WO2021239471A1 - Cancer treatment strategies using arenavirus vectors - Google Patents

Cancer treatment strategies using arenavirus vectors Download PDF

Info

Publication number
WO2021239471A1
WO2021239471A1 PCT/EP2021/062728 EP2021062728W WO2021239471A1 WO 2021239471 A1 WO2021239471 A1 WO 2021239471A1 EP 2021062728 W EP2021062728 W EP 2021062728W WO 2021239471 A1 WO2021239471 A1 WO 2021239471A1
Authority
WO
WIPO (PCT)
Prior art keywords
construct
effective amount
patient
weeks
administering
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2021/062728
Other languages
English (en)
French (fr)
Other versions
WO2021239471A9 (en
Inventor
Igor MATUSHANSKY
Andy Hwang
Kianoosh KATCHAR
Donna Edwards
Henning Lauterbach
Michael Schwendinger
Klaus Orlinger
Sarah Schmidt
Ursula BERKA
Corinne IACOBUCCI
Katia SCHLIENGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hookipa Biotech GmbH
Original Assignee
Hookipa Biotech GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hookipa Biotech GmbH filed Critical Hookipa Biotech GmbH
Priority to US17/928,098 priority Critical patent/US20230346906A1/en
Priority to JP2022573287A priority patent/JP2023527083A/ja
Priority to CR20220602A priority patent/CR20220602A/es
Priority to MX2022014725A priority patent/MX2022014725A/es
Priority to IL298420A priority patent/IL298420A/en
Priority to KR1020227044315A priority patent/KR20230046278A/ko
Priority to CN202180049174.5A priority patent/CN116490205A/zh
Priority to AU2021282287A priority patent/AU2021282287A1/en
Priority to CA3184791A priority patent/CA3184791A1/en
Priority to EP21725519.9A priority patent/EP4157342A1/en
Priority to BR112022024404A priority patent/BR112022024404A2/pt
Priority to PE2022002786A priority patent/PE20240647A1/es
Publication of WO2021239471A1 publication Critical patent/WO2021239471A1/en
Publication of WO2021239471A9 publication Critical patent/WO2021239471A9/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10023—Virus like particles [VLP]
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present application relates generally to cancer treatment strategies using arenavirus particles, and more specifically to specific treatment strategies for treating cancer, including head and neck squamous cell carcinoma, using tri-segmented arenavirus particles encoding an HPV antigen, and in some aspects administration of an immune checkpoint inhibitor.
  • HPV16 infection Human Papillomavirus 16 (HPV16) infection is associated with a substantial and rising proportion of cancers worldwide, such as cervical, head and neck, vaginal, and anal cancers (see de Martel C, et al. Int J Cancer. 2017;141 :664-670). Treatment options are limited for patients with HPV16 + recurrent or metastatic cancers, and the likelihood of long-term survival is low. The generation and maintenance of the HPV16 + malignant state requires the stable expression of HPV 16-specific E7 and E6 oncogenes, which have been shown to drive the cells’ transformation into cancer cells ( see Schmidt S, et al. Onco immunology.
  • HPV16-specific E7 and E6 can serve as immunogenic tumor-associated antigens.
  • the methods described herein satisfy need of treating HPV16 infection and provide related advantages.
  • kits for treating cancer in a patient in need thereof include administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e. two) S-segments encoding a fusion protein of human papillomavirus strain 16 (HP VI 6) E7/E6.
  • the effective amount of the arenavirus particle can be about 5 x 10 5 , about 5 x 10 6 , about 5 x 10 7 , about 1 x 10 8 , or about 5 x 10 8 replication-competent virus focus-forming units (RCV FFU).
  • the methods provided herein for treating cancer include treating an HPV 16 + cancer, regardless of origin.
  • the HPV 16 + cancer has been diagnosed as head and neck squamous cell carcinoma. In some embodiments, the HPV 16 + cancer has been diagnosed as anal cancer. In some embodiments, the HPV 16 + cancer has been diagnosed as cervical cancer. In some embodiments, the HPV 16 + cancer has been diagnosed as vaginal cancer. In some embodiments, the HPV 16 + cancer has been diagnosed as vulvar cancer.
  • the patient had tumor progression or recurrence on at least one standard-of-care therapy prior to the method.
  • the at least one standard-of-care therapy comprises pembrolizumab monotherapy.
  • the patient has only target lesions in lymph nodes.
  • the methods provided herein include administration of engineered replication-competent tri-segmented arenavirus particles using intravenous injection, intratumoral injection or a combination thereof. Accordingly, in some embodiments, administration of the engineered replication-competent tri-segmented arenavirus particle described herein includes intravenous injection. In some embodiments, administration of the engineered replication-competent tri-segmented arenavirus particle described herein includes intratumoral injection. In some embodiments, administration of the engineered replication- competent tri-segmented arenavirus particle described herein includes an intratumoral injection followed by an intravenous injection.
  • the intravenous injections are administered with a frequency of every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks.
  • the intravenous injections are ongoing or are administered for a limited number of cycles.
  • the limited number of cycles is two, three, four, five, or six.
  • the effective amount of the engineered replication-competent tri-segmented arenavirus particle administered for a limited number of cycles is one log order more than the effective amount used in the ongoing intravenous injections.
  • the intravenous injections are ongoing and are first administered with a higher frequency followed by a lower frequency.
  • the intravenous injections are ongoing and are first administered with a frequency of every 3 weeks followed by a frequency of every 6 weeks.
  • the intravenous injections are ongoing and are first administered with a frequency of every 3 weeks for 4 cycles followed by a frequency of every 6 weeks for subsequent cycles. In other specific embodiments, the intravenous injections are ongoing and are first administered with a frequency of every 4 weeks followed by a frequency of every 8 weeks. In one embodiment, the intravenous injections are ongoing and are first administered with a frequency of every 4 weeks for 4 cycles followed by a frequency of every 8 weeks for subsequent cycles. In further embodiments, the administration of the engineered replication-competent tri-segmented arenavirus particle comprises intratumoral injections.
  • the methods provided herein can also include administering an effective amount of an immune checkpoint inhibitor.
  • An exemplary immune checkpoint inhibitor that is particularly useful for use in the methods described herein include an anti -PD- 1 (programmed cell death protein 1) checkpoint inhibitor.
  • an anti-PD-1 checkpoint inhibitor can be an antibody, such as nivolumab, pembrolizumab, pidilizumab or cemiplimab.
  • the methods provided herein use engineered replication- competent tri-segmented arenavirus particles comprising Construct 1 as described herein.
  • the engineered replication-competent tri-segmented arenavirus particles are derived from LCMV, including the MP strain, WE strain, Armstrong strain, Armstrong Clone 13 strain or LCMV clone 13 strain expressing the glycoprotein of LCMV strain WE instead of endogenous LCMV clone 13 glycoprotein.
  • the effective amount of Construct 1 is about 5 x 10 6 RCV FLU, and Construct 1 is administered with a frequency of every 3 weeks.
  • the methods provided herein use engineered replication- competent tri-segmented arenavirus particles comprising Construct 2 as described herein.
  • the engineered replication-competent tri-segmented arenavirus particles are derived from Pichinde virus (PICV), including the strain Munchique CoAn4763 isolate PI 8, P2 strain, or any of the several isolates described by Trapido and colleagues (Trapido et al, 1971,
  • the methods provided herein results in a change in cytokine or chemokine levels in the serum of the patient as compared to the pre- treatment level of the patient.
  • the cytokines and chemokines comprise IFN- ⁇ , IL- 12p40, IL-15, IFN-inducible protein (IP)- 10, and TNF ⁇ .
  • the methods provided herein result in an increase of HPV16 E7/E6-specific T cells in the serum of the patient as compared to the pre-treatment level of the patient.
  • the HPV16 E7/E6-specific T cells are positive for CD8, IFN-g, TNF ⁇ , and/or CD 107a.
  • the T cells described above are detected without prior in-vitro stimulation and/or expansion.
  • the method results in more T cells infiltrating into tumor tissues as compared to the pre-treatment level of the patient or patients receiving placebo.
  • the method results in one or more improved efficacy endpoint using Response Evaluation Criteria in Solid Tumors (RECIST) and/or Immune Response Evaluation Criteria in Solid Tumors (iRECIST), compared to the pre-treatment level of the patient or patients receiving placebo.
  • the one or more improved efficacy endpoint comprises higher percentage of objective response rate, higher percentage of disease control rate, higher percentage of partial response, longer progression-free survival, and/or longer overall survival.
  • Also provided herein is a method for treating cancer in a patient in need thereof comprising: (i) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of HPV16 E7/E6 derived from a first arenavirus species, and its effective amount is about 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , or 1 x 10 9 RCV FFU; and (n) administering to the patient an effective amount of engineered replication-competent tri- segmented arenavirus particles comprising two S-segments encoding a fusion protein of HPV16 E7/E6 derived from a second arenavirus species, and its effective amount is about 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10
  • the method provided herein further comprises repeating (i) and/or (ii).
  • the arenavirus species in (i) is LCMV, and the arenavirus species in (ii) is PICV.
  • the arenavirus species in (i) is PICV, and the arenavirus species in (ii) is LCMV.
  • each session comprises: (i) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of human papillomavirus strain 16 (HP VI 6) E7/E6 derived from a first arenavirus species, wherein the effective amount is about 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , or 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and (ii) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of HPV16 E7/E6 derived from a second arenavirus species at a
  • the first arenavirus species in (i) is lymphocytic choriomeningitis virus (LCMV), and the second arenavirus species in (ii) is Pichinde virus (PICV).
  • LCMV lymphocytic choriomeningitis virus
  • PICV Pichinde virus
  • the first arenavirus species in (i) is PICV
  • the second arenavirus species in (ii) is LCMV.
  • a method comprising one or more session, wherein each session comprises: (i). administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii). administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the administration of the engineered replication-competent tri-segmented arenavirus particles in (i) and (ii) comprises intravenous injection.
  • each session lasts for 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, or 16 weeks.
  • the methods provided herein for treating cancer include treating an HPV 16 + cancer, regardless of origin.
  • the HPV 16 + cancer has been diagnosed as head and neck squamous cell carcinoma.
  • the HPV 16 + cancer has been diagnosed as anal cancer.
  • the HPV 16 + cancer has been diagnosed as cervical cancer.
  • the HPV 16 + cancer has been diagnosed as vaginal cancer.
  • the patient had tumor progression or recurrence on at least one standard-of-care therapy prior to the method.
  • the at least one standard-of-care therapy comprises pembrolizumab monotherapy.
  • the patient has only target lesions in lymph nodes.
  • the methods provided herein include administration of engineered replication-competent tri-segmented arenavirus particles using intravenous injection, intratumoral injection or a combination thereof. Accordingly, in some embodiments, administration of the engineered replication-competent tri-segmented arenavirus particle described herein includes intravenous injection. In some embodiments, administration of the engineered replication-competent tri-segmented arenavirus particle described herein includes intratumoral injection. In some embodiments, administration of the engineered replication- competent tri-segmented arenavirus particle described herein includes an intratumoral injection followed by an intravenous injection.
  • the sessions are ongoing or are repeated for a limited number of sessions.
  • the limited number of sessions is two, three, four, five, or six.
  • the effective amount of the engineered replication- competent tri-segmented arenavirus particles administered for a limited number of sessions is one log order more than the effective amount used in the ongoing sessions.
  • the intravenous injections are ongoing and are first administered in shorter sessions followed by longer sessions. In some particular embodiments, the intravenous injections are ongoing and are first administered with sessions each lasting 6 weeks followed by sessions each lasting 12 weeks. In one embodiment, the intravenous injections are ongoing and are first administered with 2 sessions each lasting 6 weeks followed by sessions each lasting 12 weeks.
  • the intravenous injections are ongoing and are first administered with sessions each lasting 8 weeks followed by sessions each lasting 16 weeks. In one embodiment, the intravenous injections are ongoing and are first administered with 2 sessions each lasting 8 weeks followed by sessions each lasting 16 weeks. [0024] In some embodiments, the method further comprises an intratumoral injection prior to the intravenous injections. In specific embodiments, the intratumoral injection is administered 3 weeks prior to the intravenous injections. In yet specific embodiments, the intratumoral injection is administered with Construct 1.
  • the methods provided herein can also include administering an effective amount of an immune checkpoint inhibitor.
  • An exemplary immune checkpoint inhibitor that is particularly useful for use in the methods described herein include an anti -PD- 1 (programmed cell death protein 1) checkpoint inhibitor.
  • an anti-PD-1 checkpoint inhibitor can be an antibody, such as nivolumab, pembrolizumab, pidilizumab or cemiplimab.
  • the methods provided herein use engineered replication- competent tri-segmented arenavirus particles comprising Construct 1 as described herein.
  • the engineered replication-competent tri-segmented arenavirus particles are derived from LCMV, including the MP strain, WE strain Armstrong strain, Armstrong Clone 13 strain or LCMV clone 13 strain expressing the glycoprotein of LCMV strain WE instead of endogenous LCMV clone 13 glycoprotein.
  • the methods provided herein use engineered replication- competent tri-segmented arenavirus particles comprising Construct 2 as described herein.
  • the engineered replication-competent tri-segmented arenavirus particles are derived from PICV, including the strain Munchique CoAn4763 isolate P18, P2 strain, or any of the several isolates described by Trapido and colleagues (Trapido et al, 1971, Am J Trop Med Hyg, 20: 631-641).
  • the methods provided herein results in a change in the levels of cytokine or chemokine levels in the serum of the patient as compared to the pre-treatment level of the patient.
  • the cytokines and chemokines comprise IFN- ⁇ , IL-12p40, IL-15, IFN-inducible protein (IP)- 10, and TNF ⁇ .
  • the methods provided herein results in an increase of HPV16 E7/E6-specific T cells in the serum of the patient as compared to the pre-treatment level of the patient.
  • the HPV16 E7/E6-specific T cells are positive for CD8, IFN- ⁇ , TNF ⁇ , and/or CD 107a.
  • the T cells described above are detected without prior in-vitro stimulation and/or expansion.
  • the method results in more T cells infiltrating into tumor tissues as compared to the pre-treatment level of the patient or patients receiving placebo.
  • the method results in one or more improved efficacy endpoint using Response Evaluation Criteria in Solid Tumors (RECIST) and/or Immune Response Evaluation Criteria in Solid Tumors (iRECIST), compared to the pre-treatment level of the patient or patients receiving placebo.
  • the one or more improved efficacy endpoint comprises higher percentage of objective response rate, higher percentage of disease control rate, higher percentage of partial response, longer progression-free survival, and/or longer overall survival.
  • a method for treating cancer in a patient in need thereof comprising: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 replication-competent virus focus-forming units (RCV FFU), and wherein Construct 1 is administered intravenously with a frequency of every 3 weeks for 4 cycles followed by ongoing cycles with a frequency of every 6 weeks; and administering to the patient 200mg of pembrolizumab intravenously with a frequency of every 3 weeks or 400mg of pembrolizumab intravenously with a frequency of every 6 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises: administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 1 x 10 8 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication- competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 6 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 6 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i. administering intravenously to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 RCV FFU; and ii.
  • RCV FFU replication-competent virus focus-forming units
  • Construct 1 administering intravenously to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 7 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 7 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 8 replication-competent virus focus-forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 replication-competent virus focus-forming units (RCV FFU), and wherein Construct 1 is administered intravenously with a frequency of every 3 weeks for 3 cycles and the method ends after 3 cycles.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising 3 sessions, wherein each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3
  • a method for treating cancer in a patient in need thereof comprising 3 sessions, wherein each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • a method for treating cancer in a patient in need thereof comprising 3 sessions, wherein each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising 3 sessions, wherein each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 1 x 10 9 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 1 x 10 9 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 9 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • RCV FFU replication-competent virus focus-forming units
  • nucleic acid comprising the nucleotide sequence of SEQ ID NOs: 1 or 2.
  • nucleic acid comprising the nucleotide sequence of SEQ ID NOs: 3, 4, 5, 6, 7, or 8.
  • the nucleic acid provided herein is RNA.
  • a host cell comprising a nucleotide sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • LCMV particle comprising the nucleotide sequences of SEQ ID NOs: 3, 4, and 5.
  • a tri-segmented Pichinde virus particle comprising the nucleotide sequences of SEQ ID NOs: 6, 7, and 8.
  • a pharmaceutical composition comprising a tri-segmented LCMV particle comprising the nucleotide sequences of SEQ ID NOs: 3, 4, and 5, or a tri-segmented Pichinde virus particle comprising the nucleotide sequences of SEQ ID NOs: 6, 7, and 8 and a pharmaceutically acceptable carrier.
  • the tri-segmented arenavirus particle comprising the dinucleotide optimized HPV16 E7E6 nucleotide sequence can have stable expression of the HPV antigen after being passaged at least 4, 5, 6, 7, 8, 9, or 10 generations, can have consistent expression of the encoded HPV fusion protein or induce strong immune responses against the encoded HPV fusion protein.
  • FIG. 1 shows HPV cancer burden and cancers related to HPV.
  • FIGS. 2 A to 2C show schematic depictions of a wild-type arenavirus (e.g., LCMV or
  • FIG. 2A shows arenavirus (LCMV and PICV) wild-type particle (left) and its genome (right).
  • the antisense RNA genome encodes 4 viral proteins: GP (glycoprotein), NP (nucleoprotein), L (RNA-directed RNA polymerase), and Z (RING finger protein Z).
  • GP glycoprotein
  • NP nucleoprotein
  • L RNA-directed RNA polymerase
  • Z RING finger protein Z
  • FIG. 2B shows an engineered tri-segmented arenavirus particle (Construct 1; LCMV -based vector, Construct 2: PICV-based vector) that contains artificially duplicated S-segments encoding a fusion protein of HPV16 E7/E6 with 5 amino acid mutations to abrogate the oncogenic potential of E7 and E6 as indicated by asterisks (*) and either GP or NP, as well as an L-segment.
  • FIG. 2C shows inefficient packaging of the 3 genome segments results in attenuation of Construct 1 and Construct 2 compared to the wild-type LCMV and PICV, respectively.
  • FIGS. 3A to 3C show results of pre-clinical studies of the engineered LCMV-based tri-segmented arenavirus particle of FIG. 2B in mice.
  • Such particles target dendritic cells and other antigen presenting cells for stimulation of the immune system in an antigen specific manner.
  • the engineered LCMV-based tri-segmented arenavirus particles induce a potent T cell response directed specifically against HPV 16 + tumor cells.
  • FIG. 3A shows immunogenicity of the engineered LCMV-based tri-segmented arenavirus particles (Construct 1) illustrated by percentages of blood HPV16 E7-specific CD8 (CD8 + B220-) T cells in healthy mice immunized with increasing doses of the particles by intravenous (IV) administration.
  • FIG 3B shows kinetics of tumor growth in HPV16 + TC-1 tumor-bearing mice treated with increasing doses of the engineered LCMV-based tri-segmented arenavirus particles following IV administration.
  • FIG 3C shows survival curves of HPV16 + TC-1 tumor-bearing mice treated with the engineered LCMV-based tri-segmented arenavirus particles (Construct 1) following intratumoral (IT) or IV administration.
  • FIG. 4 shows the treatment study design, which includes both a dose escalation and dose expansion strategy.
  • FIG. 5 shows the treatment design for a phase 1 dose-escalation study.
  • FIGS. 6A to 6B show results of distinct serum cytokine or chemokine signatures after treatment with Construct 1.
  • FIG. 6A shows results of 30-plex cytokine and chemokine analyses for twelve patients over eight time points. Day 4 data were available for ten of the twelve patients. Analytes (pg/mL) were converted to z scores. Hierarchical clustering was performed by visit day and each analyte level.
  • FIG. 6B shows effects of treatment with Construct 1 on expression of select key cytokines 4 days post-treatment. Nine of twelve patients had both samples from baseline and day 4.
  • FIGS. 7 A to 7F show results of circulating HPV E6/E7-specific poly-functional T cells after single administration of Construct 1 or Construct 2.
  • FIG. 7A shows a direct IFN- ⁇ ELISpot analysis of changes in spot-forming units from baseline to day 15 after administration of a single IV dose of Construct 1 or Construct 2.
  • FIG. 7B shows increase in E6/E7-specific T cells in patients treated with a single IV dose of Construct 1 or Construct 2.
  • FIG. 7C shows the frequency of CD4 + and CD8 + among total peripheral T cell population and the frequency of IFN- ⁇ + , TNF- ⁇ + , and CD107a + after being gated on CD8 + T cells from one patient who received one dose of Construct 1.
  • FIG. 7A shows a direct IFN- ⁇ ELISpot analysis of changes in spot-forming units from baseline to day 15 after administration of a single IV dose of Construct 1 or Construct 2.
  • FIG. 7B shows increase in E6/E7-specific T cells in patients treated
  • FIG. 7D shows the frequency of CD4 + and CD8 + among total peripheral T cell population and the frequency of IFN- ⁇ + , TNF- ⁇ + , and CD107a + after being gated on CD8 + T cells from another patient who received one dose of Construct 1.
  • FIG. 7E shows the frequency of CD4 + and CD8 + among total peripheral T cell population and the frequency of IFN- ⁇ + , TNF- ⁇ + , and CD107a + after being gated on CD8 + T cells from one patient who received one dose of Construct 2.
  • FIG. 7E shows the frequency of CD4 + and CD8 + among total peripheral T cell population and the frequency of IFN- ⁇ + , TNF- ⁇ + , and CD107a + after being gated on CD8 + T cells from one patient who received one dose of Construct 2.
  • FIG. 7F shows pie charts from each of the three patients, which represents the relative frequency of HPV 16 E6/E7- specific CD8 + T cells in combination of the three functional response markers (i.e., CD 107a, IFN- ⁇ , TNF- ⁇ ) after a single administration of Construct 1 or Construct 2.
  • FIG. 8 shows the E7E6-NP-S-segment 1 (2648 bp) of Construct 1 as per FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on LCMV ell 3 S-segment (1-78 bp) is shown without highlight;
  • the synthetic fusion protein consisting of Human Papilloma Virus type 16 (HPV 16) proteins E6 and E7 (79-846 bp) is shown in green;
