet PCV of 36% for all 6 wash cycles to improve recovery while maintaining a high theoretical washout of 99.96%.
  • the LOVO Protocol 2 parameters can be found in Table 8 below.
  • Vial filling is performed inside a biosafety cabinet. Vials are supplied sterile, fully stoppered, and ready to use. For filling, each vial is filled with 9.98 g ⁇ 5% of product (i.e., 9.5 mL, which allows for an extractable volume of 8.9 mL). Following filling, each vial is sealed, checked for weight, and subsequently capped. Once vial filling is completed, each filled vial is visually inspected for any visible defects (including large clumps or aggregates) using an inspection booth equipped with an illuminated black and white background. Subsequently, each vial is labeled with a cryogenic label.
  • the secondary storage boxes containing the labeled SQZ-AAC-HPV vials are loaded onto a rack inside the controlled-rate freezer, and subsequently cryopreserved to a product temperature of ⁇ 140 °C. Following cryopreservation, vials are provided to QC for release, characterization, stability testing and retains and placed in an isothermal LN2 tank for long term storage. The remaining cryopreserved vials are kept in the labeled secondary storage boxes and placed in an isothermal LN2 tank for long term storage.
  • a CryoMed® controlled-rate freezer with a 34L chamber capacity and a custom cryopreservation protocol were used to cryopreserve the SQZ-AAC-HPV drug product.
  • the custom freezing profile has been designed to control the latent heat released during nucleation of the drug product. Ice nucleation in the drug product is initiated at approximately -5 °C. To control the latent heat released during nucleation of the drug product, rapid cooling of the chamber must be initiated prior to the product temperature reaching the nucleation point (-5 °C).
  • the chamber temperature is rapidly cooled to -140 °C at a cooling rate of -20 °C/min after the product temperature reaches -3° C which is slightly above the nucleation point (-5 °C). Subsequently, the product is cooled to -150°C at a cooling rate of -1.5°C/min. Once the product temperature reaches -150 °C, the chamber is cooled to -170 °C at a cooling rate of -1 °C/min and then held at -170 °C for 10 minutes.
  • a maximum chamber load of 64 vials was established for cryopreservation of SQZ-AAC-HPV with one of the vials being used for product temperature monitoring. Details of the cryopreservation protocol are shown in Table 10. Chamber and product temperature profiles resulting from the cryopreservation protocol is shown in FIGs. 10 andl 1.
  • FIG. 10 shows a representative load of 48 vials of SQZ-AAC-HPV being cryopreserved using the developed protocol. As shown in the product temperature traces above, SQZ-AAC-HPV cryopreserved at different locations within the freezing chamber undergo nucleation at a similar time and reach a final product temperature ⁇ -140 °C prior to completion of the cycle. The total cryopreservation cycle time for the representative chamber load is approximately 100 minutes.
  • FIG. 11 shows a maximum load of 64 vials of SQZ-AAC-HPV being cryopreserved using a two-rack configuration.
  • SQZ-AAC-HPV placed in both racks at different locations within the freezing chamber undergo nucleation at a similar time and reach a final product temperature ⁇ -140 °C prior to completion of the cycle.
  • the total cryopreservation cycle time for the maximum chamber load is approximately 95 minutes.

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