  • the intergenic region (IGR) based on LCMV ell 3 S-segment (847-910 bp) is shown in blue;
  • the nucleoprotein (NP) based on LCMV ell 3 (911-2587 bp) is shown in gray;
  • the 3’ untranslated region (UTR) based on LCMV ell 3 S-segment (2588-2648 bp) is shown without highlight.
  • FIG. 9 shows the E7E6-GP-S-segment 2 (2648 bp) of Construct 1 as per FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on LCMV ell 3 S-segment (1-78 bp) is shown without highlight;
  • the synthetic fusion protein consisting of Human Papilloma Virus type 16 (HPV 16) proteins E6 and E7 (79-846 bp) is shown in green;
  • the intergenic region (IGR) based on LCMV ell 3 S-segment (847-910 bp) is shown in blue;
  • the glycoprotein (GP) based on LCMV WE (911- 2407 bp) is shown in yellow;
  • the 3’ untranslated region (UTR) based on LCMV cl 13 S-segment (2408-2468 bp) is shown without highlight.
  • FIG. 10 shows the L-segment (7229 bp) of Construct 1 as per FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on LCMV ell 3 L-segment (1-89 bp) is shown without highlight;
  • the matrix protein (Z) based on LCMV ell 3 (90-362 bp) is shown in green;
  • the intergenic region (IGR) based on LCMV ell 3 L-segment (363-564 bp) is shown in blue;
  • the ribonucleic acid dependent ribonucleic acid polymerase protein (L) based on LCMV ell 3 (565-7197 bp) is shown in gray;
  • the 3’ untranslated region (UTR) based on LCMV ell 3 L-segment (7198-7229 bp) is shown without highlight.
  • FIG. 11 shows the E7E6-NP-S-segment 1 (2663 bp) of Construct 2 as per FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on PICV p18 S-segment (1-52 bp) is shown without highlight;
  • the synthetic fusion protein consisting of Human Papilloma Virus type 16 (HPV 16) proteins E6 and E7 (53- 820 bp) is shown in green;
  • the intergenic region (IGR) based on PICV p18 S-segment (821-894 bp) is shown in blue;
  • the nucleoprotein (NP) based on PICV p18 (895-2580 bp) is shown in gray;
  • the 3’ untranslated region (UTR) based on PICV p18 S-segment (2581-2663 bp) is shown without highlight.
  • FIG. 12 shows the E7E6-GP-S-segment 2 (2504 bp) of Construct 2 as per FIG. FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on PICV p18 S-segment (1-52 bp) is shown without highlight;
  • the synthetic fusion protein consisting of Human Papilloma Virus type 16 (HPV 16) proteins E6 and E7 (53-820 bp) is shown in green;
  • the intergenic region (IGR) based on PICV p18 S- segment (821-894 bp) is shown in blue;
  • the glycoprotein (GP) based on PICV p18 (895-2421 bp) is shown in yellow;
  • the 3’ untranslated region (UTR) based on PICV p18 S-segment (2422-2504 bp) is shown without highlight.
  • FIG. 13 shows the L-segment (7058 bp) of Construct 2 as per FIG. 2B.
  • the following elements are indicated from 5’ to 3’ of the disclosed sequence.
  • the 5’ untranslated region (UTR) based on PICV p18 L-segment (1-85 bp) is shown without highlight;
  • the matrix protein (Z) based on PICV p18 (86-373 bp) is shown in red;
  • the intergenic region (IGR) based on PICV p18 L-segment (374-443 bp) is shown in blue;
  • the ribonucleic acid dependent ribonucleic acid polymerase protein (L) based on PICV p18 (444-7028 bp) is shown in gray;
  • the 3’ untranslated region (UTR) based on PICV p18 L-segment (7029-7058 bp) is shown without highlight.
  • FIGS. 14A to 14E show efficacy of Construct 1 and/or Construct 2 in a mice model bearing HPV16 + tumors.
  • FIG. 14A shows the correlation between the dose of Construct 1 and percentage of HPV 16 E7-specific CD8 + B220- T cells.
  • CD 8 cluster of differentiation 8
  • E7 antigenic E7 fusion protein from human papillomavirus 16.
  • FIG. 14B shows the changes of tumor volume over time in response to treatment with different doses of Construct 1 ;
  • FIG. 14A shows the correlation between the dose of Construct 1 and percentage of HPV 16 E7-specific CD8 + B220- T cells.
  • CD 8 cluster of differentiation 8
  • E7 antigenic E7 fusion protein from human papillomavirus 16.
  • FIG. 14B shows the changes of tumor volume over time in response to treatment with different doses of Construct 1 ;
  • FFU
  • E7E6 antigenic E7 and E6 fusion protein from human papillomavirus 16
  • G group,
  • FIG. 14D shows the change of percentage of HPV16 E7- specific CD8 + B220- T cells over time in mice in response to the indicated dosing regimens of Construct 1 and Construct 2 after i.v. administration of 10 5 RCV FFU of each vector with a 21- day interval.
  • CD cluster of differentiation
  • E7E6 antigenic E7 and E6 fusion protein from human papillomavirus 16
  • G group, dashes indicate the sequence of vector administration (prime/ 1st dose - boost/2nd dose).
  • 14E shows the changes of tumor volume over time in response to the indicated dosing regimens of Construct 1 and Construct 2 after i.v. administration of 10 5 RCV FFU of each vector with a 4, 7 or 10-day interval.
  • FIG. 15 shows the experiment design in Example III, which includes phase I dose escalation and phase II dose expansion.
  • FIG. 16 shows the experiment design for backfill cohorts in Example III.
  • FIGS. 17A to 171 show efficacy data of Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy.
  • FIG. 17B shows target lesion (TL) sum of diameter (SOD) change from baseline of each individual patient. Striped areas indicate decrease in target lesion change after pembrolizumab was added to therapy. # Progression with non-evaluable scans, artificially assigned 2%.
  • Non- oropharynx patients A, anal; C, cervical; NP, nasopharynx; V, vaginal.
  • IT intratumoral; IV, intravenous; SOD, sum of diameters; TL, target lesion.
  • FIG. 17C shows the best target lesion (TL) sum of diameter (SOD) change from baseline by schedule / route of administration.
  • FIG. 17D shows target lesion (TL) sum of diameter (SOD) change from baseline of each individual patient in a spider plot. Open squares represent scans performed after addition of pembrolizumab. One patient with non-evaluable efficacy scans is not shown on the spider plot.
  • Target lesion with 60% decrease is lymph node measured ⁇ 10mm, therefore unconfirmed complete response.
  • EDC data was used for some patients due to missing/incorrect data entry on TLF as of the data transfer date.
  • EDC electronic data capture
  • HNSCC head and neck squamous cell carcinoma
  • SOD sum of diameters
  • TL target lesion
  • uCR unconfirmed complete response.
  • FIG. 17E shows progression-free survival (PFS) in treated patients.
  • PFS includes time after pembrolizumab had been added prior to RECIST progression.
  • FIG. 17F shows target lesion change from baseline in patients with only lymph node lesions as well as in patients having only non-lymph node lesions or both, lymph node lesions as well as non-lymph node lesions.
  • FIG. 17G shows Sum of diameter changes for RECIST evaluable lesions in patients receiving IV administration of Construct 1 dose level 2 every three weeks and IV administration of Construct 2 / Construct 1 alternating 2-vector therapy.
  • FIG. 17H shows a correlation between best sum of diameter change and time on treatment.
  • FIG. 171 shows efficacy scans at baseline and subsequent time points during treatment.
  • FIG. 18A shows results of 30-plex cytokine and chemokine analyses after treatment with Construct 1 over nine time points. Day 4 data were available for ten of the twelve patients. Analytes (pg/mL) were converted to z scores. Hierarchical clustering was performed by visit day and each analyte level.
  • FIG. 18A shows results of 30-plex cytokine and chemokine analyses after treatment with Construct 1 over nine time points. Day 4 data were available for ten of the twelve patients. Analytes (pg/mL) were converted to z scores. Hierarchical clustering was performed by visit day and each analyt
  • Construct 1 monotherapy DL 1 (5 x 10 5 RCV FFU) or DL2 (5 x 10 6 RCV)
  • Construct 2 / Construct 1 alternating 2-vector therapy Construct 2: 1x10 6 RCV FFU, Construct 1: 5 x 10 6 RCV FFU
  • FIGS. 19A to 191 show strong immunogenicity induced by Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy.
  • FIG. 19A shows ELISpot result for six patients who received Construct 1 IV at DL2 (5 x 10 6 RCV FFU) every 3 weeks. Thawed peripheral blood mononuclear cells (PBMCs) from all patients were stimulated with overlapping HPV16 E6/E7 peptides for 24 h ( ⁇ 2 h) for direct ex vivo IFN- ⁇ ELISpot measurement. Shown is the number of spot forming units/ 1 x 10 6 PBMCs.
  • FIG. 19A shows ELISpot result for six patients who received Construct 1 IV at DL2 (5 x 10 6 RCV FFU) every 3 weeks. Thawed peripheral blood mononuclear cells (PBMCs) from all patients were stimulated with overlapping HPV16 E6/E7 peptides for 24 h ( ⁇ 2 h) for direct ex vivo IFN- ⁇ E
  • FIG. 19B shows ICS result for six patients who received Construct 1 IV at DL2 (5 x 10 6 RCV FFU) every 3 weeks.
  • PBMCs were stimulated for 6 hours with overlapping HPV16 E6/E7 peptides, washed for subsequent immunostaining for IFN- ⁇ , TNF- ⁇ , IL-2 and CD 107a, and analyzed by polychromatic flow cytometry. Shown is the percentage of IFN- ⁇ + cells among CD8 T cells.
  • FIG. 19C shows ELISpot result for three patients who received Construct 2 at DL1 (1x10 6 RCV FFU) and Construct 1 at DL2 (5 x 10 6 RCV FFU) IV every 3 weeks.
  • FIG. 19D shows ICS result for three patients who received Construct 2 at DL1 (1x10 6 RCV FFU) and Construct 1 at DL2(5 x 10 6 RCV FFU) IV every 3 weeks.
  • PBMCs were stimulated for 6 hours with overlapping HPV16 E6/E7 peptides, washed for subsequent immunostaining for IFN- ⁇ , TNF- ⁇ , IL-2 and CD 107a, and analyzed by polychromatic flow cytometry. Shown is the percentage of IFN- ⁇ + cells among CD8 T cells.
  • FIG. 19E shows flow cytometry results of CD8 + and CD4 + T cells over time in one patient receiving Construct 2 / Construct 1 alternating 2- vector therapy. PBMCs were stimulated for 6 hours with E6/E7 peptides, washed for subsequent immunostaining and analyzed by polychromatic flow cytometry.
  • FIG. 19F shows flow cytometry results of T cells that express IFN- ⁇ , TNF ⁇ , or CD 107a over time in one patient receiving Construct 2 / Construct 1 alternating 2-vector therapy.
  • FIG. 19G to 191 show changes in PBMCs from baseline to the maximal response (Max) for individual patients. Max refers to the highest E6/E7 specific T cell responses measured by IFN- ⁇ ELISpot for each individual patient.
  • FIG. 19G shows the change of white blood cell counts (WBC) from baseline to Max as WBC 10 3 / ⁇ l blood after> 2 arenaviral vector administrations (left panel).
  • WBC white blood cell counts
  • FIG. 19H shows IFN- ⁇ ELISpot results for baseline and Max as spot forming units/ 10 6 PBMCs.
  • FIG. 191 are representative pseudo color plots from PBMC samples after intracellular cytokine staining (see FIG. 19B, D and F). Left plots are from baseline, right plots are from Max.
  • the upper row is from a patient treated with Construct 1 DL2 (5 x 10 6 RCV FFU) IV-IV
  • the lower row is from a patient treated with Construct 2 DL1 (1x10 6 RCV FFU) and Construct 1 DL2 (5 x 10 6 RCV FFU) alternating 2-vector therapy.
  • cycle when used in methods for treating cancer with one species of engineered replication-competent tri-segmented arenavirus particles, is intended to refer to an administration day and the days before the next administration.
  • the term “session,” when used in methods for treating cancer with two species of engineered replication-competent tri-segmented arenavirus particles in an alternating 2-vector therapeutic approach, is intended to refer to an administration day of the first species, the days before an administration of the second species, an administration of the second species, and the days before another administration of the first species.
  • Such methods include administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6.
  • the effective amount of the particles can be about 5 x 10 5 , about 5 x 10 6 , about 5 x 10 7 RCV FFU, about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU.
  • a method for treating cancer in a patient in need thereof that includes administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 5 x 10 5 RCV FFU.
  • a method for treating cancer in a patient in need thereof that includes administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles having duplicated (i.e.
  • a method for treating cancer in a patient in need thereof includes administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of human papillomavirus strain 16 (HPV16) E7/E6, wherein the effective amount is about 5 x 10 7 RCV FFU.
  • a method for treating cancer in a patient in need thereof that includes administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of human papillomavirus strain 16 (HP VI 6) E7/E6, wherein the effective amount is about 5 x 10 8 RCV FFU.
  • a method for treating cancer in a patient in need thereof that includes administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 1 x 10 8 RCV FFU.
  • two) S-segments as described herein include replication-competent tri- segmented arenavirus particles wherein the open reading frame (ORF) encoding the NP protein is present on one S-segment, while the ORF encoding the GP protein is present on the other S- segment.
  • ORF open reading frame
  • Also provided in these publications are descriptions of pharmaceutical compositions having engineered replication-competent tri-segmented arenavirus particles that can be used in the methods described herein.
  • the methods provided herein for treating cancer include treating any HPV 16 + cancer, regardless of origin.
  • the HPV 16 + cancer has been diagnosed as head and neck squamous cell carcinoma.
  • the HPV 16 + cancer has been diagnosed as anal cancer.
  • the HPV 16 + cancer has been diagnosed as cervical cancer.
  • the HPV 16 + cancer has been diagnosed as vaginal cancer.
  • the HPV 16 + cancer has been diagnosed as vulvar cancer.
  • the methods provided herein are for treating HPV 16 + cancer (e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer) in a patient in need thereof by administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 5 x 10 5 RCV FFU, about 5 x 10 6 RCV FFU, about 5 x 10 7 RCV FFU, about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU.
  • HPV 16 + cancer e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer
  • the method provided herein include treating a patient who had tumor progression or recurrence on at least one standard-of-care therapy prior to the method.
  • the method provided herein are used to treat a patient who had failed radiation.
  • the method provided herein are used to treat a patient who had failed platinum-based therapy.
  • the method provided herein are used to treat a patient who had failed anti-PD-1 therapy. In some specific embodiments, the method provided herein are used to treat a patient who had failed anti-PD-L1 therapy. In one specific embodiment, the method provided herein are used to treat a patient who had failed pembrolizumab monotherapy. In one specific embodiment, the method provided herein are used to treat a patient who had failed nivolumab monotherapy. In one specific embodiment, the method provided herein are used to treat a patient who had failed pidilizumab monotherapy. In one specific embodiment, the method provided herein are used to treat a patient who had failed cemiplimab monotherapy. In some embodiments, the method provided herein are used to treat a patient who had failed a combination of two or more of the above-listed therapies.
  • the methods provided herein include treating a patient whose lesions involve lymph nodes or not. Accordingly, in some embodiments, the methods provided herein include treating a patient who has only target lesions in lymph nodes. In some embodiments, the methods provided herein include treating a patient who has only target lesions in non-lymph nodes. In some embodiments, the methods provided herein include treating a patient who has target lesions in both lymph nodes and non-lymph nodes.
  • the methods provided herein include administration of engineered replication-competent tri-segmented arenavirus particles using intravenous injection, intratumoral injection or a combination thereof. Accordingly, in some embodiments, the methods provided herein are for treating HPV 16 + cancer (e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar or vaginal cancer) in a patient in need thereof by intravenous injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 5 x 10 5 RCV FFU, about 5 x 10 6 RCV FFU, about 5 x 10 7 , about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU.
  • HPV 16 + cancer e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer,
  • the methods provided herein are for treating HPV 16 + cancer (e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer) in a patient in need thereof by intratumoral injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 5 x 10 5 RCV FFU, about 5 x 10 6 RCV FFU, about 5 x 10 7 , about 1 x 10 8 RCV FFU, about 5 x 10 8 RCV FFU.
  • HPV 16 + cancer e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer
  • HPV 16 + cancer e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer
  • the methods provided herein are for treating HPV 16 + cancer (e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer) in a patient in need thereof by intratumoral injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e.
  • HPV 16 + cancer e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer
  • the methods provided herein include administering intravenous injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, such as Construct 1, with a certain frequency (e.g., every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 13 weeks, every 14 weeks, etc.).
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 2 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 3 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 4 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 5 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 6 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 7 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 8 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 9 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 10 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 11 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 12 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a frequency of every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, or every 26 weeks.
  • the methods provided herein include administering intravenous injections to the patient of 5 x 10 6 RCV FFU of Construct 1 with a frequency of every 3 weeks.
  • the methods provided herein include administering intravenous injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, such as Construct 2, with a certain frequency (e.g., every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 13 weeks, every 14 weeks, etc.).
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 2 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 3 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 4 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 5 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 6 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 7 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 8 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 9 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 10 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 11 weeks. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 12 weeks.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a frequency of every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, or every 26 weeks.
  • the methods provided herein include an ongoing treatment. In other embodiments, the methods provided herein include a treatment administered for a limited number of times. Accordingly, in some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, such as Construct 1 or Construct 2, with a certain frequency and ongoing. In some embodiments, the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, such as Construct 1 or Construct 2, with a certain frequency for a limited number of times.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e.
  • a fusion protein of HPV16 E7/E6, such as Construct 1 or Construct 2 every 2 weeks for only 5 cycles, every 3 weeks for only 5 cycles, every 4 weeks for only 5 cycles, every 5 weeks for only 5 cycles, every 6 weeks for only 5 cycles, every 7 weeks for only 5 cycles, every 8 weeks for only 5 cycles, every 9 weeks for only 5 cycles, every 10 weeks for only 5 cycles, every 11 weeks for only 5 cycles, every 12 weeks for only 5 cycles.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 with a limited number of cycles, wherein the effective amount can be 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU. Accordingly, in some embodiments, the methods provided herein include administering intravenous injections to the patient of 5 x 10 6 RCV FFU of Construct 1 with a limited number of cycles as described above in the same paragraph. In some embodiments, the methods provided herein include administering intravenous injections to the patient of 5 x 10 7 RCV FFU of Construct 1 with a limited number of cycles as described above in the same paragraph.
  • the methods provided herein include administering intravenous injections to the patient of 5 x 10 8 RCV FFU of Construct 1 with a limited number of cycles as described above in the same paragraph. In some embodiments, the methods provided herein include administering intravenous injections to the patient of 1 x 10 9 RCV FFU of Construct 1 with a limited number of cycles as described above in the same paragraph. In some embodiments, the methods provided herein include administering intravenous injections to the patient of 5 x 10 9 RCV FFU of Construct 1 with a limited number of cycles as described above in the same paragraph.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 with a limited number of cycles, wherein the effective amount can be 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU.
  • the methods provided herein include administering intravenous injections to the patient of 1 x 10 7 RCV FFU of Construct 2 with a limited number of cycles as described above in the same paragraph.
  • the methods provided herein include administering intravenous injections to the patient of 1 x 10 8 RCV FFU of Construct 2 with a limited number of cycles as described above in the same paragraph.
  • the methods provided herein include administering intravenous injections to the patient of 1 x 10 9 RCV FFU of Construct 2 with a limited number of cycles as described above in the same paragraph. In some embodiments, the methods provided herein include administering intravenous injections to the patient of 1 x 10 10 RCV FFU of Construct 2 with a limited number of cycles as described above in the same paragraph.
  • the methods provided herein can also include administering an effective amount of an immune checkpoint inhibitor.
  • An exemplary immune checkpoint inhibitor that is particularly useful for use in the methods described herein include an anti -PD- 1 (programmed cell death protein 1) checkpoint inhibitor.
  • an anti-PD-1 checkpoint inhibitor can be an antibody, such as nivolumab, pembrolizumab, pidilizumab or cemiplimab.
  • the methods provided herein are for treating HPV 16 + cancer (e.g., head and neck squamous cell carcinoma, cervical cancer, anal cancer, vulvar, or vaginal cancer) in a patient in need thereof by administering to the patient an effective amount of an immune checkpoint inhibitor and an effective amount of an engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount of the particle is about 5 x 10 5 RCV FFU, about 5 x 10 6 RCV FFU, about 5 x 10 7 RCV FFU, about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU, and wherein the immune checkpoint inhibitor is an anti -PD- 1 checkpoint inhibitor (e.g ., nivolumab, pembrolizumab, pidilizumab or cemiplimab
  • the anti-PD-1 checkpoint inhibitor is nivolumab. In some embodiments, the anti -PD- 1 checkpoint inhibitor is pembrolizumab. In some embodiments, the anti-PD-1 checkpoint inhibitor is pidilizumab. In some embodiments, the anti-PD-1 checkpoint inhibitor is cemiplimab.
  • the methods provided herein include administering intravenous injection to the patient of an effective amount of engineered replication-competent tri-segmented arenavirus particles having duplicated (i.e. two) S-segments encoding a fusion protein of HPV16 E7/E6, such as Construct 1 or Construct 2, with a higher frequency followed by a lower frequency.
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 2 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 3 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 13 week.
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 4 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, or every 14 weeks.
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 5 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 6 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 3 weeks for 4 cycles followed by a frequency of every 6 weeks.
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 1 (e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU) with a frequency of every 4 weeks for 4 cycles followed by a frequency of every 8 weeks.
  • Construct 1 e.g., 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU) with a frequency of every 2 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.
  • Construct 2 e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU) with a frequency of every 3 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 13 week.
  • Construct 2 e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU) with a frequency of every 4 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, or every 14 weeks.
  • Construct 2 e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU) with a frequency of every 5 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, or every 15 weeks.
  • Construct 2 e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU
  • the methods provided herein include administering intravenous injections to the patient of an effective amount of Construct 2 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU) with a frequency of every 6 weeks for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles followed by a frequency of every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, or every 16 weeks.
  • Construct 2 e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein include the arenavirus particle of Construct 1 (LCMV-based) as described herein (FIG. 2B and Examples I and P).
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from LCMV. Specific strains of LCMV include MP strain, WE strain, Armstrong strain, Armstrong Clone 13 strain, or LCMV clone 13 strain expressing the glycoprotein of LCMV strain WE instead of endogenous LCMV clone 13 glycoprotein.
  • the engineered replication-competent tri- segmented arenavirus particles used in the methods described herein are derived from LCMV MP strain. In some embodiments, the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from LCMV WE strain. In some embodiments, the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from LCMV Armstrong strain. In some embodiments, the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from LCMV Armstrong Clone 13 strain.
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from LCMV clone 13 strain expressing the glycoprotein of LCMV strain WE instead of endogenous LCMV clone 13 glycoprotein.
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from Construct 2 ( PICV-based) as described herein (FIG. 2B and Example II).
  • Specific strains of PIC V include strain Munchique CoAn4763 isolate PI 8, P2 strain, or any of the several isolates described by Trapido and colleagues (Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-641).
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from strain Munchique CoAn4763 isolate PI 8.
  • the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from P2 strain. In some embodiments, the engineered replication-competent tri-segmented arenavirus particles used in the methods described herein are derived from any of the several isolates described by Trapido and colleagues (Trapido et al. , 1971, Am J Trop Med Hyg, 20: 631-641)
  • cytokines and chemokines are measured after the administration of the effective amount of engineered replication-competent tri-segmented arenavirus particles.
  • the cytokines and chemokines to be measured include pro-inflammatory and anti-inflammatory cytokines and chemokines.
  • a method for treating cancer in a patient in need thereof comprising administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6, wherein the effective amount is about 5 x 10 5 , about 5 x 10 6 RCV FFU, about 5 x 10 7 RCV FFU, about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU, wherein the administration of the effective amount of engineered replication-competent tri- segmented arenavirus particles results in a change (i.e., increase or decrease) in the level of a cytokine or a chemokine in the serum of the patient as compared to the pre-treatment level in the patient.
  • a change i.e., increase or decrease
  • the changed cytokines and chemokines include, but are not limited to, GM- CSF, IL-la, IL-Ib, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-12p40, IL-15, IL- 16, IL-17, IL-17A, IL-18, IL-22, IL-37, IL-38, TGF- ⁇ , IFN- ⁇ , INF- ⁇ , IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , IFN-inducible protein (IP)- 10, macrophage inflammatory protein (MIR)-Ia, MIR-Ib, monocyte chemoattractant protein (MCP)-1, MCP-4, eotaxin, eotaxin-3, thymus and activation-regulated chemokine (TARC)
  • the cytokines and chemokines described herein have pro- inflammatory and/or anti-inflammatory activities.
  • the administration of the effective amount of engineered replication-competent tri-segmented arenavirus particles results in a 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 21 -fold, 22-fold, 23 -fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, or 30-fold change (e.g., increase or decrease) in the level of a cytokine or a chemokine (e.g, having pro-inflammatory and/or anti-inflammatory activity) in the serum of the patient as compared to the pre- treatment level in the patient.
  • a cytokine or a chemokine e.g, having pro-inflammatory and/or anti-inflammatory activity
  • the administration of the effective amount of engineered replication-competent tri-segmented arenavirus particles results in a 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold change (e.g., increase or decrease) in the level of a cytokine or a chemokine (e.g, having pro- inflammatory and/or anti-inflammatory activity)in the serum of the patient as compared to the pre-treatment level in the patient.
  • a cytokine or a chemokine e.g, having pro- inflammatory and/or anti-inflammatory activity
  • the administration of the effective amount of engineered replication-competent tri-segmented arenavirus particles results in a 300- fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold change (e.g., increase or decrease) in the level of a cytokine or a chemokine (e.g, having pro-inflammatory and/or anti- inflammatory activity)in the serum of the patient as compared to the pre-treatment level in the patient.
  • a cytokine or a chemokine e.g, having pro-inflammatory and/or anti- inflammatory activity
  • the cytokines and chemokines described herein serve as biomarkers for patient population selection. Accordingly, in some embodiments, a measurement of one or more of the cytokines and chemokines described herein above a certain threshold in a patient prior to the treatment indicates the patient is suitable for the methods provided herein. In some embodiments, a measurement of one or more of the cytokines and chemokines described herein below a certain threshold in a patient prior to the treatment indicates the patient is suitable for the methods provided herein.
  • a measurement of IFN- ⁇ , IL-12p40, IL-15, IFN-inducible protein (IP)- 10, TNF ⁇ , or a combination thereof below a certain threshold in a patient prior to the treatment indicates the patient is suitable for the methods provided herein.
  • the cytokines and chemokines described herein serve as biomarkers for re-adjusting the doses and/or regimens during the treatment. Accordingly, in some embodiments, after treating a patient for a period of time, a measurement of one or more of the cytokines and chemokines described herein above a certain threshold indicates an increase of doses and/or frequency of administration. In some embodiments, after treating a patient for a period of time, a measurement of one or more of the cytokines and chemokines described herein above a certain threshold indicates keeping the same doses and/or frequency of administration.
  • a measurement of one or more of the cytokines and chemokines described herein above a certain threshold indicates a decrease of doses and/or frequency of administration.
  • a measurement of one or more of the cytokines and chemokines described herein below a certain threshold indicates an increase of doses and/or frequency of administration.
  • a measurement of one or more of the cytokines and chemokines described herein below a certain threshold indicates keeping the same doses and/or frequency of administration.
  • a measurement of one or more of the cytokines and chemokines described herein below a certain threshold indicates a decrease of doses and/or frequency of administration.
  • a measurement of IFN- ⁇ , IL-12p40, IL-15, IFN-inducible protein (IP)-10, TNF ⁇ , or a combination thereof below a certain threshold indicates an increase of doses and/or frequency of administration.
  • a measurement of IFN- ⁇ , IL- 12p40, IL-15, IFN-inducible protein (IP)- 10, TNF ⁇ , or a combination thereof above a certain threshold indicates keeping the same doses and/or frequency of administration.
  • the levels of the cytokines and chemokines can be measured at different time points before and after administering the engineered replication-competent tri- segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6. In some embodiments, the levels of the cytokines and chemokines are measured before the administration of the arenavirus particles.
  • the levels of the cytokines and chemokines are measured 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours after the administration of the arenavirus particles.
  • the levels of the cytokines and chemokines are measured 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days,
  • the levels of the cytokines and chemokines can be measured with varieties of assays, such as bioassays (e.g., tests for chemotactic activity, proliferation, or cytotoxicity), immunoassays (e.g., ELISA, and especially multiplex ELISA), flow cytometry, and aptamers- based detection methods, and molecular imaging with radiolabeled cytokines and chemokines.
  • bioassays e.g., tests for chemotactic activity, proliferation, or cytotoxicity
  • immunoassays e.g., ELISA, and especially multiplex ELISA
  • flow cytometry e.g., ELISA, and especially multiplex ELISA
  • aptamers- based detection methods e.g., aptamers- based detection methods
  • molecular imaging with radiolabeled cytokines and chemokines e.g., after the administration of the arenavirus particles encoding HPV16 E7
  • the cytokines and chemokines are measured with immunoassays. In specific embodiments, after the administration of the arenavirus particles encoding HPV16 E7/E6 the cytokines and chemokines are measured with ELISA. In some preferred embodiments, after the administration of the arenavirus particles encoding HPV16 E7/E6 the cytokines and chemokines are measured with multiplex ELISA. In some embodiments, after the administration of the arenavirus particles encoding HPV16 E7/E6 the cytokines and chemokines are measured with flow cytometry.
  • the cytokines and chemokines are measured with aptamers-based detection methods. In some embodiments, after the administration of the arenavirus particles encoding HPV16 E7/E6 the cytokines and chemokines are measured with molecular imaging with radiolabeled cytokines and chemokines.
  • a method for treating cancer in a patient in need thereof comprising administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of human papillomavirus strain 16 (HPV16) E7/E6, wherein the effective amount is about 5 x 10 5 , about 5 x 10 6 RCV FFU, about 5 x 10 7 RCV FFU, about 1 x 10 8 RCV FFU, or about 5 x 10 8 RCV FFU, wherein the administration of the effective amount of engineered replication-competent tri-segmented arenavirus particles results in a change in cytokine or chemokine levels in the serum of the patient as compared to the pre-treatment level of the patient, and wherein the administration of the effective amount of engineered replication- competent tri-segmented arenavirus particles results in an increase of HPV16 E7/E6-specific T
  • the increased HPV16 E7/E6- specific T cells described herein can be positive for different cellular markers (e.g., CD4, CD8, IFN- ⁇ , TNF ⁇ , CD 107a) alone or in combination which indicate the different functionalities of the T cells.
  • the method provided herein results in an increase of HPV16 E7/E6-specific T cells that are positive for CD4 in the serum of the patient as compared to the pre-treatment level of the patient.
  • the method provided herein results in an increase of HPV 16 E7/E6-specific T cells that are positive for CD8 in the serum of the patient as compared to the pre-treatment level of the patient.
  • the method provided herein results in an increase of HPV16 E7/E6-specific T cells that are positive for IFN- ⁇ in the serum of the patient as compared to the pre-treatment level of the patient. In some embodiments, the method provided herein results in an increase of HPV16 E7/E6-specific T cells that are positive for TNF ⁇ in the serum of the patient as compared to the pre-treatment level of the patient. In some embodiments, the method provided herein results in an increase of HPV16 E7/E6-specific T cells that are positive for CD 107a in the serum of the patient as compared to the pre-treatment level of the patient.
  • HPV16 E7/E6-specific T cells can be detected and quantified by varieties of assays, such as ELISpot and intracellular cytokine staining (ICS) followed by flow cytometry. Accordingly, in some embodiments, the method provided herein further comprises detecting and quantifying HPV16 E7/E6-specific T cells by ELISpot. In some embodiments, the method provided herein further comprises detecting and quantifying HPV16 E7/E6-specific T cells by ICS followed by flow cytometry.
  • assays such as ELISpot and intracellular cytokine staining (ICS) followed by flow cytometry.
  • any assay well known in the art can be used to test HPV16 E7/E6- specific T-cell responses.
  • the ELISPOT assay can be used (see, e.g., Czerkinsky C.C. et al, J Immunol Methods. 1983; 65:109-121; and Hutchings P.R. et al, J Immunol Methods. 1989; 120:1-8).
  • Cytokines such as but not limited to IFN- ⁇ can be measured by the ELISPOT assay.
  • the assay comprises the following steps: An immunospot plate is coated with an anti-cytokine antibody.
  • HPV16 E7/E6-specific cells secrete cytokines, which bind to the coated antibodies.
  • the cells are then washed off and a second biotyinlated-anticytokine antibody is added to the plate and visualized with an avidin-HRP system or other appropriate methods.
  • any assay well known in the art can be used to test the functionality of CD8 + and CD4 + T cells that are specific for HPV16 E7/E6.
  • the ICS combined with flow cytometry can be used (see, e.g., Suni M.A. et al, J Immunol Methods. 1998; 212:89-98; Nomura L.E. et al, Cytometry. 2000; 40:60-68; and Ghanekar S.A. et al, Clinical and Diagnostic Laboratory Immunology. 2001; 8:628-63).
  • the assay comprises the following steps: upon activation of cells via HPV16 E7/E6, an inhibition of protein transport (e.g, brefeldin A) is added to retain the cytokines within the cell. After a defined period of incubation, typically 5 hours, a washing step follows, and antibodies to other cellular markers can be added to the cells. Cells are then fixed and permeabilized. The flurochrome-conjugated anti-cytokine antibodies are added and the cells can be analyzed by flow cytometry.
  • an inhibition of protein transport e.g, brefeldin A
  • PBMCs peripheral blood mononuclear cells
  • the migration of T cells to infiltrate into tumor tissues is also an important readout for the efficacy of the methods provided herein.
  • the methods provided herein result in more T cells infiltrated into tumor tissues.
  • the methods provided herein result in more CD8 + T cells infiltrated into HPV16 + tumor tissues as compared to the pre- treatment level of the patient or patients receiving placebo (see section 11 of EXAMPLE III).
  • antigen-specific serum ELISA' s enzyme-linked immunosorbent assays
  • plates are coated with antigen (e.g, recombinant protein), blocked to avoid unspecific binding of antibodies and incubated with serial dilutions of sera.
  • bound serum-antibodies can be detected, e.g, using an enzyme-coupled anti-species (e.g, mouse, guinea pig)-specific antibody (detecting total IgG or IgG subclasses) and subsequent color reaction.
  • Antibody titers can be determined as, e.g, endpoint geometric mean titer.
  • determining the neutralizing antibodies in sera can be performed with the following cell assay using GFP-tagged viruses or cell lines expressing HPV E7/E6.
  • supplemental guinea pig serum as a source of exogenous complement is used.
  • the assay is started with seeding of 6.5 x 10 3 cells/well (50 ⁇ l/well) in a 384 well plate one or two days before using for neutralization.
  • the neutralization is done in 96-well sterile tissue culture plates without cells for 1 h at 37 °C. After the neutralization incubation step the mixture is added to the cells and incubated for additional 4 days for GFP-detection with a plate reader.
  • a positive neutralizing human sera is used as assay positive control on each plate to check the reliability of all results.
  • Titers are determined using a 4 parameter logistic curve fitting. As additional testing the wells are checked with a fluorescence microscope. Similarly, neutralizing activity of induced antibodies can be measured in clinical setting.
  • the methods provided herein result in one or more improved efficacy endpoint (e.g, percentage of objective response rate, percentage of disease control rate, percentage of partial response, progression-free survival, and/or overall survival) using Response Evaluation Criteria in Solid Tumors (RECIST) and/or Immune Response Evaluation Criteria in Solid Tumors (iRECIST), compared to the pre-treatment level of the patient or patients receiving placebo.
  • RECIST Solid Tumors
  • iRECIST Immune Response Evaluation Criteria in Solid Tumors
  • the methods provided herein result in higher percentage of objective response rate compared to the pre- treatment level of the patient or patients receiving placebo.
  • the methods provided herein result in higher percentage of disease control rate compared to the pre- treatment level of the patient or patients receiving placebo.
  • the methods provided herein result in higher percentage of partial response compared to the pre-treatment level of the patient or patients receiving placebo. In some specific embodiments, the methods provided herein result in longer progression-free survival compared to the pre-treatment level of the patient or patients receiving placebo. In some specific embodiments, the methods provided herein result in longer overall survival compared to the pre-treatment level of the patient or patients receiving placebo.
  • a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; and (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e.
  • Also provided herein is a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication-competent tri- segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; and (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU.
  • a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU, and repeating (i) and (ii) for 1 time
  • Also provided herein is a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU, and repeating (i) and (ii) for 1 time, 2 times
  • the interval between the (i) and (ii) in the preceding paragraphs is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In other embodiments, the interval between the (i) and (ii) in the preceding paragraphs is 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 23 weeks, or 24 weeks. In other embodiments, the interval between the (i) and (ii) in the preceding paragraphs is 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, or 36 weeks.
  • the interval can be the same as the original cycle of (i) and (ii), or can be different from the original cycle of (i) and (ii). Accordingly, the interval between the (i) and (ii) in the repeats can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks.
  • a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV 16 E7/E6 derived from LCMV in combination with an immune checkpoint inhibitor, wherein the effective amount of the arenavirus particles is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV in combination with an immune checkpoint inhibitor, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x
  • Also provided herein is a method for treating cancer in a patient in need thereof comprising (i) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV in combination with an immune checkpoint inhibitor, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV FFU; and (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising duplicated (i.e., two) S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV in combination with an immune checkpoint inhibitor, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , or 5 x 10 8 RCV F
  • each session comprises: (i) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of human papillomavirus strain 16 (HPV 16) E7/E6 derived from LCMV, wherein the effective amount is about 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , or 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and (ii) administering to the patient an effective amount of engineered replication- competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of HPV16 E7/E6 derived from PICV at a time point around half of the
  • each session comprises: (i) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of human papillomavirus strain 16 (HPV 16) E7/E6 derived from PICV, wherein the effective amount is about 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , or 1 x 10 9 replication-competent virus focus-forming units (RCV FFU); and (ii) administering to the patient an effective amount of engineered replication-competent tri-segmented arenavirus particles comprising two S-segments encoding a fusion protein of HPV16 E7/E6 derived from LCMV at a time point around half of the session
  • the methods provided herein comprise one or more session, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the methods provided herein comprise one or more session, wherein each session comprises: (i) administering intravenously to the patient 1 x 10 6 RCV FFU of Construct 2; and (ii) administering intravenously to the patient 5 x 10 6 RCV FFU of Construct 1, and each session lasts for 6 weeks.
  • the methods provided herein comprise one or more session, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU.
  • Session duration determines the interval between the administration of two consecutive doses of (i) in the preceding paragraphs. Accordingly, in some embodiments, each session provided herein lasts for 4 weeks. In some embodiments, each session provided herein lasts for 6 weeks. In some embodiments, each session provided herein lasts for 8 weeks. In some embodiments, each session provided herein lasts for 10 weeks. In some embodiments, each session provided herein lasts for 12 weeks. In some embodiments, each session provided herein lasts for 14 weeks. In some embodiments, each session provided herein lasts for 16 weeks. In some embodiments, each session provided herein lasts for 18 weeks. In some embodiments, each session provided herein lasts for 20 weeks.
  • each session provided herein lasts for 22 weeks. In some embodiments, each session provided herein lasts for 24 weeks. In some embodiments, each session provided herein lasts for 26 weeks. In some embodiments, each session provided herein lasts for 28 weeks. In some embodiments, each session provided herein lasts for 30 weeks. In some embodiments, each session provided herein lasts for 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 weeks.
  • the administration of (i) and (ii) in the preceding paragraphs comprises intravenous injection, intratumoral injection, or a combination of intravenous injection and intratumoral injection.
  • the methods provided herein comprise one or more session, wherein each session comprises: (i) administering intravenously, intratumorally, or a combination of intravenous injection and intratumoral injection to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering intravenously, intratumorally, or a combination of intravenous injection and intratumoral injection to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the methods provided herein comprises one or more session, wherein each session comprises: (i) administering intravenously, intratumorally, or a combination of intravenous injection and intratumoral injection to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU; and (ii) administering intravenously, intratumorally, or a combination of intravenous injection and intratumoral injection to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU.
  • the methods provided herein include an ongoing treatment. In other embodiments, the methods provided herein include a treatment administered for a limited number of times. Accordingly, in some embodiments, the methods provided herein comprise ongoing sessions or a limited number of sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the methods provided herein comprise ongoing sessions or a limited number of sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU.
  • the methods provided herein comprise only 2 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 3 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 4 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 5 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 2 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 3 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 4 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise only 5 sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 RCV FFU, and wherein each session lasts for 4, 6, 8, 10, or 12 weeks.
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 2 sessions each lasts for 4 weeks, and wherein the 3 rd session and subsequent sessions each lasts for 6,
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 2 sessions each lasts for 6 weeks, and wherein the 3 rd session and subsequent sessions each lasts for 8, 10, 12, 14, 16, 18, 20, or 24 weeks.
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 2 sessions each lasts for 8 weeks, and wherein the 3 rd session and subsequent sessions each lasts for 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 weeks.
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 2 sessions each lasts for 10 weeks, and wherein the 3 rd session and subsequent sessions each lasts for 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 weeks.
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 3 sessions each lasts for 4 weeks, and wherein the 4 th session and subsequent sessions each lasts for 6, 8, 10, 12, 14, 16 weeks.
  • the methods provided herein comprise ongoing sessions, wherein each session comprises:
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 3 sessions each lasts for 10 weeks, and wherein the 4 th session and subsequent sessions each lasts for 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 weeks
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x
  • the methods provided herein comprise ongoing sessions, wherein each session comprises: (i) administering to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU, wherein the first 4 sessions each lasts for 10 weeks, and wherein the 5 th session and subsequent sessions each lasts for 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 weeks
  • the methods provided herein comprise an intratumoral injection of Construct 1 followed by ongoing sessions, wherein each session comprises: (i) administering intravenously to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering intravenously to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the methods provided herein comprise an intratumoral injection of Construct 2 followed by ongoing sessions, wherein each session comprises: (i) administering intravenously to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU; and (ii) administering intravenously to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU.
  • the methods provided herein comprise an intratumoral injection of Construct 1 followed by ongoing sessions, wherein each session comprises: (i) administering intravenously to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU; and (ii) administering intravenously to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU.
  • the methods provided herein comprise an intratumoral injection of Construct 2 followed by ongoing sessions, wherein each session comprises: (i) administering intravenously to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 RCV FFU; and (ii) administering intravenously to the patient an effective amount of Construct 2 at a time point around half of the session, wherein the effective amount is about 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , or 1 x 10 9 RCV FFU.
  • a method for treating cancer in a patient in need thereof comprising administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 replication-competent virus focus- forming units (RCV FFU), and wherein Construct 1 is administered intravenously with a frequency of every 3 weeks for 4 cycles followed by ongoing cycles with a frequency of every 6 weeks.
  • the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 replication-competent virus focus- forming units (RCV FFU)
  • a method for treating cancer in a patient in need thereof comprising: (i) administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 5 , 5 x 10 6 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 replication-competent virus focus-forming units (RCV FFU), and wherein Construct 1 is administered intravenously with a frequency of every 3 weeks for 4 cycles followed by ongoing cycles with a frequency of every 6 weeks; and administering to the patient 200mg of pembrolizumab intravenously with a frequency of every 3 weeks or 400mg of pembrolizumab intravenously with a frequency of every 6 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 1 x 10 8 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • RCV FFU replication-competent virus focus-forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication-competent virus focus- forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 6 replication-competent virus focus- forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus- forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus- forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus- forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication-competent virus focus- forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus- forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus- forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i.
  • a method for treating cancer in a patient in need thereof comprising (1) multiple sessions of administering Construct 2 and Construct 1, wherein each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus- forming units (RCV FFU); and ii.
  • each session comprises: i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication-competent virus focus- forming units (RCV FFU); and ii.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 6 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 6 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i. administering intravenously to the patient an effective amount of Construct 2, wherein the effective amount is about 1 x 10 7 RCV FFU; and ii.
  • RCV FFU replication-competent virus focus- forming units
  • Construct 1 administering intravenously to the patient an effective amount of Construct 1 at a time point around half of the session, wherein the effective amount is about 5 x 10 6 RCV FFU, and wherein the first two sessions each lasts for 6 weeks, and the following ongoing sessions each lasts for 12 weeks.
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 7 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 7 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 1 x 10 8 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising (1) administering intratumorally to the patient an effective amount of Construct 1, wherein the effective amount of Construct 1 is about 5 x 10 8 replication-competent virus focus- forming units (RCV FFU); and (2) 3 weeks later administering to the patient multiple sessions, wherein each session comprises i.
  • RCV FFU replication-competent virus focus- forming units
  • a method for treating cancer in a patient in need thereof comprising administering to the patient an effective amount of Construct 1, wherein the effective amount is about 5 x 10 6 , 5 x 10 7 , 5 x 10 8 , 1 x 10 9 , or 5 x 10 9 replication-competent virus focus- forming units (RCV FFU), and wherein Construct 1 is administered intravenously with a frequency of every 3 weeks for 3 cycles and the method ends after 3 cycles.
  • RCV FFU replication-competent virus focus- forming units
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 7 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 7 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 8 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication- competent virus focus-forming units (RCV FFU); and administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication- competent virus focus-forming units (RCV FFU); and administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 5 x 10 8 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • RCV FFU replication- competent virus focus-forming units
  • each session comprises i. administering to the patient an effective amount of Construct 2 intravenously, wherein the effective amount is about 1 x 10 9 replication- competent virus focus-forming units (RCV FFU); and ii. administering to the patient an effective amount of Construct 1 intravenously at a time point around half of the session, wherein the effective amount is about 1 x 10 9 RCV FFU, and wherein each sessions lasts for 6 weeks, and the method ends after 3 sessions.
  • each session comprises i.
  • composition comprising an engineered replication-competent tri-segmented arenavirus particle described herein and a pharmaceutically acceptable carrier.
  • [00161] Provided herein are optimized nucleotide sequences that encode the E7/E6 fusion protein of human papillomavirus strain 16 (HPV16). These optimized open reading frames are provided as SEQ ID No: 1 and 2. The sequences are provided in the SEQUENCE TABLE below.
  • these optimized open reading frames are included into an arenaviral genomic segment.
  • SEQ ID No: 3 sets forth a first S segment derived from LCMV with the optimized open reading frame for the E7/E6 fusion protein under control of the 5’ UTR and the open reading frame for the NP protein under control of the 3’ UTR.
  • SEQ ID No: 4 sets forth a second S segment derived from LCMV with the optimized open reading frame for the E7/E6 fusion protein under control of the 5’ UTR and the open reading frame for the GP protein under control of the 3’ UTR.
  • SEQ ID No: 6 sets forth a first S segment derived from PICV with the optimized open reading frame for the E7/E6 fusion protein under control of the 5’ UTR and the open reading frame for the NP protein under control of the 3’ UTR.
  • SEQ ID No: 7 sets forth a second S segment derived from PICV with the optimized open reading frame for the E7/E6 fusion protein under control of the 5’ UTR and the open reading frame for the GP protein under control of the 3’ UTR.
  • genomic segments e.g., SEQ ID NOs: 3, 4, and 5; or SEQ ID NOs: 6, 7, and 8
  • S segments 1 and 2 SEQ ID NOs: 3, 4, 6, 7, respectively
  • L segment SEQ ID NOs: 5 and 8, respectively
  • the nucleotide sequences presented as SEQ ID NOs: 1 to 8 can be RNA or DNA sequences. Once present in a viral particle, these nucleotide sequences can be present as RNA.
  • the DNA sequences shown as SEQ ID NOs: 1-8 can be converted to RNA sequences by replacing the “T” (thymidine) with a “U” (uridine).
  • SEQ ID NOs: 1-8 can be used in the methods of treatment disclosed herein.
  • SEQ ID NOs: 3-5 can be used to generate a tri-segmented replication competent viral particle as Construct 1.
  • SEQ ID NOs: 6-8 can be used to generate a tri-segmented replication competent viral particle as Construct 2.
  • compositions comprising engineered replication- competent tri-segmented arenavirus particles comprising SEQ ID NOs: 3-5 and SEQ ID NOs: 6- 8, respectively. These pharmaceutical compositions can be used in any of the methods disclosed herein.
  • expression vectors comprising a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.
  • host cells comprising such an expression vector. Any method known to the skilled artisan can be used to generate a replication competent, tri-segmented viral particle with the genomic segments of SEQ ID NOs: 3 to 5. Any method known to the skilled artisan can be used to generate a replication competent, tri-segmented viral particle with the genomic segments of SEQ ID NOs: 6 to 8. Also provided herein are expression vectors from which any one of the genomic segments of SEQ ID NOs: 3 to 8 can be transcribed.
  • the nucleotide sequence of SEQ ID NOs: 1 and 2 encode the HPV16 E7E6 fusion protein.
  • the nucleotide sequence of the HPV16 E7E6 fusion protein of SEQ ID NOs: 1 and 2 are modified to reduce CpG dinucleotide motifs (dinucleotide optimized).
  • Arenavirus particles comprising SEQ ID No: 1 or 2 can demonstrate improved genetic stability, improved expression and improved immunogenicity in the methods provided herein. Assays to demonstrate these properties are described below.
  • the attenuated, replication-competent viral vector can be generated de novo using a cDNA rescue system comprising plasmids encoding the two short (S) genome segments including the dinucleotide optimized E7E6 nucleotide sequence as well as the gene for the arenaviral nucleoprotein (NP) or arenaviral glycoprotein (GP), respectively, and the long (L) genome segment including the genes for the RING finger protein Z and the RNA- directed RNA polymerase L.
  • the following assays may be used to demonstrate the improvement of the dinucleotide optimized sequences of SEQ ID NOs: 1 or 2.
  • the tri-segmented arenavirus particle encoding the dinucleotide optimized HPV16 E7E6 nucleotide sequence can have a stable expression of the encoded HPV antigen after being passaged multiple generations, which is necessary for larger-scale commercial production. In some embodiments, the tri-segmented arenavirus particle can have stable expression of the HPV antigen after being passaged at least 4, 5, 6, 7, 8, 9, or 10 generations.
  • Serial passaging of vector candidates in the propagation cell line Small-scale HEK293 cell cultures can be infected with replication competent vectors at MOI 0.001 (RCV FFU/ml titer). At day 4 post infection, supernatant can be cleared from cells and debris by centrifugation. Thereof determined RCV FFU titers can be used to generate the next passage by infecting fresh cells as described above. Vector stock material can be passaged for 9 sequential passages (up to passage plO).
  • Vector material generated de novo (PI) as well as derived from serial passages thereof can be subsequently analyzed for infectivity by FFU and RCV FFU assays.
  • Transgene stability of the vector can be analyzed by isolating genomic vRNA from the virus containing supernatant of different passage levels, transcription into cDNA and subsequent amplification by PCR with transgene flanking primers specific for the respective transgene and S- Segment.
  • Transgene expression of vector stocks can be confirmed by Western Blot analysis of cell lysates at different passage levels using transgene-specific antibodies.
  • the tri-segmented arenavirus particle encoding the dinucleotide optimized HPV16 E7E6 nucleotide sequence can have consistent expression of the encoded HPV fusion protein.
  • the membranes can be probed with primary antibodies directed against proteins of interest and horseradish peroxidase (HRP) conjugated secondary antibodies followed by staining with Immobilon Western Chemiluminescent HRP Substrate (Merck/Millipore) Immunogenicity
  • the tri-segmented arenavirus particle encoding the dinucleotide optimized HPV16 E7E6 nucleotide sequence can induce strong immune responses against the encoded HPV fusion protein.
  • MHC-Peptide Multimer Staining Assay for Detection of Antigen-Specific CD8 + T-cells.
  • Any assay well known in the art can be used to measure antigen- specific CD8 + T-cell responses.
  • the MHC-peptide tetramer staining assay can be used (see, e.g., Altman J.D. et al., Science. 1996; 274:94-96; and Murali-Krishna K. et al., Immunity. 1998; 8:177- 187).
  • the assay can comprise a tetramer assay used to detect the presence of antigen specific T-cells.
  • T-cells In order to detect an antigen-specific T-cell , it must bind to both, the peptide and the tetramer of MHC molecules custom made for a defined antigen specificity and MHC haplotype of T-cells (typically fluorescently labeled). T-cells that recognize the tetramer, and are thus specific for the antigen can then be detected by flow cytometry via the fluorescent label.
  • ELISPOT Assay for Detection of Antigen-Specific T-cells. Any assay well-known in the art can be used to test antigen-specific T-cell responses.
  • the ELISPOT assay can be used (see, e.g., Czerkinsky C.C. et al., J Immunol Methods. 1983; 65:109-121; and Hutchings P.R. et al., J Immunol Methods. 1989; 120:1-8) as exemplified in Table 1. Summary of Sample Collection for Central Laboratory Analyses, cytokines such as but not limited to IFN- g can be measured by the ELISPOT assay.
  • the assay comprises the following steps: An immunospot plate is coated with an anti-cytokine antibody. Cells are then incubated in the immunospot plate with peptides derived from the antigen of interest. Antigen-specific cells secrete cytokines, which bind to the coated antibodies. The cells are then washed off and a second biotyinlated-anticytokine antibody is added to the plate and visualized with an avidin- HRP system or other appropriate methods.
  • Intracellular Cytokine Assay for Detection of Functionality of CD8 + and CD4 + T-cells Any assay well-known in the art can be used to test the functionality of CD8 + and CD4 + T cell responses.
  • the intracellular cytokine assay combined with flow cytometry can be used as exemplified but not limited to Table 1. Summary of Sample Collection for Central Laboratory Analyses (see, e.g., Suni M.A. et al, J Immunol Methods. 1998; 212:89-98;
  • the assay comprises the following steps: upon activation of cells via specific peptides or protein, an inhibition of protein transport (e.g., brefeldin A) is added to retain the cytokines within the cell. After a defined period of incubation, typically 5 hours, a washing step follows, and antibodies to other cellular markers can be added to the cells. Cells are then fixed and permeabilized. The flurochrome- conjugated anti-cytokine antibodies are added and the cells can be analyzed by flow cytometry.
  • an inhibition of protein transport e.g., brefeldin A
  • Serum ELISA Determination of the humoral immune response upon vaccination of animals (e.g., mice, guinea pigs) can be done by antigen-specific serum ELISA’ s (enzyme- linked immunosorbent assays).
  • plates can be coated with antigen (e.g., recombinant protein), blocked to avoid unspecific binding of antibodies and incubated with serial dilutions of sera.
  • bound serum-antibodies can be detected, e.g., using an enzyme-coupled anti-species (e.g., mouse, guinea pig)-specific antibody (detecting total IgG or IgG subclasses) and subsequent color reaction.
  • Antibody titers can be determined as, e.g., endpoint geometric mean titer.
  • This example describes an immunotherapy treatment strategy using arenavirus based particles alone or in combination with an immune checkpoint inhibitor.
  • the arenavirus particle that can be used in this treatment strategy includes Construct 1 and Construct 2, HPV antigen constructs described in FIG. 2B.
  • the antigen constructs for the HPV16 E7/E6 antigen that can be used in the treatment strategy described herein include the antigens described in US Patent Application Publication US-2018-0179257- A1, published June 28, 2018, which are incorporated herein by reference.
  • the HPV16 E7/E6 antigen encoded by the arenavirus particles used in the described treatment strategy includes the amino acid sequence of SEQ ID NO: 10 of US Patent Application Publication US-2018-0179257-A1 (Construct 1 and Construct 2).
  • the immune checkpoint inhibitor used in this treatment strategy includes an anti-PD- 1 immune checkpoint inhibitor.
  • Immune checkpoint inhibitors that can be used in the treatment strategy described herein, including the anti -PD- 1 immune checkpoint inhibitor include those described in US Patent Application Publication US-2018-0344830-A1 , published December 6, 2018, which are incorporated herein by reference.
  • the intratumoral administration of the arenavirus particle used in this treatment strategy includes the methods described in US Patent Application Publication US-2020- 0113995-A1, published April 16, 2020, which are incorporated herein by reference.
  • Construct 1 is a replication-competent live-attenuated vector based on the arenavirus LCMV encoding a non-oncogenic E7 and E6 fusion protein. In preclinical models, both intravenously (IV) and intratumorally (IT) administered Construct 1 demonstrates potent immunogenicity by induction of HPV 16-specific cytotoxic T cells and associated efficacy (FIGS. 3A to 3C).
  • FIG. 4 The treatment strategy for using an arenavirus-based cancer immunotherapy, alone or in combination with an immune checkpoint inhibitor, in patients with HPV 16 + cancers is described in FIG. 4.
  • This treatment strategy is a first in humans, Phase I/II study of Construct 1 monotherapy or in combination with PD-1 immune checkpoint inhibitor (anti-PD-1) in HPV16 + confirmed recurrent/metastatic cancers.
  • Phase I consists of 2 treatment groups, each conducted with a 3 + 3 dose escalation design. Group 1 enrolls patients with HPV16 + head and neck squamous cell carcinoma who will receive Construct 1 IV only.
  • Group 2 enrolls HPV16 + cancer patients with a safely accessible tumor site who will receive Construct 1 IT for the first dose, followed by Construct 1 IV for subsequent doses (IT-IV).
  • Construct 1 can be administered every 21 days.
  • the Phase II component can be conducted with the recommended Phase II doses (RP2Ds) defined in Phase I and can consist of 3 groups: Group A (Construct 1 IV only), Group B (Construct 1 IV plus anti-PD-1), and Group C (Construct 1 IT-IV).
  • R2Ds Phase II doses
  • Statistical analysis that can be conducted includes each group of the Phase I Dose Escalation part following a traditional 3 + 3 design, with at least 3 DLT- evaluable patients per dose level. For this viral-based therapy, the highest dose may not necessarily be the most efficacious. Backfill of cohorts can, therefore, be used to better assess safety and potential efficacy across doses.
  • TEAEs treatment-emergent adverse events
  • CTCAE grade CTCAE grade.
  • the incidence of treatment emergent abnormal laboratory, vital signs, and ECG values can also be summarized using descriptive statistics.
  • efficacy analyses all efficacy endpoints can be determined according to RECIST vl.l and iRECIST. For Phase I, efficacy endpoints can be presented and no formal statistical testing needs to be performed. For Phase II, ORR and disease control rate can be summarized using exact 2-sided 95% CIs according to the Clopper-Pearson method. Duration of response, PFS, and OS can be performed using Kaplan-Meier curves.
  • This example describes the immunogenicity of immunotherapy using arenavirus based particles, which result in changes of cytokine and chemokine, and the induction of tumor- antigen-specific T cells, in patients with advanced HPV16 + cancers.
  • Construct 1 is a genetically engineered replication-competent tri-segmented arenavirus particle comprising two S-segments each encoding a fusion protein of HPV16 E7/E6, based on the LCMV strain Clone 13 with the viral surface glycoprotein from LCMV strain WE.
  • Construct 2 is a genetically engineered replication-competent tri-segmented PICV particle comprising two S- segments each encoding a fusion protein of HPV16 E7/E6, based on virulent strain passage 18 of PICV.
  • Different dose levels and schedules of monotherapy injections of Construct 1 alone or Construct 2 alternating with Construct 1 were analyzed. (see FIG. 5, Bonilla W, et al. Cell Rep Med. 2021 ;2(3): 1-17).
  • An IFN- ⁇ signature in serum post-treatment is an early sign of immune activation.
  • Hierarchical clustering of serum 30-plex analysis showed that IFN- ⁇ levels increased in 90% of patients after a single administration of Construct 1 (see FIG. 6A).
  • TNF ⁇ increased in nearly all 9 patients in this analysis (see FIG. 6B).
  • Direct IFN- ⁇ ELISpot analysis was conducted on five patients, using samples from baseline and day 15 after administration of a single dose of Construct 1 or Construct 2, respectively. Specifically, cryopreserved and thawed peripheral blood mononuclear cells (PBMCs) from seven patients were stimulated with overlapping HPV16 E6/E7 peptides for 24 h ( ⁇ 2 h) for direct ex vivo IFN- ⁇ ELISpot measurement. Enough cells were available from five of seven patients to be evaluated by ELISpot at the time of data cutoff. The number of circulating functional E6/E7-specific T cells in Construct 1- and Construct 2-treated patients reached levels that allowed detection in an ex vivo direct ELISpot (i.e., without in vitro expansion of T cells).
  • PBMCs peripheral blood mononuclear cells
  • FIG. 7B up to a 250-fold increase in antigen-specific IFN- ⁇ - secreting T cells from baseline to day 15 was observed in four patients who received one dose of Construct 1 monotherapy systemically (IV). Additionally, a 150-fold increase was observed in one patient after administration of a single dose of Construct 2 monotherapy (IV).
  • Intracellular cytokine staining was conducted on three patients. Specifically, samples from three patients were evaluated by ICS at baseline and day 15. Cryopreserved PBMCs from the three patients (two patients were treated with Construct 1 and one patient was treated with Construct 2) were stimulated with HPV16 E6/E7 overlapping peptides for 6 hours, and washed for subsequent immunostaining. The frequency of IFN- ⁇ + , TNF- ⁇ + , IL-2 + , CD107a + , CD4 + , and CD8 + T cells was determined by ICS followed by multicolor flow cytometry analyzed. The three patients evaluated by ICS were among the seven patients whose cells were tested by ELISpot.
  • FIGS. 7C to 7E show representative pseudocolor plots with the frequencies of CD4 + and CD8 + T cells and frequencies of IFN- ⁇ + , TNF- ⁇ + , and CD107a + cells gated on CD8 + T cells at baseline and day 15 for the three patients.
  • Two patients had an increase in T cells, predominantly CD8 + T cells, within the total peripheral T cell population after one dose of Construct 1 (8.3% vs 32.9%; see FIG. 7C) and Construct 2 (48.2% vs 69.3%; see FIG. 7E) at baseline versus day 15, respectively.
  • E6/E7-specific IFN- ⁇ + CD8 + T cells increased substantially following administration of single doses of Construct 1 or Construct 2.
  • antigen-specific IFN- ⁇ + CD8 + T cells increased from 0% at baseline to 2.8% on day 15 following a single dose of Construct 1 (see FIG. 7C).
  • antigen-specific IFN- ⁇ + CD8 + T cells increased from 0% at baseline to 8.1% on day 15 (see FIG. 7E).
  • E6/E7 specific CD8 + T cells had a higher expression of CD 107a at day 15.
  • one patient treated with Construct 1 had a slight increase in TNF- ⁇ + and CD107a + , but no increase in IFN- ⁇ + or CD8 + T cells (see FIG. 7D).
  • arenavirus vectors expressing E7/E6 constitute a new potential therapy for patients with immunotherapy and/or chemotherapy-refractory HPV16 + cancers.
  • This example describes a first-in-human Phase I/II, multinational, multi center, open- label study of Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy in patients with HPV 16 + confirmed cancers.
  • the example comprises two parts: Phase I Dose Escalation and Phase II Dose Expansion.
  • Construct 1 monotherapy and/or Construct 2 / Construct 1 alternating 2-vector therapy with or without pembrolizumab in patients with HPV 16 + head and neck squamous cell carcinoma (HNSCC) and other HPV 16 + confirmed cancers are explored during Dose Expansion.
  • HNSCC head and neck squamous cell carcinoma
  • Construct 1 is a genetically engineered TheraT® vector based on the LCMV strain Clone 13 with the viral surface glycoprotein from LCMV strain WE (Kallert et al, 2017, Nat Commun. 2017; 8:15327).
  • the Construct 1 vectors deliver a non-oncogenic synthetic fusion protein based on the HPV 16 E7 and E6 proteins (i.e., E7E6 fusion protein) in a tri-segmented, replication-competent, attenuated arenavirus vector.
  • This synthetic E7E6 fusion protein has been particularly mutated in five pivotal positions (Cassetti et al, 2004, Vaccine. 2004;22(3-4): 520-27) to eliminate its retinoblastoma protein and tumor protein p53 binding abilities, thus abrogating the oncogenicity of the parental E7 and E6 proteins while still retaining full antigenicity.
  • Construct 1 contains several fractions of vector particles that contribute to immune response.
  • TheraT® vectors were engineered to encode the mutated version of E7E6 such that one Small segment (S-segment) carries the LCMV viral surface glycoprotein from LCMV strain WE plus the mutated E7E6 fusion protein, and a second S segment carries the LCMV viral surface nucleoprotein plus a second, identical copy of the mutant E7E6 fusion protein.
  • Construct 1 contains the Large segment (L- segment) of LCMV Clone 13.
  • Construct 1 contains three genome segments (i.e., r3LCMV), two S segments, and one L segment. Inefficient packaging of the three genome segments is the basis of attenuation of Construct 1 compared to the wild type LCMV.
  • Construct 2 is a genetically engineered, attenuated replication competent tri- segmented PICV vector based on the PI 8 variant of PICV. Construct 2 delivers the same non- oncogenic HPV 16 E7E6 antigens as those in Construct 1.
  • the Construct 2 vector was designed using the same tri-segment principle as that in Construct 1 vector by segregating the essential PICV viral surface glycoprotein and nucleoprotein from the original one genomic segment onto two artificially duplicated genomic S- segments.
  • the Construct 2 vector contains three genome segments (i.e., r3PICV), including: one S segment carrying the PICV viral surface glycoprotein plus the mutated E7E6 fusion protein, a second S segment carries the PICV viral surface nucleoprotein plus a second, identical copy of the mutant E7E6 fusion protein, and an L- segment of P18 variant of PICV.
  • the genetic design of these S segments in Construct 2 absolutely prevents intersegmental recombination and reversion to a functional wild type-like single S segment encoding both PICV glycoprotein and nucleoprotein.
  • Construct 2 is administered with Construct 1 following a sequential alternating IV administration strategy, in which Construct 2 is administered IV as the prime dose, the next dose is a Construct 1 IV booster dose, and the subsequent administrations alternate between Construct 2 and Construct 1 sequentially.
  • This treatment plan is designated as “Construct 2 / Construct 1 alternating 2-vector therapy.”
  • Nonclinical studies using palpable HPV 16 + tumors in the TCI model have demonstrated that the Construct 2 / Construct 1 alternating treatment regimen resulted in suppression of tumor growth and prolonged overall survival (OS) that is superior to either vector alone (homologous Construct 1 & Construct 1 and Construct 2 & Construct 2) or prime with Construct 1 then boost with Construct 2 administrations.
  • OS overall survival
  • Construct 2 / Construct 1 alternating 2-vector therapy induced the most potent HPV 16 E7-specific CD8 T cell responses (immunogenicity) among all the possible combination regimens tested.
  • FIG. 14D among all rationally designed dosing regimens of Construct 1 and Construct 2, sequential IV administration of Construct 2 followed by Construct 1 (priming with 10 5 RCV FFU of Construct 2 and boosting with 10 5 RCV FFU of Construct 1, a regimen designated as Construct 2 / Construct 1 alternating 2-vector administration) proved to be the most immunogenic regimen, which triggered an HPV 16, E7- specific CD8 T cell response substantially higher than those induced by other combination sequence or single vector regimens.
  • the frequencies of HPV E7-specific cells reached -43% of total CD8 T cells 5 days after the boost administration of Construct 1. Furthermore, the superior immunological effect of the Construct 2 / Construct 1 alternating 2-vector therapy was sustained over the observation period (see FIG. 14D).
  • the Construct 2 / Construct 1 alternating 2-vector therapy with IV administration of each vector both at a dose of 10 5 RCV FFU (G4) also conferred superior tumor suppression capacity as compared to homologous prime-boost regimens using either Construct 1 or Construct 2 alone (G1 and G2). Together, these findings warrant further clinical testing of the Construct 2 / Construct 1 alternating 2-vector therapy in HPV 16 + cancer patients.
  • pembrolizumab (KEYTRUDA ® ) is used in this example.
  • Pembrolizumab has recently been approved by the FDA and the European Commission for the first-line treatment of patients with metastatic or unresectable recurrent HNSCC in monotherapy (for patients with tumors PD-L1 positive [CPS >1]) or in combination with chemotherapy.
  • the Phase I Dose Escalation has two treatment regimens: Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy.
  • the Construct 1 monotherapy is given to 3 different groups (1, 2, and 5, see Table 2 below).
  • Groups 1, 2, and 5 are studied to determine a safe recommended Phase II dose (RP2D) of Construct 1 for intravenous (IV) and intratumoral (IT) treatment.
  • Group 5 explores Construct 1 as an IV administration given in 3 doses.
  • the Construct 2 / Construct 1 alternating 2-vector therapy is given to 3 different groups (3, 4, and 6).
  • Groups 3, 4, and 6 are studied to determine a safe RP2D of Construct 2 for IV administration.
  • Group 6 explores administering 3 doses of Construct 2 / Construct 1 alternating 2-vector therapy such that patients receive Construct 2 administered first, followed by Construct 1, in an alternating manner until each patient has received 3 doses of Construct 2 and Construct 1 each, 6 doses in total.
  • Phase II Dose Expansion have up to six treatment groups as shown in the Table 2 below. Based on the safety, efficacy, and/or biomarker data from the Dose Escalation results, the specific Dose Expansion Treatment Groups are opened accordingly.
  • Phase II Dose Expansion Groups A and B commence upon completion of the Phase I Dose Escalation Group 1.
  • Phase II Dose Expansion Groups D and E commence upon completion of the Phase I Dose Escalation Group 3.
  • Phase II Dose Expansion Group C commence upon completion of the Phase I Dose Escalation Group 2.
  • Phase II Dose Expansion Group F commence upon completion of the Phase I Dose Escalation Group 4.
  • Groups 1, 2, 3, and 4 approximately 20 patients are enrolled in each group; Groups 5 and 6 enroll approximately 3 to 6 patients each. The actual number of patients enrolled in each group depend on when the RP2Ds are reached. Additional patients may be added in each group after a dose level has been determined to be safe to ensure sufficient biomarker data are obtained.
  • Groups A, B, C, D, E, and F approximately 20 patients are enrolled in each group. In total, approximately 200 patients are enrolled in this study, with approximately 100 in Phase I and approximately 60 to 100 in Phase II.
  • Phase I Dose Escalation is a safety and tolerability phase; there is no primary efficacy endpoint.
  • Incidence of dose-limiting toxicity (DLTs) from the first study drug administered during the DLT observation period is monitored.
  • Safety parameters e.g., types, frequency, and severity of AEs and SAEs
  • Tolerability parameters e.g., dose interruptions, reductions and dose intensity, and evaluations of laboratory values
  • the secondary efficacy endpoints for Phase I are objective response rate (ORR), and disease control rate (DCR) Response Evaluation Criteria in Solid Tumors (RECIST) and immune Response Evaluation Criteria in Solid Tumors (iRECIST).
  • ORR objective response rate
  • DCR disease control rate
  • Time to event efficacy endpoints are listed.
  • E7 and E6 antigen-specific T cell response are listed.
  • CD4 and CD 8 T cell measurements changes in SUV-based quantitative measures on CD8 tracer PET scan at baseline and post-treatment, and biomarkers in tumor specimens, blood, and serum/plasma are tested.
  • the six groups of patients are assigned and treated as below.
  • Group 1 Construct 1 as an ongoing IV administration in patients with HPV 16 + HNSCC.
  • Group 2 Construct 1 IT-IV: Construct 1 as an IT administration for the first dose, followed by ongoing Construct 1 as an IV administration for the subsequent doses in patients with HPV 16 + cancers with a safe and accessible tumor site amenable for IT administration.
  • Group 3 Construct 2 IV and Construct 1 IV: Construct 2 as an IV administration (initial) and then followed by Construct 1 IV, alternating treatment on an ongoing basis in patients with HPV 16 + HNSCC.
  • Construct 2 IV and Construct 1 IV Construct 1 as an IT administration for the first dose.
  • Treatment is followed by Construct 2 IV, then followed by Construct 1 IV, alternating treatment on an ongoing basis in patients with HPV 16 + cancers with a safe and accessible tumor site amenable for IT administration. Only one tumor site for IT administration required.
  • Group 5 (3 doses of Construct 1 IV): Assess if 3 administrations at the highest doses evaluated induce a similar immunogenicity to E7/E6 than continuous dosing. The patient would have an option to receive another 3 doses of Construct 1 IV if they progressed radiologically.
  • Group 6 (3 doses of Construct 2 IV and Construct 1 IV each): Assess 3 administrations of Construct 1 and Construct 2 each, for a total of 6 administrations, in order to test if a limited number of administrations of the construct 1 and 2 therapeutic vaccines permit to reach a level of circulating T cells compatible with control of the disease and lasting after the last injection.
  • Patients from groups 1, 3, 5, and 6 are the ones who have HPV 16 + HNSCC with tumor progression or recurrence on standard of care therapy, including more than or equal to 1 systemic therapy.
  • Patients from groups 2 and 4 are the ones who have HPV 16 + cancers with a safe and accessible tumor site amenable for IT administration, who had tumor progression or recurrence on standard of care therapy, including more than or equal to 1 systemic therapy.
  • Selected backfill cohorts require “fresh paired biopsies” for the purpose of investigating effects of Construct 1 and/or 2 treatment on molecular signaling and tumor cell responses, identifying biomarkers that may be predictive of efficacy and response.
  • tumor material is used to quantify the levels of tumor infiltrated lymphocytes by immunohistochemistry staining. The analysis provides assessment of tumor infiltration of immune cells and particularly CD8 + T cells in the tumor.
  • a schematic of the backfill cohorts is presented in FIG. 16. Backfill cohorts keep the same number as the Construct 1 monotherapy dose cohort number and are further identified by the addition of a lowercase letter for each backfill cohort. As an example, after Construct 1 monotherapy dose Cohort 1 exploring Construct 1 at dose level 1 is declared safe, the backfill cohorts are named as Cohorts la, lb, lc, Id, and le.
  • the backfill cohorts may explore Construct 1 and/or Construct 2 treatment:
  • backfill cohorts -b and -d explore Construct 1 monotherapy in an every 2 week dose (q2w) administration schedule.
  • Backfill cohorts -c and -e explore Construct 1 monotherapy and/or Construct 2 / Construct 1 alternating 2-vector therapy at a dosing schedule of every 4 weeks from Cycles 1 to 4, and every 8 weeks starting on Cycle 5. This schedule is known as “q4w, q8w.” If “q2w” or “q4w, q8w” dosing schedule in the backfill cohorts are opened, DLTs are evaluated using the same process as the Dose Escalation cohorts.
  • Backfill cohorts -f and -h allow patients on pembrolizumab treatment who have since had disease progression to continue with their pembrolizumab and add Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2-vector therapy following the “q3w, q6w” schedule.
  • backfill cohorts -g and -i allow patients on pembrolizumab treatment who have since had disease progression to continue with their pembrolizumab and add Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2-vector therapy following the “q4w, q8w.”
  • Patients’ disease progression while on pembrolizumab monotherapy should be characterized as having refractory disease or resistance to pembrolizumab accordingly.
  • the definitions of refractory and resistant disease are: Patients with refractory disease (primary resistance) are defined as having progressed within ⁇ 6 months of the first dose of pembrolizumab monotherapy by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (vl.l).
  • SD stable disease
  • Secondary resistance Patients with resistance to pembrolizumab (secondary resistance) are defined as having progressed > 6 months after the first dose of pembrolizumab monotherapy. Exception: patients who have disease progression after discontinuation due to AEs, and who did not receive at least 6 months of pembrolizumab and had no evidence of initial clinical benefit would be best classified as having primary resistance.
  • Backfill cohort -j enrolls five HPV 16 + anal cancer patients receiving IV administration only to evaluate the efficacy of either Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2-vector therapy at the q3w, q6w dose schedule. To be eligible to participate in this backfill cohort(s), patients must meet the required inclusion and exclusion criteria.
  • Phase II Dose Expansion assesses Construct 1 monotherapy and/or Construct 2 / Construct 1 alternating 2-vector therapy at the RP2D that has been defined in the Phase I Dose Escalation.
  • the patients enrolled in groups A, B, D, and E are the ones with HPV 16 + HNSCC with tumor progression or recurrence on standard of care therapy, including more than or equal to 1 systemic therapy.
  • the patients enrolled in groups C and F are the ones with HPV 16 + cancers with a safe and accessible tumor site amenable for IT administration, who had tumor progression or recurrence on standard of care therapy, including more than or equal to 1 systemic therapy.
  • the primary efficacy endpoints in the Phase II Dose Expansion groups are the ORR and disease control rate based on RECIST and iRECIST.
  • the ORR by RECIST are summarized using the point estimate together with the exact two-sided 95% CIs according to the Clopper- Pearson method.
  • the secondary efficacy endpoints for the Phase II are the duration of response, PFS and OS.
  • Safety parameters e.g., types, frequency, and severity of AEs and SAEs
  • Tolerability parameters e.g., dose interruptions, reductions and dose intensity, and evaluations of laboratory values
  • the percentage change in target lesion tumor size from baseline is summarized using descriptive statistics and presented at each timepoint.
  • HPV 16+ HNSCC and HPV 16+ cancer of any origin e.g., cervical, anal, vaginal, vulvar, or penile cancers. Only patients meeting all the inclusion criteria and none of the exclusion criteria may be enrolled into the study. The below criteria apply to both Phase I Dose Escalation and Phase II Dose Expansion.
  • Patient must have > 1 measurable lesion by computed tomography (CT) and/or magnetic resonance imaging (MRI), that is assessed for tumor response following RECIST and iRECIST during study conduct.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • Prior curative radiation therapy must have been completed > 4 weeks prior to study treatment administration.
  • Prior focal palliative radiotherapy must have been completed > 2 weeks prior to study treatment administration.
  • Activated Partial Thromboplastin Time aPTT
  • Partial Thromboplastin Time PTT
  • subject is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of anticoagulants.
  • Patient must have had tumor progression or recurrence on standard of care therapy, including > 1 systemic therapy, (e.g., failed platinum-based therapy and/or anti -PD- 1 /anti-PD-Ll therapy) or be a patient for whom standard of care therapy is contraindicated.
  • > 1 systemic therapy e.g., failed platinum-based therapy and/or anti -PD- 1 /anti-PD-Ll therapy
  • Tumor tissue (archival [no older than 2 years] or able to provide fresh biopsy specimen during Screening) collected following the patient’s progression from the last treatment, unless agreed otherwise between the Sponsor and the Investigator.
  • Patient must have had tumor progression or recurrence on standard of care therapy, including > 1 systemic therapy, (e.g., failed platinum-based therapy and/or anti-PD-1/anti-PD-Ll therapy) or be a patient for whom standard of care therapy is contraindicated.
  • > 1 systemic therapy e.g., failed platinum-based therapy and/or anti-PD-1/anti-PD-Ll therapy
  • Tumor tissue (archival [no older than 2 years] or able to provide fresh biopsy specimen during Screening) collected following the patient’s progression from the last treatment, unless agreed otherwise between the Sponsor and the Investigator.
  • Tumor progression or recurrence on standard of care treatment including > 1 systemic therapy such as platinum-based chemotherapy, or be ineligible for standard of care therapy.
  • Tumor tissue may be obtained by image-guided biopsy, such as interventional radiology, according to the institution’s own guidelines and requirements for such procedures.
  • Tumor tissue may be obtained by image-guided biopsy, such as interventional radiology, according to the institution’s own guidelines and requirements for such procedures.
  • IT Construct 1 is then administered to the tumor site.
  • the patient Apart from the tumor site(s) amenable for biopsy and IT administration, the patient must have > 1 measurable lesion, that is assessed for tumor response following RECIST and iRECIST during study conduct.
  • Tumor tissue archival [no older than 2 years] or able to provide fresh biopsy specimen during Screening) collected following the patient’s progression from the last treatment, unless agreed otherwise between the Sponsor and the Investigator.
  • Patient must be eligible, as per package insert or Summary of Product Characteristics (SmPC), to receive pembrolizumab (i.e., naive to anti-PD- 1/anti-PD-Ll therapy and have progressed on platinum-based therapy).
  • pembrolizumab i.e., naive to anti-PD- 1/anti-PD-Ll therapy and have progressed on platinum-based therapy.
  • Tumor tissue (archival [no older than 2 years] or able to provide fresh biopsy specimen during Screening) collected following the patient’s progression from the last treatment, unless agreed otherwise between the Sponsor and the Investigator.
  • corticosteroids ⁇ 10 mg prednisone or equivalent
  • Concurrent malignancy that is clinically significant or requires active intervention at the time of Screening (with the exception of adequately treated, basal or squamous cell carcinoma, non-melanomatous skin cancer), unless agreed otherwise between the Sponsor and the Investigator.
  • Toxicity attributed to systemic prior anticancer therapy including radiation and surgery, other than alopecia and fatigue that has not resolved to Grade 1 or Baseline prior to the first administration of study treatment.
  • Patients with toxicities attributed to systemic prior anti cancer therapy which are not expected to resolve and result in long lasting sequelae, such as neuropathy or ototoxicity after platinum-based therapy, are permitted to enroll.
  • Immunosuppressive doses of systemic medication such as steroids or absorbed topical steroids (doses > 10 mg/day prednisone or equivalent), within 14 days of the first administration of study treatment.
  • Active autoimmune disease that has required systemic treatment in past 2 years (i.e., with use of disease modifying agents, corticosteroids, or immunosuppressive drugs).
  • Replacement therapy e.g., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.
  • Prior anaphylactic or other severe reaction to human immunoglobulin or antibody formulation administration Prior anaphylactic or other severe reaction to human immunoglobulin or antibody formulation administration. 9. Live vaccines received within 28 days prior to the first dose of study treatment, unless agreed otherwise between the Sponsor and Investigator.
  • hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) antibody indicating acute or chronic infection.
  • Patients who test positive for HCV antibody but negative for HCV ribonucleic acid (RNA) are permitted to enroll.
  • Group 1 (Intravenous Administration of Construct 1): From Cycles 1 to 4, Construct 1 is administered every 3 weeks. Starting at Cycle 5 and onwards, Construct 1 is administered every 6 weeks. This schedule is referred to as “q3w, q6w.” For Cycles 1 to 4, a treatment cycle is defined as a period of 21 days. Construct 1 doses is administered IV on Day 1 ( ⁇ 3 days) of each cycle. For Cycle 5 and subsequent cycles, a treatment cycle is defined as a period of 42 days. Construct 1 doses is administered IV on Day 1 ( ⁇ 7 days) of each cycle.
  • Group 2 Single Intratumoral Administration of Construct 1 followeded by Intravenous Administration of Construct 1: From Cycles 1 to 4, Construct 1 is administered every 3 weeks. Starting at Cycle 5 and onwards, Construct 1 is administered every 6 weeks. This schedule is referred to as “q3w, q6w.” For Cycles 1 to 4, a treatment cycle is defined as a period of 21 days. Treatment begins with a single IT administration of Construct 1 on Day 1 of Cycle
  • Construct 1 doses are administered IV on Day 1 ( ⁇ 3 days) of Cycle 2 and thereafter.
  • a treatment cycle is defined as a period of 42 days.
  • Construct 1 doses are administered IV on Day 1 ( ⁇ 7 days) of each cycle.
  • IT administration may be performed by image-guided procedures such as interventional radiology. Methodology of IT administration is as per institutional standard. If delivery of the total volume by direct IT administration is not technically feasible, the remaining Construct 1 volume should be delivered peritumorally and/or local administration (Section 5).
  • Patients have one lesion selected for biopsy and IT administration of Construct 1.
  • the lesion selected is not assessed for tumor response following RECIST and iRECIST (e.g., lesion can be followed individually for change in diameter but should not be included in the overall sum of diameters for RECIST assessment).
  • Group 3 Intravenous Administration of Construct 1 and Construct 2: For
  • Cycles 1 and 2 a treatment cycle is defined as a period of 42 days. Patients are administered first Construct 2, then followed by Construct 1, alternating treatment every 3 weeks (21 days), and have a window of ⁇ 3 days as follows: Construct 2 is administered IV on Day 1 of Cycles 1 and
  • Construct 1 is administered IV on Day 22 of Cycles 1 and 2.
  • a treatment cycle is defined as a period of 84 days. Cycle 3, Day 1 starts following the completion of Cycle 2, Day 42.
  • Construct 2 and Construct 1 doses are administered sequentially, alternating every 6 weeks (42 days), with a window of ⁇ 7 days, as follows: Construct 2 is administered IV on Day 1 of Cycle 3 and subsequent cycles. Construct 1 is administered IV on Day 43 of Cycle 3 and subsequent cycles. This schedule is referred to as “q3w, q6w.”
  • Group 4 Single IT Administration of Construct 1, followeded by Alternating Intravenous Administration of Construct 2 and Construct 1: Treatment begins with an initial IT administration of Construct 1 on Cycle 0, Day 1.
  • a treatment cycle is defined as a period of 42 days.
  • Construct 2 and Construct 1 doses are administered sequentially, alternating every 3 weeks (21 days), with a window of ⁇ 3 days, as follows: Construct 2 is administered IV on Day 1 of Cycles 1 and 2.
  • Construct 1 is administered IV on Day 22 of Cycles 1 and 2.
  • a treatment cycle is defined as a period of 84 days. Day 1 of Cycle 3 starts following the completion of Day 42 of Cycle 2.
  • Construct 2 and Construct 1 doses are administered sequentially, alternating every 6 weeks (42 days), with a window of ⁇ 7 days, as follows: Construct 2 is administered IV on Day 1 of Cycle 3 and subsequent cycles. Construct 1 is administered IV on Day 43 of Cycle 3 and subsequent cycles. This schedule is referred to as “q3w, q6w.”
  • the IT administration is similar to the one described in group 2 above.
  • Group 5 (Three Doses of Intravenous Administration of Construct 1): Treatment Group 5 explores administering 3 doses of Construct 1 monotherapy. Patients receive IV administration of Construct 1 every 3 weeks and stop after the third dose is received. This treatment plan is referred to as “3 -dose Construct 1.” A treatment cycle is defined as a period of 21 days (3 weeks). The 3 doses of Construct 1 are given 3 weeks apart on Day 1 ( ⁇ 3 days) of Cycles 1, 2, and 3. Tumor scan for efficacy assessment is performed every 42 days (6 weeks) starting from the first dose of Construct 1 administered. Tumor response is measured using RECIST until disease progression. Upon disease progression per RECIST, iRECIST is used to assess tumor response.
  • patient may receive another 3 doses of Construct 1 administered 3 weeks apart. Patients with disease progression during the 3 -dose regimen are not eligible to receive the additional 3 doses.
  • the efficacy assessment is re-baselined to RECIST. Tumor scan(s) continue every 42 days (6 weeks).
  • iRECIST is used to assess tumor response. Following disease progression per iRECIST, the patient proceeds to study EOT visit and complete the required assessments.
  • Group 6 (Three Doses of Intravenous Administration of Construct 2 and Construct 1): Treatment Group 6 explores administering 3 doses of Construct 2 / Construct 1 alternating 2-vector therapy. Patients receive 3 doses of Construct 2 and Construct 1 each, of which they receive 6 doses in total. A treatment cycle is defined as a period of 42 days. In each cycle, Construct 2 is administered first, followed by Construct 1, in an alternating manner. Each dose is given 3 weeks apart, with a window of ⁇ 3 days as follows: Construct 2 is administered IV on Day 1 of Cycle 1, 2, and 3. Construct 1 is administered IV on Day 22 of Cycle 1, 2, and 3. This treatment plan is referred to as “3 -dose Construct 1 & Construct 2.”
  • Tumor scan for efficacy assessment is every 42 days starting from the first dose of Construct 2 administered. Tumor response is measured using RECIST until disease progression. Upon radiological progression defined by RECIST or iRECIST and after the patient has received the full 3-dose regimen, another “3-dose Construct 2 & Construct 1” treatment may be given. Patients with disease progression during the “3-dose Construct 2 & Construct 1” regimen would not be eligible to receive the additional 3 doses. The efficacy assessment is re-baselined to RECIST. Tumor scan(s) continue every 42 days (6 weeks). Upon disease progression per RECIST, iRECIST is used to assess tumor response. Following disease progression per iRECIST, the patient proceeds to study EOT visit and complete the required assessments.
  • the dosing schedule for Phase II Dose Expansion is similarly selected based on the review of the available data from the safety, efficacy, and/or biomarker results of the Dose Escalation Treatment Groups.
  • the study treatment dosing schedule for Dose Expansion could be one of the following:
  • Construct 1 and/or 2 study treatment administered every 4 weeks from Cycles 1 to 4 and every 8 weeks starting in Cycles 5 and after. This schedule is referred to as “q4w, q8w”.
  • Group A Phase II Dose Expansion Group A of Construct 1 monotherapy can commence upon completion of Phase I Dose Escalation Group 1 (with determination of the RP2D of Construct 1 when administered IV).
  • a treatment cycle is defined as a period of 21 days: Construct 1 doses are administered IV on Day 1 ( ⁇ 3 days) of each cycle.
  • a treatment cycle is defined as a period of 42 days: Construct 1 doses are administered IV on Day 1 ( ⁇ 7 days) of each cycle.
  • Group B Intravenous Administration of Construct 1 and Pembrolizumab:
  • Phase II Dose Expansion Group B of Construct 1 monotherapy and pembrolizumab can commence upon completion of Phase I Dose Escalation Group 1 (with determination of the RP2D of Construct 1 when administered IV).
  • the patient are receiving Construct 1 monotherapy and pembrolizumab.
  • a treatment cycle is defined as a period of 21 days.
  • Construct 1 doses are administered IV only on Day 1 ( ⁇ 3 days) of each cycle.
  • a treatment cycle is defined as a period of 42 days.
  • Construct 1 is administered IV on Day 1 ( ⁇ 7 days) of each cycle.
  • Pembrolizumab is administered on a q3w or q6w schedule, overlapping with study visits.
  • Group C Single IT Administration of Construct 1 followeded by Intravenous Administration of Construct 1: Phase II Dose Expansion Group C of Construct 1 monotherapy can begin upon completion of Phase I Dose Escalation Group 2 (with determination of the RP2D of Construct 1 IV and IT).
  • a treatment cycle is defined as a period of 21 days.
  • Treatment begins with a single IT administration of Construct 1 on Day 1 of Cycle 1.
  • Subsequent Construct 1 doses are administered IV on Day 1 ( ⁇ 3 days) of each cycle.
  • a treatment cycle is defined as a period of 42 days.
  • Construct 1 doses are administered IV on Day 1 ( ⁇ 7 days) of each cycle.
  • IT administration may be performed by image-guided procedures such as interventional radiology. Methodology of IT administration is as per institutional standard. Ideally, all the volume should be delivered via direct IT administration. If delivery of the total volume by direct IT administration is not technically feasible, the remaining Construct 1 volume should be delivered peritumorally and/or local administration (see Section 5).
  • Patients have one lesion selected for biopsy and IT administration of Construct 1.
  • the lesion selected is not assessed for tumor response following RECIST and iRECIST (e.g., lesion can be followed individually for change in diameter but should not be included in the overall sum of diameters for RECIST assessment). All other lesions do not receive Construct 1 or be biopsied.
  • Group D (Sequential Alternating Intravenous Administrations of Construct 2 and Construct 1): Phase II Dose Expansion Group D of Construct 2 / Construct 1 alternating 2- vector therapy can begin upon completion of the Phase I Dose Escalation Group 3 (with determination of the RP2D of Construct 2 when administered IV with Construct 1 in a sequential alternating schedule).
  • a treatment cycle is defined as a period of 42 days. Treatment begins with IV administration of Construct 2 on Day 1 of Cycle 1 , followed by Construct 1 alternating every 3 weeks (21 days) as specified below. Construct 2 and Construct 1 dose administrations have a window of ⁇ 3 days. Construct 2 is administered IV on Day 1 of Cycles 1 and 2.
  • Construct 1 is administered IV on Day 22 of Cycles 1 and 2.
  • a treatment cycle is defined as a period of 84 days.
  • Day 1 of Cycle 3 starts following the completion of Day 42 of Cycle 2.
  • Construct 2 and Construct 1 dose administrations in Cycle 3 and subsequent cycles have a window of ⁇ 7 days.
  • Construct 2 and Construct 1 doses alternate every 6 weeks (42 days) as follows: Construct 2 is administered IV on Day 1 of Cycle 3 and subsequent cycles.
  • Construct 1 is administered IV on Day 43 of Cycle 3 and subsequent cycles.
  • Group E (Sequential Alternating Intravenous Administration of Construct 2 and Construct 1, and Pembrolizumab) : Phase II Dose Expansion Group E Construct 2 / Construct 1 alternating 2-vector therapy and pembrolizumab can begin upon completion of the Phase I Dose Escalation Group 3 (with determination of the RP2D of Construct 2 when administered IV with Construct 1 in a sequential alternating schedule).
  • a treatment cycle is defined as a period of 42 days. Treatment begins with IV administration of Construct 2 on Day 1 of Cycle 1.
  • Construct 2 and Construct 1 dose administrations in Cycles 1 and 2 have a window of ⁇ 3 days.
  • Construct 2 and Construct 1 Patients are administered the first two doses of Construct 2 and Construct 1 alternating every 3 weeks (21 days) as follows: Construct 2 is administered IV on Day 1 of Cycles 1 and 2. Construct 1 is administered IV on Day 22 of Cycles 1 and 2. For Cycle 3 and subsequent cycles, a treatment cycle is defined as a period of 84 days. Day 1 of Cycle 3 starts following the completion of Day 42 of Cycle 2. Construct 2 and Construct 1 dose administrations in Cycle 3 and subsequent cycles have a window of ⁇ 7 days. Construct 2 and Construct 1 doses alternate every 6 weeks (42 days) as follows: Construct 2 is administered IV on Day 1 of Cycle 3 and subsequent cycles. Construct 1 is administered IV on Day 43 of Cycle 3 and subsequent cycles. Pembrolizumab is administered on a q3w or q6w schedule, overlapping with study visits.
  • Group F Intratumoral Administration of Construct 1 followeded by Sequential Alternating Intravenous Administrations of Construct 2 and Construct 1:
  • Phase II Dose Expansion Group F can begin upon completion of the Phase I Dose Escalation Group 4 (with determination of the RP2D of Construct 1 when administered IT and followed by a sequential alternating schedule of the RP2D of Construct 2 and Construct 1 when administered IV).
  • Treatment begins with an initial IT administration of Construct 1 on Day 1 of Cycle 0.
  • IT administration may be guided by image guided procedures such as interventional radiology.
  • 21 days later treatment continues with IV administration of Construct 2 on Day 1 of Cycle 1 and IV administration of Construct 1 on Day 22.
  • all the volume should be delivered via direct IT administration. If delivery of the total volume by direct IT administration is not technically feasible, the remaining Construct 1 volume should be delivered peritumorally and/or local administration (see Section 5).
  • a treatment cycle is defined as a period of 42 days.
  • Construct 2 and Construct 1 dose administrations in Cycle 2 have a window of ⁇ 3 days.
  • Patients are administered Construct 2 and Construct 1, alternating every 3 weeks (21 days) as follows: Construct 2 is administered IV on Day 1 of Cycles 1 and 2.
  • Construct 1 is administered IV on Day 22 of Cycles 1 and 2.
  • a treatment cycle is defined as a period of 84 days. Day 1 of Cycle 3 starts following the completion of Day 42 of Cycle 2.
  • Construct 2 and Construct 1 dose administrations in Cycle 3 and subsequent cycles have a window of ⁇ 7 days.
  • Construct 2 and Construct 1 doses alternate every 6 weeks (42 days) as follows: Construct 2 is administered IV on Day 1 of Cycle 3 and subsequent cycles. Construct 1 is administered IV on Day 43 of Cycle 3 and subsequent cycles. IT administration is similar to the one in group C above.
  • pembrolizumab can be added to the Construct 1 and/or 2 treatment. Eligibility to receive pembrolizumab should be assessed using inclusion and exclusion criteria pertaining to the pembrolizumab cohorts (Refer to Section 3). Dosing of pembrolizumab should overlap with study visits and follow the q3w or q6w schedule depending on the Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2- vector therapy assigned treatment schedule.
  • the total volume of Construct 1 for IT administration depends on the provisional Construct 1 dose prescribed ( see Table 3).
  • One lesion and/or site of disease is selected for Construct 1 IT administration. This should be the same lesion/site of disease that was selected for biopsy pre- and post Construct 1 IT administration.
  • all the volume should be delivered via direct IT administration. If delivery of the total volume by direct IT administration is not technically feasible, the remaining Construct 1 volume should be delivered peritumorally and/or local administration for the primary purpose of treating one specific lesion or site of disease. To ensure the entire volume of Construct 1 dose prescribed is administered, the following types of administrations are allowed:
  • the delivery of the total IT volume may occur via one injection or via more than one injection, or via one injection and multiple re-positioning of the needle without withdrawal, or a combination.
  • Pembrolizumab should be administered per institutional guidelines or per standard of care, such as the appropriate KEYTRUDA® SmPC or Package Insert.
  • Table 3 below describes the Construct 1 starting dose and the dose levels that may be evaluated during Phase I Dose Escalation (Groups 1 and 2).
  • Phase I Dose Escalation Group 1 Construct 1 IV only
  • the starting dose of Construct 1 starts at 5 x 10 5 RCV FFU.
  • the subsequent dose of Construct 1 is increased to the next sequential dose level as listed in Table 3.
  • Phase I Dose Escalation Group 2 Construct 1 IT-IV
  • the starting dose for IT administration of Construct 1 starts at 5 x 10 5 RCV FFU.
  • the starting dose for IV administration also starts at 5 x 10 5 RCV FFU.
  • the subsequent doses of Construct 1 for IT and IV administration are the same and both are increased to the next sequential dose level as listed in Table 3.
  • the dose can start one log order up from highest dose level declared safe in Group 1.
  • the Construct 1 dose level Group 5 can explore is 5 x 10 7 RCV FFU.
  • the subsequent dose level of Construct 1 is increased to the next sequential dose level as listed in Table 3
  • FFU focus-forming units
  • RCV replication-competent virus
  • the proposed human starting dose of Construct 2 is 1 x 10 6 RCV FFU.
  • the proposed human starting dose of Construct 1 is the highest dose level declared safe in Group 1 or Group 2. If RP2D is declared for Construct 1 from monotherapy, then Construct 1 dose level in Construct 2 / Construct 1 alternating 2-vector therapy remains at the RP2D, while Construct 2 provisional levels are explored. Table 4 describes the starting dose for the Construct 2 / Construct 1 alternating 2-vector therapy and the dose levels that may be evaluated during Phase I Dose Escalation (Groups 3 and 4).
  • the dose of Construct 2 and Construct 1 can start one log order up from highest dose level declared safe in Group 3.
  • the dosages for Group 6 can be at 5 x 10 7 RCV FFU for Construct 1 and 1 x 10 7 RCV FFU for Construct 2.
  • the subsequent dose levels of Construct 2 and Construct 1 are increased to the next sequential dose level as listed in Table 4.
  • FFU focus-forming units
  • RCV replication-competent virus
  • RP2D recommended Phase II dose.
  • the Construct 1 or Construct 2 dose explored in the cohort may be a dose level lower than indicated in the provisional dose table.
  • the dose of Construct 1 in Cohort 1 is the RP2D determined in Groups 1 and 2, or the highest dose determined to be safe if the RP2D has not been reached.
  • Pembrolizumab is administered on a 200mg once every 3 weeks or 400mg once every 6 weeks schedule for Groups B and E of Phase II.
  • Efficacy is assessed utilizing CT or MRI scans of the chest/ abdomen/ pelvis and all suspected anatomic regions involved with the disease and are performed to assess tumor response.
  • CT modality is mandatory. Ultrasound should not be used to measure sites of disease. If a CT/MRI scan is scheduled on the same day as study treatment administration, the CT/MRI should be performed prior to dosing.
  • CR Complete response
  • PR Partial response
  • samples from saliva, feces (e.g., fecal swab), blood, and urine are collected for viral shedding analysis.
  • Viral shedding is analyzed by quantitative reverse transcription PCR to quantify the copies of nucleoprotein RNA, and may be coupled with infectivity assay to characterize the shed material to confirm absence of infectious virus.
  • samples collected should not be from areas potentially containing viable cancer cells.
  • blood samples including serum and plasma are collected for phenotypic, genomic, proteomic, and transcriptional analyses.
  • Blood samples are collected for phenotypic characterization of lymphocyte subsets.
  • Messenger RNA expression profiling in blood is performed to evaluate gene signatures associated with clinical response and/or resistance.
  • Plasma is collected for circulating tumor DNA assessment.
  • Neutralizing and binding antibodies against study treatment essentially, E7E6 fusion protein
  • pro inflammatory, Thl/Th2 cytokines such as IL 1, IL 12, and IL-18, TNF, and IFN- ⁇ are assessed in serum.
  • IHC immunohistochemistry
  • MHC major histocompatibility complex
  • RNA ribonucleic acid
  • TCR T cell receptor
  • TIL tumor-infiltrating lymphocyte
  • Tumor tissue samples are collected with the purpose of investigating effects of Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy on molecular signaling and tumor cell responses, identifying biomarkers that may be predictive of efficacy and response.
  • Tumor tissue are obtained by image-guided biopsy, such as interventional radiology. All patients submit tissue from either a core or excisional biopsy (fine needle aspirate not accepted) to central laboratory for biomarker assessment.
  • Next generation sequencing provides the opportunity to assess at the genetic level in the tumor.
  • Next generation biomarkers such as gene expression profile signatures by RNA sequencing, microsatellite instability, tumor mutational burden, and human leukocyte antigen loss of heterozygosity are examined in tumor tissues obtained from patients to understand the potential biomarker of clinical response and/or resistance.
  • tumor material is used to quantify the levels of tumor infiltrated lymphocytes by immunohistochemistry staining.
  • the analysis provide assessment of tumor infiltration of immune cells and particularly CD8 + T cells in the tumor.
  • Tumor samples are collected at Screening or on the day of their first study drug administration and post dose. Fresh tumor biopsy should be provided, if accessible. If tumor biopsy and CT/MRI scan are performed on the same visit, the CT/MRI scan should be performed first, followed by the tumor biopsy.
  • paired fresh tumor biopsies are required prior to first study drug administration and post dose before the first efficacy assessment CT/MRI scan for participation in the backfill cohorts.
  • Tissue samples must be newly obtained from either a core or excision biopsy (fine needle aspirate not accepted) for biomarker assessment.
  • submission of the tumor block with largest tumor focus (minimum of two cores) or highest tumor cellularity is required. Tumor blocks of resection/excision specimens are preferred over slides. 9.3 Other Exploratory Biomarker Assessments
  • exploratory biomarker research may be conducted on any tumor tissue, serum/plasma, and peripheral mononuclear cells (PBMC) samples collected during the example.
  • PBMC peripheral mononuclear cells
  • additional exploratory biomarkers include, tetramer sorted antigen specific T cell profiling, T cell receptor sequencing and chromatin changes on antigen specific T cells.
  • Immunogenicity testing is done in all patients to monitor patients’ CD8 + T cells functionality and antigen recognition by measuring IFN- ⁇ , TNF- ⁇ , IL-2, CD 107a via intracellular staining and secreted IFN- ⁇ specific cells in peripheral blood mononuclear cells as an antigen specific immune response against Construct 1 and/or Construct 2, with and without pembrolizumab (see Table 7).
  • CD4 cluster of differentiation 4
  • CD8 cluster of differentiation 8
  • E7E6 antigenic E7 and E6 fusion protein from human papillomavirus 16
  • ELISpot enzyme-linked immune absorbent spot
  • ICS intracellular cytokine staining
  • IFN- ⁇ interferon-gamma
  • IL-2 interleukin-2
  • LMCV lymphocytic choriomeningitis virus
  • NP nucleoprotein
  • PBMC peripheral blood mononuclear cell
  • PICV Pichinde Virus
  • TNF- ⁇ tumor necrosis factor alpha.
  • the aim of this exploratory immune imaging objective is to capture the distribution and influx of CD8 + cells into tumor tissues upon treatment with Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2-vector therapy.
  • the distribution of CD8 + cells by assessing whole body PET/CT images using CD8 PET Tracer is measured to evaluate changes before and after treatment with Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2-vector therapy.
  • Clinical outcome is correlated through quantification of CD8 PET Tracer signal.
  • evaluating the change in CD8 PET Tracer signal before and after treatment is used to predict treatment efficacy, and true radiological progression and pseudo- progression during the early phase of Construct 1 monotherapy and Construct 2 / Construct 1 alternating 2-vector therapy are also distinguished.
  • a sub-study is carried out to include exploratory immune imaging with positron emission tomography (PET)/CT scan to assess 89 Zr-Df-IAB22M2C (CD 8 PET Tracer, an anti- CD 8 minibody (IAB22M2C), conjugated with deferoxamine (Df) and radiolabeled with Zirconium-89 ( 89 Zr-Df-IAB22M2C)) in patients with HNSCC receiving Construct 1 monotherapy or Construct 2 / Construct 1 alternating 2- vector therapy.
  • a dose of 1.0 ( ⁇ 20%) mCi of CD8 PET Tracer 1.5mg of API is administered IV over 5-10 minutes.
  • CD 8 PET Tracer uptake in tumors is determined by standardized uptake value (SUV)-based quantitative measures (SUVmax, SUVpeak, SUVmean, CD8 tumor volume). Volume of tumor tissues with increased CD8 uptake with SUV > 20% SUVmax is quantified.
  • SUV uptake value
  • PET/CT scans PET Baseline and PET Post-Treatment are obtained at 24 ⁇ 3 hours after each infusion of CD 8 PET Tracer.
  • FIG. 17C demonstrate superiority of intravenous (IV) over intratumoral (IT) administration. Furthermore, a three-weeks interval between vector administrations appeared to be superior to vectors being administered every two weeks. Data further suggest a superior anti-tumor effect in patients treated with the Construct 2 / Construct 1 alternating 2-vector therapy as compared to patients receiving the Construct 1 single vector treatment at the same dose. As illustrated in FIG. 17D and Table 9, a significant subset of patients demonstrated stable disease following arenaviral vector therapy. Several endpoints, such as response rate, disease control rate and progression-free survival, of patients receiving arenaviral vector therapy are summarized in Table 9 below. Overall, objective response rate was 6.9% with 2 patients achieving partial response and 16 patients experiencing stable disease.
  • FIG. 17E shows the progression-free survival for all HNSCC patients.
  • ORR objective response rate
  • PR partial response
  • SD stable disease
  • PD progressive disease
  • DCR disease control rate
  • PFS progression-free survival.
  • PFS includes time after pembrolizumab had been added prior to RECIST progression.
  • FIG. 18A as an updated FIG. 6 A, demonstrates distinct serum cytokine or chemokine signatures after administration of Construct 1. Increased IFN- ⁇ levels are already observed on day 4 after the first treatment in 90% of analyzed patients. Besides IFN- ⁇ other immune stimulatory cytokines and chemokines are also upregulated in treated patients demonstrating early signs of NK and T cell activation. E.g., a single dose of Construct 1 increased levels of IFN- ⁇ , IFN-inducible protein (IP)- 10, interleukin (IL)-12p40, IL-15 and tumor necrosis factor TNF- ⁇ in patients at day 4 after treatment.
  • FIG. 18B as an updated FIG.
  • FIG. 6B shows increased levels of IFN- ⁇ , IL-12p40, IL-15, IFN-inducible protein (IP)- 10, and TNF ⁇ in patients on the 4 th day after treatment with Construct 1 monotherapy at DL 1 (5 x 10 5 RCV FFU) or DL2 (5 x 10 6 RCV) or Construct 2 / Construct 1 alternating 2-vector therapy (Construct 2: 1x10 6 RCV FFU, Construct 1: 5 x 10 6 RCV FFU).
  • FIG. 19D shows an increase of circulating total CD8 + T cells.
  • FIG. 19F shows an increase in functional and cytotoxic E6/E7-specific CD8 T Cells that expressed IFN- ⁇ , TNF ⁇ , or CD 107a in the same patient. Production of IFN- ⁇ , TNF ⁇ , or CD 107a demonstrates that induced E6/E7- specific CD8 T cells are multifunctional and not exhausted.
  • FIG. 19G to FIG. 191 further show that treatment with either Construct 1 alone or Construct 2 / Construct 1 alternating 2-vector therapy induces substantial antigen-specific T cell responses in patients, with up to 40% of circulating CD 8+ T cells being E6/E7 specific. IFN- ⁇ and TNF- ⁇ production indicates that the respective T cells are not exhausted. Furthermore, expansion of E6/E7-specific CD8+ T cells in patients mirror the results observed in murine models.
  • Construct 1 and Construct 2 induced type 1 cytokine secretion in serum.
  • Direct IFN- ⁇ ELISpot and ICS performed without prior in-vitro expansion captured high- magnitude T-cell responses.
  • Single doses of Construct 1 and Construct 2, respectively were capable of driving strong E6/E7 specific CD8 T cells with up to 9.9% IFN- ⁇ + CD8 T cells.
  • three patients after a single dose of Construct 1 produced more than 3% antigen specific CD8 + T cells.
  • One patient after a single dose of Construct 2 responded with about 10% antigen specific CD8 + T cells.
  • Construct 2 / Construct 1 alternating 2-vector therapy achieved unprecedented 40% of E6/E7 specific IFN- ⁇ +CD8 T cells, which is the highest response ever reported for an in-vivo therapy such as a cancer vaccine by a wide margin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/EP2021/062728 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors Ceased WO2021239471A1 (en)

Priority Applications (12)

Application Number Priority Date Filing Date Title
US17/928,098 US20230346906A1 (en) 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors
JP2022573287A JP2023527083A (ja) 2020-05-29 2021-05-12 アレナウイルスベクターを使用するがんの治療方法
CR20220602A CR20220602A (es) 2020-05-29 2021-05-12 Estrategias de tratamiento del cáncer mediante el uso de vectores de arenavirus
MX2022014725A MX2022014725A (es) 2020-05-29 2021-05-12 Estrategias de tratamiento del cáncer mediante el uso de vectores de arenavirus.
IL298420A IL298420A (en) 2020-05-29 2021-05-12 Cancer treatment strategies using RNA virus vectors
KR1020227044315A KR20230046278A (ko) 2020-05-29 2021-05-12 아레나바이러스 벡터를 이용한 암 치료 전략
CN202180049174.5A CN116490205A (zh) 2020-05-29 2021-05-12 使用沙粒病毒载体的癌症治疗策略
AU2021282287A AU2021282287A1 (en) 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors
CA3184791A CA3184791A1 (en) 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors
EP21725519.9A EP4157342A1 (en) 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors
BR112022024404A BR112022024404A2 (pt) 2020-05-29 2021-05-12 Método para tratar câncer, sequência de nucleotídeos, célula hospedeira, partícula de lcmv, partícula de picv, composição farmacêutica, partícula de arenavírus
PE2022002786A PE20240647A1 (es) 2020-05-29 2021-05-12 Estrategias de tratamiento del cancer mediante el uso de vectores de arenavirus

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202063032362P 2020-05-29 2020-05-29
US63/032,362 2020-05-29
US202163173155P 2021-04-09 2021-04-09
US63/173,155 2021-04-09
US202163175842P 2021-04-16 2021-04-16
US63/175,842 2021-04-16

Publications (2)

Publication Number Publication Date
WO2021239471A1 true WO2021239471A1 (en) 2021-12-02
WO2021239471A9 WO2021239471A9 (en) 2022-01-13

Family

ID=75914543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2021/062728 Ceased WO2021239471A1 (en) 2020-05-29 2021-05-12 Cancer treatment strategies using arenavirus vectors

Country Status (12)

Country Link
US (1) US20230346906A1 (https=)
EP (1) EP4157342A1 (https=)
JP (1) JP2023527083A (https=)
KR (1) KR20230046278A (https=)
AU (1) AU2021282287A1 (https=)
BR (1) BR112022024404A2 (https=)
CA (1) CA3184791A1 (https=)
CR (1) CR20220602A (https=)
IL (1) IL298420A (https=)
MX (1) MX2022014725A (https=)
PE (1) PE20240647A1 (https=)
WO (1) WO2021239471A1 (https=)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11401528B2 (en) 2007-12-27 2022-08-02 Universität Zürich Replication-defective arenavirus vectors
US11407790B2 (en) 2015-06-10 2022-08-09 Hookipa Biotech Gmbh HPV vaccines
US11554169B2 (en) 2013-12-03 2023-01-17 Hookipa Biotech Gmbh CMV vaccines
WO2023152116A1 (en) * 2022-02-08 2023-08-17 Hookipa Biotech Gmbh Combination therapy with arenavirus particles and immune checkpoint modulators or cytokines
WO2025056782A1 (en) 2023-09-15 2025-03-20 Hookipa Biotech Gmbh Arenavirus formulations, methods and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016198531A2 (en) * 2015-06-10 2016-12-15 Hookipa Biotech Ag Hpv vaccines
US20170319673A1 (en) 2014-11-13 2017-11-09 Université De Genève Tri-segmented arenaviruses as vaccine vectors
WO2018185307A1 (en) * 2017-04-07 2018-10-11 Hookipa Biotech Ag Arenavirus particles to treat solid tumors
US20180344830A1 (en) 2015-11-12 2018-12-06 Hookipa Biotech Ag Arenavirus particles as cancer vaccines
US20190135875A1 (en) 2016-05-18 2019-05-09 Hookipa Biotech Gmbh Tri-segmented pichinde viruses as vaccine vectors

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170319673A1 (en) 2014-11-13 2017-11-09 Université De Genève Tri-segmented arenaviruses as vaccine vectors
WO2016198531A2 (en) * 2015-06-10 2016-12-15 Hookipa Biotech Ag Hpv vaccines
US20180179257A1 (en) 2015-06-10 2018-06-28 Hookipa Biotech Ag Hpv vaccines
US20180344830A1 (en) 2015-11-12 2018-12-06 Hookipa Biotech Ag Arenavirus particles as cancer vaccines
US20190135875A1 (en) 2016-05-18 2019-05-09 Hookipa Biotech Gmbh Tri-segmented pichinde viruses as vaccine vectors
WO2018185307A1 (en) * 2017-04-07 2018-10-11 Hookipa Biotech Ag Arenavirus particles to treat solid tumors
US20200113995A1 (en) 2017-04-07 2020-04-16 Hookipa Biotech Gmbh Arenavirus particles to treat solid tumors

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
ALTMAN J.D. ET AL., SCIENCE, vol. 274, 1996, pages 94 - 96
ANONYMOUS: "History of Changes for Study: NCT04180215: A Phase I/II Study of TheraT Vector(s) Expressing Human Papillomavirus 16 Positive (HPV 16+) Specific Antigens in 16+ Confirmed Cancers (version 7)", 7 May 2020 (2020-05-07), XP055826375, Retrieved from the Internet <URL:https://www.clinicaltrials.gov/ct2/history/NCT04180215?V_7=View#StudyPageTop> [retrieved on 20210721] *
BONILLA W ET AL., CELL REP MED, vol. 2, no. 3, 2021, pages 1 - 17
BURMAN BHARAT ET AL: "A phase I/II study of HB-201, an arenavirus-based cancer immunotherapy, alone, or in combination with anti-PD-1 in patients with HPV16+ cancers", JOURNAL OF CLINICAL ONCOLOGY, 25 May 2020 (2020-05-25), XP055826341, Retrieved from the Internet <URL:https://ascopubs.org/doi/10.1200/JCO.2020.38.15_suppl.TPS3171> [retrieved on 20210721] *
CASSETTI ET AL., VACCINE, vol. 22, no. 3-4, 2004, pages 520 - 27
CZERKINSKY C.C. ET AL., J IMMUNOL METHODS, vol. 65, 1983, pages 109 - 121
DONG Z ET AL., FRONT IMMUNOL, vol. 11, 2021, pages 586796
GHANEKAR S.A. ET AL., CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, vol. 8, 2001, pages 628 - 63
HUTCHINGS P.R. ET AL., J IMMUNOL METHODS, vol. 120, 1989, pages 1 - 8
KALLERT ET AL., NAT COMMUN, vol. 8, 2017, pages 15327
KALLERT SANDRA M. ET AL: "Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy", NATURE COMMUNICATIONS, vol. 8, no. 1, 26 May 2017 (2017-05-26), XP055772613, Retrieved from the Internet <URL:http://www.nature.com/articles/ncomms15327> DOI: 10.1038/ncomms15327 *
MARTEL C ET AL., INT J CANCER, vol. 141, 2017, pages 664 - 670
MURALI-KRISHNA K. ET AL., IMMUNITY, vol. 8, 1998, pages 177 - 187
NOMURA L.E. ET AL., CYTOMETRY, vol. 40, 2000, pages 60 - 68
SCHMIDT S ET AL., ONCOIMMUNOLOGY, vol. 9, no. 1, 2020, pages 1809960
SELINA KHAN ET AL: "Development of a replication-deficient adenoviral vector-based vaccine candidate for the interception of HPV16- and HPV18-induced infections and disease : Development of a HPV16/18 specific Adenovirus vector based vaccine", INTERNATIONAL JOURNAL OF CANCER, vol. 141, no. 2, 15 July 2017 (2017-07-15), US, pages 393 - 404, XP055624376, ISSN: 0020-7136, DOI: 10.1002/ijc.30679 *
SUNI M.A. ET AL., J IMMUNOL METHODS, vol. 212, 1998, pages 89 - 98
TRAPIDO ET AL., AM J TROP MED HYG, vol. 20, 1971, pages 631 - 641
TRICKETT ET AL., J IMMUNOL METHODS, vol. 275, no. 1-2, 1 April 2003 (2003-04-01), pages 251 - 5

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11401528B2 (en) 2007-12-27 2022-08-02 Universität Zürich Replication-defective arenavirus vectors
US12227755B2 (en) 2007-12-27 2025-02-18 Universität Zürich Replication-defective arenavirus vectors
US11554169B2 (en) 2013-12-03 2023-01-17 Hookipa Biotech Gmbh CMV vaccines
US11407790B2 (en) 2015-06-10 2022-08-09 Hookipa Biotech Gmbh HPV vaccines
WO2023152116A1 (en) * 2022-02-08 2023-08-17 Hookipa Biotech Gmbh Combination therapy with arenavirus particles and immune checkpoint modulators or cytokines
WO2025056782A1 (en) 2023-09-15 2025-03-20 Hookipa Biotech Gmbh Arenavirus formulations, methods and uses thereof

Also Published As

Publication number Publication date
IL298420A (en) 2023-01-01
JP2023527083A (ja) 2023-06-26
AU2021282287A1 (en) 2023-01-05
WO2021239471A9 (en) 2022-01-13
KR20230046278A (ko) 2023-04-05
EP4157342A1 (en) 2023-04-05
CR20220602A (es) 2023-05-16
PE20240647A1 (es) 2024-04-04
MX2022014725A (es) 2023-02-09
BR112022024404A2 (pt) 2023-02-07
CA3184791A1 (en) 2021-12-02
US20230346906A1 (en) 2023-11-02

Similar Documents

Publication Publication Date Title
US20230346906A1 (en) Cancer treatment strategies using arenavirus vectors
Yarchoan et al. Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial
Galon et al. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies
US11065285B2 (en) Biomarkers and combination therapies using oncolytic virus and immunomodulation
AU2017268822B2 (en) Neoepitope vaccine compositions and methods of use thereof
Brahmer et al. JNJ-64041757 (JNJ-757), a live, attenuated, double-deleted Listeria monocytogenes–based immunotherapy in patients with NSCLC: results from two phase 1 studies
EP4031573A1 (en) Combination of a poxvirus encoding hpv polypeptides and il-2 with an anti-pd-l1 antibody
Pham et al. First-in-human phase I clinical trial of a combined immune modulatory approach using TetMYB vaccine and Anti-PD-1 antibody in patients with advanced solid cancer including colorectal or adenoid cystic carcinoma: The MYPHISMO study protocol (NCT03287427)
JP2021516957A (ja) パラポックスウイルスベクター
US20240047009A1 (en) Nant cancer vaccine strategies
AU2016263147A1 (en) Recombinant oncolytic viruses and uses thereof
TW201038943A (en) Biomarker for monitoring patients
CN116490205A (zh) 使用沙粒病毒载体的癌症治疗策略
Tan et al. 269 Tumoral and peripheral landscape of Viral-versus Carcinogen-Driven Head and Neck Cancer
Rossi et al. Therapeutic vaccines targeting HPV epitopes in human papillomavirus–positive oropharyngeal cancer: a critical review
JP2025500258A (ja) 変異型ｃａｌｒ及びｊａｋ２に基づくワクチン並びにこれらの使用
US20150177241A1 (en) Biomarkers of immune response in mucosal lesions and their use with therapeutic vaccination
Hellner et al. A Phase 1b/2, Randomised, Placebo-controlled Study to Evaluate Safety, Tolerability, Efficacy, and Immunogenicity of VTP-200, a Chimpanzee Adenovirus and Modified Vaccinia Ankara-vectored Multigenotype High-Risk Human Papillomavirus Vaccine in Women with Low-grade Cervical Lesions
Makrinos et al. A multivalent mRNA-LNP therapeutic vaccine with broad cross-genotype immunogenicity elicits clearance of HBV infected hepatocytes
AU2024309282A1 (en) Engineered chimeric fusion protein compositions and methods of use thereof
WO2026046208A1 (zh) 中性粒细胞数在预测溶瘤病毒治疗肿瘤的疗效中的应用
WO2024138754A1 (zh) 一种hla-a*11:01限制性抗原点替换方法、获得的多肽及其应用
WO2025149656A1 (en) Treatment and prevention of lung cancer
JP2023539332A (ja) 個別化された免疫原性組成物及びその製造方法及びその使用
CN120137934A (zh) 一种aurkc抗原hla-a2限制性ctl表位肽及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21725519

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3184791

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022573287

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022024404

Country of ref document: BR

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021282287

Country of ref document: AU

Date of ref document: 20210512

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2021725519

Country of ref document: EP

Effective date: 20230102

WWE Wipo information: entry into national phase

Ref document number: 202180049174.5

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 112022024404

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20221129